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Development and application of high throughput molecular methods for...

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Titel Development and application of high throughput molecular methods for studying human gut microbiota in relation to diet and health
Looptijd 04 / 2002 - 06 / 2007
Status Afgesloten
Dissertatie Ja
URL http://library.wur.nl/wda/abstracts/ab4208.html
Onderzoeknummer OND1309786

Samenvatting (EN)


[Introduction]:
The human gastrointestinal tract inhabits up to 1014 total bacteria, termed the microbiota or microflora, which are influencing human health in many ways. The composition of microflora is host dependent, but still affected by environmental factors such as diet, age, and lifestyle. It may also be altered due to intestinal or other diseases. A so-called balanced microbiota has a positive influence on the health of the host. The majority of gut microbes have not yet been cultivated and described. Thus methods to analyze large numbers of human fecal samples with respect to the microbial composition, that do not rely on cultivation methods, will result in significant insight on the effect of the diversity of the microbiota on our health. High throughput molecular methods such as DNA array technology would allow large numbers of samples to be analyzed. In addition to the diversity the activity of the microbiota may also be estimated with such a technique.
[Aim]:
The main aim of this project is to develop high throughput molecular methods for gut microflora monitoring, and to find links between intestinal dysfunction and the microbiota.
[Research topics]:
A diversity DNA array for detection of numerically dominant microorganisms present in the human gastrointestinal tract is being developed. Initially a macroarray is being constructed based on a modification of the dot blot hybridisation technique. The 16S ribosomal DNA of a known sequence spotted on an array is hybridised to different variable regions of 16S rDNA or probes for an unknown organism. Hybridisation conditions are being optimised to give specific and strong signals, which can also give an information about quantification ratio of microbiota. The DNA macroarray will be scaled up to high-density microarrays.
Molecular methods will be used to investigate specifically the role played by intestinal sulfate reducing bacteria (SRB) and methane producing archaea (MPA) which have been implicated in the aetiology of ulcerative colitis (UC). Genetic, environmental and immune system interactions result in the development of UC and components of the intestinal microflora play a crucial role. In situ hybridization of 16S rRNA-based specific probes will be used to determine the location of SRB and MPA in biopsy material from UC patients. The microbial activity of SRB and MPA will be analyzed in biopsies by focusing on the expression of the genes for dissimilarity sulfite reductase and the methyl CoM reductase. The microflora composition in mucosa of UC patients that are acutely diseased and patients in remission will be compared by denaturing gel electrophoresis (DGGE). Ultimately this research can provide a grater understanding of the intestinal microbial influence on human health.

Betrokken organisaties

Betrokken personen

Promotor Prof.dr. W.M. de Vos
Co-promotor Prof.dr. H. Smidt
Co-promotor Dr. E.E. Vaughan
Onderzoeker M. Rajilic (MSc)
Projectleider Prof.dr. W.M. de Vos
Promovendus M. Rajilic (MSc)

Bovenliggende onderzoeksactiviteit(en)

Themagebied VLAG3-D1 Cell

Classificatie

A71000 Voeding
D21300 Biochemie
D21700 Fysiologie
D23340 Biofarmaceutische wetenschappen, toxicologie

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