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The most abundant microflora is present in the distal parts of gut and the majority of the bacteria are Gram-negative anaerobes. Toll-like receptor 4 (TLR4), one of the ¡§pathogen-recognition molecules¡¨, recognize the lipopolysaccharide (LPS), derived from Gram-negative bacteria. TLR4 recognized the intestinal microflora and triggers the inflammatory responses. Although the effect of intestinal microflora on enhancing host to response the challenge from bacteria has been established, the mechanism has not been well studied. In this study, the relationship between TLR4 expression and the inflammatory response under intestinal microflora depletion was investigated. Mice fed with antibiotics for 4 weeks to delete the intestinal commensals and supplemented with or without LPS to stimulate TLR4 at week 3 were under sham or burn treatment. Results showed that thermal injury intestinal permeability, bacterial translocation to mesenteric lymph nodes, and neutrophil deposition in lung. Also, the activation of NF-£MB, expression of HSP70 and TLR4 were induced in intestinal after thermal injury. Moreover, TLR4 expression was increased in lung and peritoneal cells, ICAM and TNF-£\ expression were increased in peritoneal cells after thermal injury. However, NF-£MB activation, expression of TLR4, ICAM, and HSP70 were decreased in intestinal mucosa of mice with microflora depletion after thermal injury. Microflora depletion also significantly decreased the MPO activity in lung, and the phagocytic activity, the TLR4, ICAM, and TNF-£\ expression of peritoneal cells after thermal injury. Interestingly, LPS supplement reversed the effect of microflora depletion, suggest that intestinal microflora can trigger the host defense through TLR4 signaling pathway in thermal injured mice.

The human colon is an anaerobic environment densely populated with bacterial species, creating what is known as the human intestinal microbiome; an ecosystem imperative to physiological function with regards to metabolism of non-digestible residues, growth of cells and immune protection from invading organisms. As such, quantifying, and subsequently developing an understanding of the behaviour of this microbial population can be of great value. Unfortunately, because of the physical inaccessibility of many parts of the gastro-intestinal (GI) tract, routine experimentation with this environment is not practical. However, theoretical modelling techniques including in vitro and in silico simulation/experimental platforms provide a means by which further studying of intestinal microflora can be approached. Perfecting these theoretical models is an important step in further understanding colon microbiota. An existing in silico model of carbohydrate digestion in the colon, developed by Munoz-Tamayo et al. (2010) has been used as a platform for experimentation with the intention of of discovering features which may be removed and/or added to improve the performance and reliability of the design. The model is an adaptation of the waste-water engineering based mathematical model ADM1 (Anaerobic Digestion Model 1), developed to incorporate biochemical and environmental specifications as well as physical structures particular to the human colon. The model is then a system of 102-ordinary differential equation with 66 parameters.Simulations with the default model configuration as well as variations of input variables, namely dietary fiber consumption and system flow rate, were completed to study the effect on average biomass concentration, demonstrating significant sensitivity to input variables and an unexpected linearity based on the non-linearity of the original complex system. Simulations and further study suggest that advancements in in silico modelling of the colon rely on the development of a metric or scheme that can effectively compare mathematically generated data with that collected through traditional experimentation. Also, experimenting with various reactor configurations as a basis for mathematical modelling may prove simpler configurations capable of generating comparable data to more complicated designs which may then also be applicable to existing in vitro representations of the colon.

The objectives of this study were first, to investigate the effect of stress and ascorbic acid (AA) supplement during stress on intestinal microflora of broilers and layers, and secondly, to determine nitric oxide (NO) production in intestinal tract of broilers during stress, when supplemented with L-arginine, and NO production in cecal bacteria. The intestinal microflora from broilers and layers were analyzed for bacterial populations during stress and when supplemented with ascorbic acid. In both studies, stress response was induced by adrenocorticotropic hormone (ACTH) via a mini osmotic pump for 7 days, and intestinal samples were collected before and after stress response was induced. During stress, there were no significant effects on intestinal bacterial populations, but changes in intestinal microflora were found in stressed layers and broilers. When AA was supplemented during stress, both short-term and long-term, the microbial population was changed. Cecal NO production during stress, cecal bacterial NO production, and large intestinal NO production when L-arginine was supplemented in broilers were determined as nitrite using Griess reagents. The stress response was induced as in the previous studies. The cecal pouches were collected at day 7 after ACTH insertion. Nitric oxide production by the ceca of broilers during stress was decreased. The cecal bacterial NO production was determined in vitro. The cecal bacteria that produced NO were identified as Lactobacillus fermentum, and Clostridium butyricum. Supplementing with L-arginine, Nù -nitro-L- arginine methyl ester (L-NAME), and sodium nitrate did not affect bacterial NO production on MRS agar incubated anaerobically, but sodium nitrate did affect bacterial NO production on tryptic soy and anaerobic agar incubated aerobically and anaerobically, respectively. L-arginine was supplemented in broiler diet to determine the effect on intestinal NO production and microbial populations. Supplemented with L-arginine affected cecal NO production, but did not affect large intestinal NO production or microbial populations. The positive correlation coefficient between NO contents and bacterial populations was only observed in the large intestine when L-arginine was supplemented in the diet.

Serotonin (5-hydroxytryptamine, 5-HT) has been shown to activate carbohydrate metabolism in adult female Ascaris suum. Serotonin may be either absorbed directly from the environment or synthesized de novo from the absorbed L-tryptophan in adult female A. suum. The enzymes necessary for the synthesis of 5-HT have been identified in both intestine and muscle tissues. The serotonin absorbed from the environment is obtained either from the host's gastrointestinal contents or from the 5-HT producing bacteria in the intestine of A. suum. Numerous 5-HT producing bacteria were identified in the intestinal microflora. The physiological contributions of 5-HT producing bacteria to the 5-HT level, turnover and binding sites in the intestinal tissue of A. suum were investigated.

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The increasing prevalence of allergic diseases in affluent societies has been associated with changes in microbial exposure early in life and a less diverse gut flora. The objective of this thesis was to assess the development of allergic sensitisation and symptoms during the first four years of life in a non-selected birth cohort in relation to environmental factors, family history, gut microbiota and salivary IgA antibodies. The cohort comprised all 1,228 infants living in a Swedish county who were born over a one-year period. The parents replied to questionnaires, and 817 children (67 %) were skin prick tested both at 1 and 4 years of age. Saliva (n=279), faecal (n=139) and blood (n=253) samples were collected at 1 year of age from children with a positive skin prick test at 1 year and from a sample of children with a negative skin prick test. Faecal samples were also obtained from 53 children at 4 years of age. Dog keeping during infancy was associated with a decreased risk of sensitisation to pollen and late-onset wheezing at age 4, and the reduced odds ratios persisted after adjustment for heredity and avoidance measures, OR 0.3, 95% CI 0.1-0.9 and OR 0.5, 95% CI 0.2-1.0, respectively. In contrast, early dog keeping was associated with an increased risk of earlyonset transient wheezing but only in children with parental asthma (OR 2,8, 95% CI 1.3-6.4). Levels of short chain fatty acids (SCFAs) in faeces were assessed both at 1 and 4 years of age and related to the development of sensitisation and symptoms. The levels of acetic (p<.01) and propionic (p<.01) acids decreased from one to four years of age, whereas valeric acid (p<.001) increased which is in line with a more complex gut microbiota with age. Allergic children, compared with non-allergic children, had lower levels of i-butyric, i-valeric and valeric acid in faeces both at 1 and 4 years of age. Low levels of secretory IgA (SIgA) in saliva were associated with wheezing but only in sensitised children. In children with positive SPT to at least one allergen both at 1 and 4 years of age and in children with circulating IgE antibodies to egg or cat at one year of age, those who developed late-onset wheezing had lower levels of SIgA than those who did not, p=.04 and p=.02 respectively. Of 9 children with levels of SIgA in the upper quartile and persistent sensitisation, none developed wheezing, compared with 10/20 children with lower levels, (p=. 01). Having older siblings, more than three infections during infancy, at least one smoking parent and male gender were all associated with high levels (in the upper quartile) of total IgA and SIgA. The findings in this thesis indicate that the microbial load early in life could affect the development of allergy. A functional assessment of the gut flora demonstrated differences between allergic and non-allergic children both at 1 and 4 years of age. Salivary IgA was associated with infections during infancy, and high levels of secretory IgA protected from symptoms in sensitised children. Finally, dog keeping in infancy may offer protection from allergy, but the mechanism is uncertain.

<p>Allergic diseases, for example asthma and eczema, are nowadays considered belonging to the most common chronic diseases amongst children in the West, but the cause for this increase in allergy prevalence is unknown. Since studies have indicated a connection between children's exposure of microorganisms during infancy and risk of developing allergic disease, it is suggested that this exposure is a crucial factor in question of allergy development or not. Other studies have established differences in microflora composition between healthy children and children with allergic disease, and several studies have shown that probiotic therapy can give positive results in both prevention and treatment of allergic diseases.</p><p>The aim of this master's thesis was to develop a method, using PCR and denaturing gradient gel electrophoresis, to study the diversity of <em>Lactobacillus spp</em>. in fecal samples retrieved from a study of the probiotic strain<em> L. reuteri</em> ATCC 55730. The developed method was successful in detecting lactobacilli in fecal samples, but three other bacterial genera commonly found in humans were also amplified. Comparison of average numbers of detected bacterial strains and lactobacilli strains between samples belonging to the probiotics and placebo groups, respectively, showed higher numbers for the probiotics group. Also, the only fecal samples that contained <em>L. reuteri</em> belonged to the probiotics group. Although the results are far from statistically significant, they support the theories that probiotics may influence the intestinal microbiota.</p>

The intestinal microflora is an extensive ecosystem of microorganisms that consists of symbiotic and pathogenic species. The microflora is responsible for many important functions in the human body. An unquestionable function is that it affects the health state of the host. The higher the biodiversity, the greater the benefit for the host. However, it is necessary to point out that this should not include a high diversity of pathogenic bacterial species. There are many "beneficial" species, especially from the Bifidobacterium and Lactobacillus families. In recent decades, the popularity of supplementing these "beneficial" species with various supplementary diets (e.g. probiotics) has been growing a lot. The presented diploma thesis deals with pilot studies of liquid commercial probiotic preparations from American companies Ascended Health (not available on the Czech market) and their effects on the human microbiome. The study involved 9 volunteers who provided 70 fecal samples before, during, and after use of the studied products. Two methods were used to determine the biodiversity of intestinal bacterial species. Both are based on the identification by bacterial DNA that encodes gene 16S rRNA. The first method uses PCR-DGGE technique and then identification by Sanger sequencing. The second method...

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