[...]L'ensemble de nos études soulignent le rôle important du stress oxydant qui est lié à la stimulation du SRAA dans la survenue de la dysfonction endothéliale chez le rat présentant une cirrhose biliaire. L'action sur des facteurs pouvant induire le stress oxydant comme la translocation bactérienne et le traitement par des antioxydants (polyphénols) ont clairement montré leurs actions bénéfiques chez nos rats cirrhotiques. Le rôle du NO, en tous cas en tant que vasodilatateur pur, n'est peut-être pas primordial ce qui peut expliquer les résultats décevants en clinique publiés parcertains auteurs. / [...]The important role of oxidative stress in the development of endothelial dysfonction in rats with biliary cirrhosis is emphasized in our studies. Oxidative stress is associated with the stimulation of the RAAS and bacterial translocation. Treatment with antioxidants (polyphenols) and probiotics have clearly demonstrated their beneficial effects on the endothelial dysfonction in our cirrhotic rats. The role of NO as a pure vasodilator is perhaps not essential which may explain the disappointing clinical results published by some authors.

The prevalence of allergy is increasing and it is becoming a serious problem not on- ly in medicine, but also in social and economic terms. The most effective way to minimize the development of allergic diseases is preventive measures. In recent years, many studies have attempted to confirm or rebut the hypothesis that early administration of probiotic bacteria to newborns and pregnant women before birth could have preventive effects on the development of allergy. In the Czech Republic, the probiotic strain Escherichia coli O83:K24:H31 (EC O83), being registered with the State Health Institute for Drug Control under the name Colinfant Newborn, has long been used to prevent allergies and paediatri- cians have long been known and used it against various diarrhoea. The aim of this work was to elucidate the effect of EC O83 on CBMC (cord blood mononuclear cells) and to compare the ability of CBMC of healthy mothers (children with a relatively low risk of developing allergic disease) and allergic mothers (children at high risk of developing allergies) to form cytokines in response to EC O83 stimulation. Phytohemagglutinin was used as a positive control, Escherichia coli Nissle 1917 was used as a reference probiotic strain, which is much more known abroad than EC O83. Cytokine production was detected by...

(EN) Reduced microbial stimulation of an immature neonatal immune system can lead to a poor balance adjustment of immune responses, thus contributing to the development of allergic diseases, whose incidence continues to rise. One of the promising precautionary measures seems to be an early preventive administration of probiotic bacteria to pregnant or nursing mothers, or to newborns. Previous works have described a beneficial effect of Escherichia coli O83:K24:H31 (E. coli O83) in the prevention of allergic diseases. In order to contribute to the clarification of E. coli O83 effects on the neonatal immune system, its immune- modulating properties were tested in vitro on umbilical cord blood cells. The ability of E. coli O83 to support the maturation of in vitro-derived dendritic cells from cord blood precursors (moDCs) of the children of healthy (children with a relatively low risk of allergy) and allergic (children at a relatively high risk of developing allergies) mothers was tracked by flow cytometry, qPCR and ELISA. Probiotic bacteria-stimulated moDCs were subsequently cultured with autologous naive CD4+ T lymphocytes and immune response polarization was also characterised by flow cytometry, qPCR, and ELISA. It was evident from the results that E. coli O83 promoted moDCs maturation. The presence of...

Physiological stress in farmed abalone can lead to immunosuppression and increase the susceptibility to bacterial, viral and parasitic disease, often followed by mortality. Thus, handling and poor water quality can reduce farm production efficiency. Probiotics in aquaculture have been effective in a wide range of species in enhancing immunity, survival, improving feed utilisation and growth. Three putative probionts identified as a result of in vitro screening had been beneficial to laboratory-reared abalone in a previous study. The aim of this study was to produce an abalone feed that contains a suite of probionts that may promote abalone growth and health under farming conditions. The objectives were to compare growth and physiological responses (i.e., haemocyte and phagocytosis counts) of abalone fed a commercial feed (Abfeed®S 34, Marifeed, Hermanus) supplemented with probiotics (i.e., the probiotic diet) to abalone fed the commercial feed without probiotic supplementation as a control treatment in a factorial design with handling method as an independent variable. This experiment was conducted at HIK Abalone Farm (Pty Ltd) for a period of eight months with initial weight and length 36.1 ± 0.05 g and 58.6 ± 0.06 mm abalone-1. Another experiment was carried out at Roman Bay Sea Farm (Pty) Ltd with initial weight and length 34.7 ± 0.17 g and 62.3 ± 0.18 mm abalone-1, but this experiment included one factor only, i.e. the presence and absence of the probionts in the feed. At HIK there was no significant interaction between diet and handling on average length and weight gain month-1 after four (p=0.81 and p=0.32) and eight (p=0.51 and p=0.53) months, respectively. Average length (additional handling = 73.9 ± 0.52 mm, normal farm handling = 75.8 ± 0.57 mm) and weight gain (mean: additional handling = 68.5 ± 1.20 g, normal farm handling = 74.3 ± 1.86 g) increased significantly in animals that were handled under normal farm procedure and were either fed probiotic or control diet after eight months (p=0.03 and p=0.02, respectively). There was no iii difference in length gain or weight gain of abalone fed the probiotic diet and those fed the control diet (ANOVA: F(1,16)=0.04, p=0.84; F(1,16)=0.14, p=0.71, respectively). After four months phagocytotic count was significantly different between dietary treatments with mean values of 74.50 ± 10.52 and 63.52 ± 14.52 % phagocytosis count per sample for the probionts and control treatment, respectively (p=0.04), there was no difference after eight months at HIK Abalone Farm. There was no effect of stressor application (p=0.14) and no interaction between dietary treatment and stressor application for this variable i.e., phagocytosis count (p=0.61). There was no difference in feed conversion ratio between treatments with values ranging from 2.9 to 3.8. At Roman Bay Sea farm, there was no significant difference in mean length gain between abalone fed the probiotic and control diet after eight months (repeated measures ANOVA: F(4,28)=16.54. Mean weight gain of abalone fed the probiotic diet was significantly greater than those fed the control diet after eight months (repeated measures ANOVA: F(4,28)=39.82, p(0.00001). There was no significant difference in haemocyte counts between animals fed either probiotic or control diet after four and eight months at Roman Bay Sea farm (p>0.05).

Les probiotiques sont des « microorganismes vivants qui, lorsqu’ils sont ingérés en quantité suffisante, exercent un effet positif sur la santé de l’hôte ». Ils agissent en modulant le système immunitaire, en empêchant l’adhésion et/ou la croissance des bactéries pathogènes, en renforçant la barrière intestinale et en stabilisant sa microflore. Cependant, les mécanismes d’action de ces bactéries restent encore peu connus.Ce travail de thèse propose d’élucider les modes d’action de deux souches de Bacillus probiotiques humains : Bacillus clausii O/C et de Bacillus subtilis CU1.L’adhésion des probiotiques aux surfaces intestinales est un facteur important pour leur persistance dans l’organisme, l’immunomodulation et la compétition envers les agents pathogènes. B. clausii et B. subtilis présentent de fortes capacités d’adhésion sous forme de spores grâce à leurs protéines de surface préférentiellement impliquées dans les interactions avec l’hôte. En effet, celles-ci jouent un rôle prépondérant dans la stimulation de l’expression des gènes codant les cytokines dans les cellules Caco-2 et la production de cytokines par les cellules immunitaires, induite par les souches probiotiques, via la liaison avec des récepteurs de l’hôte. Des protéines S-layers, protéines ribosomales et protéases ont été identifiées à la surface de ces souches, ainsi qu’une grande quantité de flagelline à la surface de B. subtilis.Par ailleurs, les composés sécrétés par les souches stimulent également la production de cytokines chimiotactiques et anti-inflammatoires. B. clausii sécrète une protéase capable de neutraliser plusieurs types de toxines dont celles sécrétées par C. difficile et B. cereus. B. subtilis n’a montré aucune propension pour l’inhibition de l’adhésion des agents pathogènes testés, mais un essai clinique a démontré sa capacité à moduler le système immunitaire et la composition du microbiote intestinal. / Probiotic are ”live microorganisms, which when administered in adequate amounts confer a health benefit on the host”. They act by modulating the immune system, preventing the adhesion and / or growth of pathogenic bacteria, reinforcing the intestinal barrier and stabilizing the microbiota.However, the mechanisms of action of these bacteria are still poorly understood.This thesis proposes to elucidate the mode of action of two human probiotic strains of Bacillus : Bacillus clausii O / C and Bacillus subtilis CU1.The adhesion of probiotics to intestinal surfaces is an important factor for their persistence in the host, immunomodulation and competition with pathogens. B. clausii and B. subtilis have strong abilities to adhere as spores, through their surface-associated proteins which are preferentially involved in interactions with the host. Indeed, they play a key role in the up-regulation of gene expression encoding cytokines in Caco-2 cells and in the stimulation of cytokine production by immune cells, induced by probiotic strains, through binding with host receptors. Some S-layers proteins, ribosomal proteins and proteases have been identified on the surface of these strains, and a large quantity of flagellin on the surface of B. subtilis.In addition, secreted compounds of these probiotics also stimulate the production of chemotactic and anti-inflammatory cytokines. B. clausii secretes a protease able to neutralize several types of toxins, including those secreted by C. difficile and B. cereus. B. subtilis has not predisposition to compete with the adhesion of pathogens tested, but a clinical trial has demonstrated its ability to modulate the immune system and the composition of the intestinal microbiota.

Dans les écosystèmes marins côtiers, l‘activité anthropique et les paramètres naturels induisent chez lesorganismes aquatiques des situations de «multistress». Parmi ces paramètres, deux d‘entre eux ont été étudiés:La contamination métallique et l’infection bactérienne. Dans ce contexte la compréhension des réponsesmoléculaires et physiologiques de l’ormeau rouge Haliotis rufescens a été étudiée, en particulier, face l’impactmétallique (cuivre), et au pathogène (Vibrio parahaemolyticus) en interaction avec des bactéries probiotiquespendant le stade juvénile de ce mollusque en conditions contrôlées. Dans un première temps, une évaluation surl’effet d’un consortium probiotique formé de trois souches bactériennes: Vibrio sp C21, Agarivorans albus F1et Vibrio sp F15 a été testée et les paramètres de survie et de croissance des juvéniles de l’ormeau ont été suivis.Les résultats montrent que les probiotiques augmentent les performances de ces deux paramètres de trait de viecomparés aux individus témoins sans probiotiques. Dans un second temps, une approche génomique parbanques soustractives a été réalisée afin d’identifier les transcrits régulés par l’impact métallique. Cetteméthode a permis d’identifier 108 gènes potentiellement impliqués dans la réponse cellulaire et physiologiquedes organismes face au stress. La cinétique de l’expression génique a été suivie à l’aide de 14 transcrits et leurvariation temporelle pendant la phase critique révèle des régulations parfois complexes et différentielles chezles organismes. Ensuite, les réponses immunitaires des hémocytes d’ormeau face l'exposition au cuivre ont étérecherchées. Les résultats montrent que l’exposition au métal provoque des disfonctionnement importants desactivités hémocytaires. Enfin, l’interaction entre les bactéries pathogènes et probiotiques ont étés étudiées etleurs effets sur le mollusque ont été également recherchés. La présence des bactéries pathogènes régulel’expression génique des transcrits impliqués dans des fonctions physiologiques clés des organismes (lesdéfenses immunitaires et l’état énergétique). De plus, les résultats de cette interaction nous renseignent sur lerôle capital accompli par les bactéries probiotiques sur les performances en termes d’amélioration de la survieet de la croissance des jeunes ormeaux en condition de stress. / In coastal ecosystems, anthropogenic activity and natural factors induce “multi-stress” situations. Inthis study, we focused on two such stress-inducing factors: metal contamination and bacterial infection.Specifically, under controlled conditions, we examined the molecular and physiological responses of redabalone Haliotis rufescens toward the impact of a metal (copper) and a pathogen (Vibrio parahaemolyticus)with respect to this mollusk’s interaction with probiotic bacteria during the juvenile stage. First, we assessed theeffect of a consortium of three probiotic bacterial strains—Vibrio sp. C21, Agarivorans albus F1, and Vibrio sp.F15—on two abalone lifecycle-related parameters, namely, survival and growth. The results showed that,compared to the control treatment (no probiotics), the probiotics improved the performance of these twolifecycle parameters. In a second step, the molecular mechanisms underlying the juvenile abalone response tocopper stress were studied under experimental conditions, using a suppression subtractive hybridizationmethod. This method identified 108 partial sequences of genes involved in the cellular and physiologicalresponses to stress. The kinetics of gene expression were followed using 14 of these transcripts, and theirtemporal variations during the critical phase showed complex and differential regulation exerted by copper inthese organisms. We then investigated the immune response of abalone hemocytes to copper exposure. Theresults showed that exposure to the metal causes a significant dysfunction of hemocyte activities. Finally, theinteraction between probiotics and pathogenic bacteria (transcriptomic and cellular aspects) was studied and theeffects of these bacteria on mollusk performance were also investigated. The presence of pathogenic bacteriawas found to regulate gene expression of transcripts involved in key physiological functions, including thosethat regulate immune responses and energy metabolism. Examination of these interactions thus indicates therole of probiotic bacteria in performance in terms of the improved survival and growth of juvenile abaloneunder stress conditions.

La nutrition postnatale précoce conditionne la santé du futur adulte du fait de son rôle déterminant dans l’implantation du microbiote intestinal et le développement de la physiologie de l’hôte. L’objectif de mon travail de thèse a été d’évaluer les conséquences de la réintroduction de matière grasse laitière associée ou non au probiotique Lactobacillus fermentum dans des préparations pour nourrissons, sur le microbiote, la physiologie intestinale et le métabolisme de l’adulte, en utilisant le miniporc Yucatan comme modèle de l’Homme. Nos travaux ont montré un renforcement des défenses non spécifiques intestinales chez le jeune et une amélioration des fonctions endocrine et immunitaire intestinales de l’adulte soumis à un régime hyper-énergétique, réduisant ainsi le risque de développer une inflammation et des désordres métaboliques. Ces effets ont été associés à une modification de la digestion des préparations chez le jeune et à une modulation de la composition et de l’activité métabolique du microbiote intestinal. Des effets spécifiques de la matière grasse laitière et de L. fermentum, et d’autres complémentaires, ont été observés, suggérant des mécanismes d’action différents. Les modifications induites par la composition des préparations pour nourrissons étaient site- et âge-spécifiques, le comportement du microbiote caecal se rapprochant du microbiote fécal. En conclusion, l’optimisation des préparations pour nourrissons pourrait passer par l’ajout de matière grasse laitière et du probiotique L. fermentum. / Early postnatal nutrition programs adult health owing to its crucial role in gut microbiota colonization and host physiology development. The objective of my thesis was to investigate the consequences of dairy lipid addition with or without probiotic Lactobacillus fermentum in infant formulas on adult gut microbiota, physiology and metabolism, using Yucatan minipig as a model for humans. We demonstrated increased non-specific intestinal defences in piglets and improved intestinal endocrine and immune functions in adults submitted to a high-energy diet, which may protect them from inflammation and metabolic disorders. These effects were associated in piglets to changes in digestion and gut microbiota composition and metabolism. We observed specific and complementary effects of dairy lipids and L. fermentum, suggesting different mechanisms of action. The infant formula composition had site- and age-specific effects, the caecal microbiota being closer to the faecal one. To conclude, the addition of dairy lipids and L. fermentum in infant formulas is an effective way to improve infant formulas.

In this work the functionalised magnetic particles were tested with streptavidin to selective DNA isolation. The method of selective DNA isolation was tested by using DNA probiotic strain Lactobacillus paracasei subsp. paracasei CCDM 211/06. A test was done on the biotinyl oligonucleotic particles, which was immobilised by containing streptavidin and it was used like a DNA probe for isolation complementary DNA chain by means of DNA/DNA hybridization. The primer R 5´ bio and the biotinyl denatured specific PCR product were tested for species Lb. paracasei as a DNA probe. These following experimental conditions were optimized for selective DNA isolation: temperature and time of hybridization, amount of DNA and the release of DNA from microspheres. Isolation of DNA was verified by PCR with specific generic primers. The specific generic PCR product was amplified in extent 250 bp, which was detected by using electrophoresis in agarose gel. This optimized method was successfully used in selective isolation of DNA Lactobacillus from a complementary sample of supplementary food (BIFI pangamin).

The thesis deals with a theme of probiotic mikroorganisms which are aplicated as a dietary supplement. Health benefit on the host have been known for a long time, however, there might be some negative effects affecting a consumer. In connection with this observation defining requirements and evaluating of safety of probiotics in food was necessary. The new probiotic dietary supplement can not be expanded on the market without these processes. Every strain must be tested separately on each property. The ability of rezistance against conditions inside human gastrointestinal tract belongs to the most basic tests. The aim of the practical part is testing this ability of rezistance in vitro by simulated conditions. The model of gastrointestinal juices was designed according to the Czechoslovak codex. The form contributes to the higher rezistance of probiotics within implementing to the host. Also a dosage form of probiotics was involved to a in vitro testing.

The aim of this diploma thesis was to introduce real-time PCR with high-resolution melting analysis for Bifidobacterium species. Currently a small number of publication, dealing with identification of Bifidobacterium species using high-resolution melting analysis, is available. According to publications dealing with identification of lactic acid bacteria were selected primers P1V1 and P2V1, LAC1 and LAC2, LsppUPF and LsppUPR, V3F and V3R, V6F and V6R. Using this primers bacterial DNA was amplified by real-time PCR with high-resolution melting analysis. After evaluation of the measured results efficiency of selected primers was verified on DNA izolated from complex sample of probiotic product. After further optimisation real-time PCR with high-resolution melting analysis could be suitable using selected primers for Bifidobacterium species.