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Small Intestinal Transporters in Two Species of Galliformes: Male and Female Turkey (Meleagris gallopavo) and Chicken (Gallus gallus)Weintraut, Melodie Lynn 12 June 2015 (has links)
The objective of the first study was to characterize amino peptidase N (APN), peptide (PepT1), amino acid (ASCT1, bo,+AT, CAT1, EAAT3, LAT1, y+LAT2), and sugar transporter expression (GLUT2, GLUT5, SGLT1) in the small intestine of male and female turkeys. Small intestine samples were collected during embryonic development (E21, E24) and DOH. In a separate experiment during post-hatch development (DOH, D7, D14, D21, D28). APN, bo,+AT, PepT1, y+LAT2, GLUT5 and SGLT1 were expressed most on DOH. Post-hatch, all genes except GLUT2 and SGLT1 were expressed greater in females than males. SGLT1 was expressed greater in males. Basolateral transporters were expressed more during early development; while there was more expression of brush border transporters EAAT3, GLUT5 and SGLT1 later in development. In chickens, there are alternatively spliced exons of the PepT2 gene that encode proteins with four different N-termini (Variants 5-8). The objective of this study was to characterize the patterns of expression of these PepT2 variants. Brain, kidney, liver and intestine were analyzed at E18 and D7 (n=5). Expression of Variant 5 was most prominent in the brain and variant 6 was most prominent in the kidney. Variant 8 appeared in all tissues on E18 and D7. Variant 7 was only expressed in late embryonic development in the ileum. Results from these studies demonstrate that there are differences in gene expression of nutrient transporters in two agriculturally important avian species from the same order Galliformes. These differences can be used to improve feed efficiency and enhance the growth of both species. / Master of Science
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Nutrient Transporter Inhibition Disrupts Mammary and Intestinal Polarized Epithelial Function2016 February 1900 (has links)
The transporters primarily responsible for transporting important nutrients involved in energy metabolism have a wide substrate specificity setting up the potential for drug-nutrient transporter interactions. Pharmacological inhibition of nutrient transport across the lactating mammary and neonatal intestinal epithelial barrier can directly and indirectly affect growth and maturation of the developing neonate by either reducing the uptake of important nutrients by the neonate or by disrupting epithelial barrier integrity. My thesis focused on two transporters, OCTN2 and MCT1, expressed in immortalized intestinal and mammary epithelial cell cultures to assess the effects of their pharmacological inhibition on L-carnitine and butyrate flux, respectively, and polarized epithelial barrier integrity.
Human colorectal adenocarcinoma (Caco-2) and bovine mammary (BME-UV) cell lines were grown into monolayers on 12-well tissue culture plates and subsequently exposed to the presence or absence of OCTN2 and MCT1 inhibitors for 6, 12, and 24 hours as well as 7 days. Failure to obtain a polarized mammary monolayer prevented the analysis of the direct effects of nutrient transport inhibition on nutrient flux forcing the focus on the indirect effects. To assess polarized epithelial barrier integrity, transepithelial electrical resistance and Lucifer yellow rejection rates were measured at each time point. No trend was noted between control and treated groups. To assess the acute and chronic effects of pharmacological exposure on polarized epithelial function, a limited appraisal of nutrient transporter expression and cellular homeostasis parameters was conducted. Following exposure at each time point, mRNA expression of OCTN1, OCTN2, MCT1, MCT2 and GADPH were measured using qPCR. Low mRNA yields resulted in an inability to assess transporter expression levels in the epithelial systems. Cellular homeostasis parameters were analyzed using the CellTiter-Glo Luminescent Cell Viability Assay, pH-Xtra Glycolysis Assay and MitoXpress Xtra Oxygen Consumption Assay. These assays measured ATP synthesis, glycolytic flux and cellular respiration, respectively. No significant trend was noted in ATP synthesis between control and treated groups. An upward trend in both glycolytic flux and cellular respiration was noted in treatment with both inhibitors in both cell lines.
Complications in obtaining polarized monolayer forced the focus on the indirect affects, therefore, obtaining and utilizing a more accurate portrayal of the lactating mammary and neonatal intestinal epithelium is critical in answering this research question as both of these systems are highly synthetic and complex. By doing so, a more accurate representation of the effects of pharmacological inhibition of nutrient transporters essential for energy metabolism can be identified.
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Temporal and Tissue Specific Changes in Expression of Nutrient Transporters and Host Defense Peptides in Young Broilers during Salmonella and Campylobacter infectionsGarcia, Javier S. 13 June 2017 (has links)
Salmonella and Campylobacter are the leading causes of bacterial foodborne illness in the United States. Commonly found in the gastrointestinal tract of poultry, Salmonella and Campylobacter may show little to no signs of infection in birds. The objective of this dissertation was to evaluate the influence on mRNA abundance of nutrient transporters and host defense peptides (HDPs) during a Salmonella or a Campylobacter challenge in young commercial broilers. Comparisons were made between non-challenged and challenged (106, 107, or 108 colony forming units of Salmonella or Campylobacter) broilers on expression of nutrient transporters and host defense peptides in the duodenum, jejunum, ileum and cecum at various days after inoculation. During a Salmonella challenge, changes in mRNA abundance of nutrient transporters and avian beta-defensins (AvBD) vary by day, tissue and challenge dose. ZnT1 may play an important role during a Salmonella challenge as mRNA abundance of ZnT1 significantly increased (P<0.05) by day 7 in the 108 group compared to the control. Early changes in LEAP2 mRNA abundance were observed in the 106 group than the 107 and 108 groups. However, at a later time point post challenge, a lower abundance of almost all AvBD mRNA (P<0.05) was observed in the lower gastrointestinal tract especially in the 107 and 108 groups compared to the control group, indicating that the pathogen may be influencing intestinal expression of AvBD mRNA. In Campylobacter, analyses revealed that expression of zinc transporter 1 (ZnT1) increased (P<0.05) in the duodenum, ileum and ceca in the 106 group on day 7. An increase (P<0.05) in the expression of avian beta-defensins were observed on day 14 in the ileum and ceca in the 106 group compared to the control group. Pathogens like Salmonella and Campylobacter may have an influence on the mRNA abundance of nutrient transporters and HDPs. Manipulation of these genes may ensure the survivability of these pathogens. Through sequestration of nutrients, the pathogen would have the ability to colonize the host and replicate. However, it must evade the host immune system as well. The processing of infected poultry with these pathogens may lead to foodborne illness in humans. Further research is needed to investigate possible methods to counter the influence these pathogens have on host immunity genes. / Ph. D.
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Gene Expression of Nutrient Transporters and Digestive Enzymes in the Yolk Sac Membrane and Small Intestine of the Developing Embryonic ChickSpeier, Jacqueline S. 20 September 2011 (has links)
Chick embryos derive nutrients from the yolk during incubation and transition to intestinal absorption posthatch. Nutrient uptake is mediated by digestive enzymes and membrane bound transporter proteins. The objective of this study was to determine expression profiles of nutrient transporters and digestive enzymes during incubation in the yolk sac membrane (YSM) and small intestine of Leghorn and Cobb chickens derived from 22–30 wk (young) and 45–50 wk (old) breeder flocks. Genes examined included peptide transporter PepT1, amino acid transporters EAAT3, CAT1, and B0AT, monosaccharide transporters SGLT1 and GLUT5, and digestive enzymes APN and SI. Expression of these genes was measured in YSM at embryonic day (e) 11, 13, 15, 17, 19, 20, and 21 and small intestine at e15, e17, e19, e20, and e21. Absolute quantification real-time PCR assessed gene expression. PepT1, APN, and B0AT expression in YSM peaked between e15 and e17 and then decreased until e21, while expression increased over time in the small intestine. SGLT1 and EAAT3 expression increased over time in the small intestine and YSM. There was minimal gene expression of SI in the YSM, while the small intestine had high expression. GLUT5 and CAT1 expression decreased in the YSM, while peaking at e19 then decreasing in the small intestine. Breed and flock age affected expression levels in some genes. These results demonstrate that these genes show tissue- and development-specific patterns of expression and that the YSM expresses many digestive enzymes and nutrient transporters associated with the small intestine. / Master of Science
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Metabolic adaptation of inflammatory neutrophils in human diseases revealed by retroviral envelope-derived ligands : focus on cystic fibrosis / Adaptation métabolique des neutrophiles inflammatoires dans les maladies humaines révélée par des ligands dérivés d'enveloppes rétrovirales : étude dans la mucoviscidoseLaval, Julie 09 October 2013 (has links)
Notre étude est consacrée à la caractérisation des mécanismes d'adaptation métabolique des neutrophiles au cours de l'inflammation et plus particulièrement au cours de leur recrutement dans les voies aériennes des patients atteints de mucoviscidose (cystic fibrosis ou CF en anglais). Dans la mucoviscidose, nous avons précédemment décrit que les neutrophiles présents dans la lumière du poumon sont viables et soumis à une reprogrammation, notamment via l'activation de la voie anabolique mTOR. Ce travail est fondé sur les propriétés spécifiques de ligands solubles dérivés des domaines liant le récepteur (RBD, pour Receptor-Binding Domain) des glycoprotéines d'enveloppes rétrovirales permettant leur utilisation pour la détection de transporteurs métaboliques à la surface des cellules. Dans un premier temps, nous avons validé l'utilisation de ce nouveau groupe de marqueurs pour identifier et caractériser le phénotype métabolique des leucocytes CF obtenus à partir d'échantillons de différents compartiments (sang et expectorât pulmonaire). Dans un deuxième temps, l'étude de l'expression de ces transporteurs métaboliques sur les neutrophiles sanguins de patients atteints de CF ou de polyarthrite rhumatoïde et de sujets contrôles, a permis la distinction de phénotypes métaboliques au niveau systémique selon différents états inflammatoires. Ensuite, nous avons comparé l'expression des transporteurs métaboliques entre les neutrophiles sanguins et pulmonaires de patients CF et révélé une adaptation métabolique de ces cellules lors de leur recrutement vers les poumons. Enfin, les neutrophiles présents dans les voies aériennes des patients CF présentent une modulation de leur activité transcriptionnelle. De façon surprenante, nos travaux démontrent que malgré leur reprogrammation, les neutrophiles recrutés vers les poumons CF sont fonctionnellement compétents, ajoutant ainsi un nouvel angle d'approche dans l'étude des neutrophiles au cours de l'inflammation, notamment dans le cas de la pathologie pulmonaire liée à la mucoviscidose. / The present study focuses on adaptive metabolic steps adopted by neutrophils during inflammation, particularly during their recruitment into the cystic fibrosis (CF) airways. In CF, we previously described that airway neutrophils are alive and undergo reprogramming, featuring notably the activation of the anabolic mTOR pathway. The present work is based on specific properties of soluble ligands derived from the receptor-binding domains (RBD) of retroviral glycoprotein envelopes, which can be used for the detection of metabolite transporters at the cell surface. First, we validated the use of this new set of markers for the identification and characterization of the metabolic phenotype of CF leukocytes obtained from distinct compartments (blood and sputum). Second, by studying the metabolite transporter expression on blood neutrophils from CF or rheumatoid arthritis patients and control subjects, we distinguished metabolic phenotypes characteristic of specific inflammatory states. Then, we compared metabolite transporter expression between CF blood and airway neutrophils and showed that neutrophils undergo significant metabolic adaptation upon recruitment into the lungs. Finally, we demonstrated that CF airway neutrophils display significant transcriptional modulation and that despite their metabolic reprogramming, they remain functionally competent, thus adding an additional angle of approach to neutrophil studies with regard to inflammation, notably during CF airway disease.
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Metabolic adaptation of inflammatory neutrophils in human diseases revealed by retroviral envelope-derived ligands : focus on cystic fibrosisLaval, Julie 09 October 2013 (has links) (PDF)
The present study focuses on adaptive metabolic steps adopted by neutrophils during inflammation, particularly during their recruitment into the cystic fibrosis (CF) airways. In CF, we previously described that airway neutrophils are alive and undergo reprogramming, featuring notably the activation of the anabolic mTOR pathway. The present work is based on specific properties of soluble ligands derived from the receptor-binding domains (RBD) of retroviral glycoprotein envelopes, which can be used for the detection of metabolite transporters at the cell surface. First, we validated the use of this new set of markers for the identification and characterization of the metabolic phenotype of CF leukocytes obtained from distinct compartments (blood and sputum). Second, by studying the metabolite transporter expression on blood neutrophils from CF or rheumatoid arthritis patients and control subjects, we distinguished metabolic phenotypes characteristic of specific inflammatory states. Then, we compared metabolite transporter expression between CF blood and airway neutrophils and showed that neutrophils undergo significant metabolic adaptation upon recruitment into the lungs. Finally, we demonstrated that CF airway neutrophils display significant transcriptional modulation and that despite their metabolic reprogramming, they remain functionally competent, thus adding an additional angle of approach to neutrophil studies with regard to inflammation, notably during CF airway disease.
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