25/03/2015 In Vitro Scientist ­ Cambridge job with Proximagen Limited | 1401561216

http://jobs.newscientist.com/en­gb/job/1401561216/in­vitro­scientist­cambridge/?TrackID=1345&cmpid=PRC|JOBS|NSNJJ­INDEED­SPONSORED­LIFE 1/3

In Vitro Scientist ­Cambridge

Recruiter

ProximagenLimited

Location

Cambridge,Cambridgeshire, UK

Salary Highlycompetitivepackage

Posted 23 Mar 2015

Closes 13 Apr 2015

ResultType

Jobs

Discipline ,

,,

LifeSciencesBiochemistryCell BiologyMolecularBiology

PositionType

Permanent

Hours Full Time

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In Vitro ScientistCompetitive salary plus benefitsPermanent

Proximagen is a drug discovery anddevelopment company focused ondeveloping small molecule therapeuticsaddressing diseases of the CNS.

Proximagen is a rapidly expanding businesswith research facilities near Cambridge andits head office in London. It provides anexciting and rewarding environment forindividuals wanting a challenging career in thepharmaceutical industry. We offer a highlycompetitive salary and benefits package.

Proximagen is looking to expand its biologylaboratory team at our site near Cambridgethrough the appointment of an scientist with assay development expertise.

in vitro

The successful candidate will be responsiblefor:

Development and establishment of robustassays suitable for compound screeningfor various targets;Routine screening, including instructingothers as appropriate;Scientific input to project team andproviding technical expertise for variousprojects; andRecording and analysing experimentaldata and presenting results in written andoral form, including at meetings withresearch partners.

Additionally they will have:

Scientific and drug discovery backgroundwith experience in assay development and

25/03/2015 In Vitro Scientist ­ Cambridge job with Proximagen Limited | 1401561216

http://jobs.newscientist.com/en­gb/job/1401561216/in­vitro­scientist­cambridge/?TrackID=1345&cmpid=PRC|JOBS|NSNJJ­INDEED­SPONSORED­LIFE 2/3

optimization for a wide range of formats forboth biochemical and functional assays;Significant experience with plate basedpharmacological assays (radioligand andcell signalling) is essential;Also essential to the role is previousexperience with cell culture and cell basedassays;Experience with medium to highthroughput screening techniques andequipment (preferred);Experience of designing and executingspecial studies such as mechanism ofaction studies, species translation andsupporting competitor differentiationstrategies (preferred);Expertise in biochemistry & molecularpharmacology (preferred);Background knowledge and prior workexperience in the area of diseases of theCNS is desirable (preferred);An ability to work independently as well aspart of a team; andStrong organisational skills with excellentattention to detail.

The successful candidate will also be a self­starter, and possess excellentcommunication and interpersonal skills.

Qualifications

Required Qualifications

Degree in biological/life sciences(required), and

PhD in a life science subject (preferred butnot essential)

Package

Proximagen is offering a highly competitivepackage. Benefits will include a contributorypension scheme, life cover and privatemedical insurance.

How to apply

If you would like to be considered for this role,

Christopher Francois Phone number : (+44) 075 30185017

Email: chrisrobfranc@yahoo.co.uk

26th March 2015 New Hall, 076

NG11 8PT, Nottingham

UK

Dear Sir/Madam

Please find enclosed a copy of my CV and skills portfolio in application of the in vitro

scientist job posted on the newscientist jobs webpage on the 23rd

of March.

As a current student of a Bachelor of Science (BSc) in Pharmacology, I am about to finish my

fourth and final year at Nottingham Trent University in the UK. I will graduate in June 2015,

but would already like to start investigating opportunities for my future career. During my 4

years of study I spent my third year of my university “sandwich” course as an intern in the

Quality department of a subsidiary of Baxter, an international healthcare company giving me

some entry level experience in the pharmaceutical industry. During this time I assisted in the

quality control audit, ensuring the reliability of Good Manufacturing Practices by running

tests on raw materials, re-evaluating key systems and updating the documentation.

During my studies I studied many subjects, of which antibody and DNA technologies was

particularly interesting and relevant to the post enabling me to know how to perform ELISA’s

(see skills portfolio skill 3) and western blots. In addition, I am well versed in oral

presentations and paper critiques where expressing your opinion and knowledge was

important. Out of the 2 optional modules available to students I chose current topics in

Neuroscience where I learnt to perform radioactive in situ hybridization studies, a very

interesting area of research

Having studied your company’s website I would be very interested in the possibility of

working with you or meeting a member of the lab research team in order to determine

whether I could play any role in this company. I am a fast and accurate writer, with a keen eye

for detail and I should be very grateful for the opportunity to progress in my career with this

company. I am also able to take on the responsibility of this position as of June 6th, and have

the enthusiasm and determination to ensure that I make a success of it.

I look forward to hearing from you

Sincerely

Christopher François

Christopher François

CORE SKILLS & EXPERIENCE

As a pharmacology student finishing my 4th and final year of studies, I spent my 3rd year doing an industrial

placement in FASONUT, a subsidiary of the pharmaceutical company Baxter Healthcare. During this time I

participated in audits, learnt about the international pharmacopeia and Good Manufacturing Practices

(GMP), as well as the role of risk assessments. This gave me a better understanding of science in a

commercial environment, notably the importance of scientific rigour and the necessity of thorough

documentation. My final year research project: ‘Comparison of intron sizes of GABA(A) receptor subunit

genes between primitive and complex vertebrate species’ will allow me to advance in the use of genomic

and database analysis. I have also taken part in the home office licences 1-3 allowing me to allowing me to

apply for a personal license.

Other work experience has allowed me to take responsibility for work areas, where my attention to detail

and work ethic, in addition to meeting strict deadlines to a high standard was critical.

EDUCATION

Sept 2011– June 2015: University of Nottingham Trent (UK)

QUALIFICATIONS: Pharmacology BSc (Hons)

- 2011-12: Practical techniques, Essentials of living systems, Intro to Pharmacology, Human

Systems, Genetics/Immunology. Year End result: 1st

- 2012-13: Drugs of addiction and abuse, Antibody/DNA technologies,

Cancer/chemotherapy, Pathophysiology and Pharmacology of cells and tissues,

Pathophysiology and Pharmacology of organs systems, Neuroscience. Year End result: 2.1

- Practical techniques: polymerase chain reaction, in situ hybridisation, RNA isolation,

bacterial transformation, sectioning of brain tissue, gel electrophoresis, LD50/EC50 toxicity.

- 2013-14: Placement year (see below)

- 2013-14: Final year: Current topics in Neuroscience, Current topics in Pharmacology,

Clinical Pharmacology, Toxicology and dissertation (Comparison of the GABA(A) receptor

subunit intron sizes between simple and complex vertebrate species).

- Home office licence for small animal handling obtained

Sept 2006-June 2011: Zurich International School (ZIS) Switzerland

QUALIFICATIONS: International Baccalaureate (max points per subject 7)

- Higher level in Chemistry (4), Biology (4) and French (6).

- Standard levels: Economics (4), English (5) and Maths (5).

- Theory of knowledge, CAS (Creativity, Action & Service) requirements achieved through

sport involvement (swimming competition) and sport coaching to lower school children

- Extended essay “The change of pigment quality in the petal colour of the Hydrangea flower

when the pH of the soil is changed.”

WORK EXPERIENCE

Industrial Placement year: September 2013 / June 2014 JOB TITLE: Quality intern COMPANY: FASONUT (France)

RESPONSIBILITIES - Assisting with the quality department at FASONUT

Researching the effect of temperature on raw materials Researching through data analysis a new matrix for product shelf life Testing/calibrating the spectrophotometers’ accuracy by creating a concentration deviation

curve Translation of professional documents based on the pharmacopeia

SUMMER JOB: July/ Aug 2012 JOB TITLE: Maintenance

COMPANY: Zurich International School (Switzerland)

RESPONSIBILITIES - Assisting with maintenance at a local school

Ensuring classrooms were prepared for the new school year Maintaining the garden and grounds Working in an international team on a tight schedule

INTERNSHIP: July 2011

JOB TITLE: Intern in quality control area of a manufacturing plant (UK based)

COMPANY: Baxter Healthcare LTD (Norfolk UK)

RESPONSIBILITIES - Assisting the quality control teams of the manufacturing plant

Support activities in the microbiology, finished goods chemistry and raw materials labs Working as an integral part of a large team Data entry on the company database Learning how to operate in a working environment

INTERNSHIP: April 2010

JOB TITLE: Sales Assistant

COMPANY: IT SHOP, ‘VIRTUAL SPACE’ (South France)

RESPONSIBILITIES - Helping to set up shop, manage store orders, product placement

Manning the till Managing customer service Organizing stock presentation

VOLUNTEER WORK:

Habitat for Humanity

Helping coach children’s football

ADDITIONAL SKILLS/ATTRIBUTES

Open water PADI + French NIVEAU 2 diving permit

Swimming: Many years competing with the school swim team

NBLQ (National Beach Lifeguard Qualification) lifesaving course in England, summer 2010

Choir/Honour choir

PERSONAL DETAILS

ADDRESS (to June 2015) New Hall, Clifton Campus, Clifton Lane, NG11 8NS, Nottingham (parents‘ home address) Im Lätten 11, 8802 Kilchberg, Switzerland TEL: mobile: +447530185017 Home: +41434435963 EMAIL chrisrobfranc@yahoo.co.uk

NATIONALITY British/French, Swiss C permit LANGUAGES English/French: Bilingual, German: Intermediate Date of Birth: 26 November 1993 in Singapore.

REFERENCES

Dr Christian Thode, Dissertation Tutor (Nottingham Trent University)

Stephanie Roure, internship tutor at FASONUT (Montpellier, France)

Pharmacology Portfolio

Student name: Christopher Francois

Student Number: N0379018

Job: Proximagen – In vitro scientist

Skills/Knowledge necessary for the position:

1. Ability to perform a variety of bioscience techniques

2. Experience working individually and as a team

3. Commercial awareness

4. Ability to critically analyze experimental data

5. Attention to detail

6. Ability to present data visually

7. Ability to present data in a written format

8. IT skills

9. Hands on approach

10. Effective oral communication

Evidence

Number : Title

Source Skills in evidence

1 - Poster

Presentation

BIOL22111 - Pathophysiology and

Pharmacology of Cells and Tissues

2, 4, 5, 6, 7, 8, 10

2 – Powerpoint

presentation BIOL33211 - Toxicology

4, 6, 7, 8, 10

3 - ELISA BIOL22011 – Antibody/DNA

technologies

1, 3, 4, 5, 6, 8, 9

4 – PV92 screen BIOL33141 – Current topics in

Pharmacology 1, 2, 4, 5, 9

5 – SPC BIOL33201 – Clinical Pharmacology 2, 3, 4, 5, 7, 9

6 – Gut bath BIOL22111 – Pathophysiology and

Pharmacology of cells and tissues 1, 2, 4, 5, 6, 9

7 – Effects of

Organophosphate

on axon growth

BIOL33211 - Toxicology 1, 2, 4, 5, 6, 9

8 – Home office

Licensee training

course

BIOL33201 – Clinical Pharmacology 1, 3, 5, 9

9 – Radioactive

in situ

hybridisation

BIOL33131 – Current Topics in

Neuroscience 1, 2, 5, 9

10 – LC50 lab BIOL33211 - Toxicology 1, 2, 4, 5, 7, 8, 9

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th

March 2015

Source: Pathophysiology and Pharmacology of Cells and Tissues

Experiment: Poster presentation

Portfolio Skills:

Bioscience skills Transferable skills

1. Demonstrate the ability to

summarise scientific research to fit in a

poster format

2. Working as a team to create an

outcome to be presented to a group of

researchers

3.To summarise scientific papers

visually

1. Effective oral communication

2. Experience working individually

and as a team

3. Ability to present complicated data

visually

4. IT skills

Title: Insulins role in Alzheimer’s poster presentation

Aim: The aim was to, in pairs, read, understand and delve deeper into an existing area of research:

Insulins role in Alzheimer’s disease, and create a poster that would present the information well.

Summary of evidence

The poster presentation shown below was an assignment where my partner and I had to design a poster

around a topic of choice: we chose Insulins role in Alzheimer’s disease, and present the current scientific

pathophysiological evidence of the topic as well as any pharmacological solutions to an audience of our

fellow peers. However, the poster needed to be designed with a specific type of target audience in mind.

Would the audience we wanted to target be scientists who worked in the same field versus scientists who

worked in the same general area but not in the same field. We chose the middle way.

We had to consider what title would be most appropriate to entice someone to read it vs what would

capture the attention. We had to compromise and figure out how a very complicated subject can be

presented easily in a short amount of time and put on a poster with a short amount of space. We had to

work as a team and communicate to each other where some compromises had to be made due to limited

space and we then separated the work equally to the agreement of both parties and present it as a team.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Reflection

This piece of evidence supports my ability to take complicated data and present them in such a way that

they will be understood by a select audience, whether they are familiar with the research or not. It also

demonstrates the ability to work together splitting workloads equally to achieve a goal.

Whilst this piece of work illustrates the ability to work as a pair, share ideas, present complicated theories

to a select audience it also illustrated some serious drawbacks that needed to be addressed. Comments on

the piece of work were that the presentation was not as clear and as colourful. This made me think that

increased used of 3dimensional structures that I could have used or changing the poster to have a

horizontal poster structure that may have allowed for more information to be displayed and better charts,

tables and diagrams to be used and understood.

I still believe that this piece of work is a valuable piece of evidence due to the fact that poster and

PowerPoint presentations often go far in explaining how you interact with people in an “on the spot”

situation as well as how you would interact with someone daily when trying to use complicated data to

explain what you might be doing to a layman or your supervisor.

Portfolio Evidence:

A -

Early stage

1. Decrease

In BBB transport of insulin (Blood

Brain Barrier)

2. Decrease In

insulin concentration

in the brain

3. Decrease

in insulin signaling

4. Increase in

Amyloid-β oligomers

binding

5. Synaptic dysfunction (Long Term Potentials affected)

Could Insulin be affecting your Memory?

Pathogenesis of Alzheimer’s Disease and Treatments N0379018 with N0447271

New research has highlighted the potential role of Insulin resistance in neurodegenerative disease, such as Alzheimer’s Disease. ‘High glucose concentration, a major pathological characteristic of diabetes, may have toxic effects on neurons’[2]. In This presentation, we will be exploring two possible mechanisms of insulin resistance and how these mechanisms relate to the two main characteristics of Alzheimer’s Disease and its range of current and future treatments of Alzheimer’s Disease .

B -

Late stage

1. Increased levels

of GSK-3β (Glycogen

Synthase Kinase-3β)

2. Increased Hyper-phosphorylation of

Tau protein

3.

Neurofibrillary Tangle

Formation

4. Synaptic and

axonal dysfunction

Fig 1. Mechanisms of Insulin Resistance In Alzheimer’s Disease

References: [1] ‘Molecular Pathways to Neurodegeneration’ by Bossy-wetzel, Schwarzenbacher & Lipton; Published in ‘Nature Medicine,vol 10 2004’ http://www.nature.com/nm/journal/v10/n7s/full/nm1067.html#f1 [2] ‘Tau phosphorylation by GSK-3β promotes tangle-like filament morphology’ by Rankin, Sun & Gamblin; published in ‘Molecular Neurodegeneration 2007’ http://www.molecularneurodegeneration.com/content/2/1/12 [3] ‘Neurodegenerative Diseases’ by Shamim I. Ahmad; published in ‘ Advances in Experimental Medicine and Biology Vol 724, 2012’ http://www.springerlink.com/content/978-1-4614-0653-2#section=1046741&page=2&locus=60 [4] Neuroscience: Exploring the Brain book by Bear, Connors & Parradiso, third edition, box 2.3, page 36, chapter 2. [5] ‘Efficacy of rosiglitazone in a genetically defined population with mild-to-moderate Alzheimer's disease’ by M. E. Risner et al ; Published in ‘The Pharmacogenomics Journal (2006) http://www.nature.com/tpj/journal/v6/n4/full/6500369a.html [6] ‘Rosiglitazone increases dendritic spine density and rescues spine loss caused by apolipoprotein E4 in primary cortical neurons‘ by Jens Brodbeck et al; Published in Proc Natl Acad Sci U S A. 2008 January 29; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234140/ [7] fig 2. diagram of paired helical filaments by Goegerd 1996

It is a neurodegenerative disease that is ‘characterized by; the presence of extracellular amyloid plaques and intracellular tangles of hyper-phosphorylated tau protein’[2]. The resulting symptoms of Alzheimer’s Disease include; 1) Increased confusion 2) Memory loss (particularly of short term memory) 3) Decreased cognitive ability

‘An inadequate response by insulin targets tissues, such as skeletal muscle, liver and adipose tissue, to the physiological effects of circulating insulin and often is accompanied by raised insulin levels’[3]. Role of Insulin in the brain [3]

1.Regulating food intake via receptors on the Olfactory blub and Thalamus 2.Effects on cognitive functions e.g. memory 3.Prevent binding of Amyloid-β oligomers (clumps of amyloid-β peptides cut from Amyloid Precursor Protein)

The first mechanism In detail:

1-3. ‘In insulin resistance, it is suggested that the transport of insulin into the brain across the BBB (Blood Brain Barrier) is reduced and therefore levels of insulin (and insulin signaling) in the brain is decreased’[2].

4. Amyloid-β40 and Amyloid-β42 are residues on the APP (Amyloid Precursor Protein) which are ‘cut’ varying lengths via an enzyme call γ and β secretase. These peptides aggregate forming oligomers[1].

5. ‘Recent research has demonstrated that insulin signaling in the CNS prevents the binding of Amyloid-β oligomers. However, with low levels of insulin signaling, the Amyloid-β oligomers attach with specificity to particular synapses, acting as pathogenic ligands. They attach on to synapses and inhibit long-term potentials (LTP)’[3]. This therefore may lead to cognitive dysfunction and memory loss.

The second mechanism in detail: What Is Tau?

‘Tau is a microtubule-associated protein that normally functions as a bridge between microtubules in axons – ensuring they are straight/parallel to one another’[4]. 1. In Alzheimer’s, It has been proposed that ‘less insulin signaling may also induce increased activity of GSK-3β (Glycogen Synthase Kinase-

3β)[2]. 2. This leads to the enhanced phosphorylation of tau protein which detaches from the microtubules and accumulates in the soma. 3. In the soma, they aggregate to form ‘Paired Helical Filaments’ and hence the formation of Neurofibrillary Tangles (NFTs)’[2] 4. This results in the disruption of the cytoskeleton that ‘causes the axons to wither’, thus the normal flow of information (action potentials) is disrupted[4]. This could explain memory loss and cognitive dysfunction.

3 - What is ‘Insulin Resistance’?

1 - Introduction

2 - What is Alzheimer’s Disease?

4b - How Insulin Resistance can lead to Alzheimer’s Disease late stage Alzheimer's

4a - How Insulin Resistance can lead to Alzheimer’s Disease Early stage Alzheimer's

The diagram (Fig 1.) below, shows two proposed mechanisms of how insulin resistance can lead to the development of Alzheimer’s disease, amyloid plaque formation and tangles of tau protein.

Existing cures for Alzheimer's are based on medication based on

differing hypotheses on the cause of Alzheimer's.

- The outdated Cholinergic hypothesis has numerous acetyl

cholinesterase inhibitors and various other forms of drugs used to

control the acetyl choline deficiency that was thought to induce

Alzheimer's. There is little or no evidence that indicates that these

drugs have had any effect in stopping or slowing the disease.

- The Amyloid hypothesis a modern hypothesis for which several

drugs have been created to inhibit/modulate and eject enzymes or vast

amounts of the beta amyloid peptide. Ex. Gamma Secretase

inhibitor/modulator*

- The Immunotherapeutic approach is the hope that through a series

of gradual immunotherapies or direct vaccines, the immune system

would be sufficiently stimulated to attack beta-amyloid formations. *

- Angiotensin-receptor blockers, a type of anti-hypertensive

medication was found to have an effect on people in regard to

Alzheimer's. Patients taking ARB’s were found to have a 35%-40%

lower chance of getting the disease.

- Anti-bacterial/ Anti-viral medication might also have an impact.

5 - Pharmacology of Alzheimer's: Existing Medication

The main problem with insulin resistance is its limited quantities within the neuronal cells in the brain. A drug that has been trialed and perceived to have an effect has been Rosiglitazone, an insulin sensitizer anti-diabetic drug. The drug works by binding to the PPAR- gamma nuclear receptors and, acting like a transcription factor, affects the expression of Nuclear DNA. This treatment has been found to benefit AD patients without the ApoeE4 allele, however further clinical trials are needed to establish that fact[5][6].

6 - Insulin Sensitizers

Fig 2. Paired Helical Filaments of Tau [7]

10

0 n

m

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th March 2015

Source: Toxicology

Experiment: Toxicology Powerpoint presentation

Portfolio Skills:

Bioscience skills Transferable skills

1. Demonstrate the ability to

summarise scientific research to fit in a

PowerPoint format

2. To summarise scientific papers

visually

3. Project management

1. Effective oral communication

2. Ability to present complicated data

visually

3. IT skills

4. Ability to critically analyse

experimental data

Title: Neuroinflammatory response in rats chronically exposed to Cs-137

Aim: The aim of this project was create a powerpoint presentation on a peer-reviewed

Summary of evidence

The aim of this PowerPoint presentation was to find a recently published, peer-reviewed scientific paper

on a toxicological compound. It was up to the individual doing the presentation whether that was a toxin

in the traditional sense in the form of a natural compound (and not a poison) or an environmental toxin

harming things other than humans.

In my presentation I chose to focus on the radioactive isotope caesium 137, one of the more prominent

radio-isotopes created as a by-product of nuclear fission reactions in nuclear blasts or reactors. The paper

that I chose investigated the role of the isotopes toxicology by looking at the neuro-inflammatory

response in rats chronically exposed to it.

The presentation had to last 5 minutes where a basic understanding of the isotope had to be given, its

mechanisms of action as described by other sources and scientific papers discovered, the context of the

study had to be given, and the scientific results given concisely towards the end. The results in the

conclusion found that the caesium affected the hippocampus more than the frontal cortex. It also found

the inflammation was minimal even though the isotope did modify the pro and anti-inflammatory

cytokine responses possibly perturbing Neurotransmitter synthesis.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Reflection

A PowerPoint presentation is a crucial way of passing information easily in a limited amount of time to a

group of individuals, a skill vital in the reporting of results, quarterly reports and so on found in the

commercial environment. This piece of evidence adequately demonstrates the ability to critically analyse

a scientific paper, to summarise concise scientific data from the paper, to create an electronic presentation

of the data, discussion and results and present it to a group. The presentation includes several figures and

diagrams making the theory easier to understand. It also introduces the topic well with the mechanisms of

action being described at length.

Things that could be improved about the presentation is the inclusion of more colour in the text as well as

less text on some slides containing complicated information. The main difficulty of the presentation was

to get all the information across in such a short amount of time and this is where, in the future,

improvements could be made.

Portfolio Evidence:

Christopher Francois, N0379018

NEURO-INFLAMMATORY RESPONSE IN

RATS CHRONICALLY EXPOSED TO CS-137

CS-137 WHAT IS IT?

• Isotope of an Element = Caesium 133 is the

stable isotope , alkali metals

• Radionuclides = nuclear fission products,

Nuclear tests and Chernobyl, Fukushima

Daiichi

• Radioisotopes : Cs-137 (½ life 30.17 yrs)

153 sec

MECHANISMS OF ACTION

• Found primarily in soft tissues

(muscles)

• Action resembles potassium

• Biological ½ life = 70 days

• Very soluble in water

• Reacts with H2O into cesium

hydroxide

• External/internal: Beta (electron

release)/Gamma decay(fast photon)

• Potassium-40 = 0.000071Curies/g

• Cesium-137= 86 Curies/g

153 sec

NEURO-INFLAMMATORY RESPONSE IN

RATS CHRONICALLY EXPOSED TO CS-137

Philippe Lestaevel *, Line Grandcolas, Franc¸ois Paquet, Philippe Voisin,

Jocelyne Aigueperse, Patrick Gourmelon NeuroToxicology, Volume 29, Issue 2, March 2008, Pages 343–348

• Chronic effects of cs-137 on the brain unknown, Cs-137 didn’t affect CNS

• Experimental evidence showed metabolic changes to neurotransmitter in rat with chronic

exposure (low dose 45 Bq)

• Aim: to measure the impact of chronic ingestion of 137Cs, at post-Chernobyl doses, on the

brain inflammatory response”

• 3 months, 12 rats, Contaminated drinking water (400Bq /kg), Frontal cortex/hippocampus

observed/ inflammatory status via cytokines/NO-ergic pathway

• Hippocampal/Frontal cortex extracted

• mRNA extraction, cDNA produced, PCR amplification of cytokines/Cytokine assay IL-

6/TNF-α, NOS monitored

• In hippocampus exposure to 137Cs led

to increase in mRNA levels (+32%,) and

increase in concentrations (41.7 3.2 pg

mg1 protein for 137Cs group versus 32.4

2.1 pg)).

• 137Cs also led to a significant increase

of +56% in IL-6 mRNA levels

• Maybe part of an adaptive survival

response by tissues to pathological

situations

NEURO-INFLAMMATORY RESPONSE IN

RATS CHRONICALLY EXPOSED TO CS-137

• Anti-inflammatory cytokines

IL1beta/IL-8 levels not altered

• Anti-inflammatory IL-10 was

increased +79% in frontal

cortex (modifies microglial

function) (protective against

Cs137)

NEURO-INFLAMMATORY RESPONSE IN

RATS CHRONICALLY EXPOSED TO CS-137

• Effect of 137Cs exposure on the gene expression (A and B) and enzymatic activities

(C and D) of constitutive NOS (cNOS) and inducible NOS (iNOS) in the frontal

cortex and hippocampus.

• Mainly performed on hippocampus/frontal cortex due to implication with mental disorders

• The hippocampus appeared more affected than frontal cortex

• Neuro-inflammation observed in hippocampus could be potential factor in possible neurodegeneration

• The inflammation witnessed in the study is minimal.

• The present in vivo study demonstrated sub chronic exposure to Cs-137 leads to

modification in pro/anti-inflammatory cytokine responses and on NO-pathway. These are

cerebral structure dependent. It could perturb Neurotransmitter synthesis.

• More analysis should be done

CONCLUSION

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th

March 2015

Source: Antibody and DNA technologies

Experiment: ELISA

Portfolio Skills:

Bioscience skills Transferable skills

1. Accurate use of pipettes

2. Preparation of serial dilutions

3.Ability to perform an ELISA

(enzyme linked immunosorbent assay)

1. Attention to detail

2. Ability to present data visually

3. Ability to present data in a written

format

4. IT skills

5. Understanding theories from

experimental data

Title: ELISA (Enzyme Linked ImmunoSorbent Assay)

Aim: The first half of the experiment was to estimate the 50% maximal titre for each of the 3 ‘bleeds’

of the immunisation schedule and to see how the titres change from 1 bleed to the next allowing us to

calculate the antibody concentration after each immunisation.

The second half of the experiment was to determine the specificity of the antibodies used in the 3rd

bleed

(so at the end of the last immunisation) and use them with human albumin, goat albumin and rabbit

albumin in order to

Summary of evidence

The pictures, tables and figures below illustrate my ability to perform the ELISA protocol that allows the

user to determine the presence of an antibody and to determine the amount of it (the titre) the antibody’s

specificity. Here we monitor changes in the titre of antisera during the immunisation schedule and

determine whether the antiserum, taken after the third immunisation, is specific to the human immunogen

(i.e. to human albumin).

In the 1st part, the ELISA is performed and passed through a spectrophotometer giving the raw data, the

raw data is analysed in excel to give data points that enable a graph of log dilution concentration vs

absorbance to be plotted. From this graph the titre of the antisera can be determined where the 1st bleed

had less antibody than the 2nd

bleed, and the 2nd

bleed had less than the 3rd

bleed, illustrating what should

be expected during an immunisation.

The 2nd

part determines the specificity of the antibody at the 3rd

bleed and its abilty to bind to the

immunogen. The results show it was very reactive to the human albumin, minimally reactive to the goat

antigen and not reactive to the rabbit antigen (since the anti-sera came from the rabbit in the 1st place.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Portfolio Evidence – Part 1: Determining the antibody 50% titres

Figure 1: The figure above shows the 96 well plate with the serum albumin. The first 2 rows contains

Anti-human albumin antiserum from bleed 1, then bleed 2 of the same, then bleed 3. After the dotted line

there is 2 rows of Non-immune antiserum bleed 1, then the next 2 rows are bleed 2, then bleed 3.

Figure 2: After having finished the procedure the plate was analysed using a spectrophotometer giving

these raw results.

Bioscience – Skills Portfolio Page 3

Dilution Log10

[dilution]

Corrected Mean Abs

(Average Immune Abs@450nm - Average Non-Immune

Abs@450nm)

BLEED 1 BLEED 2 BLEED 3

2500 3.398 0.8045 0.8365 1.0265 5000 3.699 0.5965 0.856 0.942

10000 4 0.4325 0.7035 0.8895 25000 4.398 0.2675 0.5 0.7715 50000 4.699 0.1165 0.448 0.6055 100000 5 0.004 0.2365 0.4575 200000 5.301 -0.0485 0.1235 0.3275 400000 5.602 -0.011 -0.026 0.11

Table 1: By taking the average of each bleed (1, 2 ,3) for both sections and then subtracting the non-

immune from the immune, a table could be created with the corrected Mean absorbance.

Figure 3: Using the data from the table, a graph of absorbance versus the log of dilution could be plotted

to determine the Antibody 50% titre. Giving the final results for the antibody 50% titres in the table

below:

Table 2. Calculation of Corrected Antibody Titre for each bleed BLEED 100% Max. binding

value (Abs @ 405nm)

50% Max. binding value

(Abs @ 405nm)

Log10 (dilution) value (Reading on x-axis)

Titre

BLEED 1 0.8045 .40225 4.050 BLEED 2 0.8365 .41825 4.758 BLEED 3 1.0265 .51325 4.870

Bioscience – Skills Portfolio Page 4

Part 2: Determining Antibody specificity

Figure 4: The figure above shows the 96 well plate with the serum albumin from the 3rd

bleed. The first 2

rows contains Anti-human albumin antiserum with human albumin (represented with the H), then the

next 2 rows with goat albumin, then the next 2 with rabbit albumin. After the dotted line each of the 2

rows contains the same order of human, goat and rabbit albumin but with non-immune antiserum from

bleed 3.

Figure 5: After having finished the procedure the plate was analysed using a spectrophotometer giving

these raw results.

Bioscience – Skills Portfolio Page 5

Dilution Log10

[dilution]

Corrected Mean Abs

(Average Immune Abs@450nm - Average Non-Immune

Abs@450nm)

HUMAN GOAT RABBIT

2500 3.398 0.8925 0.1505 0.006 5000 3.699 0.8365 0.069 -0.0015

10000 4 0.7805 0.0745 0.011 25000 4.398 0.7275 0.019 -0.016 50000 4.699 0.5855 0.002 0.002

100000 5 0.3605 -0.0015 -0.0355 200000 5.301 0.221 -0.026 -0.0215 400000 5.602 0.139 -0.036 0.004

Table 3: By taking the average of each bleed (1, 2 ,3) for both sections and then subtracting the non-

immune from the immune, a table could be created with the corrected Mean absorbance.

Figure 3: Using the data from table 4, a graph of absorbance versus the log of dilution could be plotted to

determine the Antibody specificity. The graph enabled the discovery of the 50% biding value (=the

specificity) seen in the table below:

SPECIES 100% Max.

binding value (Abs @ 405nm)

50% Max. binding value

(Abs @ 405nm)

Log10 (dilution) value

Titre

Human 0.8925 .44625 4.848 7.047 * 104

Goat 0.1485

.07425 4.048 1.117 * 104

Rabbit 0.0115

.00575 - -

Bioscience – Skills Portfolio Page 6

Reflection

The main strengths of this piece of coursework was that it enabled me to learn a new skill, I practiced the

ability to manipulate data in excel, to create graphs using excel and interpret data from them to give an

understandable answer to a certain problem. To critically analyse the data that I was given in a raw

context, to understand it and create something meaningful from the results.

The things I would do better would be to perhaps redo the experiment as this experiment was the 1st time

I had done it and as such the learning curve was steep and not everything was understood at first, if given

a second chance I may do things differently. I might also give a better format to the results and make it

look more appealing as the graphs seem simplistic and might be better if created on another program

however, they are well done to demonstrate the ability to create graphs on excel

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th

March 2015

Source: Current topics in Pharmacology

Experiment: PV92 screen kit

Portfolio Skills:

Bioscience skills Transferable skills

1. Ability to conduct an agarose gel

electrophoresis

2. Ability to conduct PCR

3.Analysis of DNA ladder after UV

fluorescence

4. Accurate use of pipetting technique

5. Ability to critically analyse data

1. Ability to perform a variety of

biosciences techniques

2. Experience working individually

and as a team

3. Hands on approach

4. Ability to critically analyse data

5. Attention to detail

Title: PV92 laboratory

Aim: To perform a screening procedure using PCR and gel electrophoresis to analyse for short

repetitive interspersed elements (SINE’s) such as the intronic Alu sequence in an individuals DNA. Once

the genotypic results of the class are pooled analysis can be performed using the Hardy-Weinburg

equation to determine the frequency of the genotype in the population tested.

Summary of evidence

The screen is to determine whether the individuals DNA contains alleles within the PV92 locus (found on

chromosome 16) containing the Alu insert that occurs randomly and is found on either 0, 1 or both

chromosomes. The screening process utilizes the fact that the Alu insert is 300bp long, the normal PV92

locus is 641 bp long and chromosomes with both the PV92 locus and the insert are 941 bp long. The

screen isolates the DNA, specifically that of the PV92 insert, multiplies it exponentially using PCR and

separates the different sized fragments. If found on 1 allele on 1 on the 2 chromosomes the individual has

a heterozygous genotype (+/-), if 1 is found on both of the chromosomes the individual has a homozygous

positive (+/+) genotype or they can be found on neither chromosomes thus making homozygous negative

(-/-) genotype.

The evidence shown below are pictures of some of the various procedures that had to be performed to

investigate the prevalence of the Alu intronic inserts resulting with figure 5. Then from that the frequency

at which the insert is found in the population can be determined using the Hardy-Weinberg equation. The

result of which show that the class was too small to have a correlation of our data with the equations

probability theory of genetic equilibrium.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Portfolio Evidence

Figure 1: Illustrates some of the supplies (pipettes, centrifuge) used in the screening process.

Figure 2: These water baths shown (56C on the left and 100C on the right) above are only used once the

instagene matrix and the cells have been in mixed. The instagene mix used is a mixture of beads bound to

bivalent ions to stop the DNAase enzyme from breaking down the DNA when the cells are perforated

which they then are in the 56C water bath due to the higher temperatures of the water baths which

denature the DNAases and other enzymes, disrupt any connective tissue and extracellular matrix holding

the cells together. The 100C water bath is used to disrupt the cells and nuclear membrane to get the DNA

out.

Bioscience – Skills Portfolio Page 3

Figure 3: The centrifuge is used to separate the intra cellular components from the DNA that we are

looking for. The DNA is isolated and put into a PCR tube with some mastermix (a main mixture of

primers meant for the sequences we are looking for, DNA Taq polymerase, Mg2+ cofactor for the Taq

polymerase, salt buffer and nucleotides).

Figure 4: Illustrates a thermocycler for use in PCR reactions. The thermocycler allows the mixture of

isolated DNA from the centrifuge and master mix to heat cool and heat again to denature the DNA

strands, to then anneal the primers and then to elongate the strands using the primers as a base. Once done

the DNA is dyed in purple.

Bioscience – Skills Portfolio Page 4

Figure 5: An Agarose Gel electrophoresis where the dyed DNA fragments are gently inserted into small

wells (to make sure that the dye does not overflow into the water above). Once everything is prepared the

electricity is switched on and the device is left alone. The electricity makes the smaller, lighter and more

negatively charged fragments travel faster than the heavier fragments that travel slower. The result is the

separation of different DNA sizes.

Figure 6: The machine used to analyse the agarose gel, it illuminates the gel with UV therefore exciting

the SYBR safe fluorescent part of the DNA making the DNA fragments visible. It then takes a picture.

Bioscience – Skills Portfolio Page 5

Figure 7: The final results of the PV92 experiment showing (from left to right) the homozygous positive

(+/+) control first, then the DNA ladder (DNA standard MMR), then the homozygous negative (-/-)

control, then the heterozygous (+/-) control followed by the DNA of 4 individuals of which I was the 2nd

last one.

Analysis:

Table 1: Observed genotypic frequencies of the class

Genotype Number Frequency (# of genotypes/total)

Homozygous (+/+) 1 1/13 = 0.077

Heterozygous (+/-) 1 1/13 = 0.077

Homozygous (-/-) 11 11/13 = 0.84

Total = 13 Total (13/13) = 100%

Table 2: Calculated allelic frequency of the class

Allele Category Number Frequency, Total = 1

(+) alleles 3 p (3/26) = 0.115

(-) alleles 23 q (23/26) = 0.885

Total = 26

The Hardy-Weinburg equation for genotype frequency in a population is an algebraic expansion of p + q

= 1 giving: p2 + 2pq + q

2 = 1. This equation is used in population genetics to predict whether the

population is at genetic equilibrium meaning that the frequencies of the alleles are stable from generation

to generation where p2 is

the expected frequency of the (+/+) genotype in the population, q

2 is the

expected frequency of the (-/-) genotype in the population and 2pq is the expected frequency of the (+/-)

genotype of the population.

If the frequencies in table 2 give a partition that is expected at 25%/50%/25% then the population is at

genetic equilibrium. Using our data: (0.115)2 + 2(0.115)(0.885) + (0.885)

2 = 1, the result:

0.013225 + .20355 + .783225 = 1 the equilibrium is not seen. This is simply because the sample size was

too small.

Bioscience – Skills Portfolio Page 6

Reflection

This experiment demonstrates my ability to perform a variety of bioscience techniques, which included

the use of PCR and gel electrophoresis techniques that I had done before but quite enjoy doing. These

skills are vital due to the fact that DNA isolation and analysis utilizing these techniques is becoming more

and more important with the advent of pharmacogenomics and personalised medicine. These screening

kits are becoming cheaper and easier to produce and implement on an industrial scale and the knowledge

of how to perform these procedures and analyse the data is crucial.

One thing that could have been done better on his experiment is the fact that the DNA ladder, supposed to

be found all the way on the left of the Agarose gel, was mixed up with the homozygous positive control

which is why it found itself first and the DNA ladder standard next.

Another problem was that the sample size, our class, was too small for the Hardy Weinberg equation to

give a positive result showing that with larger numbers of people the frequencies of alleles might be

partitioned in the theorised 25%/50%/25%.

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th March 2015

Source: Clinical Pharmacology

Experiment: SPC

Portfolio Skills:

Bioscience skills Transferable skills

1. Ability to write an SPC

2. Ability to analyse lots of scientific

data and summarize

1. Ability to present data in a written

format

2. Ability to critically analyse

experimental data

3. Commercial Awarness

4. Attention to detail

5. Hands on approach

Title: SPC (Summary of Product characteristics)

Aim: To create an SPC, a summary of product characteristics, for a pharmaceutical drug utilizing 147

scientific paper summaries.

Summary of evidence

The work presented shows a fake SPC document similar to what would be the legal documentation that is printed

given in all pharmaceutical drugs that are administered to patients. Using medical case studies and abstracts of

scientific findings on this fake drug the information required was taken and summarized to form the legal SPC

document in the same manner as SPC’s are created in the pharmaceutical industry.

The document has several sections that summarize the drugs look, colour, form, feel and composition followed

by the clinical particulars. The clinical particulars includes the method of administration, dosage, warnings and

precautions for use including whether or not the drug should be administered to pregnant women or any other

patient with special circumstances. It also includes possible adverse effects and what to do in case of an overdose.

This is followed by the next section illustrating the pharmacological properties, pharmacokinetic and dynamics of

the drug which is then followed by other legal and logistical matters.

Reflection

The piece of work presented shows my ability to analyse data that was published in a scientific context and

summarize it in a simpler form for consumption by the general public. This is a vital skill in any industry as

database searching for information has become widespread. It also presents a skill very particular to any

pharmaceutical companies as SPCs regularly have to be created with each new drug. The scientific reports need

to be understood and summarized in the same way.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Areas of improvement with this particular work should focus on increasing the amount explained in certain areas

such as the interactions with other medicinal products and other forms of interactions. In addition more studies

should have been cited in the different sections of the SPC.

Portfolio Evidence:

Bioscience – Skills Portfolio Page 3

Breaking Bad Limited Nottingham Trent University

Burton Street

Nottingham

NG1 4BU

Telephone:+44 (0) 1707 366 000

Facsimile: +44 (0) 1707 338 297

WWW: https://www.breakingbaduk.com

Medical information direct line: +44 (0) 800 328 1629

Customer care direct Line: +44 (0) 800 731 5711

Medical information email: medinfo.uk@breakingbad.com

Medical information facsimile: +44 (0)1707 390 378

Document last updated on the eMC: Friday 24th October 2014

NTU2699T Hydrochloride

1. NAME OF THE MEDICINAL PRODUCT NTU2699T Hydrochloride Tablets 12.5 mg

2. QUALITATIVE AND QUANTITATIVE COMPOSITION Each Tablet contains 12.5mg NTU2699T Hydrochloride

3. PHARMACEUTICAL FORM Lilac tablets with a diameter of 6mm. Marked with NTU on the top face. Oval cross

section.

4. CLINICAL PARTICULARS

4.1. Therapeutic indications NTU2699T is a secondary amine. It is a dual action drug being both anti-cholinergic

(competitive anti-muscarinic) (144) and acting as a calcium antagonist. These properties

act as a non-dose related spasmolytic (49) to inhibit muscle contractions therefore relaxing

the detrusor muscle of the urinary bladder enabling it to store urine more efficiently.

Drug therapies using this treatment are mainly used for 2 categories of disorders (95). It

can be administered to counteract the disorders that involve the storage function of the

bladder or the disturbances of the emptying function of the bladder. The drug therapy is

mainly used to improve the contractile activity of the detrusor muscle or can also be used

to reduce the urethral outflow resistance.

This enables the treatment of adult patients with detrusor instability, neurogenic bladder

disorders (such as uninhibited bladder and reflex bladder disease), motor urge

incontinence, urinary frequency, urgency, nocturia (77) and nocturnal enuresis (88).

NTU2699t increases bladder capacity (42) and it can reduce the frequency of micturition.

It can also reduce incontinent episodes for those suffering of motor urge incontinence.

Bioscience – Skills Portfolio Page 4

4.2. Posology and method of administration Adults: The drug is meant to be administered orally in a single dose (1 tablet of

12.5mg/kg) twice daily. This dose can be increased to 2 tablets twice daily if the

symptoms of the condition are found to be serious.

It should be noted that this treatment should continue for a period of 2-4 (20) weeks until

the optimal response has occurred as steady state levels of drug in the blood plasma occur

only 10-14 days after administration of the treatment. The final dose eventually depends

on the response and tolerance of body to the drug and its side effects.

Elderly: It should not be necessary to exceed the 1 tablet twice a day in elderly patients

and a lower dose should be administered to them as elderly patients often have lower

affinities and higher retention of the drug in their bodies (105).

Children: Although the drug has been found to be effective in the treatment of nocturnal

enuresis in children (40), it has been advised that more tests are needed for the safety of

the drug to be established and as such this Drug is not recommended for child

consumption.

4.3. Contraindications Use of NTU2699t in groups of patients that have these disorders: those with bladder

outflow obstruction, flaccid bladder or high residual urine, obstructive lesions particularly

of the upper gastrointestinal tract, severe hepatic dysfunction or biliary tract dysfunction,

glaucoma is contraindicated due to the adverse effects would occur from their

administration of such a drug into their system.

Use of the NTU2699t drug would also be contraindicated to patients with cardiac

problems, or where an increase in cardiac activity would not be beneficial because of the

danger of the drugs calcium antagonistic properties prolonging the QTc interval. For more

information (see 4.8 Undesirable effects).

4.4. Special Warnings and precautions for use As with other drugs with anticholinergic effects as well as calcium antagonistic effects

caution should be observed in patients with gastric retention, incipient obstruction of the

gastrointestinal tract, fever, thyrotoxicosis. Although experience with such patients is

limited, there is no evidence for a required adjustment of the dose given to these patients.

Those with heart disease where an increase in heart rate is undesirable should also be

attentive as there is evidence of NTU2699t causing a number of ventricular cardiac

arrhythmias: torsade de pointes, bradycardia (15) (see 4.8 Undesirable effects).

Caution should also be observed in patients with hepatic dysfunction and in frail or elderly

patients due to the

4.5. Interaction with others medicinal products an other forms of

interaction

Bioscience – Skills Portfolio Page 5

Potential of clinical effects may be expected when NTU2699t is administered to patients

who also are receiving other drugs with anticholinergic (anti-muscarinic) or calcium-

antagonist effects (such as iproveratril, or prenylamine, Nicardipine). It is recommended

that a patient not be given differing drugs with the same pharmacodynamic actions due to

the possible adverse effects that could occur. This is especially the case when a dual drug

treatment is being given.

There is evidence to support the claim that administration by a GP of NTU2699t does not

require additional monitoring when given to a patient also taking daily doses of warfarin

treatment(82). The drugs do not interact and NTU2699t has been shown to NOT influence

the effect of the anti-coagulant or the enantiomer plasma levels of the warfarin (82).

4.6. Pregnancy and lactation Pregnancy: The use of this drug has shown no teratogenic effects during tests, however,

no clinical trials have been specifically conducted to assess the viability of the drug in

pregnant women. As such there is too little clinical evidence to administer this drug to

pregnant women and general use of the drug whilst pregnant should be avoided especially

during the first trimester unless the potential benefits are important enough to outweigh

the possible risks.

Lactation: Drug Administration to Breast feeding mothers should be avoided unless

considered essential.

4.7. Effects on ability to drive and use machines NTU2699t has been shown, in clinical trials, to cause varying side effects (see section 4.8

Undesirable effects) which are due to the anti-cholinergic and calcium antagonistic effects

of the drug. These include risk of cardiac arrhythmia, dizziness, Uticaria, and rare

instances of nausea and tremor have been reported. It is therefore recommended not to

conduct tasks requiring high degrees of coordination or spatial awareness after NTU2699t

administration. These types of tasks should be avoided during the 2-4 weeks of taking the

drug.

4.8. Undesirable effects At the doses of drug considered to be safe and recommended (12.5mg tablets twice a day

or 25mg (2 tablets) once a day) various adverse effects and risks may still affect the

patient. It is recommended that you review these common, uncommon and rare adverse

effects and insure that you do not have any conditions that might worsen these side

effects. For the most part during clinical trials patients’ data supported the claim that the

Drug can be well tolerated (95).

Common adverse effects: These include the anti-cholinergic effects of NTU2699t seen

that may cause dryness of mouth, visual accommodation disturbances, reduced lacimation,

constipation, dizziness, tachycardia and retention of urine. Rare instances of nausea and

tremor have been reported. Incidences of dysuria, thirst, sense of residual urine,

orthostatic hypotension and arrhythmia (although rare). Dizziness, nasal obstruction (104)

uticaria (115), pupillary motility and heart rate variation adverse effects were also

observed (102). These side effects disappeared once the treatment had been discontinued

(43).

Bioscience – Skills Portfolio Page 6

Signs of Bronchodilation as a result of the anticholinergic effects were also observed in

certain patients.

Uncommon side effects:

The administration of NTU2699t, however caused exclusively by the R(+) enantiomer

(11), does pose a considerable risk to elderly patients giving cardiac problems such as

bradycardia (15) or more prominently: polymorphous ventricular cardiac arrhythmia (1,

10, 34) called Torsade de pointe (2, 7, 25, 46, 56). This is due to the side effects of K

channel blockers and the calcium antagonist properties of the drug.

At 10mg/kg ECG readings showed a significantly prolonged QTc interval caused by the

muscle preparation losing its excitability in relation to the calcium antagonists. This

greatly prolonged the QTc interval by 6-8%. Ventricular arrhythmias (10, 11)) causes QT

prolongation, increased QT interval and QRS duration. NTU2699t increases QTc and QTd

(heart rate corrected QT and QT dispersion) in a concentration dependent manner (16).

ECGs obtained before a 4 weeks treatment show peak effects occurring 8 hours after

administration (with dose ranges of 2.5-10mg) in elderly men.

This is particular to the frail and elderly, this drug should be avoided with other people

that are affected or have other risk factors for polymorphic VT particularly hypokalaemia

and cardiac disease (46). These also disappeared once the treatment was finished or

discontinued.

Drug intolerance is a possibility but has only been observed once and can be considered

rare (133).

4.9. Overdose In cases of overdose the symptoms precluding an overdose may be see (see 4.8

undesirable effects).

Countermeasures: Perform Gastric lavage followed by symptomatic treatment. If there is

a predominate atropine-like central anti-cholinergic side effects give the patient a dose of

physostigmine 1.0 – 2.0 mg by slow intravenous injection (ca 2 mins). In Children give

0.02-0.04 mg/kg. Titrate to an effective dosage, this dose may be repeated in 30-60mins.

If the cases of pronounced excitation or convulsions diazepam 10mg should be

administered intravenously (children 0.1-0.2mg/kg).

In respiratory insufficiency give artificial respiration.

Tachycardia should be treated by administration of propranolol 1-5mg by slow

intravenous injection.

Urinary retention may be treated by catheterisation. The patient should be placed in a

dark, quiet room.

If mydriasis is troublesome treatment with pilocarpine eye drops should be considered.

If there are predominant calcium antagonist effects, bradycardia and hypotension should

be treated by intravenous infusion of 10ml calcium gluconate solution 10% and then

Bioscience – Skills Portfolio Page 7

repeat if necessary, until the patient’s circulation has improved. An Adequate fluid

intravenous substitution should be administered.

So far only 1 Fatal overdose has occurred on NTU2699t. The blood and urine

concentrations were greater than 10mg/L, whereas therapeutic concentrations are usually

not more than 1mg/L. (92)

5. PHARMACOLOGICAL PROPERTIES

5.1. Pharmacodynamic properties The effectiveness of NTU2699t can be attributed its nonselective competitive

anticholinergic profile and calcium locking effects within the same concentration range

with the anti-cholinergic effect predominating at low concentrations and the calcium

antagonist effects predominating at higher concentrations. Due to its suitable lipophilicity

and low binding properties that enable easy topical application and absorption throughout

the body leading to a bioavailability of around 90% (14) it has been deemed a useful drug

in the treatment of the various bladder disorders.

Anti-cholinergic: The (+)-NTU2699t enantiomer is the main contributor to the relaxant

detrusor effects of the racemate and this part of the drug action is anticholinergic (93). The

(+/-) NTU2699t enantiomer has an affinity and selectivity of muscarinic receptor subtypes

that has been determined from functional in vivo responses of rabbit vas deferens (M1),

guinea pig atria (M2), bladder detrusor muscle (M3). It inhibits the intravesical bladder

pressure and induced salivary secretions as well as increasing pupil diameter (53). It

appears that the action of the NTU2699t drug at the M3 receptors mediating bladder

contraction may not be separable from its actions at receptors mediating mydriasis and

salivation. This is what causes many of the common adverse effects observed.

NTU2699t also acts as a calcium channel blocker and inhibits the generation of a

transmembrane proton electrochemical gradient driven by the vesicular H(+)-ATPase, this

is done principally by the uncoupling of this ATPase to not allow ATP induce action

potentials (57). The calcium channel blockers also primarily block the uptake of calcium

(126) as well as blocking the use of intracellular stores of calcium.

It has been shown in clinical trials that NTU2699t at 10^-4M, the drug being a calcium

antagonist, inhibited the contraction evoked by both nerve selective stimulation and

exogenously induced ATP stimulation (36). This gives it the ability to stop the stimulation

of nerves required for muscle contraction. Efferent nerve discharges from the pelvic nerve

on the micturition reflex were inhibited by NTU2699t (at 3mg/kg). Moreover, both the

bladder contractions evoked by the electrical stimulation of the peripheral or central cut

end of the pelvic nerve were dose-dependently inhibited by the drug (at concentrations of

1-10mg/kg). At the 3mg/kg or more concentration the contraction was significantly

inhibited and the effect was long (117). This is how the Calcium channel blocker partially

inhibit the electrically as well as agonist induced detrusor contractions (122).

It should be noted however that all calcium antagonists due to their inhibition of calcium

uptake into the myocardium can block the excitation and contraction of vascular smooth

muscle (139). This is what may be causing the increasing QTc intervals observed during

clinical trials in elderly geriatric patients. The calcium blocking also leads to the

myocardial necrotization arising from deleterious intracellular calcium overload.

Bioscience – Skills Portfolio Page 8

5.2. Pharmacokinetic properties Absorption: Human clinical trials have given evidence for an oral dose of 25mg, the (+) –

NTU2699t enantiomer had a lag time of absorption of 0.6 +/- 0.5hr, an absorption half-life

of around 0.9hr. The time until a maximum serum concentration was obtained was at 5.6

hr. The maximum corresponding blood serum concentration was found to be 62

microgram/L. These figures characterize the (+)-NTU2699t as well as its racemate as low

clearance long half-life drugs meaning that they have a low measure of renal excretion

ability followed by a longer half-life (17, 19, 23).

Absorption began within the 1st hour and this was followed by the maximum rate of

absorption which was determined at 1 and 3h. By the 3.4h mark 90-93% of available dose

had been absorbed.

Elimination: The systemic clearance of the drug from the patients system was deemed to

be measured at: 86-93ml/min, the renal clearance of the drug at 14ml/min with a total

volume of distribution measured at 407L. At a steady state the terminal half-life was 54-

56h with a mean residence time of 77h-81h (23, 19,17).

The Bioavailability of the NTU2699t drug was 93%.

5.3. Preclinical safety data NTU2699t, when administered in dogs leads to blockade of the sodium and calcium

channels as well as muscarinic receptors in canine cardiac tissue, resulting in heart

problems.

The administration of NTU2699t in rabbits has given results concluding that the pH and

concentration of the solution are factors of major importance for the amount of drug

absorbed in the bladder. (26)

A dose of 10mg/kg of NTU2699t induced significant increase in bladder volume capacity

in conscious rats, as well as reduced Micturition pressure(62).

The Bioavailability of NTU2699t was shown to be in beagle dogs: 25%, the results gave

a terminal half life of 3hr, a systemic clearance of 40ml/min, a volume of distribution

71/kg. The unbound fraction of serum was 15-40% and respectively there was 25%

excreted in urine (which happened to be 60% of the dose). Increased heart rate was also

observed due to large doses tested in this pre-clinical trial (79).

The drug in oral pill form was also shown to produce slight to moderate oesophageal

lesions in cats. (111)

6. PHARMACEUTICAL PARTICULARS

6.1. List of excipients Lactose

Sucrose

Bioscience – Skills Portfolio Page 9

6.2. Incompatibilities None stated

6.3. Shelf life 3 years

6.4. Special precautions for storage Store in the original package. Keep in a warm and dry area.

6.5. Nature and contents of container A sealed carton containing 4 blister strips with 25 tablets each

6.6. Instructions for use, handling and disposal Not Applicable

7. MARKETING AUTHORISATION HOLDER Breaking Bad Limited, Nottingham Trent University, Burton Street, Nottingham, NG1 4BU,

United Kingdom

8. MARKETING AUTHORISATION NUMBER(S) PL 0042/0524

9. DATE OF FIRST AUTHORISATION/RENEWAL OF THE

AUTHORISATION 24th October 2014

10. DATE OF REVISION OF THE TEXT 24th October 2014

11. LEGAL STATUS

POM

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th

March 2015

Source: Intro to pharmacology

Experiment: Guinea Pig Ileum Gut Bath

Portfolio Skills:

Bioscience skills Transferable skills

1. Ability to perform a tissue bath

2. Ability to analyse experimental data

3. Ability to plot a dose-response curve

4. Ability to determine the pA2 value

of an antagonist and plot schild plot

1. Ability to perform a variety of

bioscience techniques

2. Ability to critically analyse

experimental data

3. Attention to detail

4. Hands on approach

Title: Guinea pig ileum Gut Bath

Aim: 1) To understand the response of guinea pig ileum in a gut bath experiment to the administration

of agonists (histamine, carbachol, propionylcholine) and antagonists (atropine, mepyramine)

2) Draw up a dose response curve for carbachol and histamine, and to determine the pA2 of the results for

carbachol and atropine

Summary of evidence

The guinea pig ileum gut bath lab was a lab to learn how to set up a gut bath and once the tissue inside the

bath how to properly administer the compounds being tested and interpret the results that can be produced

mechanically on paper (figure 1) or electronically (on the computer in figure 2). After administration of

the drug, the tissue must be washed so that the drug would not remain in the tissue and generate false

readings.

After this trial period the tasks were to produce from the raw data a dose response curve for the agonist

(histamine, figure 3) and use that to determine the pA2 value, a pharmacological value that determines the

affinity of the antagonist for its receptor (mepyramine, figure 4). The dose response curve is

understandable with % response on y-axis and log concentration on the x axis. The second graph is

another method to determine the pA2 value other than a schild plot. It utilizes a theoretical dose (x) that is

doubled (2x) and both dose maximal responses are marked on the graph. The maximal dose of the agonist

is given with increasing amounts of the antagonist giving you a curve to graph. The –log of the

intersection of the line demarcating the x dose with the curve gives you the pA2 value.

However, the pA2 value is not accurate as the concentration for the theoretical 2x dose is supposed to

give only a 67% maximal response not a 120%. This error was caused by improper washes and a dying

tissue. This piece of evidence helps illustrate how to determine the pA2 and my ability to identify

problems in the raw data.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Portfolio Evidence

Figure 1: The drug responses charted on this graph record the stretches of muscle (induced by the drug)

that create varying voltage levels. The voltage levels are charted on either specialized software or

mechanically giving slightly larger responses as the dosage was increased. These are responses to .1mls

and .3mls of propionylcholine at 10^-3, -2, and -1 Molar concentrations.

Figure 2: The image above shows the gut bath set up with the guinea pig ileum stretched in the

oxygenated centre (pointed out with the red arrow). The computer uses specific software to measure the

voltage created by the guinea pig ileum stretches and charts it.

Bioscience – Skills Portfolio Page 3

Figure 3: This graph shows a dose-response curve for the drug histamine. The increasing concentrations

of histamine gradually induce a larger contraction of the ileum that registered larger voltages.

Bioscience – Skills Portfolio Page 4

Figure 4: This graph illustrates a different way to determine the pA2 value of an antagonist without

having to go through the lengthy procedure of creating a Schild plot. The dose (x) that gives 50% of

maximal % response is doubled giving you 2x. Using this dose alone you gradually increase the amount

of antagonist administered. This lowers the % response of the ileum when the agonist is administered that

you can graph. The x dose is marked and where the intersection occurs with the lowering agonist

response curve the pA2 value can be determined. Unfortunately, due to the ileum dying and improper

washes of the tissue, when the 2x dose was administered the voltage was registered as 116% when the

theoretical % response should have been 67% making the pA2 value unreliable.

Bioscience – Skills Portfolio Page 5

Reflection

This piece of evidence illustrates the ability to perform a gut bath, how to take the data generated from the

experiment and analyse it to create a % dose response curve for agonists. It also illustrates the ability to

use that data to potentially generate a schild plot or an equivalent method that can be utilized to determine

the pA2 value of the antagonist being tested.

I am aware that figure 4, illustrating the method to find the pA2 value is not accurate due to the raw data.

In the experiment the Guinea pig ileum showed a % response closer to 120% than the theoretical 67%

when the double dose (2x) of the agonist (histamine) was administered. The cause of this was probably

due to the improper washing of the tissue as well as the ileum slowly dying. This led to the curve being

far steeper than it should be meaning that the calculated pA2 value does not accurately determine the

affinity of the antagonist for its receptor.

The value of this graph is to show the ability to determine the pA2 value as well as to determine when the

pA2 value is not accurate or when data is giving false readings. These skills are ever important in a

laboratory context as experiments that generate abnormal results can occur and the employee needs to be

able to interpret what went wrong, how to fix the situation in a time frame and re-do the experiment

correctly.

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th

March 2015

Source: Toxicology

Experiment: Effects of organophosphates on axon outgrowth and acetylcholinesterase activity

Portfolio Skills:

Bioscience skills Transferable skills

1. Ability to use a spectrophotometer

2. Ability to analyse raw data and plot

a graph

3. Knowledge of microscopy

4. Ability to differentiate neuronal cell

morphology

1. Ability to perform a variety of

bioscience techniques

2. Ability to critically analyse

experimental data

3. Ability to present data visually

Title: Effects of organophosphates on axon outgrowth and acetylcholinesterase activity

Aim: To 1) identify major changes in in neuronal cell morphology that occur when cells differentiate

in the presence of chlorpyrifos and 2) Demonstrates how this relates to the activity of an enzyme that play

a central role in cholinergic neuron function.

Summary of evidence

The experiment involved pictures of different microscope slides that showed neurons. It was up to us to

use these slides to learn how to differentiate the neurons that were in a normal condition (the controls)

and the neurons that had changed due to the presence of the organophosphate chlorpyrifos, a neurotoxin

that inhibits neuronal development.

After the exercise in differentiated was complete, the 2nd

pat of the experiment was to determine how this

morphological change in the neuronal cells relates to the activity of an enzyme. To do this an

acetylcholinesterase assay had to be done to determine acetylcholinesterase activity by mixing cell

extracts originating from differentiated and undifferentiated neurons with substrate that changed colour as

time went on that could be measured by putting the cuvet into the spectrophotometer. The results can be

plotted onto a graph to determine rate of reactions.

The results show that the acetylcholinesterase activity is much higher and has a faster rate of reaction in

the untreated control neurons than the chlorpyrifos treated neurons indicating that the organophosphate

does inhibit the enzymes activity and that organophosphates are neurotoxins.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Portfolio Evidence

Figure 1: This graph shows the rate of reaction of the Control neurons compared with the chlorpyrifos

treated neurons. It is clear that the untreated, control neurons have a faster rate of reaction than the

chlorpyrifos neurons.

Bioscience – Skills Portfolio Page 3

Figure 2: This photo shows the spectrophotometer as well as other basic bioscience tools that were used

to generate the data required to plot the graph above.

Reflection

This is a simple piece of evidence illustrating the ability to use a pipette, to use a spectrophotometer, to

recognize and analyse microscopy slides and what is on them. It illustrates the ability to differentiate

between cells types and how to interpret the data to turn it into a plotted graph followed by interpretation

of the results. The evidence shows the basics skills required to work in any laboratory based job that

utilizes simple microscopy and spectroscopy as well as how to interpret data generated from the 2

methods.

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th

March 2015

Source: Home office training course

Experiment: Modules 1-3

Portfolio Skills:

Bioscience skills Transferable skills

1. Ability to handle animals correctly

and in accordance with the law

2. Knowledge of ASPA and the laws

relating to experimentation on animals

3. Knowledge of animal care

1. Ability to perform a variety of

bioscience techniques

2. Hands on approach

3. Commercial awareness

Title: Home office training course

Aim: To complete modules 1 to 3 in the Home office course, a course where the act of animal

handling for experimentation will be taught as well as the laws that govern hat type of research.

Summary of evidence

The modules 1 to 3 are part of a British qualification that once done allows the participant to apply for a

home office license which is a government license allowing that individual to perform experiments on

certain specific animals in a laboratory setting (where the laboratory also has obtained their license). The

course teaches you about the laws that have been implemented to regulate the practice of animal

experimentation. It familiarises the participant with knowledge of the 3 cardinal ethical rules of animal

experimentation, the 3 R’s: replacement, reduction and refinement all to minimise the animals suffering.

It also allows for hands on experience in handling the animals.

The course also teaches how to care for the animals, how to determine whether or not they are feeling

well from their behaviour and appearance. It also defines the importance of knowing what the definition

of a protected animal is, what species are held within those regulations and what severity limits are and

how to determine them.

The evidence shown below is the booklet that contained all the presentations I had to go through in order

to then pass the exam. The screenshot of the email is my teacher saying that I passed the exam along with

others who also went through the same course and passed.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Portfolio Evidence

Figure 1 (left): The booklet for the theory part

of the home office modules 1 to 3. The figure

below shows the inside which shows a set up

of PowerPoint slides that can be written on to

take notes.

Figure 2 (below): The inside of the booklet: it

illustrates the many PowerPoint slides that are

mandatory for the course. Each presentation

had a slightly different theme. These slides

detail the implementation of ASPA (animals

scientific procedures act, 1986) differences

between procedure establishment licenses, the

Procedure Project license and the

responsibilities that come with each.

Bioscience – Skills Portfolio Page 3

Figure 3: This shows a print screen of an email that was sent by the teacher hosting the home office

modules at our university saying that those in the email had passed the exam set at the end of the theory

part of the course. It then says that the practical session will be organised at a later date.

Reflection

This piece of evidence shows that I can take an opportunity when presented with one and go with it right

to the end. It shows that I have the capacity to adapt quickly when there is a steep learning curve and to

pay attention to details that are either spoken or, on the animals in the lab, seen. The home office modules

enabled us to learn a critical skill that is sometimes a barrier to entry in the pharmaceutical industries. It

also taught us a great deal on the practical implementation of the law in the biosciences field, expanding

on the idea of ethics and doing no harm. In all it was well worth going through the course to learn new

ideas that might apply later on.

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th

March 2015

Source: Current topics in Neuroscience

Experiment: Radioactive in situ hybridisation

Portfolio Skills:

Bioscience skills Transferable skills

1. Ability to perform radioactive in situ

hybridisation techniques

2. Ability to lead a team

3. Some experience with

immunohistochemistry,

Autoradiography and fluorescent

microscopy

1. Ability to perform a variety of

bioscience techniques

2. Experience working in managing a

team

3. Hands on approach

Title: Radioactive in situ hybridisation of the Malat-1 a splicing non-coding RNA

Aim: In a team we were 1) To research the MALAT -1 gene sequence and design a anti-sense

oligonucleotide sequence, 45bp, 2) To section several slices mouse brain tissue and fix them to

microscope slides 3) To undergo a labelling reaction to label the sequence we chose with a radioactive

isotope of [α-S35

]dATP as well as measuring the probe and purifying it. 4) Lastly take the sections,

hybridise the labelled probe to the sections and wash them.

Summary of evidence

The experiment was a radioactive in-situ hybridisation utilizing the most effective anti-sense

oligonucleotide probe that we were to identify in order to distinguish the concentrations of the non-coding

RNA MALAT-1 in sections of a mouse brain. There were several parts to the procedure parts of which

were performed in a group which I was the leader of therefore the piece of evidence shows my ability to

lead a team as this was a group effort with the different stages of the ordering, sectioning, purifying,

hybridisation, fixing and washing of the slides done by select individuals and the work pooled together.

The evidence illustrates some of the procedures the first of the process of researching the MALAT-1 gene

and finding an appropriate gene sequence that would allow an anti-sense strand to bind to it. Unseen are

the processes of fixing the tissue section to the slides, and purifying the probe, figure 2 shows the

machine that gives the results (figure 3) of how pure the probe is, allowing it o be used for actual

hybridisation and then visualisation.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Portfolio Evidence 4801 gagctgctca ggactttgca tataagcgct tgcctctgtc ttctgttctg ctagtgagt

4861 tgtgatgtga gaccttgcag tgagtttgtt tttcctggaa tgtggaggga gggggggat

4921 gggcttactt gttctagctt tttttttaca gaccacacag aatgcaggtg tcttgactt

4981 aggtcatgtc tgttctttgg caagtaatat gtgcagtact gttccaatct gctgctatt

5041 gaatgcattg tgacgcgact ggagtatgat taaagaaagt tgtgtttccc caagtgtttg 5101 gagtagtggt tgttggagga aaagccatga gtaacaggct gagtgttgag gaaatggctc 5161 tctgcagctt taagtaaccc gtgtttgtga ttggagccga gtccctttgc tgtgctgcct 5221 taggtaaatg tttttgttca tttctggtga ggggggttgg gagcactgaa gcctttagtc 5281 tcttccagat tcaacttaaa atctgacaag aaataaatca gacaagcaac attcttgaag 5341 aaattttaac tggcaagtgg aaatgttttg aacagttccg tggtctttag tgcattatct 5401 ttgtgtaggt gttctctctc ccctcccttg gtcttaattc ttacatgcag gaacattgac 5461 aacagcagac atctatctat tcaaggggcc agagaatcca gacccagtaa ggaaaaatag 5521 cccatttact ttaaatcgat aagtgaagca gacatgccat tttcagtgtg gggattggga 5581 agccctagtt ctttcagatg tacttcagac tgtagaagga gcttccagtt gaattgaaat 5641 tcaccagtgg acaaaatgag gacaacaggt gaacgagcct tttcttgttt aagattagct 5701 actggtaatc tagtgttgaa tcctctccag cttcatgctg gagcagctag catgtgatgt 5761 aatgttggcc ttggggtgga ggggtgaggt gggcgctaag ccttttttta agatttttca 5821 ggtacccctc actaaaggca ctgaaggctt aatgtaggac agcggagcct tcctgtgtgg 5881 caagaatcaa gcaagcagta ttgtatcgag accaaagtgg tatcatggtc ggttttgatt 5941 agcagtgggg actaccctac cgtaacacct tgttggaatt gaagcatcca aagaaaatac

Code found through http://www.ncbi.nlm.nih.gov/nuccore/NR_002847.2 Section chosen:5’ gaccacacag aatgcaggtg tcttgacttc aggtcatgtc tgttc 3’ GC ratio

22:23 3’ CTGGTGTGTC TTACGTCCAC AGAACTGAAG TCCAGTACAG ACAAG 5’

(complementary sequence) 5’ GAACA GACATGACCT GAAGTCAAGA CACCTGCATT CTGTGTGGTC 3’

(antisense oligonucleotide probe sequence)

Figure 1: Above is part of the gene sequence for the MALAT-1 RNA with the section containing an

appropriate (highlighted in yellow) for the anti-sense nucleotide probe (highlighted in green) to bind to.

The anti-sense oligonucleotide sequence is simply the complementary sequence reversed.

Figure 2: The Liquid Scintillation Analyser measures the radioactivity of the probe to determine

The probes activity and purity that would give us the results in figure 3 illustrating the how effective the

purification of the labelled probe was.

Bioscience – Skills Portfolio Page 3

Figure 3: Raw data from the scintillation machine giving results as to how pure the probes were. Not

everyone on this list was working on the MALAT-1 sequence. The most pure probe used in our

experiment is marked with the red arrow. The screenshot of the email proves that I was team leader on

his occasion.

Bioscience – Skills Portfolio Page 4

Figure 4: Above are 3 images of the brain sections that have been labelled with the MALAT-1 antisense

oligonucleotide radioactive label. The images were taken by an x-ray machine that had different imaging

function giving these different variations of the pictures, allowing for some pictures to outline a specific

section more than another picture. It shows that MALAT-1 has increased concentrations in the

cerebellum as well as in the hippocampus.

Reflection

Although this piece of evidence focuses on the use of the Hybridisation techniques to label a tissue

section, hybridisation techniques are regularly used in a variety of other applications such as the FISH

techniques where the radioactive probes are replaced with a fluorescent one. Their main utility is to

analyse the locations of certain molecules by “lighting up” the region of the DNA or RNA, thereby

locating the main areas where that piece of RNA/DNA is located. In the current environment, drugs have

started targeting the RNA and DNA of cells a lot more (certainly in oncogenic cases) and this is true in

the case of the CNS.

The work was very interesting and I learnt the dynamics of how to delegate tasks and how to effectively

manage a team, qualities imperative in a laboratory context.

Bioscience – Skills Portfolio Page 1

Name: Christopher Francois Date: 26th

March 2015

Source: Toxicology

Experiment: LC50 – Comparison of the selective toxicity of a natural and synthetic insecticide

Portfolio Skills:

Bioscience skills Transferable skills

1. Ability to determine and plot an

LC50/LD50 graph electronically

2.Ability to write up the results in a

formal report

3. Determine which statistical analysis

(parametric/non-parametric) is best for

the results

4. Ability to perform statistical

analysis such as trimmed spearman

Karber

1. Ability to critically analyse

experimental data

2. IT skills

3. Hands on approach

Title: LC50 – Comparison of the selective toxicity of a natural and synthetic insecticide

Aim: To compare the selective toxicity of a natural (rotenone) and synthetic (pirimicarb) insecticide by

determining the LC50 of both on 3 differing animals (blow fly (larvae and adults), wood lice and

Daphnia).

Summary of evidence

The toxicity bioassay to determine the LC50 of the 2 insecticides rotenone and pirimicarb helped to

illustrate the sort of experiments that are very often seen when working in a toxicological context. The

experiment involved taking the 3 species, separating them into separate groups including the control

group and dosing them with varying concentrations of the insecticide. After the peak effect of the

insecticide was reached the mortality of the insects was calculated. Concentration of the insecticide vs %

mortality curves could be plotted and for the experiments that generated results that could be processed

using statistical tests the Trimmed Spearman-Karber test was used to determine the LC50 with confidence

intervals. The piece of evidence below is the report that analyses the results and discusses them in detail.

The evidence also illustrates why the Trimmed Spearman-Karber analysis was used: because it is a non-

parametric statistical test compared to a similar test, the Probit test that analyses data for the same thing,

the LC50 value but with parametric data.

BIOSCIENCE PORTFOLIO

Bioscience – Skills Portfolio Page 2

Reflection

Although far from perfect the written report illustrates some important skills that can be of use in the

pharmaceutical industry, toxicological studies of any kind and other potential areas. It shows my ability to

perform a toxicity bioassay, to analyse the data generated by plotting graphs that represent the data

graphically, to analyse the data to see whether the data is parametric or non-parametric to then later

process the data with a non-parametric statistical test.

Things that could be improved to get a better mark, outlined by the teacher grading the piece of work,

was that the introduction needed more work with additional information and citations. The discussion

could have done with more depth where more could be written on the possible mechanisms that may have

accounted for differences in results at the different life stages of the organisms and the differences

between different species. In addition, the discussion section could also have had a few sentences cut out

since they re-iterate what had been shown in the results section.

Portfolio Evidence

N0379018, Christopher Francois BIOL 33211 Toxicology

Comparison of the selective toxicity of a natural and

synthetic insecticide Abstract

The aim of this experiment is to determine the LC50 values for two different insecticides:

Rotenone, a natural insecticide often found in nature and Pirimicarb, a synthetic insecticide,

using a toxicity bioassay. These 2 insecticides were applied at varying concentrations to a

variety of organisms, (Daphnia, Woodlice, Blowfly larvae and Adult blow flies) with the

results being analysed using mean % dead vs concentration curves and Spearman-Karber

LC50 statistical analyses. From the values calculated the results were compared to determine

the selective toxicity of each. Graphical analysis showed that the Rotenone showed a large %

dead (beyond 50% dead) for Daphnia, Woodlice where the woodlice LC50 was calculated to

be 0.02mM. It showed a medial response for %dead Adult blow fly and a poor response for %

dead Blowfly larvae. It was found that the pirimicarb demonstrated a LC50 value of 2.37µM

to % dead in Daphnia. Other organisms did not give a dose response curve that intersected the

50% %dead line. To conclude, the LC50 trimmed spearman-karber method determined that

pirimicarb was more potent than the rotenone and the lower %dead yields of pirimicarb for

the Maggots and flies made the insecticide more selective than the Rotenone.

1. Introduction

When using an insecticide it is important to use one that does not endanger beneficial insects

and does not kill everything that it is used on such as the plants it is meant to protect. An

insecticide needs to be selective. In the development of insecticides and other toxicological

compounds the compounds are tested in toxicity bioassays that use biological organisms to

test how toxic the compound is. These are used to determine how selective the compound is in

relation to a varying number of insects, how it kills the insects (its biological mechanism of

action) and how potent it is. The potency of the toxicological compound is measured as a

LD50 (Lethal dose for compounds given to animals on land) or LC50 (Lethal concentration

given to any material that is toxic and soluble and therefore drinkable to both humans and

aquatic organisms). This number is the concentration or dose that causes a 50% death rate in

a given test population. A toxicological bioassay allows for a LC50 value to be calculated and

if several insecticides are tested it also allows a comparison of how selective these

insecticides are.

To perform a toxicological bioassay 2 differing insecticides must be administered to varying

organisms. In this experiment the organisms used to determine the LC50 will be: Daphnia,

Woodlice, Maggots/Blowfly larvae and Adult Blowfly. Daphnia are small aquatic organisms

that are often used in toxicological and drug testing due to their reproductive capacity, short

lifespans, transparent skin and thin membranes. This makes them ideal for the experiments we

will be conducting. Woodlice are an ideal vertebrate organism for eco-toxicological testing

due to their prevalence around the world, their size and their similar traits in short life span

and reproductive capacity (M. F. L. Lemo et al, 2010). Additionally maggots and blow flies

have been used in toxicological cases for a long time.

N0379018, Christopher Francois BIOL 33211 Toxicology

One insecticide that will be used during this experiment is Pirimicarb. Pirimicarb is a

synthetic carbamate insecticide that is produced in a factory through a chemical process. It

works as an acetylcholinesterase inhibitor (Pesticide Residues in Food, 2004) that if taken in

large enough amounts can cause neurological damage.

Another insecticide that will be used in this experiment is a natural insecticide called

Rotenone. Rotenone is found naturally in plants roots and seeds and is used by the plants as a

naturally occurring piscicide or fish killer which means that it will be the drug that has the

most effect on the Daphnia.

The aim of this paper is to compare these 2 insecticides and determine how selective they are.

Differing groups of the organisms will be administered different doses of the 2 insecticides.

After obtaining the average tabulated results and having graphed them with the use of excel, a

statistical software like LC50 will be used to perform a trimmed Spearman Karber test, a

statistical test that allows you to estimate an LC50 value for a data set that has 1 value above

50% dead and one value below 50% dead. In addition the program allows for the calculating

of confidence intervals which determine that if the experiment is repeated how probable it is

that the same is LC50 value is discovered. From these graphs and the LC50 values, an

adequate comparison should be possible to determine whch of the 2 is more potent and which

is more selective.

2. Methods:

Experiment: The method for the experiment was similar to the protocol.

Basic concentration and dilution calculations determined that from a stock bottle solution of

8g/L with a relative molecular mass of 394.4, the molarity was 20.3mM. with a subsequent

dilution equivalent to 5ml of the bottle solution in 1.1 litres the field strength (1X) of the

rotenone was determined to be 9.220 x 10-5

and therefore the remaining concentrations of 3x,

0.3x, 0.1x, 0.03x were 2.768x10-4

, 9.22x10-5

, 2.768x 10-5

, 9.22x10-6

, 2.768x 10-6

respectively.

Using the concentration obtained and the tabulated mean results logarithmic scales were

created and the results of the experiment were graphed.

The mean tabulated results were also statistically analysed by analysis programs such as a

program named LC50 originating from Montana State university which were both used to

determine the LC50 values (Hamilton, 1978). The LC50 program used the Trimmed

Spearman-Karber method. Each of these statistical tests has its own benefits and drawbacks.

For more information see 4. Discussion)

3. Results

Graph 1: Pirimicarb and Graph 2: Rotenone

N0379018, Christopher Francois BIOL 33211 Toxicology

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

-8 -7.5 -7 -6.5 -6 -5.5

% M

ort

alit

y

Log10 PIRIMICARB concentration (M)

Effect of Pirimicarb concentration vs % mortality of varying species

% dead Daphnia % dead Woodlice % dead Maggots % dead Flies

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

-6 -5.5 -5 -4.5 -4 -3.5

% M

ort

alit

y

Log10 ROTENONE concentration (M)

Effect of Rotenone concentration vs % mortality of varying species

% dead Daphnia % dead Woodlice % dead Maggots % dead Flies

N0379018, Christopher Francois BIOL 33211 Toxicology

The graphs above show the mean % dead on the y-axis, and the absolute values of

log10(Pirimicarb/Rotenone) concentrations (M) that were administered on the x-axis. The

curves give the dose response of each organism with the Daphnia seen as the blue, the

Woodlice seen in red, the maggots/blowfly larvae seen in green and the Adult blow flies seen

in light blue. Each data point is supported by a positive and negative standard error bar.

The daphnia population being administered pirimicarb, and the woodlice populations being

administered rotenone both had data points that bisected the 50% mark and therefore allowed

for the data to be analysed using the Trimmed Spearman-Karber LC50 statistical tests.

The Daphnia population being administered Rotenone did have multiple values above 50%

but none of the values except for the control were beneath 50% mean % dead. Since all

trimmed Spearman-Karber analysis require one value above and one value below the 50%

mark this meant that the program for trimmed Spearman-Karber LC50 analysis, which does

not function for concentrations of 0, would be unable to calculate a reasonable LC50 value

that corresponded with the above graphs.

Fig1: LC50 for Daphnia with pirimicarb determined by Trimmed Spearman-Karber

Pirimicarb Mean # killed Total tested

Concentration (M) Concentration (µM)

Program input Raw data

input Program

Raw data input

Program

0 N/A 0.2 N/A 11.9 N/A

3 x 10-8 0.03 2.5 2 12.3 12

1 x 10-7 0.10 2.5 2 12.6 12

3 x 10-7 0.30 3.7 3 12.2 12

1 x 10-6 1.00 4 4 12.6 12

3 x 10-6 3.00 6.1 6 11.9 11

LC50: 2.37µM LOWER 95%

LIMIT: 0.67

UPPER 95% LIMIT:

8.35

PERCENT TRIM:

45.45

This is a table showing the results of the LC50 analysis of the pirimicarb concentrations on

the Daphnia populations at the bottom with the Lower and upper confidence limits as well as

the percent trim. In the columns it shows the raw mean data and next to these numbers are the

numbers that have been rounded up by the user or the program. This is to highlight that the

program rounds up the data that is put in and therefore makes the statistical results

questionable (See 4. Discussion). The N/A is there because the program would not accept

control values.

Fig2: LC50 for Woodlice with Rotenone determined by Trimmed Spearman-Karber

Rotenone # killed Total tested

Concentration (M) Conc results (mM) Raw data Program Raw data and program 0 N/A 0.833333333 N/A 10

2.768 x 10-6 0.00 3 3 10

9.220 x 10^-6 0.01 4.333333333 4 10

2.768 x 10^-5 0.03 5.833333333 5 10

9.220 x 10^-5 0.09 8.333333333 8 10

2.768 x 10^-4 0.28 9.6 9 10

LC50: 0.02mM LOWER 95%

LIMIT: 0.01

UPPER 95% LIMIT:

0.07

PERCENT TRIM:

30.00

N0379018, Christopher Francois BIOL 33211 Toxicology

Figure 2 is very similar to figure 1. It illustrates the results of the LC50 analysis of the

Rotenone concentration on the Woodlice populations at the bottom with the Lower and upper

confidence limits as well as the percent trim. It also shows the raw mean data and next to

these numbers are the numbers that have been rounded up by the user or the program.

4. Discussion

At first glance the first graph makes it seem that it is the less potent chemical, but upon closer

inspection, a lower concentration is given than the concentrations in Graph 2: Rotenone. It

shows that at a relatively small concentration the curves for %dead daphnia, woodlice and

Maggots all seem to rise to 10-20%. This is followed by a continuous increase of % dead in

the curves for %dead woodlice and Daphnia. The % dead Daphnia does go past the 50% dead

mark and subsequently enables a Trimmed Spearman-Karber LC50 analysis to be performed

on the data giving an LC50 value estimated at: 2.37µM. The curves for the %dead maggots

and %dead flies show a marginal decrease and never rise up past the 20% line. However, the

standard error bars are quite large.

In the second graph: Rotenone, when the % dead Daphnia rises up quickly and plateaus

relatively quickly in comparison to the other organism curves. It should be noted that the high

% found at the control level for both the Daphnia and the woodlice in the rotenone graph may

have influenced the results by increasing the % dead and therefore increasing the efficiency of

the drug. The curve for % dead woodlice does rise up, and reach 96% dead at the end of the

experiment and with subsequent analysis of the LC50 for that drug with the woodlice being

estimated at 0.02mM. The curve for the %dead flies shows a gradual increase but not reaching

50% and then decreases slightly at the end. On the bottom are the undulating results of the %

dead maggots which never rise up above 10% dead.

When comparing the 2 graphs, a trend can be spotted. The high % dead in rotenone for the

Daphnia, the Woodlice and the rising % dead of the flies indicates that rotenone’s mechanism

of action allows it to be more unselective in its effect on varying species. The mechanism of

action of Rotenone: the interference of the electron transport chain by inhibiting the iron-

sulphur centers in complex 1 specifically at the Q10 coenzyme known as ubiquinone (de

Groot et al, 2014) therefore stopping oxidative phosphorylation may be why the daphnia died

so quickly. This leads to falling rates of ATP that in turn cause cell death. An additional effect

to the oxidative phosphorylation stopping is that it creates a backflow of electrons that then

reduce oxygen into ROS (reactive oxygen species) which subsequently disrupts DNA and

RNA processes in the cell and the mitochondria (El-wakeil N.E, 2013). The combination of

these 2 effects leads to cellular apoptosis in normal and neuronal cells indicating symptoms of

parkinsons disease in larger animals (Rang et al, 2012) eventually resulting in death of the

organism. The data in graph 2 for Rotenone emphasizes the point that rotenone is a natural

insecticide found in seeds, roots and stems of plants among other things. Its use in these

organism’s gives it purpose in necessitating it to be as lethal as possible in order for the host

producing it to survive. In other words, Rotenone shows that a natural insecticide can be built

to be unselective and so that the host may survive. A good example seen is that the daphnia

die very quickly when exposed to certain concentrations of the compound, this may be due to

N0379018, Christopher Francois BIOL 33211 Toxicology

a possible ease of entry through their thin membranes, it is logical that a plant would want to

make sure over-reproductive feeder species like Daphnia do not take up all the nutrients

around it.

Comparing the data in the 1st graph to the 2nd shows that pirimicarb acting an

acetylcholinesterase antagonist (Pesticide Residues in Food – 2004, p252) has a less

devastating effect than rotenone. The low levels of % dead are attributable to low levels of

pirimicarb tested but also due to pirimicarb being a selective systemic insecticide whose mode

of action is only through direct contact with the insect (Tomlin, C.D.S, 1994). The

antagonistic effects of the pirimicarb to the organism’s muscarinic and nicotinic receptors

leads to a breakdown of the organism’s neuronal activity that affects the motor systems

through paralysis, perhaps convulsions and spasms eventually leading to brain death

(Francisco Sánchez-Bayo, 2011).

Figure 1 principally highlights the LC50 value found with Pirimicarb having an LC50 of 2.37

µM in Daphnia. The comparison of this to Figure 2 highlights the difference between the 2

substances. In figure 2 the LC50 value found for the administration of Rotenone on the

woodlice is 0.02 mM. This shows that the Rotenone is less potent than the pirimicarb since

pirimicarb requires a concentration of 2.37 µM to function as a lethal concentration to kill

50% of the population versus the 0.02mM that once turned into µM is 20 µM. However, it

must be noted that the test illustrated in figure 1 is on Daphnia, the test illustrated in figure 2

is a woodlouse. The differences between the 2 organisms, namely that one is aquatic and the

other not, as well as the Daphnias thin-membrane like skin, its small size compared to the

woodlouse and the general difference in their biochemistry due to one living in the water.

The same figure also presents raw data as well as data that has been rounded up. Some of this

data was rounded up by the user (Concentration) to be able to run the calculation, or was done

by the program (Mean # killed/Total tested) at the end of the test. The difference between the

actual data and the input data leads to the LC50 result shown having a high trimmed percent

(45.45) and the higher the trimmed percent is, the less reliable the confidence intervals ,here

shown as the lower and upper 95% limits, are (Hamilton et al., 1978). The confidence levels

help determine the possible range of where the LC50 values will be if the calculations are

repeated in another experiment. In this case the range has an upper limit of +8.35 from the

mean LC50 value of 2.37 µM and a lower limit of 0.67 away from the mean LC50 value. If

the confidence levels are unreliable then a repeated LC50 level would potentially find a

different LC50 value that is not within this range and giving a different answer.

The effect of the differences between the raw data and input data are minimal in figure 2 since

the lower 95% limit is 0.01 and the upper 95% limit is found to be 0.07. The trimmed percent

is also much lower than the previous figure at a 30% rather than 45.45% with 50% being the

limit for unreliable trimmed percent (Hamilton et al., 1978).

Probit and Trimmed Spearman-Karber Statistical tests for LC50 both have advantages and

disadvantages. They both require 2 values to bracket the 50% mean dead, they both give

LC50 values and confidence intervals however it is how they do this that they differ. In this

paper, Trimmed Spearman-Karber is the main Statistical test done on the organisms with 2

N0379018, Christopher Francois BIOL 33211 Toxicology

data points above and below the 50% mean % dead. This is because Probit is a parametric test

that assumes that the null hypothesis is made based on knowledge of the data parameters and

it makes assumptions about the data’s distribution, specifically in this case: that the data is

normally distributed. When the data is put into minitab and a probit analysis is performed, the

Goodness of fit Probability value (p) gives a value higher than 0.05, therefore the data is not

normally distributed and a nonparametric test is better suited. Another reason for this choice is

that non-parametric tests such as the Trimmed Spearman-Karber are more comfortable if the

data deviates strongly from assumptions, something that with this set of data makes it more

favourable to work with than a parametric test that might give incorrect results due to a

deviation. The last reason a non-parametric test should be used in this situation is the small

sample size, something that would make a parametric test less precise but something for

which the non-parametric was made for. In contrast however, non-parametric tests are less

statistically powerful. They are also not as efficient as parametric tests.

5. Conclusion

The analysis of the data seems to suggest that pirimicarb is the more potent insecticide due to

its LC50 value being smaller than the LC50 value of rotenone. Rotenone, with its major

graphical elimination of Daphnia and woodlice, its medial elimination of Adult blow flies and

its minor elimination of maggots shows that its mechanism of action is more widespread than

an acetylcholinesterase inhibitor like pirimicarb making Rotenone the less selective of the 2

insecticides. Pirimicarb is therefore found to be the more potent and the more selective of the

2 insecticides.

6. References

de Groot M. W. G. D .M ., Westerink R.H.S. (2014), Chemically-induced oxidative stress

increases the vulnerability of PC12 cells to rotenone-induced toxicity, NeuroToxicology 43

(2014) 102–109.

El-wakeil N.E.(2013), Botanical Pesticides and Their Mode of Action, Gesunde Pflanzen

(2013) 65:125-149

Francisco Sánchez-Bayo. (2011), Insecticides Mode of Action in Relation to Their Toxicity to

Non-Target Organisms, J Environment Analytic Toxicol 2011, S:4

Hamilton, M.A., R.C. Russo, and R.V. Thurston, 1977. Trimmed Spearman-Karber method

for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol.

11(7): 714-719; Correction 12(4):417 (1978)

Hayes' Handbook of Pesticide Toxicology, Volume 1, pg 135-136, Academc Free Press, 2010

M. F. L. Lemos., C. A. M. van Gestel., A. M. V. M. Soares (2010) Developmental Toxicity of

endocrine Disrupters Bisphenol A and Vinclozolin in a terrestrial isopod, Enviromental

Contamination an toxicology 59(2): 274-281

N0379018, Christopher Francois BIOL 33211 Toxicology

Pesticide Residues in Food - 2004: Evaluations 2004, Part 2: Toxicological, p 252

Rang H.P., Dale M.M., Ritter J.M., Fowler R.J., Henderson G. (2012) Rang and Dale’s

Pharmacology Seventh Edition. Elsevier Limited.

Tomlin, C.D.S. (ed.). The Pesticide Manual - World Compendium. 10th ed. Surrey, UK: The

British Crop Protection Council, 1994., p. 820