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Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans.

In the Xenopus embryo, cell rearrangements during early development require the dynamic modulation of adhesion. Cells primarily use the integrin family of transmembrane receptors for attachment to and interpretation of the extracellular environment. While acting as adhesion receptors, integrins also have bidirectional signalling properties essential for driving cellular movements. The regulation of integrin activity is thought to stem from cytoplasmic assemblies of constitutively expressed molecules. PINCH (Particularly Interesting New cysteine-histidine rich protein), an adapter protein, is part of an IPP complex that has emerged as a key signalling scaffold indispensable for integrin function in vitro. As such, I tested the hypothesis that PINCH regulates integrin function in the Xenopus embryo. Xenopus PINCH was successfully cloned using RT-PCR. The predicted amino acid sequence of PINCH shares a 98% similarity with mammalian orthologs, and comprises of five highly conserved LIM domains. PINCH mRNA and protein are ubiquitously expressed throughout embryogenesis. In situ hybridization indicates that PINCH mRNA is expressed in the blastocoel roof and the pre-involution mesoderm. The localization and temporal expression of PINCH suggests a role in mediating cell adhesive events during gastrulation. A functional approach was used to examine the role of PINCH during gastrulation. I used site-directed mutagenesis to generate non-functional LIM1 (LIM1mut) and LIM4 (LIM4mut) domains that have been proposed to bind ILK and Grb4 respectively. Over-expression of PINCH leads to a delay in blastopore closures, while the expression of both LIM1mut and LIM4mut relieve this inhibition at lower concentrations. Further analysis indicates that PINCH, LIM1mut, and LIM4mut inhibit FN matrix assembly independent of integrin adhesion. Contradictory to in vitro studies, co-immunoprecipitation analysis indicates that endogenous PINCH does not bind ILK, confirming an integrin-independent role during gastrulation. Furthermore, in the embryo PINCH is found at cell boundaries but does not appear to directly modulate cadherin adhesion. As such this thesis provides evidence that PINCH regulates cell intercalation movements independent of integrin and cadherin receptors and raises the possibility that the LIM4 domain is involved in PINCH regulation of cell adhesion during early development.

Amphibian populations are in mass decline on a global scale. Various explanations have been considered, including harmful effects from exposure to toxicants. Using Xenopus laevis adults and tadpoles, potential sublethal effects of atrazine, a herbicide, were investigated in this thesis. I also investigated the toxicity of an organic herbicide compared this with the toxicity of a synthetic herbicide, using LC50 values. Whether X. laevis adult frogs could communicate chemically was tested experimentally. The results suggest that adult female X. laevis communicate chemically, but there was no evidence that male individuals did so. For testing tadpoles I used a kin-preference assay. An encouraging trend for kin preference was evident, for both an outbred and an inbred line. Tadpoles changed their behaviour after exposure to l0μg/L of atrazine for 24 hr. Kin preferences in the control tests were reversed after exposure. A hypothesis of altruistic kin avoidance was suggested by these results. However, when individuals were isolated and then exposed, these individuals had more pronounced preference for kin compared to controls. X. laevis tadpoles exposed to Organic Interceptor (organic herbicide) had a LC50 that was more than 7000 times lower than the 20% recommended dose, whereas Roundup Renews' (a synthetic herbicide) LC50 was around 8 times lower than the 1% recommended dose. This research adds to evidence that toxicants have a negative impact on amphibian populations, and suggests that more research needs to be conducted to identify other sublethal effects of toxicants and to clarify the implications these effects might have for the amphibian populations in nature.

This work examines the ecological, social and genetic factors that predispose amphibians to infection. In the last 30 years many amphibian populations have declined due to infectious disease, although few researchers have studied the factors involved in mediating amphibian infection. My research is designed to explore some of these factors. I first examined the effects of crowding, kin composition (the relatedness of individuals in a group), and habitat complexity on the growth and survival of Xenopus laevis tadpoles exposed to the bacterial pathogen Aeromonas hydrophila. In tadpoles, stress, and in particular corticosterone, a hormone associated with stress, is known to inhibit growth. Crowding, kin composition, and habitat complexity have all been linked to tadpole growth. As corticosterone exposure is also linked to reduced immune function, I examined how these ecological factors influence tadpoles' disease resistance. Tadpoles exposed to the bacterium were significantly smaller and more likely to die than control tadpoles. Tadpoles reared only with siblings (pure sibship groups) were larger, less variable in size, and had lower mortality rates than tadpoles reared in mixed sibship groups. The size difference between pure and mixed sibship groups was greatest when they were exposed to the pathogen. Habitat complexity reduced size variation within tanks but did not affect mean tadpole size. Mixed kinship composition and high tadpole density can increase competition, reduce growth, and increase disease susceptibility. These results indicate that growth was inhibited by pathogen exposure but kin association may ameliorate this effect. The Major Histocompatibility Complex (MHC) is an integral part of the vertebrate adaptive immune system. To determine the importance of the MHC in conferring disease resistance in amphibians, I challenged X. laevis tadpoles, bearing different combinations of four MHC haplotypes (f, g, j, and r), with A. hydrophila in two experiments. Exposure to A. hydrophila affected the growth and survival of these tadpoles and that the MHC moderated these effects. Tadpoles with two MHC haplotypes (r and g) were susceptible to this pathogen and tadpoles with the other two haplotypes (f and j) were resistant. Heterozygous tadpoles with both susceptible and resistant haplotypes were always intermediate to either homozygotes in size and survival. These results demonstrate that MHC genotype plays a major role in determining the impact of bacterial pathogens on the growth and survival of X. laevis tadpoles. To test whether the effect of exposure to pathogens differs according to the similarity of the hosts I challenged tadpoles with natural levels of the microorganisms associated with different MHC genotypes by exposing the tadpoles to water preconditioned by adults of different MHC genotypes. If the pathogens are adapted to the MHC genotype of their hosts, tadpoles exposed to water from adults with which they shared MHC haplotypes would be more susceptible than those exposed to water from MHC-dissimilar adults. Alternatively, if the hosts are adapted to their pathogens tadpoles may be more resistant to pathogens from MHC-similar frogs than those from MHC-dissimilar frogs. I found that tadpoles exposed to water from MHC-dissimilar animals developed faster, but without increased growth, and were more likely to die than those exposed to water from MHC-similar animals. Furthermore, there was an optimal difference between the tadpoles’ and the donors’ MHC where tadpoles were sufficiently different to the donor to defend against its locally adapted pathogens, and sufficiently similar to not be exposed to especially virulent foreign pathogens. Finally, I present an inventory of bacteria found in the gut and skin (nonsystemic sites) and heart, muscle, and abdominal cavity (systemic sites) of captive frogs. I found several species of bacteria previously identified as amphibian pathogens and many bacteria in systemic sites that have not been considered pathogenic to amphibians. None of the frogs tested positive for the amphibian chytrid fungus, Batrachochytrium dendrobatidis. I discuss the potential importance of these species of bacteria as amphibian pathogens and as protective probiotics, using New Zealand frogs as a case study. In its sum, this work describes some of the factors that can affect amphibians’ ability to resist disease. I show that the genetic constitution of an individual, specifically in terms of the MHC, affects the impact of a disease, and so too does its social and ecological conditions, including the level of crowding, the kinship of its groupmates and the specific microbial challenges of its immediate environment. I also show that many of the factors linked to tadpole growth and development that are well described in other amphibians also affect Xenopus tadpoles.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [74]-88).

Thesis (M.S.)--Duquesne University, 2004. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 106-115 ) and index.

Thesis (Ph. D.)--University of Virginia, 2002. / Includes bibliographical references (leaves 153-174). Also available online through Digital Dissertations.

Thesis (Ph.D.)--Georgetown University, 2009. / Includes bibliographical references.

Thesis (Ph.D.)--Georgetown University, 2009. / Includes bibliographical references.