| Summary: |
Helicobacter pylori is the main cause of several gastroduodenal diseases, but recent data showed the presence of
non-H. pylori bacteria in the gastric mucosa and gastric juice, which means that interactions between non-H. pylori
population and H. pylori are possible, if not likely. Hence, the possible impact of the microbiota in gastric diseases
needs to be defined.
To clarify this paradigm, the Fluorescence In Vivo Hybridization (FIVH), was recently applied directly in the stomach,
but this detection was limited to a single bacterium (H. pylori). In addition, as the number of microorganisms that
can be discriminated by conventional FISH technique is usually limited to 3 targets, another new technology, called
Combinatorial Labeling and Spectral Imaging-FISH (CLASI-FISH), is emerging. However, these FISH techniques
suffer from limitations associated to the use of DNA probes, which might be overcome taking advantage of the
enhanced hybridization properties of nucleic acid mimics (NAMs). Taking into account this, the main goal of this
proposal is to combine the FIVH and CLASI-FISH using NAMs probes (NAM-CLASI-FIVH) in order to create a more
straightforward procedure to study the composition and spatial organization of the microbiota directly in the stomach.
In future, the application of the method together with sotisfied equipments (e.g. confocal laser endomicroscopy) will
then be useful to track over time the gastric microbiota behavior directly in the stomach of animal models without
sacrificed them.
First, to accomplish our goal, the NAMs probes used in NAM-CLASI-FIVH technique will be developed and optimized
in conditions similar those found in stomach (e.g. low pH, 37°C); then, the technique will be validated in gastric biopsies
samples. Once this has been achieved, the developed protocol will be tested and adpated in a mouse model. Then,
as a case study, we will analyse the microbial changes over time in a mouse model in terms of composition  |
Summary
Helicobacter pylori is the main cause of several gastroduodenal diseases, but recent data showed the presence of
non-H. pylori bacteria in the gastric mucosa and gastric juice, which means that interactions between non-H. pylori
population and H. pylori are possible, if not likely. Hence, the possible impact of the microbiota in gastric diseases
needs to be defined.
To clarify this paradigm, the Fluorescence In Vivo Hybridization (FIVH), was recently applied directly in the stomach,
but this detection was limited to a single bacterium (H. pylori). In addition, as the number of microorganisms that
can be discriminated by conventional FISH technique is usually limited to 3 targets, another new technology, called
Combinatorial Labeling and Spectral Imaging-FISH (CLASI-FISH), is emerging. However, these FISH techniques
suffer from limitations associated to the use of DNA probes, which might be overcome taking advantage of the
enhanced hybridization properties of nucleic acid mimics (NAMs). Taking into account this, the main goal of this
proposal is to combine the FIVH and CLASI-FISH using NAMs probes (NAM-CLASI-FIVH) in order to create a more
straightforward procedure to study the composition and spatial organization of the microbiota directly in the stomach.
In future, the application of the method together with sotisfied equipments (e.g. confocal laser endomicroscopy) will
then be useful to track over time the gastric microbiota behavior directly in the stomach of animal models without
sacrificed them.
First, to accomplish our goal, the NAMs probes used in NAM-CLASI-FIVH technique will be developed and optimized
in conditions similar those found in stomach (e.g. low pH, 37°C); then, the technique will be validated in gastric biopsies
samples. Once this has been achieved, the developed protocol will be tested and adpated in a mouse model. Then,
as a case study, we will analyse the microbial changes over time in a mouse model in terms of composition and spatial
organization during H. pylori infection.
From our knowledge, this is the first time that the fusion between combinatorial labelling and in vivo detection and
location of the gastric microbiota directly in the stomach, is suggested, making this a very innovative and challenge
project.
This projet is based on the complementary know-how of both junior and senior researchers with an excellent track
record in their specific areas of expertise. The principal investigator (PI), Andreia Azevedo, experienced on probes
design (see CV), has already developed and applied FISH methods for the in situ detection/location of microorganisms.
Nuno Azevedo (Co-PI), from FEUP/LEPABE, has recently expand his research interests to new NAMs and their
application against H. pylori (see CV). His work has led to the initiation of a biotech company, Biomode SA; Céu
Figueiredo, from IPATIMUP/i3s, is internationally recognized for her work on H. pylori-associated gastric diseases
(see CV). |