Introduction. The adverse health effects of cigarette smoking are well established, including the direct effects of nicotine on human endothelial cells and fibroblasts. However, the direct effects of nicotine in the nasal cavity remain uninvestigated. The study aim was to ascertain the direct effect of chemical components in cigarette smoke on the microbial flora or microbiota of the nasal cavity. Methods. We enrolled 40 participants from the Benson Idahosa University’s Health clinics to conduct this study. Information on demographic characteristics including age, sex, and smoking duration was obtained at baseline. We collected nasal swabs of 20 smokers and 20 non-smokers. The wool content of the swap stick was immediately stored in mac Cathy bottles containing 9 ml of normal saline and transported to the laboratory in less than 45 minutes after collection for microbiological analysis under aseptic condition. The enumeration of the microorganisms was carried out using the streak and pours plate method on Nutrient agar, Blood agar, Chocolate agar, Mac coney agar and Potato Dextrose agar (PDA). A two-sample t-test was used to determine differences in enumeration of microorganisms and isolates between smokers and non-smokers. Results. Total bacterial counts for the smokers ranged from 177 - 307 x 102 cfu/ml and 223 - 551 x 102 cfu/ml for non-smokers with (p = 0.046 Nutrient agar, and p = 0.011 PDA). The bacteria isolates were identified as Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Proteus vulgaris and Streptococcus pneumonia, and the fungi isolates were identified as Aspergillus niger, Rhizopus stolonifer, Tricoderma viridae, respectively. The mean of nutrient agar in smokers is significantly lower (12.10) than that in nonsmokers (19.35), and the mean of PDA in smokers is significantly higher in smokers (5.75) than in nonsmokers (2.35). Higher percentages of gram-negative rods including Klebsiella pneumoniae (20%), Escherichia coli (20%) and Proteus vulgaris (20%) were identified in the smokers, which is in contrast to higher percentage of gram-positive cocci including Staphylococcus aureus and Streptococcus pneumonia identified in non-smokers. The most commonly isolated fungus in smokers was Aspergillus niger (45%) and nonsmokers was Rhizopus stolonifer (60%). Conclusions. Study findings demonstrated increased bacterial count and isolates in nasal cavity of smokers than non-smokers. Future studies should be warranted to understand the mechanistic role of nicotine in influencing microbiota of the nasal cavity.

Environmental contamination is a public health concern. In particular persistent organic pollutants like Polychlorinated Biphenyls (PCBs) have been associated with multiple chronic inflammatory diseases, including non-alcoholic fatty liver disease (NAFLD). NAFLD prevalence has steadily increased and is expected to continue to rise with an estimated 25% of the world’s population and 80-100 million people affected in the United States alone. Importantly, the liver is the primary site for endobiotic and xenobiotic metabolism, hence its proper function is critical for the body’s response to innate and extrinsic molecules. One way to combat the deleterious effects of PCB toxicity and fatty liver disease is by increasing consumption of beverages and foods that contain beneficial bioactive nutrients, like dietary polyphenols. However, the biological properties of these dietary compounds are subject to their bioavailability which is directly dependent on the activity of the liver. The first aim of this dissertation was to test the hypothesis that in the presence of a compromised liver, PCB-126 toxicity is altered. Indeed, hepatic and systemic PCB-126 toxicity was exacerbated in this severe liver injury mouse model with an observed increase in hepatic inflammation, systemic inflammation, and early markers of endothelial cell dysfunction. Interestingly, we also observed an increase in the novel gut-liver axis derived cardiovascular disease (CVD) marker trimethylamine-N-oxide (TMAO). Taken altogether, aim 1 proved that a compromised liver can alter PCB toxicity, with implications of the gut microbiota in disease pathology. In aim 2 we investigated whether GTE can protect against MCD-induced hepatic toxicity and development of NAFLD. Results indicated that MCD mice exhibited severe liver injury and gut dysbiosis and unexpectedly, GTE had no protective effects. Interestingly MCD mice displayed differential epigallocatechin-3-gallate (EGCG) metabolism at the hepatic and gut microbiota level, which may alter polyphenol bioavailability and therapeutic potential. Overall, the results provide insight into how a dysfunctional liver and gut dysbiosis can alter polyphenol metabolism, possibly reducing its therapeutic efficiency. In aim 3 we sought to determine potential protective effects of a prebiotic in this mouse model. MCD-fed mice were exposed to PCB-126 with or without inulin supplementation. Although findings from this study are preliminary, our evidence indicates that inulin restores body weight and body composition in this MCD+PCB mouse model and alters the expression of Cyp1a1 in PCB exposed mice, suggesting that inulin’s protective effects may be a result of its ability to interact with the AhR pathway. However further analysis will need to be done to examine the effects of inulin on hepatic, systemic, and gut microbiota endpoints. Overall the data contained in this dissertation suggests that in the presence of a compromised liver both pollutant toxicity and nutrient metabolism are altered, with implications of the gut-microbiota in disease risk. These findings suggest that individuals with end stage liver injury may be more susceptible to pollutant-induced toxicity and nutritional intervention may be unsuccessful at mitigating disease risk.

Neurological disorders, such as epilepsy, often result from inherited or newly acquired genetic mutations. However, individuals possessing the exact same disease-causing mutations can exhibit dramatic differences in the severity of their symptoms. These differences can be explained in part by environmental factors, such as the microbes in our gut, that play an important role in the manifestation of disease symptoms. Within the last decade, microbes living in the gut have established themselves as an environmental factor with profound effects on our health and well-being. Of special interest is the relationship between the gut microbiota and neurological disease. The goal of my thesis was to: 1) characterize the gut microbiota composition and 2) understand how the gut microbiota modulates seizure-like behavior using Shudderer, a fruit fly (Drosophila melanogaster) model of epilepsy. Shudderer flies possess a mutation in the voltage-gated sodium channel gene and display seizure-like behavioral abnormalities including spontaneous tremors and heat-induced seizures. We identified differences in the microbial composition of the gut microbiota between Shudderer and control (healthy) flies. We also found that by removing the gut microbiota we could improve seizure-like behavior of Shudderer flies as well as another Drosophila mutant harboring a similar genetic mutation. Together, these findings provide evidence that a bidirectional interaction exists between the gut microbiota and neurological function. Since the molecular and cellular mechanisms controlling basic biological processes are highly conserved between fruit flies and humans, these findings are expected to be applicable to mammalian systems, including humans, and may lead to the future development of novel therapeutics to treat epilepsy and other neurological disorders.

Orientador: Flávio Dutra de Resende / Resumo: O objetivo desse estudo foi avaliar o efeito da suplementação de extrato de taninos (ET) e a inclusão de diferentes níveis de ureia na alimentação de bovinos Nelores na fase de crescimento durante o período da seca. A área de pastejo era formada por forrageira Urochloa brizantha cv. Marandu (PB = 5,9% e FDN = 74,8%). Os suplementos proteico energéticos eram ofertados, diariamente, na quantidade de 3 g/kg PC (PB = 26%). O arranjo dos tratamentos foi em fatorial 2 × 2: Fator 1 - baixa (3%) e alta (5%) de inclusão de ureia; Fator 2 - inclusão ou não de ET (0,7 g/kg MS de suplemento; SilvaFeed ByPro®). No primeiro experimento (n = 8, 293 kg ± 5,6 duplo quadrado latino), foi observada tendência de aumento da digestibilidade da PB com a inclusão de ET (P = 0,06). A alta ureia no suplemento promoveu uma tendência de aumento da ureia sérica nas primeiras horas após a suplementação (P = 0,08). Além disso, o tratamento com ureia interagiu com o tempo para N-NH3 no rúmen (P < 0,01), sendo maior às 6 horas pós-suplementação para alta ureia. O ET aumentou a riqueza e reduziu a diversidade bacteriana. A análise de componentes principais indicou que a variação da microbiota ocorreu principalmente em função das diferentes inclusões de ureia nos suplementos. No segundo experimento, [n = 64, 294 kg ± 15,2, delineamento em blocos casualizados, blocagem por peso corporal (PC)], verificou-se que o desempenho não foi influenciado pelos tratamentos (P ≥ 0,21). Em conclusão, o desempenho e os parâme... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the effect of tannins extracts (ET) supplementation and different levels of urea inclusion in Nellore cattle feed in the growing phase during the dry season. The grazing area was constituted by Urochloa brizantha cv. Marandu (CP = 5.9% and NDF = 74.8%). The energy protein supplements were offered daily in the amount of 3 g/kg BW (CP = 26%). The treatment arrangement was in factorial 2 × 2: Factor 1 - low (3%) and high (5%) inclusion of urea; Factor 2 – With or without inclusion of ET (0.7 g / kg MS of supplement, SilvaFeed ByPro®). In the first experiment (n = 8, 293 kg ± 5.6 double latin square), a trend of increased digestibility of PB was observed with the inclusion of ET (P = 0.06). High supplemental urea promoted a tendency for serum urea to increase in the first hours after supplementation (P = 0.08). In addition, treatment with urea interacted with time for N-NH3 in the rumen (P <0.01), being greater at 6 hours post-supplementation for high urea. ET increased richness and reduced bacterial diversity. Principal component analysis indicated that microbiota variation occurred mainly due to the different urea inclusions in the supplements. In the second experiment, [n = 64, 294 kg ± 15.2, randomized block design, blocking by body weight (PC)], it was found that performance was not influenced by treatments (P ≥ 0.21). In conclusion, performance and digestive parameters were not affected by higher urea inclusion in the dry period and ET ... (Complete abstract click electronic access below) / Mestre

The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.

The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.

An investigation was conducted to determine the contribution of the gastrointestinal microbiota to variation in bioefficacy of methionine sources and the interrelationship between intestinal microbiota and cereal grain type with respect to gastrointestinal physiology. Apparent gastrointestinal absorption of DL-methionine (MET) and 2-hydroxy-4-methylthiobutanoic acid (MHA-FA), post-weaning intestinal morphology, digestive physiology, mucin dynamics and digesta flow were studied in a series of experiments using conventional and gnotobiotic pigs. At 14 d of age, sow - reared conventional (CON) pigs and isolator - reared monoassociated gnotobiotic pigs (EF) were weaned to corn or wheat/barley based diets supplemented with MET or MHA-FA. At 24 d of age, after an overnight fast, pigs were fed experimental diet supplemented with 107 Bq of either 3H-L-MET or 3H-L-MHA-FA per kg of feed and chromic oxide (0.5% wt/wt). Pigs were killed 3 h after consuming the meal to collect digesta and tissue samples from the stomach and along the small intestinal (SI) length. Conventional pigs fed a wheat/barley-based diet had increased (P < 0.05) total aerobes, whereas supplementation with MHA-FA increased (P < 0.05) total aerobes and lactobacilli populations in proximal SI. Among the gnotobiotic pigs, 8 pigs (2 isolators) were monoassociated with a bacteria closely related to <i>Providencia</i> spp. and 16 pigs (4 isolators) were monoassociated with <i>Enterococcus faecium</i> (EF). Species of bacterial contaminant and diet composition did not affect residual MET or MHA-FA in digesta. Decreased (P < 0.05) apparent residual MET in digesta compared with MHA-FA in CON but not monoasscoiated pigs, along with significantly higher (P<0.05) MET associated radioactivity at 5% SI tissue suggested that microbial metabolism of MHA-FA increases its retention in small intestinal digesta and contributes in part to the lower bioefficacy of MHA-FA compared to MET. A comparison of CON and EF pigs showed that wheat/barley diets increased digesta viscosity (<i>P</i> < 0.01) and proliferating cell nuclear antigen (PCNA) expression (<i>P</i> < 0.001) and tended to decrease (<i>P</i> < 0.07) aminopeptidase N (APN) activity. Monoassociation decreased (<i>P</i> < 0.01) body weight, relative spleen weight, crypt depth, PCNA expression, caspase-3 activity, sucrase expression, total goblet cells in crypts and mucin gene expression and increased (<i>P</i> < 0.01) relative SI length, digesta viscosity, villus height, APN and sucrase activity. Interactive effects between cereal grain type and microbial status were observed only as trends (<i>P</i> < 0.1) for PCNA, Muc2, APN and sucrase suggesting these effects were mediated indirectly through microbial changes. Decreased % retained chromic oxide in digesta at all SI locations and no chromic oxide at 95% SI length in monoassociated pigs indicated slower small intestinal transit of digesta in monoassociated pigs. We successfully developed the chromic oxide microassay for estimating chromic oxide in 1/20th of original sample size (2.0 g). Results of this study indicate that microbial metabolism of MHA-FA contributes in part to the lower bioefficacy of MHA-FA compared to MET. Monoassociation had major effects on intestinal physiology whereas limited indirectly mediated effects of cereal type were observed suggesting no major influences of cereal grain type during the short early post-weaning phase.

An investigation was conducted to determine the contribution of the gastrointestinal microbiota to variation in bioefficacy of methionine sources and the interrelationship between intestinal microbiota and cereal grain type with respect to gastrointestinal physiology. Apparent gastrointestinal absorption of DL-methionine (MET) and 2-hydroxy-4-methylthiobutanoic acid (MHA-FA), post-weaning intestinal morphology, digestive physiology, mucin dynamics and digesta flow were studied in a series of experiments using conventional and gnotobiotic pigs. At 14 d of age, sow - reared conventional (CON) pigs and isolator - reared monoassociated gnotobiotic pigs (EF) were weaned to corn or wheat/barley based diets supplemented with MET or MHA-FA. At 24 d of age, after an overnight fast, pigs were fed experimental diet supplemented with 107 Bq of either 3H-L-MET or 3H-L-MHA-FA per kg of feed and chromic oxide (0.5% wt/wt). Pigs were killed 3 h after consuming the meal to collect digesta and tissue samples from the stomach and along the small intestinal (SI) length. Conventional pigs fed a wheat/barley-based diet had increased (P < 0.05) total aerobes, whereas supplementation with MHA-FA increased (P < 0.05) total aerobes and lactobacilli populations in proximal SI. Among the gnotobiotic pigs, 8 pigs (2 isolators) were monoassociated with a bacteria closely related to <i>Providencia</i> spp. and 16 pigs (4 isolators) were monoassociated with <i>Enterococcus faecium</i> (EF). Species of bacterial contaminant and diet composition did not affect residual MET or MHA-FA in digesta. Decreased (P < 0.05) apparent residual MET in digesta compared with MHA-FA in CON but not monoasscoiated pigs, along with significantly higher (P<0.05) MET associated radioactivity at 5% SI tissue suggested that microbial metabolism of MHA-FA increases its retention in small intestinal digesta and contributes in part to the lower bioefficacy of MHA-FA compared to MET. A comparison of CON and EF pigs showed that wheat/barley diets increased digesta viscosity (<i>P</i> < 0.01) and proliferating cell nuclear antigen (PCNA) expression (<i>P</i> < 0.001) and tended to decrease (<i>P</i> < 0.07) aminopeptidase N (APN) activity. Monoassociation decreased (<i>P</i> < 0.01) body weight, relative spleen weight, crypt depth, PCNA expression, caspase-3 activity, sucrase expression, total goblet cells in crypts and mucin gene expression and increased (<i>P</i> < 0.01) relative SI length, digesta viscosity, villus height, APN and sucrase activity. Interactive effects between cereal grain type and microbial status were observed only as trends (<i>P</i> < 0.1) for PCNA, Muc2, APN and sucrase suggesting these effects were mediated indirectly through microbial changes. Decreased % retained chromic oxide in digesta at all SI locations and no chromic oxide at 95% SI length in monoassociated pigs indicated slower small intestinal transit of digesta in monoassociated pigs. We successfully developed the chromic oxide microassay for estimating chromic oxide in 1/20th of original sample size (2.0 g). Results of this study indicate that microbial metabolism of MHA-FA contributes in part to the lower bioefficacy of MHA-FA compared to MET. Monoassociation had major effects on intestinal physiology whereas limited indirectly mediated effects of cereal type were observed suggesting no major influences of cereal grain type during the short early post-weaning phase.

The mammalian intestine contains a dense and diverse community of microorganisms. The resident microbiota makes contributions to host to promote proper immune system development and limit pathogen colonization. In this study, the effects of microbiota disruption with or without TLRs stimulation on intestinal permeability and immunity were examined in C57BL/6 mice receiving antibiotic treatment for 6 days and in antibiotics-treated mice received dead E. coli or S. aureus at day 4. The results showed that antibiotic treatment significantly decreased the total number of bacteria including specific aerobic group Enterobacteriaceae and Enterococcus, and specific anaerobic group Lactococcus/Bifidobacterium in intestinal mucosa and lumen. Although only a slight increase in the intestinal permeability and no change in caspase-3 activity of intestinal mucosa were observed after antibiotic treatment, the bacterial translocation (BT) to mesenteric lymph nodes (MLN) increased significantly. Subsequent experiments showed that antibiotic treatment decreased the mucosal killing activity and the expression of non-defensin family including RegIII£], RegIII£^, CRP-ductin and RELM£] but not the defensin family, and increased the translocation of pathogen K. pneumoniae significantly, suggesting that the increase of BT to MLN after antibiotic treatment is likely due to a reduction in gut immunity rather than an increase of intestinal permeability. Moreover, stimulation of TLR4 reversed the effect of antibiotic treatment, suggesting that the functioning of TLR4 in intestinal epithelium is required to prevent pathogenic invasion and maintain intestinal homeostasis.

A series of four experiments were conducted to evaluate whether a preconditioning period, in which adult leghorn cockerels are allowed 3 weeks to adapt to diets containing relatively high levels of guar meal (GM) resulted in changes in digestive tract morphology and ecology. It was expected that these changes would result in increased utilization of GM as birds became acclimated to consuming it at high levels. In the 1st experiment, 28 birds were fed diets containing 0, 6, 12, or 24% GM. Consuming the 24% GM diet resulted in decreased body weight, increased small intestine, liver, and pancreas weight, increased ceca and villus length, and increased severity of injury to the intestinal mucosa (P < 0.05). In the 2nd experiment, 28 cockerels were subjected to a partial cross-over experiment. Preconditioning to the 24% GM diet resulted in decreased TMEn of GM, decreased body weight, and decreased absorptive capacity of the GI tract as evidenced by increased energy lost via the excreta (P < 0.05). The 3rd experiment evaluated how differences in endogenous energy losses (EEL) from 30 cockerels affected the TMEn content of GM in birds consuming 0, 6, or 12% GM. Decreased EEL was observed in birds conditioned to 6 and 12% GM relative to the control group (P < 0.05). Further, decreased TMEn of GM was observed in birds consuming 6% GM relative to those consuming the control diet (P < 0.05). In the 4th experiment, changes in cecal microbiota were evaluated in 24 cockerels consuming 0, 6, or 12% dietary GM using denaturing gradient gel electrophoresis of amplified bacterial DNA sequences. Cecal microbiota of birds consuming 12% GM was considerably different from the control group (similarity coefficient = 84%) with an apparent decrease in the complexity of microbial communities. Results of these studies show that consuming 12% dietary GM results in changes in the cecal microbial community which may be responsible for modest increases in the TMEn of GM when compared to 6% dietary GM, which consistently resulted in decreased TMEn values of GM. Further, determining the TMEn value of GM appears to be highly dependent upon factors such as preconditioning diet and timeframe and innate physiological "thresholds" which seem to initiate or limit the maximum physiological response to preconditioning that the bird is capable of.