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.

<p>The peritrophic matrix (PM) lines the insect midgut and is composed of chitin and protein. It is required for organization of digestion and for protection of epithelial cells from mechanical damage, pathogens, and toxins. The PM of <i>Mamestra configurata</i> (Lepidoptera: Noctuidae), bertha armyworm, a serious pest of cruciferous oilseed rape, was studied. The multilayered PM is delaminated from the anterior midgut epithelium during molting Phase II by periodic pulses and degraded during the molting Phase I stage. These events are controlled by chitin synthase-B, and chitinolytic enzymes, such as chitinase and β-<i>N</i>-acetylglucosaminidase. Eighty-two PM proteins were identified and classified as: i) peritrophins, ii) enzymes and iii) other proteins. Peritrophins were further classified as simple, binary, complex and repetitive according to their structural organization and phylogenetic analysis of peritrophin A domains. The expression of most genes encoding PM proteins was specific to the midgut and independent of larval feeding status, developmental stage, or PM formation.</p> <p>This study includes the first report of chitin deacetylase (CDA) activity in the insect midgut suggesting that the PM may contain chitosan. Digestive enzymes, such as insect intestinal lipases (IILs) and serine proteases were also associated with the PM. The IIL genes differed in their expression during larval development; however, serine protease genes were expressed continuously and serine protease activity was present in the midgut of feeding and nonfeeding stages. <i>M. configurata</i> IIM4, a complex peritrophin, was susceptible to degradation by Mamestra configurata nucleopolyhedrovirus-A challenge, as the first evidence of IIM degradation by an alphabaculovirus enhancin. <i>M. configurata</i> IIM2, a binary peritrophin, was unaffected by baculoviral challenge and such resistance of an IIM has not been reported previously. The current study is also the first demonstration of silencing by RNA interference (RNAi) of any gene encoding a PM protein, in this case <i>M. configurata</i> CDA1 (McCDA1) and McPM1. In addition, both <i>in vitro</i> and <i>per os</i> feeding experiments revealed <i>McCDA1</i> silencing starting at 24 or 36 hours posttreatment, as one of the most successful demonstrations of RNAi in a lepidopteran.</p>

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.

Les oomycètes phytopathogènes ont co-évolué avec les microbiotes des plantes hôtes. Il en résulte la formation de biofilms et des réseaux complexes d’interactions dont nous commençons juste à comprendre l’incidence sur la biologie et la virulence des oomycètes. Déterminer la nature de ces interactions et leur rôle dans le contexte d’une infection est aujourd’hui un enjeu cognitif qui concerne la caractérisation des mécanismes moléculaires et communautaires sous-jacents. C’est également une opportunité en termes d’innovation pour élaborer des méthodes de lutte alternative à l’usage de fongicides. Dans ce contexte, ce travail de thèse a consisté à étudier, d’une part, la formation de biofilm chez Phytophthora parasitica, un oomycète polyphage et tellurique, ainsi que, d’autre part, les interactions au sein de ce biofilm entre P. parasitica et le microbiote procaryote rhizosphérique de la plante hôte Solanum lycopersicum. L’analyse du génome de P. parasitica et du transcriptome du biofilm a conduit à la caractérisation d’une nouvelle famille de mucines restreinte à la lignée des oomycètes, les protéines MUCL. Ces 315 protéines sécrétées (25-30 kDa) sont réparties en 15 groupes et possèdent deux domaines : un domaine hautement conservé de fonction inconnue ; un domaine caractéristique des mucines, riche en résidus Sérine et Thréonine avec de très nombreux sites putatifs d’O-glycosylation. Chez P. parasitica, les 3 gènes PPMUCL1/2/3 sont exprimés et co-régulés spécifiquement au stade biofilm. / The interactions between a pathogen and the host surface resident microbiota are critical to disease outbreak. These interactions shape the distribution, the density and the genetic diversity of inoculum. However for most plant pathogens how each of these interactions acts on disease as a single one or as a member of a functional network remains to be specified. This issue is addressed here through the analysis of two types of interactions involving the polyphagous oomycete P.parasitica : (i) the intraspecific interaction that leads to monospecific biofilm formation by P. parasitica zoospores on plant surface; (ii) the interspecific interactions that occur between P. parasitica biofilm and the prokaryotic microbiota of Solanum lycopersicum rhizosphere. The biology of monospecific biofilm is investigated through the characterization of MUCL, a new oomycete-specific Mucin-like Protein family. Gene profiling, biochemical and immunohistological analyses define the extent of this family and lead to identify three members, PPMUCL1/2/3, as residing in P. parasitica biofilm. The Phytophthora parasitica-Microbiota interaction is explored using first a metagenomic approach. Two microbial metagenomes derived from a soil of a tomato greenhouse is defined and compared after 16S RNA gene sequencing: M1 which corresponds to the sub-rhizospheric microbiota able to colonize the roots of axenic tomato seedlings; M2, the sub-microbiota able to colonize the tomato seedling roots previously coated with P. parasitica monospecific biofilm. A representative collection of microorganisms from M2 were also obtained through in vitro selection on a medium prepared from P. parasitica extract.

In this thesis, I present a detailed analysis of the role cell morphology, solution rheology, and swimming mechanism has on the motility of Helicobacter Pylori. H. Pylori, the bacterium that causes gastric ulcers, has a helical cell shape that has long been believed to provide an advantage in penetrating the viscous mucus layer protecting the stomach lining, its niche environment. I present results obtained by performing optical microscopic live cell bacteria tracking of wild-type H. Pylori and cell shape and flagella mutants of H. Pylori. Bacteria tracking experiments show that helical shaped bacteria swim faster than straight rod-shaped bacteria, and bacteria with larger number of flagella swim faster. Altering cell shape is found to have a smaller effect on swimming speed than altering the number of flagella a bacterium has. These experimental observations are then compared to resistive force theory predictions. Resistive force theory shows qualitative agreement to our experimental observations, but overestimates the increase in swimming speed for a helical cell when compared to straight rod cell. In addition to effect of cell morphology on motility, I explore how motility is altered in different polymer environments by tracking bacteria in pig gastric mucin, methylcellulose, and gelatin solutions and gels. Bacteria are found to increase their swimming speed non-monotonically with increasing polymer concentration, while the number of mobile bacteria is found to decrease with increased polymer concentration. I also present an analysis of the swimming mechanism used by H. Pylori. H. Pylori is found to use a run-reverse swimming mechanism which I model as a random walk. This random walk model fits well to the experimental data and provides a theoretical tool for interpreting H. Pylori’s swimming mechanism. Taken together these results provide a detailed description of the motility of H. Pylori in different media and are applicable to the broad question of how H. pylori infects and colonizes the mucus layer of the stomach.

O estudo da relação entre os aditivos utilizados e os nutrientes é fundamental para o aprimoramento da utilização desses insumos e o máximo desempenho dos animais. O objetivo do presente estudo foi avaliar o desempenho e saúde intestinal (morfometria, microbiota do intestino e produção de mucina) em frangos de corte desafiados com Eimeria spp. alimentados com rações com níveis crescentes de treonina digestível e a inclusão de uma combinação de óleos essenciais. Os tratamentos foram arranjados em esquema fatorial 3x2, com três níveis de treonina digestível (100, 110 e 120% das recomendações) e suplementação ou não de óleos essenciais na ração, em um experimento no delineamento inteiramente casualizado. As rações baseadas em milho e farelo de soja foram formuladas para atender as necessidades de todos os nutrientes e fornecidas à vontade. Cada tratamento tinha seis repetições com 45 aves cada, totalizando 1620 animais. Aos 14 dias de idade, todas as aves foram desafiadas com Eimeria spp. via oral. O desempenho das aves foi avaliado semanalmente até 40 dias e, aos 21 e 40 dias de idade, foram coletadas amostras dos intestinos para análise de morfometria das vilosidades. Aos 21 dias de idade foram coletadas amostras do conteúdo do intestino delgado e cecos para análise da microbiota. Aos 40 dias, o conteúdo do íleo foi coletado para a determinação da produção de mucina. Os dados foram submetidos à análise de variância a 5%, avaliando a interação e os efeitos dos fatores principais. Aos 40 dias de idade, a inclusão do blend de óleos essenciais na dieta diminui o consumo de ração dos animais, sem alterar o ganho de peso e a conversão alimentar. Aos 21 dias, houve efeito significativo (p<0,05) no nível de 120% de treonina digestível comparado ao nível de 110%, onde os animais que receberam um maior nível de treonina na dieta obtiveram uma maior altura de vilos no íleo. Não houve efeito dos tratamentos sobre a quantidade de mucina no conteúdo ileal de frangos de corte aos 40 dias de idade. A inclusão de níveis crescente de treonina digestível e óleos essenciais na dieta, não modifica a população microbiana, podendo ser observado apenas tendências de alterações nas frequências de alguns gêneros no ceco dos animais. / The study of the relationship between the additives and nutrients is essential for improving the use of these substances and the maximum animal performance. The purpose of this study was to examine the effects of levels of threonine and the inclusion of blends of essential oils on the performance and intestinal health (morphology, intestinal microbiota and mucin production) in broilers challenged with Eimeria spp. The treatments followed a 3x2 factorial arrangement with three levels of digestible threonine (100, 110,120% of the Brazilian Tables recommendations), and supplementation or not of a blend of essential oils in the feed in a completely randomized design experiment. The diets based on corn and soybean meal to meet the nutritional requirements were fed ad libitum. Each treatment had six replicates of 45 birds each, totaling 1620 animals. On day 14, all birds were orally gavaged with 10 times the recommended dose of a commercial coccidial vaccine with sporulated live oocysts of Eimeria spp. . Performance was evaluated weekly until 40 days of age. At 21 and 40 days of age, samples of the intestines were collected for morphometry. At 21 days of age samples of the contents of the small intestine and the cecum were collected for analysis of microorganisms. At 40 days of age, the contents of the ileum were collected for determining the mucin production. The data were submitted to analysis of variance at 5%, studying the interaction and the effects of the main factors. At 40 days of age the inclusion of essential oils in the diet decrease the feed intake of the animals, without changing growth and feed conversion . At 21 days of age, 120% of digestible threonine in the diet increased the height of villus in the ileum when compared with 110% of digestible threonine (p<0,05). There was no effect of treatments on the production of mucin in the contents of the ileum. The increasing levels of digestible threonine and essential oils in the diet did not modify the microbiota.It is possible to observe a tendency of change on the frequency of some genera in the cecum of the animals.

La mucine MUC4 est un acteur important de la cancérogenèse pancréatique. Elle favorise la progression tumorale et son expression est associée à un mauvais pronostic. Il a également été montré l’implication de la mucine MUC4 dans la résistance aux chimiothérapies. L’ensemble de ces données souligne l’intérêt de la mucine MUC4 comme cible thérapeutique. De plus, sa néoexpression dès les stades précoces de la cancérogenèse pancréatique lui confère un rôle potentiel de marqueur précoce de la carcinogenèse. Les mécanismes moléculaires responsables de l’induction précoce de l’expression de MUC4 sont toutefois encore peu connus.L’étude de la régulation de l’expression de la mucine MUC4 contribuerait donc mieux à comprendre son rôle dans la cancérogenèse. Ainsi, il a été montré que le gène MUC4 est régulé in vitro par des mécanismes transcriptionnels et par des mécanismes épigénétiques de méthylation de l’ADN et des modifications post-traductionnelles des histones. En revanche, la régulation post-transcriptionelle de l’expression de MUC4 notamment par les miARN est peu connue. Nos travaux ont pour but d’identifier les miARN dérégulés dans le cancer du pancréas et/ou ciblant potentiellement MUC4, de déterminer le(s) miARN inhibant l’expression de la protéine oncogénique MUC4 et l’impact de l’administration de ce(s) miARN dans la cancérogenèse pancréatique, et d’identifier les miARN impliqués dans la chimiorésistance médiée par MUC4 dans le cancer du pancréas.Dans un premier temps, nous avons dressé le profil d’expression des miARN dans des lignées cellulaires pancréatiques humaines normales et cancéreuses par puces miARN. Nous avons pu mettre en évidence une signature d’expression de miARN qui a permis de valider nos modèles cellulaires. Puis, à l’aide des bases de données TargetScan, Microcosm et MiRanda, nous avons identifié les miARN ciblant potentiellement MUC4. L’analyse par PCR quantitative a permis de montrer que seuls le miR-145 et miR-219-1-3p étaient sous-exprimés dans l’ensemble des lignées cancéreuses étudiées. Finalement, uniquement miR-219-1-3p est capable d’inhiber l’expression protéique de MUC4 ; c’est pourquoi nous nous sommes intéressés à son rôle dans le cancer du pancréas.Nous avons observé une perte d’expression du miR-219-1-3p dans des tissus de patients atteints d’adénocarcinome pancréatique. Par deux approches complémentaires de surexpression (transitoire ou stable) ou d’inhibition de miR-219-1-3p, nous avons montré qu’il était capable de réprimer l’expression de MUC4 au niveau protéique en se fixant directement sur son 3’-UTR. Nous avons observé une inhibition de la migration et de la prolifération cellulaires associées à une diminution de l’expression de la cycline D1 et de l’activation des voies de signalisation Akt et Erk. In vivo, la croissance tumorale est fortement ralentie après l’injection intratumorale de miR-219-1-3p. Grâce au modèle murin Pdx1-Cre;LSL-KrasG12D de lésions PanIN, nous avons pu mettre en évidence que la perte d’expression du miR-219-1-3p intervient précocement dès les stades PanIN-1/2 de la cancérogenèse pancréatique et qu’il existait une corrélation inverse entre l’expression de miR-219-1-3p et celle de la mucine Muc4.Par ailleurs, nous avons observé que le traitement des cellules cancéreuses pancréatiques humaines par la gemcitabine induit une forte surexpression du miR-219-1-3p qui laisse penser à un rôle potentiel de ce miARN dans la sensibilité des cellules à la chimiothérapie. Cependant, cette surexpression n’a montré aucun effet sur la survie cellulaire après traitement. Nous avons par la suite mis en évidence un profil d’expression différentiel des miARN entre les cellules invalidées pour MUC4 et les cellules contrôles. [...] / The MUC4 mucin is an important actor of pancreatic tumorigenesis as it contributes to tumor progression and its expression correlates with a poor prognosis. It has also been shown that MUC4 is involved in resistance of cells to chemotherapies. In this context, MUC4 is a potential therapeutic target in pancreatic cancer. MUC4 neoexpression at early stages of carcinogenesis confers to this mucin a potential interest as an early marker. Molecular mechanisms responsible for MUC4 induction of expression are not well defined. Thus, studying MUC4 gene expression regulation would contribute to better understand its role in tumorigenesis. MUC4 gene is regulated at the transcriptional level and epigenetically by DNA methylation and histone modifications mechanisms. However, MUC4 post-transcriptional regulation notably by miRNA is largely unknown. Our work aimed at (i) identifying miRNA dysregulated in pancreatic cancer and/or potentially targeting MUC4, (ii) determining miRNA(s) inhibiting MUC4 expression and its (their) impact on pancreatic carcinogenesis and finally (iii) identifying miRNAs involved in chemoresistance mediated by MUC4 in pancreatic cancer.First, using miRNA microarrays we established the miRNA expression profile of normal and cancerous pancreatic cell lines. We showed a cancer-specific miRNA signature which allows us to validate our cellular models. Then, performing in silico studies with TargetScan, Microcosm and MiRanda databases led us to identify miRNA potentially targeting MUC4. Analysis by qRT-PCR showed that miR-145 and miR-219-1-3p were downregulated in human pancreatic cancer cell lines. Finally, only miR-219-1-3p inhibited MUC4 expression thus we focused on its role in pancreatic cancer.We observed a loss of miR-219-1-3p expression in pancreatic cancer tissues. Complementary approaches overexpressing (transiently or stably) and inhibiting miR-219-1-3p expression, led us to show that miR-219-1-3p represses MUC4 protein expression by interacting directly with its 3’-UTR. We observed a decrease of cell migration and cell proliferation which was associated with an inhibition of cyclin D1 expression and an inhibition of Akt and Erk activation. Tumor growth in scid mice was strongly slowed down following miR-219-1-3p intratumoral injection. In the Pdx1-Cre;LSL-KrasG12D mouse model of PanIN, loss of miR-219-1-3p expression was an early event as soon as PanIN1/2 and miR-219-1-3p expression was conversely correlated to Muc4 expression.While the strong induction of miR-219-1-3p following gemcitabine treatment of pancreatic cancer cells suggests a potential role of this miRNA in sensitivity of cells,miR-219-1-3p has no effect on survival rate of cells treated with gemcitabine. We then established the miRNA expression profile of MUC4 knocked-down (MUC4-KD) cells and control cells (Mock) and showed a dysregulation of miRNA expression in MUC4-KD compared to Mock cells.To conclude, our results indicate that miR-219-1-3p, downregulated in pancreatic cancer, negatively regulates MUC4 expression, alters cancer cell biological properties and has an antimoral effect in vivo. Altogether, these results propose miR-219-1-3p as tumor suppressor in pancreatic cancer. Loss of MUC4 leads to an aberrant miRNA expression profile suggesting a potential role of miRNA as markers of chemoresistance in pancreatic cancer.

The human gut microbiome is a vast ecosystem of microorganisms that play an important role in human metabolism, immunological function, and even inflammatory gut diseases. Metagenomics research on the human gut microbiome has demonstrated the presence of DNA from dietary yeast species like Saccharomyces cerevisiae. However, it is unknown if the S. cerevisiae detected in metagenomics studies is solely from dead dietary sources or if they can live and colonize the human gut like their close relative Candida albicans. While S. cerevisiae can adapt to low oxygen and acidic environments, it has yet to be explored whether it can metabolize mucin, the primary carbon source found in the mucus layer of the human gut. Mucins are large, gel-forming, highly glycosylated proteins that make up a majority of carbohydrate sources in the gut mucosa. This work determined that S. cerevisiae can utilize mucin as their main carbon source which results in a significant reduction in cell size. Additionally, an aspartyl protease named Yps7, part of a family containing known homologues to mucin-degrading C. albicans proteins in S. cerevisiae, is important for growth on mucin media. To further identify biological pathways required to grow optimally in mucin, both a transcriptome analysis on wild type cells (BY4743) and a chemogenomics screen was performed. In total, 2131 genes demonstrated significant differential expression in mucin media, and 30 genes upon their deletion impacted their growth on mucin. Both these screens suggest that mitochondrial function is required for proper growth in mucin, which was further elucidated by the change in mitochondrial morphology and oxygen consumption in yeast cells upon mucin treatment. Indeed, the uncharacterized open reading frame YCR095W-A is required for growth on mucin as the deletion mutant showed dysfunction in mitochondrial morphology and cellular respiration, further suggesting a potential role in mitochondrial function. Importantly, this project serves as the initial step towards establishing if our most common dietary fungus can survive in the mucus environment of the human gut.

The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. Mucin 1 (MUC1), the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern in many cancers. The presence of truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play key roles in tumor initiations, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with escape of immune defenses. Human macrophage galactose-type lectin (hMGL, HML, CD301 or CLEC10A), a C-type lectin expressed by antigen presenting cells (APC), is a receptor of mucin-type TACAs, -GalNAc (Thomsen nouvelle antigen; Tn; CD175) and its 2,6-sialylated derivative (sTn; CD175s). To date, the relative contributions of these glycans, as well as underlying peptide backbone, and different degrees of valency, on binding thermodynamics and kinetics with hMGL remains elusive. In order to discern the subtle utility of these distinct features, chemical syntheses of the MUC1, HGVTSAPDTRPAPGSTAPPA tandem repeat sequence, and its site-specific serine (Ser) and threonine (Thr) glycosylated analogs were carried out. Circular dichroism (CD) spectroscopy experiments detected increasing structural order of the Thr glycopeptides compared to its nonglycosylated analogs. Isothermal titration calorimetry (ITC) data analysis of lectin binding to the Thr glycopeptides invariably showed enthalpy-driven processes. Affinity enhancement of the Thr glycopeptides for hMGL occurred relative to free GalNAc, revealing an increasing trend in affinity by one order of magnitude, for mono- (KD = 6-8 μM) to triglycosylated (KD = 600 nM) MUC1 peptides. To delineate the relevance of the solvent structure in the protein carbohydrate recognition process, experiments in D2O were performed, exposing enthalpy-entropy compensation differences. KinITC analysis highlighted prolonged complex lifetimes. Furthermore, atomic force microscopy (AFM) based dynamic single-molecule force spectroscopy (SMFS) provided molecular level insight into the energy landscapes governing recognition of the MUC1(Tn)-hMGL complexes. In summary, our results suggest that contact with hMGL critically depends on the type of TACA, nature of the vicinity surrounding the glycan, and its density. This highlights the importance and current efforts in design of prophylactic and therapeutic cancer vaccines with special emphasis on the synthetic glycopeptide vaccines. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

As a result of alcohol consumption being highly prevalent in today’s society, research has been done to investigate the effects of alcohol on the body’s physiological systems. Research has indicated that heavy alcohol consumption is detrimental to the normal structure and function of some organs, especially the liver. However, little research has focused on the effects of chronic alcohol consumption on the female reproductive system. To investigate these effects, the uterine tissues of mice fed an ethanol diet (the NIAAA model also known as the Lieber-DeCarli diet) and mice fed a control diet were compared. The NIAAA model was chosen for this research because it simulates the drinking pattern that is known to cause liver disease in alcoholic hepatitis patients. This is achieved by incorporating both chronic and binge drinking patterns of alcohol consumption. In this study, the mucin layer that lines the endometrial surface of the uterus was analyzed in mice separated into ethanol and control fed groups. The ethanol fed mice were put on the Lieber-DeCarli 5% (v/v) ethanol diet ad libitum for 10-days followed by a single high dose of ethanol (5g/kg) on the 11th day. The control fed mice were placed on an ethanol free isocaloric diet (supplemented with maltose dextrin to match the calories of ethanol). After the 11th day, the mice were sacrificed, and uterine tissues were harvested. The tissues were then embedded in paraffin, sectioned, stained via the Hematoxylin and Eosin (H&E) technique, and examined under a microscope. The thickness of the uterine mucin layer was then measured for each animal and the average thicknesses were calculated. A one-way ANOVA test was employed to compare the mucin thickness between the two groups of animals. The test revealed no statistically significant difference between the thicknesses of the uterine mucin layer in the control and ethanol fed animals (P-value: 0.774).