Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is a common and potentially lethal infectious human disease. Rifampicin is a front line anti-tuberculosis drug usually prescribed in combination with isoniazid, pyrazinamide and streptomycin for a period of six to seven months. When given orally for the treatment of MTB, rifampicin exhibits low bioavailability. Recent attempts to increase bioavailability and decrease dosage of anti-tuberculosis drugs have focused on creating polymer coated rifampicin nanoparticles. The research effort presented in this thesis evaluates the formation, characterization and relative bioavailability of rifampicin loaded carboxymethylcellulose acetate butyrate (CMCAB) particles using two different formulation techniques. Multi inlet vortex mixer (MIVM) and manual spray drying techniques were used to form the rifampicin containing CMCAB particles. Characterization studies and analyses of particles revealed differences in particle sizes, shapes and drug loading between the different particle formulation techniques. In vivo pharmacokinetic studies in BALB/c mice indicate that a single dose of rifampicin laden CMCAB spray dried particle formulations are able to improve pharmacokinetic parameters including relative bioavailability of rifampicin compared to that of the free drug form at the same concentration. / Master of Science

Two experiments were conducted to determine the effects of various alternatives to antibiotics in broilers grown on used litter on performance, intestinal lesion scores, body composition, and cecal volatile fatty acid (VFA) concentrations. The first experiment used a corn-soybean meal based basal diet to generate a negative control (NC) diet (without treatment), a Bacillus licheniformis probiotic containing diet (NC + 0.10% DFM1), and four diets that contained various concentrations of a Bacillus subtilis probiotic (NC + 0.05% DFM2, NC + 0.10% DFM2, NC + 0.20% DFM2, and NC + 0.40% DFM2). Experimental diets were fed to broilers over a 42-day period. Body weight gain (BWG) was depressed from d 0-28 and d 0-42 with supplementation of 0.10% DFM1 and 0.05% DFM2 compared to NC fed birds (P ≤ 0.05), with no differences among the remaining treatments and the NC fed birds (P > 0.05). Feed intake (FI) and mortality corrected feed efficiency (FEm) were not different over the 0-42 day period. No differences in oocyst shedding, lesion scores, body composition or cecal VFA production were observed (P > 0.05). Consistently low lesion scores were indicative of a mild coccidial challenge. The second experiment utilized a corn-soybean meal basal diet to generate experimental treatment diets. The basal diet without additional supplements was fed to two groups of control birds, one on clean pine shaving litter (PC) and a second on used litter (NC). Treatment groups were fed the same diet, supplemented with butyrate for the first 14 days (NC + But 0-14), botanicals from d 15-43 (NC + Bot 15-43), butyrate from d 0-14 and botanicals from d 15-43 (NC + But 1-14/Bot 15-43), and butyrate from d 0-43 (NC + But 1-43). Butyrate supplementation reduced BWG from d 0-14 (P ≤ 0.05), but there were no differences in BWG from 0-43 d (P > 0.05). Feed intake was reduced for the NC + But 0-14 group from d 0-43 in comparison to both PC and NC. Lesion scores in the jejunum were reduced with supplementation of butyrate alone, fed for either d 0-14 or d 0-43 (P ≤ 0.05) and is an indication of a mild coccidial infection due to the used litter. Although there were no significant differences among treatments, overall performance was above industry expectations, likely due to the mild fall weather. These two experiments indicate that there are numerous factors involved in the efficacy of antibiotic alternatives, at least partially explaining the inconsistent results observed in the published literature. / Master of Science / Recently, increased regulations and customer demand have restricted and reduced the use of antibiotics in the poultry industry. Historically, antibiotics were used in poultry feed at sub-therapeutic levels to improve performance and prevent the incidence of clinical and subclinical disease. Since the reduction of antibiotic use, many producers have experienced reduced performance resulting in reduced profits. The limited use of antibiotics can also present an animal welfare issue associated with increased sub-clinical and clinical disease. Many researchers are investigating alternative feed additives that will both improve performance and prevent disease, including probiotics, organic acids, and botanical products. Previous research has demonstrated the ability of these alternative to positively, if inconsistently, influence the performance and health of broiler chickens. Therefore, the objective of this thesis is to investigate the effects of two probiotic bacteria fed at various concentrations and the effects of butyric acid and botanicals, fed alone, or in combination, on the performance, intestinal lesion scores, body composition, and cecal volatile fatty acid concentrations of broiler chickens raised on used litter. In these experiments, the used litter is representative of a mild disease challenge that would likely be present in a commercial poultry setting. Overall, there were few differences with the treatment of broilers with probiotic bacteria, but the lowest inclusions slightly reduced body weight gain compared to the control fed broilers. Butyric acid supplementation reduced body weight gain over the first 14 days, but these differences were no longer observed over the 0 to 42 day period. Although performance was not improved, butyrate did result in reduced intestinal lesion scores from the middle section of the small intestine, indicating potential health benefits with butyrate treatment. Overall, the data presented in this thesis suggest that there are a variety of factors that can alter the effectiveness of these alternatives in broiler production and care should be used in selection of antibiotic replacement tools.

Pervaporation is a selective membrane technique in which a liquid feed mixture is separated by means of partial vaporization through a non-porous perm-selective membrane. This method can be used for the recovery of heat sensitive aroma compounds to avoid them from thermal damage in beverage industries. The main objective of this study was to determine the effects of feed temperature (30, 40, 50&deg / C), composition (different model solutions, strawberry essence), concentration (50, 100, 150 ppm) and permeate pressure (4, 8 mbar) on the recovery of aroma compounds of strawberry by pervaporation in terms of mass flux and selectivity. In addition, it was aimed to optimize the extraction conditions (extraction time, temperature, agitation speed, strawberry matrix) of Solid-phase microextraction (SPME), which is used for the analysis of strawberry aroma compounds. Optimum results for SPME were obtained at 40&deg / C, 700 rpm for 30 min and no matrix effect was observed. Pervaporation experiments were performed using a hydrophobic membrane, PERVAP 1070 (PDMS). As the feed temperature increased, the mass flux and selectivity increased and the total mass flux followed an Arrhenius type relation. Decreasing downstream pressure increased both total flux and selectivity, while increase in feed concentration led to higher organic fluxes but lower selectivities. In general, PERVAP 1070 showed a higher selectivity towards Methyl butyrate (MTB) than Ethyl butyrate (ETB) and MTB flux was affected negatively by the presence of ETB in the feed solution. Pervaporation experiments were also performed with a strawberry essence and strawberry model solution. The selectivities of MTB and ETB were negatively affected by the presence of other aroma compounds.

Au cours des dernières années, l’immunothérapie a révolutionné le paysage en oncologie. L’anti-CTLA-4 a montré son efficacité sur la survie globale des patients atteints de mélanome métastatique. Cependant, ce traitement présente des limites à son utilisation telles que l'efficacité clinique obtenu chez seulement 20% des patients et la survenue fréquente de colites pouvant être sévères. La recherche de biomarqueurs prédictifs de réponse clinique et/ou de développement de toxicité devient maintenant un enjeu majeur pour sélectionner les patients pouvant avoir un bénéfice à l’utilisation de ces traitements. En partant de l’observation que les colites induites par l’anti-CTLA-4 présentent des similitudes avec les maladies inflammatoires chroniques de l'intestin, nous avons émis l’hypothèse de l’existence d’un microbiote intestinal associé à une dysrégulation du système immunitaire pouvant prédire la réponse clinique et/ou la survenue d’une colite induite par l’anti-CTLA-4. Nous avons montré dans une cohorte de patients atteints de mélanome métastatique et traités par ipilimumab, qu'un microbiote intestinal enrichi en Faecalibacterium et autres Firmicutes est associé à une meilleure survie globale et sans progression ainsi qu'un risque accru de développer une colite. Les patients avec une flore enrichie en Firmicutes présentent également après traitement par ipilimumab, une activation lymphocytaire plus efficace. Par la suite, nous nous sommes intéressés aux métabolites issus du microbiote fécal et leur implication dans la réponse à l'anti-CTLA-4. Le butyrate est le principal métabolite produit par les Firmicutes. Nous avons observé chez la souris, une inhibition de l'efficacité anti-tumorale de l'anti-CTLA-4 lorsqu'elles étaient supplémentées en butyrate. In vivo, nous avons montré que le butyrate inhibe la surexpression sur les cellules dendritiques, des molécules CD80 et CD86 (molécules B7) induite par l'anti-CTLA-4. Cette immaturité des cellules dendritiques entraine un défaut d'activation des lymphocytes T spécifiques d'antigènes dépendant de l'axe CD28/B7 réduisant ainsi l'efficacité anti-tumorale. Chez l'Homme, nous avons valider cette hypothèse en montrant qu'une concentration sérique élevée en butyrate est associée à une diminution de la survie globale et sans progression comparativement aux patients avec un faible niveau de butyrate sérique.Ces travaux mettent en évidence le lien entre la composition du microbiote et les réponses immunologiques au blocage du CTLA-4. Ils apportent une explication sur un lien indirect via le butyrate entre la composition du microbiote intestinal et la réponse anti-tumorale aux immunothérapies. / In the last years, immunotherapy has revolutionized the landscape in oncology. The efficacy of anti-CTLA-4 has been demonstrated by improving overall survival of patients with metastatic melanoma. However, this treatment has limitations to its use such as the clinical efficacy obtained in only 20% of patients and the high incidence of severe colitis. Predictive biomarkers of clinical response and / or toxicity development are mandatory for a better selection of patients who will benefit from this treatment. Based on the observation that anti-CTLA-4-induced colitis has similarities with inflammatory bowel disease, we hypothesized that the gut microbiota associated with dysregulation of the immune system may predict the clinical response and / or occurrence of anti-CTLA-4-induced colitis.In a cohort of patients with metastatic melanoma treated with ipilimumab, we have shown that a gut microbiota enriched with Faecalibacterium and other Firmicutes is associated with a better of overall and progression-free survival as well as an increased risk of developing colitis. Firmicutes-driven microbiota is also associated with an improvement in lymphocyte T activation after ipilimumab treatment. Subsequently, we were interested in microbial metabolites and their involvement in the clinical response to anti-CTLA-4. Butyrate is the main metabolite produced by the Firmicutes. In mice, we observed an inhibition of anti-tumor effect of anti-CTLA-4 in butyrate-supplemented mice. In vivo, we have shown that butyrate inhibits the overexpression on dendritic cells, of CD80 and CD86 molecules (B7molecules) induced by anti-CTLA-4. This immaturity of the dendritic cells leads to a poor signaling of CD28 / B7 axis and activation of antigen-specific T-cells, thereby reducing anti-tumor efficacy. In humans, we validated this hypothesis by showing that a high serum concentration of butyrate is associated with decreased overall and progression-free survival compared to patients with low serum butyrate levels.This studie highlights the link between the composition of gut microbiota and the immunological responses to CTLA-4 blockade. They provide an explanation of an indirect link via butyrate, between the composition of the gut microbiota and the anti-tumor response to immunotherapies.

Introduction : Un hypométabolisme du glucose au cerveau peut apparaître des décennies avant le diagnostic de maladie d’Alzheimer. Pourtant, le métabolisme des cétones, le carburant alternatif principal au glucose, n’est pas altéré, autant chez les personnes plus âgées que chez les personnes atteintes de la maladie d’Alzheimer. Les méthodes connues (diète cétogène, jeûne, huile de triglycérides de moyennes chaînes) pour augmenter la production de cétones comportent leurs inconvénients. L’objectif de ces projets était de vérifier l’efficacité de produits commerciaux vendus à cet effet et d’évaluer d’autres molécules qui pourraient être utilisées pour leur propriété cétogène. Ce sont des suppléments alimentaires pouvant servir de substrat pour la cétogenèse (butyrate, monoglycéride d’octanoate (O-MAG), leucine) ou de facilitateur du transport des acides gras (carnitine). Méthodes : Pour ce faire, les participants devaient se présenter au laboratoire pour plusieurs demi-journées d’étude d’une durée de 4 heures. Lors de ces journées, un des suppléments leur était servi avec un déjeuner et des prises de sang étaient effectuées toutes les 30 minutes. Résultats : Le produit commercial contenant des MCT, le butyrate, l’O-MAG et la leucine ont permis d’augmenter respectivement les concentrations maximales en cétones plasmatiques de 568, 226, 67 et 70 µmol/L (p≤0,05) par rapport au contrôle. De plus, la concentration plasmatique en octanoate était corrélée significativement avec la concentration sanguine d’acétoacétate (r=0,85 ; p <0,0001) et de β-hydroxybutyrate (r=0,58 ; p=0,01). L’aire sous la courbe par gramme de produit ingéré montre que le butyrate était le supplément cétogène le plus efficace et était plus cétogène que le produit commercial, l’O-MAG et la leucine (p=0,009). Discussion : Le butyrate était 3 fois plus cétogène que tous les autres suppléments testés. Par contre, son ingestion a causé quelques effets secondaires à certains participants. La leucine a été le seul supplément à ne créer aucun inconfort chez les participants. Il faudrait donc étudier la possibilité de combiner plusieurs molécules dans un supplément chimiquement pensé pour être le plus efficace possible et pour être intéressant lors d’une utilisation quotidienne.

Ketosis is a metabolic condition that occurs during heat stress, prolonged exercise, fasting, and in obese and diabetic individuals. The major ketone body, β-OH butyrate, affects cellular signaling pathways in a hormone-like manner through its receptor GPR109a. While physiological ketosis is often adaptive, chronic hyperketonemia may contribute to the metabolic dysfunction of diabetes. To understand how β-OH butyrate signaling affects hepatic metabolism we compared the fasting response in control and 3-hydroxy-3-methylglutaryl-CoA Synthase II (HMGCS2) knockdown mice that are unable to elevate β-OH butyrate production, and in GPR109a +/+ and -/- mice. To directly assess the response to ketone signaling we gave intraperitoneal injections of the GPR109a agonists niacin (0.8mmol/kg) and β-OH butyrate (5.7mmol/kg) to HMGCS2 knockdown mice over the final 9 hours of a 16 hour fast. Ketogenic deficient mice showed a more robust increase in hepatic carnitine palmitoyltransferase 1 (CPT1) and uncoupling protein 2 (UCP2) mRNA than control mice in response to fasting. Accordingly, niacin and β-OH butyrate administration decreased fasting CPT1 mRNA expression in knockdown mice, while fasting knockdown UCP2 mRNA and control HMGCS2 mRNA were lowered by β-OH butyrate. GPR109a knockout did not alter the hepatic response to fasting, although the effect of niacin on fasting HMGCS2 mRNA expression was dependent on GPR109a expression. While the role of GPR109a and how chronic hyperketonemia alters β-OH butyrate signaling require further study, these data suggest that endogenous β-OH butyrate signaling during a fast regulates the transcript levels of hepatic genes directly involved in its own synthesis.

Combined intakes of specific dietary fiber and fat subtypes protect against colon cancer in animal models. We evaluated associations between self-reported individual and combinations of fiber (insoluble, soluble, and pectins, specifically) and fat (omega-6, omega-3, and docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), specifically) and colorectal cancer (CRC) risk in the Women's Health Initiative prospective cohort (n = 134,017). During a mean 11.7 years (1993-2010), 1952 incident CRC cases were identified. Cox regression models computed multivariate adjusted hazard ratios to estimate the association between dietary factors and CRC risk. Assessing fiber and fat individually, there was a modest trend for lower CRC risk with increasing intakes of total and insoluble fiber (p-trend 0.09 and 0.08). An interaction (p = 0.01) was observed between soluble fiber and DHA + EPA, with protective effects of DHA + EPA with lower intakes of soluble fiber and an attenuation at higher intakes, however this association was no longer significant after correction for multiple testing. These results suggest a modest protective effect of higher fiber intake on CRC risk, but not in combination with dietary fat subtypes. Given the robust results in preclinical models and mixed results in observational studies, controlled dietary interventions with standardized intakes are needed to better understand the interaction of specific fat and fiber subtypes on colon biology and ultimately CRC susceptibility in humans.

As intestinal epithelial cells mature, they continuously transition from proliferation to differentiation to apoptosis under the influence of cell signalling pathways including c-Jun N-terminal kinase (JNK). Glutathione S-transferases (GSTs) are cytoprotective detoxification enzymes, some of which, including GSTA1, also sequester and inhibit JNK through complex formation. Thus, GSTA1 may be a key sensor of cellular state and regulator of responses to cell stress stimuli. The focus of this research study was to investigate the functional importance of GSTA1 in two contexts: 1) modulating complex integrity with JNK and activation of JNK by oxidative stress, 2) controlling cellular transitioning between proliferation, differentiation and apoptosis. In the first study, the impact of GSTA1 levels on dissociation of GSTA1-JNK complexes and JNK activation in response to cellular stress was investigated in the human colonic adenocarinoma Caco-2 cells. The pro-oxidant menadione caused GSTA1-JNK complex dissociation in preconfluent Caco-2 cells, whereas postconfluent cells were relatively resistant to this effect. Preconfluent cells were more sensitive than postconfluent cells to menadione-induced cytotoxicity. Additionally, menadione-induced JNK activation was transient since removal of the stimulus resulted in re-association of GSTA1 with JNK and significantly reduced cytotoxicity. Over-expression and knockdown of GSTA1 affected the degree of GSTA1-JNK complex association without altering the JNK activation. However, enhanced GSH levels by N-acetyl cysteine blocked menadione-induced complex dissociation and JNK activation in Caco-2 cells. The results suggest that the mechanism of menadione-mediated JNK activation involves the production of reactive oxygen species, likely superoxide anion, and that the level of intracellular GSH plays an important role in preventing menadione-induced GSTA1-JNK complex dissociation and subsequent JNK activation. The functional importance of GSTA1 in controlling cellular proliferation, differentiation and apoptosis was investigated. Sodium butyrate (NaB) is a short-chain fatty acid, physiologically present in the human large intestine and modulates transitioning of cell states in colon cancer cell lines. GSTA1 levels increased in association with differentiation markers in postconfluent Caco-2 cells. Forced expression of GSTA1 significantly reduced cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increased cells in S-phase and associated cell proliferation. NaB (1 mM) reduced Caco-2 cell proliferation, increased differentiation and up-regulated GSTA1 activity. In contrast, higher dose of NaB (10 mM) caused toxicity in preconfluent cells via apoptosis through caspase-3 activation in association with reduced GSTA1 activity. GSTA1 down-regulation by siRNA did not alter NaB-induced differentiation or the sensitivity of Caco-2 cells to NaB-induced apoptosis. Furthermore, NaB (10 mM) caused GSTA1-JNK complex dissociation but did not affect JNK activation. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis.

Universiẗat, Diss., 2004--Hohenheim.

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No. of bitstreams: 1 peixoto_mc_me_jabo.pdf: 2637809 bytes, checksum: 171595b1d99f68cdfbe44623d1901fda (MD5) Previous issue date: 2015-12-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O aumento da expectativa de vida dos animais de companhia tem estimulado as pesquisas em nutrição para cães e gatos seguir a tendência da nutrição humana, que visa estudar intervenções que proporcionem melhor qualidade de vida e favoreçam a saúde como um todo, retardando ou até mesmo minimizando o aparecimento de doenças relacionadas ao envelhecimento. Cães idosos apresentam inflamação de baixo grau na mucosa do trato gastrointestinal, evidenciada por enterites e colites discretas a moderadas. O consumo de dieta contendo carboidratos fermentáveis, como o amido resistente (AR) pode amenizar essas alterações provocadas pela idade. O fornecimento de substrato fermentável para a microbiota intestinal, com correspondente produção de ácidos graxos de cadeia curta, em especial o butirato favorece melhor suporte nutricional à mucosa, influenciando sua estrutura e função, com potenciais benefícios à saúde e diminuição de inflamação. Este estudo avaliou os efeitos do consumo de amido resistente (AR) em cães idosos sobre a digestibilidade dos nutrientes, produtos de fermentação, IgA fecal e as características histológicas da mucosa intestinal. Uma mesma formulação foi processada de dois modos diferentes, originando ração com baixo (0,21%) e elevado (1,46%) teor de AR. Os resultados de consumo, digestibilidade, produtos de fermentação e IgA foram avaliados pela função GLM do SAS, considerando esquema fatorial 2x2, com dois tempos e dois tratamentos. Os dados de morfometria das criptas do intestino grosso foram analisados pelo teste T-pareado. Os resultados histológicos das biopsias intestinais foram avaliados por estatística não paramétrica, utilizando-se o teste de Wilcoxon para dados pareados (P<0,05). A digestibilidade da proteína (P=0,002) e do extrato etéreo (<0,001) foi maior na dieta com menos AR. Quando alimentados com a ração com mais AR, as fezes dos cães apresentaram menor pH, maiores teores de propionato, butirato, ácidos graxos voláteis totais e lactato (P<0,05). À análise histológica, não foi verificado diferença significativa em nenhum parâmetro analisado quando comparado os valores obtidos no período final de consumo da ração baixo AR e elevado AR. À análise morfométrica, foi observada tendência ao aumento do tamanho das criptas do cólon descendente dos cães que consumiram a ração com elevado AR (P=0,083). No presente estudo, mesmo apresentando efeitos sobre os produtos de fermentação bacterianos e pH fecal, não houve alterações benéficas significativas na mucosa gastrointestinal, que demonstram ser necessários mais estudos sobre o assunto em cães. / The increase in life expectancy of pets has stimulated researchs in dogs and cats nutrition that follow the tendency of human nutrition ones, aim and to study interventions that can improve quality of life and promote health, slowing or even minimizing the onset of diseases related to the aging process. Elderly dogs present low degree of inflammation in the mucosa of the gastrointestinal tract, evidenced by discrete to moderate enteritis and colitis. The consumption of diet containing fermentable carbohydrates such as resistant starch (RS) can reduce these changes caused by age. The supply of fermentable substrate for the intestinal microbiota, stimulates short chain fatty acids production, specially butyrate, that favors a better nutritional support to the mucosa influencing its structure and function with potential health benefits and reduction of inflammation. This study evaluated the effects of resistant starch (RS) consumption on nutrient digestibility, fecal concentration of fermentation products, histologic and morphometric characteristics of the intestinal mucosa of elderly dogs. A basal diet was processed in two different extrusion conditions to obtain two treatments: diet with low (0.21%) and high (1.46%) RS content. Feed intake, nutrient digestibility, fermentation products and fecal IgA were performed by GLM procedure of SAS considering a 2x2 factorial arrangement of treatments, two times and two treatments. Morphometric characteristics from large intestine were evaluated by T-paired test. Histological evaluation of intestinal biopsy were assessed by non-parametric statistic using Wilcoxon test for paired data (P<0.05). Protein (P=0.002) and fat (P<0.001) digestibility were higher for diet with low RS. Dogs receiving high-RS diet had lower fecal pH and higher concentration of values for propionate, butyrate, total volatile fatty acid and lactate (P<0.05). There was no significant differences in either parameter analyzed the histological analysis compared the values obtained in the final period feed consumption of low and high AR. According to the morphometric evaluation there was increase in the crypt size in descending colon of dogs receiving high-RS diet (P=0.083). Even with effects on bacteria fermentation products and fecal pH, there were no significant beneficial changes on gastrointestinal mucosa, that suggests that more studies on this issue are required.