molecularbiologyseminar

Valentina Serna

Medicine studentMolecular Biology 

INTRODUCTION

NECROSIS• Morphological expression of cell death in a living tissue. (different of apoptosis)

•Pathological character.

•The most serious manifestation of disease at cellular level.

• When Atrophy, hypertrophy, metaplasia, hiperplasia are insufficient

•Irreversible.

Frequent causes : Ischemia, hypoxia, trauma, chemical or toxic substances, infection.

1. Inability to maintain the integrity of the membrane and escape of cytoplasm elements.

2. Denaturation of proteins (autolysis or heterolysis)

3. The cell loses the function

4. phagocytosis by macrophages.

INTRODUCTION

Microbial invasion, necrotic root canal, periapical disease, affect permanent tooth germ.

Sterile and isolated from microbiota

Loss of the integrity of these layers :

•Caries -> Risk of infection of the pulp (permeability dentin)

•Trauma -> exposure of dentinal tubules maybe be a route of entry for microorganisms in the oral cavity . An ischemic stroke occurs.

BACTERIA VS PULP

•SYMPTOMS•DX•TYPES

Endodontics

.Endodontics

.

INTRODUCTION

the endodontic diseases are caused by a large and diverse community of bacteria

pulp tissue necrosis and periapical diseases

Clinical and radiological data

PCR DGGE

INTRODUCTION

PCR:

• Acellular cloning process•In vitro amplification of a gen or DNA fragment •Exponential.

UTILITY: Identify virus or bacterias causing the disease

Prenatal Dx of genetic diseases and polymorphisms.

DNA typing for transplants Identify persons or bodies

Tracking mutationsEvolutionary studies

Detection of tumor cells

MATERIALS:•DNA•2 primers ( specific sequence you want to amplify)•dNTPs•Thermostable DNA polymerase (Taq)•Buffer and MgCl2•Thermocycler.

1. Denatured DNA 2. Alignment 3. Synthesis4. Electrophoresis

techniques agarosa gel or acrylamide.

20-40 cycles duration: 2H

INTRODUCTION

INTRODUCTION

Denaturing gradient gel electrophoresis (DGGE) •Is a molecular fingerprinting

method that separates polymerase chain reaction DNA products

•according to their mobility in increasingly denaturing conditions during the DNA migrate through a polyacrylamide gel.

Different bacterial species have different nucleotide sequences in the variable regions of the 16S rRNA gene, and migrate differently in the gel

Each band representing a different bacterial population

UTILITY: Comparing the diversity of microbial communities and to monitor changes in the dynamic over time.

INTRODUCTION

GENOME SEQUENCING:

•Restriction enzymes cut de DNA•Use of DNA fragments •Labeling by chemistry or radioactive form with fluorescent.•Electrophoretic methods to separate.

CHEMICAL METHOD Maxam and Gilbert (<250nucleotids)

ENZYMATIC METHOD Sanger DNA is not degrades but is interrupted synthesis of complementary strand during in vitro replication

.

Fingerprints can be upload into databases in which similar fingerprint can be view to determine microbial structural and form differences between treatments

Fingerprints can be upload into databases in which similar fingerprint can be view to determine microbial structural and form differences between treatments

OBJECTIVES

• “Possible association between clinical and radiological data from patients with community profiles of bacteria involved in cases of necrosis in the primary root canals.”

• Knowledge of the diversity of pathogens that infect the root canals of teeth could help develop more effective therapies.

• Characterize the profiles of the community of bacteria involved in cases of necrosis.

MATERIALES

Este estudio fue aprobado por el Comité de Investigación y Ét ica, El consentimiento por escrito se obtuvieron de los famil iares de los menores.

MATERIALES

RESULTADOS

Todas las muestras presentaron bandas, indicando comunidades polimicrobianas. Estas bandas presentaban una gran variación entre los individuos, por lo cual no fue

posible determinar un patrón asociado a las condiciones clínicas.

RESULTADOS

Los resultados estadíst icos señalaron que: Dientes anteriores < 20 bandas Dientes posteriores>20 bandas

>4 años relacionados con posteriores < 4 años relacionados con dientes anteriores

Se identif icaron 4 clusters A: 3-8 años, con caries en dientes anteriores y posteriores

B:> 4 años de edad con caries en posteriores

RESULTADOS

Hubo una diferencia signif icativa en: # bandas y caries (<20 bandas) o trauma (>20 bandas) como causa de

infecciones endodónticas.

No hubo diferencia signif icativa entre:

# bandas dolor, hinchazón, la movilidad, la fístula o reabsorción radicular.

DISCUSSION

CONCLUSSION

• DGGE technique is useful for investigate the endodontic microbiota in primary root canal with necrosis.

• To know the etiology of root canal infection is a good tool to treat this problem in the correct form and to avoid the necrosis in children.

• The root canal infection in children is a problem that can be avoided by using appropriate cleaning measures objects and proper cleaning of teeth.

• This seminar was a way to learn more about a topic related to medicine that allows us to broaden our scope and form of treating a patient.

CARIES OR TRAUMA

Root canal infection Pulp tissue necrosis

Cell death with pathological

character

Clinical data

Relation with:

Radiography

Produce:

PCR DGGE

•Gen 16S rRNA.•U968f-GC1•L1401

Dendograms

MAP

Develop:

Is:

Bacterial invasion

Stimulate:

Bibliography

• Martínez, L. (2012) Libro de clase Biología Molecular séptima edición

• Andrade, V. (2014) Clinical signs and bacterial communities ofdeciduous necrotic root canals detected byPCR-DGGE analysis: Research association retrieve agost 22,2014 from

http://www.sciencedirect.com/science/article/pii/S0003996914001149

THANK YOU!