Research group of

Functional Genomics and Proteomics

http://bio.kuleuven.be/df/ls/people

Input

Environ

mental

stimuli

Output

Fysiological

processes

Behaviour

Processing ?

Basics of wiring, neuronal circuits: understanding

intercellular signalling

small model organisms

Neuropeptides, neuronal communication

and neuroplasticity

Humane hersenen

~100 miljard neuronen C. elegans

302 neurons

Mechanisms of biological processes and behaviours conserved during evolution.

200 neuropeptides

• Learning and memory

• Ageing

• Feeding behaviour • Reproduction

• Sleep

• Addiction

Extensive genetic toolkit: knockouts, cell-

specific knockouts, overexpression, cell

specific rescues

• Small (1 mm) freeliving soil nematode

• Feeds on bacteria

• 959 transparant cells

• Easy to cultivate

• Genome sequenced

At the level of the genotype Big ideas from small brains

C. elegans TOPICS

BRAIN CONTROL: what and how do we learn/forget? * * *

- neuropeptides and learning & memory

- sleep wakefulness mechanism

NEURODEGENERATION: *

why don’t we know why? (Huntington, Alzheimer Disease)

AGEING: WHO WANTS TO LIVE FOREVER? * * *

neuron-specific -omics in long-lived worms-

WEIGHT CONTROL * *

- why and how do we gain weight?

REPRODUCTION

what is happening in puberty?

Basics of wiring, neuronal circuits,

understanding intercellular

signalling

oxytocin trust

bonding Love hormone

Nieuwsblad november 2012:

Oxytocin ('the Love Hormone') houdt

uw man weg van andere vrouwen.

Ron Paul Won't Use Brain Chemical

Oxytocin to Influence Voters, Obama and

Romney Will

Nematocin mutants can’t associate sensory stimuli with presence/absence of food. They can’t learn from earlier experiences.

Nematocin: Pavlov’s dog revisited

Wormen leren op... | Facebook

www.facebook.com/faculteitwetenschappen/

Wormen leren op dezelfde manier als mensen! Tot die bevinding

kwam Isabel Beets, doctoraatsstudente in de on

Wormen leren op dezelfde manier als mensen... Tof. - Topsy

In het nieuws – KU Leuven DE MORGEN. Wormen kunnen net als

mensen verbanden leggen tussen prikkels uit hun omgeving en zo

Worm leert zoals mens | Eos Wetenschap - P1

29 okt. 2012 – Wormen kunnen net als mensen verbanden leggen

tussen prikkels uit hun omgeving en zo leren uit hun ervaringen.

Wormen leren uit hun fouten - Wetenschap - Nieuws -

Knack.bewww.knack.be ›

Signaalmolecule om te leren ontdekt bij de worm –

Wetenschapsforum www.wetenschapsforum.nl › ... ›

Wormen leren op dezelfde manier als mensen - HLN.be

www.hln.be/... 26 okt. 2012 – Wormen kunnen net als mensen

verbanden leggen tussen prikkels uit hun omgeving en zo leren uit

opgedane ..., lees meer op Kanaal Planet.

Twitter: houbi‏@houbi27 oktober

„Wormen‏leren‏op‏dezelfde‏manier‏als‏mensen”‏lees‏ik‏in‏DM‏. Maar

is dat dan met of zonder iPads in de klas?

oxytocine

Molecular pathways

• molecular signals used by

VP/OT during learning ?

• up- and downstream players ?

Neuronal circuit dynamics

• neuronal circuit behind

vasopressin/oxytocin effects on

learning ?

• changes in cellular response upon

learning?

• evolutionary conserved circuit?

calcium imaging optogenetics

BRAIN CONTROL neuropeptides and learning and short-term memory

The behaviour of the worm can be genetically manipulated by light by making transgene worms in which specific neurons express light-sensitive ion channels, Channelrhodopsine-2 (ChR2, depolarisation, activation) and Halorhodopsine.

Photoactivation of defined neurons in the head let the worms crawl in the shape of a triangle

From genetics to behaviour: Understanding experience-based learning behaviour

Optogenetic tools.

BRAIN CONTROL neuropeptides and long-term memory

Info: Isabel Beets, Liesbet Temmerman

Is vasopressin/oxytocin involved in longterm learning & memory?

Are there gender-specific differences?

- Set up longterm memory test (spaced training)

- identify cells involved via GFP reporter constructs and confocal microscopy

- identify downstream players via single-cell RNA sequencing

Build model for learning & memory

Alzheimer’s disease • World’s most common neurodegenerative disease • Dementia: dramatic emotional and financial consequences • Amyloid-β and Tau known toxic aggregates in brain

roel.vanassche@bio.kuleuven.be

Techniques

• Single copy insertion @ known place in

genome (MosSCI)

• Microinjection

• Confocal microscopy

• Behavioural/learning assay

• Molecular genetics

Aim • Generate humanized C. elegans Alzheimer

models • Measure toxic effects of Amyloid-β and Tau • Analyse learning/forgetting behaviour,

movement

Neurodegeneration: why don’t we know why?

Everyone ages.

Why?

Naakte molrat 28 jaar

Huismuis 4 jaar

AGEING: WHO WANTS TO LIVE FOREVER? * * *

Ageing

Before 1990:

ageing = random accumulation of

damage

The “rate‏of‏living”‏and oxidative

damage theory

Antioxidants have no

lifespan extending effect

Ageing 1993:

Knocking out one gene doubles life span in

Caenorhabditis elegans

Ageing is a biological proces contralled by

signalling molecules and pathways!

Uit Kenyon et al., 1993

Ageing: Who wants to live forever? Neuron-specific -omics in long-lived worms

Techniques:

Many life-extending interventions available in C. elegans

insulin pathway mutations, dietary restriction, life-extending‏→

compounds (metformin, spermidine,…)

Topic 1: Neuronal control of longevity: Neurons play a central role in the endocrine regulation of aging which neuronal proteomic & peptidomic changes allow

animals to live longer?

Proteomics & Peptidomics Fluorescent labelling

of neurons

Info: Wouter De Haes, Liesbet Temmerman

Timing lifespan

Contact: Wouter.DeHaes@bio.kuleuven.be

Topic 2: Cognitive decline in ageing

• Ageing leads to cognitive decline; process is poorly understood

• The applicant will profile the age-related decline of cognition in C. elegans and identify the molecular mechanisms involved.

• Techniques: Associative learning assays, RNAi

• Several naturally occurring compounds can increase lifespan • The applicant will screen promising compounds from

medicinal herbs on their ability to increase lifespan in C. elegans.

• Techniques: Lifespan assays, RNAi,…

Topic 3: Effect of medicinal plant extracts on ageing

Ageing: Who wants to live forever?

All animals sleep

What is the molecular mechanism? Is it evolutionarily conserved?

BRAIN CONTROL: SLEEP-WAKEFULNESS MECHANISMS

IN C. ELEGANS

NLP-22

Induction of sleep and

molting

GNRR-

3

In vivo localisation

Overexpression , rescue and double mutants

(molecular biology, PCR, cloning...)

sleeping worm movie.mov

Assesing sleep

behavior

Feeding behaviour and metabolism

- Hart en vaataandoeningen - Diabetes - Hoge bloeddruk - Galblaas dysfunctie - Ademhalingsproblemen - Sommige vormen

van kanker

1.6 miljard met overgewicht

400 miljoen obese

1/3 in US

1/7 in België

Onderzoekers hopen met de toepassing van genomics-technieken de sleutelprocessen in kaart te brengen die leiden tot een verstoorde stofwisseling.

Curing obesity: unravelling the CK-signalling system

Signaling pathways regulating food intake and fat storage are highly conserved in C. elegans

Molecular techniques

- (qRT)-PCR

- receptor de-orphanization

- in vivo localisation, cell specific rescue

- in vivo calcium imaging

Phenotyping

- Food intake

- Fat content

- Live motility tracking

CCK/CK Aim: molecular and genetic

dissection of CK-pathway

controlling satiety and fat storage

Contact: kevin.vancalster@bio.kuleuven.be

Restoring (ab)normal feeding behavior in Caenorhabditis elegans through genetic engineering

Supervisor: Lise Peeters (Lise.Peeters@bio.kuleuven.be)

blanco DM Nlp-12 Ckr-2 Eat-2 N2

Feeding

assay

Techniques • generation of transgenic lines by MosSci-biotic

• Localisation of receptor by fosmid recombineering and

modified pSM vectors

• Starvation and & refeeding experiments, real-time PCR

Mutagenesis

GFP fluorescent reporter worms for oocyte maturation

Fluorescent worms

worm sorter

Non fluorescent worms

Sequencing van genoom en vergelijken met wildtype

Molecular genetic deciphering of the reproductive pathway in the model organism Caenorhabditis elegans

Aim: identify genes that influence oocyte maturation

1) FORWARD GENETICS

TARGET PHENOTYPING

2) defects with respect to r

egg-laying behavior and

development

3) qRT-PCR analysis of the effects on steroidogenesis

1) one-step WGS and SNP

mapping,

genetic rescues

vit-2::gfp

reporter

EMS

mutagenesis mutant identification

2) REVERSE GENETICS

fundamental insights in genetics of

reproduction

RNAi of candidate genes

TECHNIQUES

Molecular genetic deciphering of the reproductive pathway in themodel organism Caenorhabditis elegans

Supervisor: Liesbeth Van Rompay (liesbeth.vanrompay@bio.kuleuven.be)

TECHNOLOGY DEVELOPMENT

2D-DIGE

Differential

proteomics

Microarray-

analysis

Transcript-

omics

Mass

spectrometry

Peptidomics

RNA

interference

Gene Silencing

Next

generation

sequencing

Phenotypic plasticity in locusts:

epigenetic regulation

Elucidation of locust phase polyphenism with gel based proteomics.

2 phenotypes

1 Genome

Epigenetic component?

DNA

Methyl-Cytidine

Differential DNA methylation?

Differential PROTEOME?

Workflow:

Tissue dissection

Protein extraction

Differential analysis: 2D-DIGE

Protein identification

- MALDI TOF/TOF mass spectrometry

- Bioinformatics de novo protein identification

Brain

Bart.Boerjan@bio.kuleuven.be

More info?

Towards a novel insect pest control strategy with endogenic parasites

Endogenic

gut parasites

Isolation and

characterisation

of the locust-parasite

mutualism

Genome sequencing project

of the parasite

Modification of the transgenic

parasites

-Fluorescent tags

- study of infection dynamics

Modification of the parasite

which is lethal for the host

More info:

Bart.Boerjan@bio.kuleuven.be

Creativity

Genetic

engineering

Proteome analysis of FFPE colon tumor tissue (Valerie.Broeckx@bio.kuleuven.be)

Seperation of proteins

FFPE tissue Protein extraction Proteomics Mass spectrometry

Formalin Fixed Paraffin Embedded tissue

PROBLEM:

Formalin-induced protein cross-links pose challenges for

proteome analyses of FFPE tissue

OBJECTIVES:

• Develop a reliable proteomics workflow enabling the analysis of the FFPE tissue

proteome

• Identify prognostic biomarker candidates for colorectal cancer

TECHNIQUES:

• Proteomics:

Gel Enhanced Liquid Chromatography, gel free approach, 2D gel electrophoresis

• Mass spectrometry

Aphid = agricultural pest, easy to breed, endosymbionts, diverse

host-pathogen interactions, reproductive plasticity, viviparous ...

epigenetic control

microRNA control

.

. International context: Erasmus .

Summertime: asexual

reproducing females

LD SD

Photoperiod shortening

Fall: switch towards sexual reproducing females

.

ERASMUS FRANCE: APHID PHENOTYPIC PLASTICITY

EPIGENETIC AND MICRO-RNA CONTROL

OVERVIEW ALL TOPICS & CONTACTS

BRAIN CONTROL: neuropeptides and learning&memory (2) Isabel Beets

sleep and wakefulness Lotte Frooninckx NEURODEGENERATION: why don’t we know why? (Huntington or Alzheimer Disease)

Roel Van Assche WHO WANTS TO LIVE FOREVER? neuron-specific -omics in long-lived worms

Wouter De Haes Cognitive decline in ageing

Liesbet Temmerman Effect of medicinal plant extracts on ageing

WEIGHT GAIN: role of cholecystokinin (curing obesitas) Kevin Van Calster restoring (ab)normal feeding behaviour by genetic engineering

Lise Peeters MOLECULAR GENETICS OF REPRODUCTION: forward and reverse genetics

Liesbeth Van Rompay LOCUST PEST CONTROL: from harmless parasite to Trojan horse

Epigenetic cause of solitary-migratory behavior switches in locusts?

Bart Boerjan CLINICAL: proteome of FPPE tissue Valerie Broecks ERASMUS: Université de Rennes: Aphid phenotypic plasticity: epigenetics and microRNA’s

info: website http://bio.kuleuven.be/df/ls/ Liliane.Schoofs@bio.kuleuven.be or xxx.yyyy@bio.kuleuven.be