Our project was aimed at the development of novel CB1 cannabinoid receptor antagonists that may have clinical applications for the treatment of cannabinoid and psychostimulant addiction. In this study, we designed, synthesized, and established the CB1 affinity for the 1,5-diaryl-1,2,3- triazole esters, a series of 4,5-diaryl-1-substituted-1,2,3-triazole analogues and a series of 4,5- diaryl-2-substituted-1,2,3-triazoles. Our research group has been interested in the synthesis of amphibian alkaloids due to their interesting biological activities. We have recently developed a general synthetic strategy which can rapidly prepare a few amphibian alkaloids simply from the abundant natural product (-)- cocaine This strategy was first successfully applied to the synthesis of (-)-monomorine. More recently, this strategy has also been utilized in the syntheses of both of the enantiomers of cispyrrolidine 225H and (+)-gephyrotoxin 287C.

Au cours de ma thèse, j'ai étudié l'impact des stress associés aux vols spatiaux sur l'immunité humorale du pleurodèle et de la souris. Chez le pleurodèle adulte, j'ai d'abord étudié l'utilisation des gènes VH lors de la synthèse des chaînes lourdes d'anticorps suite à une immunisation pendant 5 mois à bord de Mir (expérience Genesis en 1999). J'ai ensuite étudié le processus de maturation de l'affinité des anticorps chez ces mêmes animaux. Ce processus s'effectue par l'apparition d'hypermutations somatiques dans les segments variables des gènes d'anticorps. Ces travaux ont permis de montrer que les segments VH sont utilisés différemment sur Terre et dans Mir et que la fréquence des hypermutations est diminuée suite au vol. Ensuite, j'ai étudié l'impact des stress rencontrés lors d'un autre vol spatial sur la synthèse des premiers anticorps (IgM) chez le pleurodèle en développement (expérience AMPHIBODY en 2006). Le taux d'IgM étant modifié suite à cette expérience, nous avons recréé sur Terre chacun des stress rencontrés en vol (microgravité, hypergravité, choc thermique, radiations, perturbation du rythme circadien et confinement) afin de connaître le(s) stress responsable(s) de cette modification. Ainsi, seule la gravité modifiée affecte l'expression des IgM. Enfin, j'ai étudié l'impact de l'hypergravité (2G et 3G) sur la réponse au stress et le système immunitaire de la souris. Nous avons mis en évidence une réponse physiologique et comportementale au stress à 3G mais pas à 2G. Pourtant, des modifications du système immunitaire sont constatées dès 2G. Cela montre qu'une modification de la gravité, associée ou non à une réponse au stress, affecte le système immunitaire / During my PhD, I studied the impact of spaceflight-associated stresses on Pleurodeles waltl and Mus musculus humoral immunity. In adult P. waltl immunized during 5 months onboard the Mir space station (Genesis experiment in 1999), I first determined how individual VH genes are used. Then, I studied antibodies affinity maturation in these animals. This maturation implies the introduction of somatic hypermutations (SHM) in DNA encoding the variable segments of antibodies genes. These two pieces of work have shown that variable segments of heavy chain gene are differently used and that SHM frequency is reduced when immunization occurs in space. Then, I studied antibodies production during animal development onboard the international space station (ISS) (AMPHIBODY experiment in 2006). The antibodies production being increased in larvae that developed in the ISS, we recreated in the laboratory each stress encountered during the spaceflight (hypergravity, microgravity, heat shock associated to the re-entry in the atmosphere, radiations, perturbation of circadian rhythm and confinement) to determine their impact on IgM heavy chain transcription. This allowed to observe that only gravity changes affect this transcription. Finally, I studied the impact of hypergravity (2G and 3G) on the murine immune system. I observed physiological and behavioural stress responses in mice exposed to 3G but not in 2G mice. However, immune system alterations were observed in both the 2G and 3G groups, suggesting that gravity modifications, associated or not with stress responses, are responsible for immune system modifications

Changes in the structure, content and morphology of chromosomes accumulate over evolutionary time and contribute to cell, developmental and organismal biology. The axolotl (Ambystoma mexicanum) is an important model for studying these changes because: 1) it provides important phylogenetic perspective for reconstructing the evolution of vertebrate genomes and amphibian karyotypes, 2) its genome has evolved to a large size (~10X larger than human) but has maintained gene orders, and 3) it possesses potentially young sex chromosomes that have not undergone extensive differentiation in the structure that is typical of many other vertebrate sex chromosomes (e.g. mammalian XY chromosomes and avian ZW chromosomes). Early chromosomal studies were performed through cytogenetics, but more recent methods involving next generation sequencing and comparative genomics can reveal new information. Due to the large size and inherent complexity of the axolotl genome, multiple approaches are needed to cultivate the genomic and molecular resources essential for expanding its utility in modern scientific inquiries. This dissertation describes our efforts to improve the genomic and molecular resources for the axolotl and other salamanders, with the aim of better understanding the events that have driven the evolution of vertebrate (and amphibian) chromosomes. First, I review our current state of knowledge with respect to genome and karyotype evolution in the amphibians, present a case for studying sex chromosome evolution in the axolotl, and discuss solutions for performing analyses of large vertebrate genomes. In the second chapter, I present a study that resulted in the optimization of methods for the capture and sequencing of individual chromosomes and demonstrate the utility of the approach in improving the existing Ambystoma linkage map and generating targeted assemblies of individual chromosomes. In the third chapter, I present a published work that focuses on using this approach to characterize the two smallest chromosomes and provides an initial characterization of the huge axolotl genome. In the fourth chapter, I present another study that details the development of a dense linkage map for a newt, Notophthalmus viridescens, and its use in comparative analyses, including the discovery of a specific chromosomal fusion event in Ambystoma at the site of a major effect quantitative trait locus for metamorphic timing. I then describe the characterization of the relatively undifferentiated axolotl sex chromosomes, identification of a tiny sex-specific (W-linked) region, and a strong candidate for the axolotl sex-determining gene. Finally, I provide a brief discussion that recapitulates the main findings of each study, their utility in current studies, and future research directions. The research in this dissertation has enriched this important model with genomic and molecular resources that enhance its use in modern scientific research. The information provided from evolutionary studies in axolotl chromosomes shed critical light on vertebrate genome and chromosome evolution, specifically among amphibians, an underrepresented vertebrate clade in genomics, and in homomorphic sex chromosomes, which have been largely unstudied in amphibians.

Global climate change is projected to impact multiple levels of biodiversity by imposing strong selection pressures on existing populations, triggering shifts in species distributions, and reorganizing entire communities. The Lower Guineo-Congolian region in central Africa, a reservoir for amphibian diversity, is predicted to be severely affected by future climate change through rising temperatures and greater variability in rainfall. Geospatial modelling can be used to assess how environmental variation shapes patterns of biological variation – from the genomic to the community level – and use these associations to predict patterns of biological change across space and time. The overall goal of this dissertation is to examine potential impacts of climate change on amphibian diversity in central Africa. Geospatial modeling is used to: 1) map the distribution of the amphibian fungal pathogen, Batrachochytrium dendrobatidis (Bd) in a biodiversity hotspot in Cameroon under current and future climate; 2) assess phenotypic and adaptive genomic variation in a widespread frog species, Phrynobatrachus auritus, in order to predict areas where populations may best adapt under climate change; 3) determine how amphibian community composition may shift with climate change and which areas may experience greatest loss of functional groups. Findings show that most Bd samples belong to a globally hypervirulent lineage. However, areas of highest predicted environmental suitability for Bd are predicted to shrink under warming temperatures. Within P. auritus, most phenotypic and genomic turnover occurred across known ecological gradients and are heavily influenced by seasonal precipitation. Current amphibian beta diversity is greatest throughout the Cameroonian highlands and forest-savanna ecotones flanking the central Congolian lowland forests. Greatest shifts in community composition under climate change are predicted to occur in coastal Cameroon and its eastern border whereas the greatest predicted loss of functional richness was in central Gabon. Overall, this dissertation shows that areas of elevated environmentally-associated phenotypic, genomic, and community turnover are associated with key ecological gradients. Regions predicted to experience high genomic mismatch, large shifts in community composition, and high loss of functional richness resulting from climate change may warrant conservation attention.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 149-168). Also available on the World Wide Web.

Amphibian populations worldwide are currently in decline. One approach to preventing extinction of some of the affected species is to create assurance colonies. These sustainable captive populations might some day be used to reestablish wild populations. One issue with creating assurance colonies is successful breeding; often difficulties arise when attempting to breed exotic animals in captivity. Sexual conditioning, a form of Pavlovian conditioning, has been shown to improve breeding behavior. In this project the efficacy of sexual conditioning to improve breeding behavior in the dyeing dart frog (Dendrobates tinctorius) was tested. The frogs were trained with a stimulus that was either predictive of or independent of exposure to a member of the opposite sex. The group trained with the predictive stimulus showed shorter latencies to a variety of breeding behaviors and produced more eggs than the control groups. The sexual conditioning procedure also increased expression of various breeding behaviors allowing for careful examination of calls and visual signaling within this species.

Urodele amphibians have the remarkable ability to re-grow lost body parts. This regenerative response after injury in urodeles involves dedifferentiation of fully differentiated cells into proliferative cells. One well-studied example of this is the dedifferentiation of multinucleated muscle cells into mononucleate cells resembling their precursors, the myoblasts. To form these mononucleate cells the differentiated myotubes in vivo must re-enter and complete the cell cycle; they again proliferate and produce progeny. A key question is what factors induce the myotubes to re-enter the cell cycle and proliferate. Early events of cell cycle re-entry can be studied in the A1 cell line, a myogenic cell line isolated from the Notophthalmus viridescens hindlimb, which traverses cell cycle until G2 in response to serum. In particular, it was found that thrombin cleavage induces a factor in serum of all animals tested so far to promote S phase re-entry in A1 myotubes. We have used this S phase re-entry of the A1 cell line to purify the serum activity and developed a 5-step purification protocol that enriches the activity almost 2 000 fold over the starting material, or 40 000 fold over serum. To conveniently produce and test potential candidates for their ability to induce S phase re-entry in A1 myotubes, we also developed an overexpression- and purification system for emerging candidates. Candidates were then tested for this activity with or without prior incubation with thrombin. We identified Bmp proteins as the first pure molecules that were found in fractions across the purification of the activity and that could also induce cell cycle re-entry in a dose-dependent manner when recombinantly added to the A1 myotubes. Furthermore, this response could be blocked in a dose-dependent manner by the known bmp-inhibitor noggin. Finally, we showed that inhibition of Bmp signaling in vivo causes defects in axolotl tail regeneration.

The geographic isolation of populations can result in a metapopulation effect where regional dynamics of extinction and colonization are more important to population viability than local dynamics of individuals' birth or death. When this partial isolation is maintained for many generations genetic variability can be geographically structured. Populations of temperate-zone anuran amphibians are often considered to be geographically isolated on relatively small spatial scales due to the animals presumed high site fidelity and strict dependence on moisture for respiration and breeding. As a result, temperate, pond-breeding anuran amphibian populations are considered likely candidates to test hypotheses of metapopulation theory, movement and phylogeography. Using data from the Fowler's toad, (Bufo fowleri) I test the applicability of metapopulation theory, the likelihood of limited movement and the strength of phylogeographic structure. Specifically, I show that the generalization of the amphibians-as-metapopulations paradigm, due to their limited dispersal capabilities, is not supported (I). Bufo fowleri movement is well described by an inverse power function. Whereas most individuals do not move, some move long distances. There is no sex bias to this movement and I propose the hypothesis that the animals moving the longest distances are aided by the passive action of lake currents (II). B. fowleri juveniles are not a dispersal stage. They move neither farther nor faster than adults. The observation of predominantly juvenile contribution to a dispersal pool is due to their abundance---there are simply many more juveniles than adults (III). My observations of amphibian population turnover do not support the predictions of two specific metapopulation models when parameterized on a local (<10km) or regional scale (~300km) (IV). B. fowleri populations in Canada exhibit both shallow and deep phylogeographic structure. The shallow divisions are geo

Hypothesised causes of the observed world-wide decline of amphibian populations are varied and in some cases contentious. Insufficient information relating to the autecology of many amphibian species can cause erroneous speculations regarding critical habitat requirements and hence management programs designed to enhance population viability are often unsuccessful. Most amphibians display a bi-phasic life history that involves occupation of an aquatic breeding habitat and terrestrial habitats that are used for foraging, and shelter from predation and environmental stress. However, the focus of most amphibian research is centred on the breeding habitat, with limited research being conducted into the terrestrial habitat requirements of most amphibian species. Barakula State Forest is a large continuous area of open woodland situated in the semi-arid region of Queensland. The forest supports 21 species of endemic anurans, many of which use ephemeral waterbodies for breeding. This area is, therefore, an ideal location to test the relative importance of terrestrial habitat on the distribution of a suite of frogs that display different morphological and physiological characteristics. On the landscape scale, the attributes of the terrestrial environment at three survey areas within Barakula were similar. However, at the patch scale, ground truthing showed there were considerable variations in vegetation and ground cover attributes within and between each survey site. Measured properties of the soil also tended to vary within and between sites. Soil texture ranged from sandy to heavy clay, soil pH ranged from 3.9 to 6.4 and soil moisture varied considerably. Agar models, used for testing evaporative moisture loss at different microhabitats, retained significantly higher levels of moisture when positioned in the buried microhabitat during summer, but in winter, models that were placed under leaf litter retained higher levels of moisture. Variations in levels of moisture loss at the five different microhabitats were evident within and between the survey sites. Despite a prolonged drought, 1844 native frogs representing 17 species were pitfall trapped. Members from the family Myobatrachidae comprised 94% of these captures, and burrowing species accounted for 75% of total captures. Species were not randomly distributed within or between the survey sites. Vegetation attributes and soil properties played a significant role in influencing the catch rates and traplines that supported similar vegetation and soil attributes also tended to catch similar species. Capture rates of six of the seven burrowing species were significantly influenced by soil properties. When given a choice of four different microhabitats created in enclosures, individuals from five species showed varying responses to habitat choice during night time activity. During daylight all species tended to avoid bare areas and burrowing species tended to burrow under some form of cover. Pseudophryne bibronii metamorphs showed a significant avoidance to soils with high pH. The number of Limnodynastes ornatus metamorphs was significantly and positively correlated with moisture levels surrounding a breeding area. Limnodynastes ornatus metamorphs tended to avoid areas that did not support some form of cover. Embryos from the terrestrial egg laying P. bibronii translocated to sites with varying levels of soil pH, suffered increased mortality where the soil pH was &gt4.8. In the laboratory, embryonic survival was not significantly different between the four pH treatments. There was a significant influence of fungal infection on survival rates and ranked fungal infection was significantly different between the four pH treatments. The terrestrial environment at the three survey sites has provided sufficient protection from environmental elements to allow a large diversity of anurans to persist for long periods without access to permanent water. Management must consider the importance of the non-breeding habitat when defining buffer zones, restoration programs and conservation strategies to ensure that the complete set of ecological requirements for frog species are provided.

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 53-58). Also available on the World Wide Web.