Reptiles can often experience perturbations that greatly influence their metabolic status (e.g., temperature, exercise, digestion, and ontogeny). The most common cause of fluctuations in metabolic status in post-embryonic reptiles is arguably digestion and physical activity (which will be further referred to as exercise). The objective of this thesis is to determine the mechanisms involved in CO2 transport during digestion, determine the mechanisms that allow for the maintenance of acid-base homeostasis during digestion, and observing the effect of an understudied form of exercise in semi-aquatic reptiles on the regulation of metabolic acidosis and base deficit. This dissertation provided evidence for potentially novel and under investigated mechanisms for acid-base homeostasis (e.g., small intestine and tissue buffering capacity; Chapters 3 & 4), while also debunking a proposed hypothesis for the function of an anatomical feature that still remains a mystery to comparative physiologist (Chapter 2). This thesis is far from systematic and exhaustive in its approach, however, the work accomplished in this dissertation has become the foundation for multiple distinct paths for ecologically relevant investigations of the regulation of metabolic acidosis/alkalosis in reptiles.

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Since the removal of in feed antibiotics in the past few years commercial poultry production is especially sensitive to the health of the small intestine. Healthy small intestines balance nutrient absorption and defensive barrier functions to ensure the chicken is able to meet the whole-body nutritional needs and is able to help prevent internalization of pathogens or potentially toxic components. This balance can only be maintained under stable conditions. When a disturbance event occurs the intestine imbalances until a new, and less efficient, balance can be achieved. The objective of this dissertation is to propose a novel model to understanding intestinal homeostasis in the face of various disturbance events. Chapter 2 investigated the effects of Runting Stunting Syndrome on broiler chickens in four different groups of chicks displaying clinical symptoms. The major finding in this study was that in two of the four groups the expression of stem cell gene Olfactomedin 4 was absent from the crypt though other functional genes were found to still be expressed there. Chapter 3 characterized intestinal gene expression following a single challenge of Eimeria acervulina in broiler chickens. During Eimeria infection gene expression of multiple host defense peptide genes were decreased compared to uninfected chickens. Further, Eimeria infected chickens increased cell proliferation within the crypt and post-peak infection showed signs of intestinal recovery. Additionally, chapter 3 developed a novel method for visualizing Eimeria as it infects the intestine. In chapters 4 and 5 cell type population changes during the peri-hatch intestinal maturation process were evaluated. Peri-hatch intestinal maturation is critical for the successful transition from embryonic to post-hatch life. Chapter 4 profiled changes in proliferative cells and gene expression of various stem cell marker genes during the peri-hatch period: the last three days of embryogenesis and the first week post-hatch. The stem cell marker gene Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (Lgr5) decreased during the post-hatch period while Olfactomedin 4 increased post-hatch. Both stem cell genes were expressed within the intestinal crypt, though prior to hatch Lgr5 was expressed in the lamina propria and villi as well. Additionally, the marker of proliferation Ki67 gene was expressed in cells throughout the intestine prior to hatch but became restricted to the crypts and along the center of the villi. Chapter 5 assessed the effect of providing probiotics to late term embryos via in ovo feeding (IOF). The effects of IOF were primarily observed on embryonic day 20 (e20), roughly 48 hours after IOF. On e20 the embryos in ovo fed probiotics in saline had increased expression in the ileum of Peptide Transporter 1 (PepT1) a marker gene for enterocytes and Mucin-2 (Muc2) a marker gene for goblet cells compared to non-injected control embryos. Also, on e20 the embryos in ovo fed saline only had numerically increased PepT1 and Muc2 compared to non-injected control embryos. The difference in responses between the probiotic and saline fed embryos on e20 suggests different routes of stimulation. These investigations illustrate various possible scenarios and means of investigating intestinal homeostasis during disturbance events. / Doctor of Philosophy / In healthy birds, the small intestine absorbs nutrients while preventing the free passage of microbes or toxic chemicals into the body. The two functions: absorption and barrier exclusion seem contradictory, but a balance is struck to ensure both functions continue. This balance-point, homeostasis, persists until an event disturbs it. Once disturbed the balance-point is changed and the intestine is unable to maintain both functions, until a new balance is found following recovery. The objective of the dissertation is to better understand intestinal homeostasis, through four different research projects. Experiment 1 characterized the intestinal cell population changes in broiler chickens during Runting Stunting Syndrome, a viral infection. The major finding of this chapter was that a stem cell gene, that is normally robustly expressed was not expressed in some groups of infected chicks but not all. Experiment 2 investigated the intestinal response of broiler chickens to Eimeria acervulina, an intestinal parasitic infection. Eimeria, which infects intestinal enterocytes, caused a decrease in defensive genes during the peak of infection. Then after the peak the intestine began to recover, as indicated by increased cell proliferation. Experiment 3 profiled changes in the expression patterns of stem cell and proliferation genes in the small intestine during the last days before hatch and the first week post-hatch. Pre-hatch stem and proliferative gene expression occurred in the crypt and villus, but became restricted to the crypt early during the post-hatch period. Experiment 4 assessed the effect of feeding probiotics to embryos before hatching on intestinal gene expression. Embryos fed probiotics had increased Mucin-2 and Peptide Transporter 1 gene expression in the last segment of the intestine, the ileum compared to non-fed embryos 48 hours after feeding. Additionally, treatments fed saline also showed increased gene expression, though to a lesser extent. Together these projects illustrate various disturbances to intestinal homeostasis and how intestinal cells change and respond during the disturbance and recovery periods.

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

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In vertebrates the cornea must maintain its transparency throughout adult life to ensure sight, and understanding the mechanisms underpinning corneal homeostasis are fundamental to developing new treatments to cure or prevent blindness. This study investigated the role the planar cell polarity pathway plays in the directed migration of adult corneal epithelial cells, in maintaining the homeostatic environment of the eye and during wound healing. RT-PCR confirmed, for the first time, the expression of multiple core PCP genes within human corneal epithelial (HCE) cells. Components of the PCP pathway were pharmacologically and genetically manipulated during wound healing of corneal epithelial cells and the importance of the downstream target JNK, and core PCP gene Vangl2, during wound healing was demonstrated. Manipulation of core PCP components was found also to directly affect the ability of HCE cells to realign and migrate in response to physical topographical cues in vitro. This study therefore indicated that PCP may regulate the directed migration of corneal epithelial cells as they travel over the basement membrane. Using conditional knockout mice the loss of Vangl2, a core PCP gene, and its effect on both planar and the apical-basal polarity of the corneal epithelium was investigated. Severe morphological defects were observed in Vangl2-null mice indicative of underlying problems in apical-basal polarity of the epithelial cells. The basement membrane of Vangl2-null cells was largely absent in vivo, which suggested that at least some of the planar defects were secondary to an unexpected failure of apical-basal polarity. This study has shown for the first time that PCP plays a crucial role in the maintenance of an adult vertebrate tissue, particularly during wound healing and maintenance of the corneal epithelium. It has also indicated a role for the core PCP gene, Vangl2, in setting up apical-basal polarity of these adult cells.