Amphibian alkaloids are attractive targets for synthesis due to their biological activity. An important class of amphibian alkaloids is the 2,5-disubstituted pyrrolidine-based family of compounds. There are many synthetic approaches for the preparation of the trans-2,5- disubstituted pyrrolidines, but methods for the construction of the cis-2,5-pyrrolidines are limited. Therefore, it was desired to develop an enantioselective approach for the preparation of cis-2,5-disubsituted pyrrolidines. (+)-Tropin-2-one derived from cocaine was used as starting material to exploit the inherent stereochemistry for construction of the cis-pyrrolidine ring. This permitted the unequivocal assignment of the absolute configuration of the target pyrrolidine. The structurally simple pyrrolidine alkaloid, 225H, was selected as a target to develop a general synthetic approach. The enantioselective synthesis of 225H was achieved in nine steps and good overall yield. The search for potent cannabinoid receptor partial agonist ligands as potential marijuana addiction therapeutic agents has led to an investigation of the synthesis of diaryl ether hybrid analogues of BAY 59-3074. A series of 2-(3-alkyl-5-hydroxyphenoxy)-6- (trifluoromethyl)benzonitriles, 3-(2-cyano-3-(trifluoromethyl)phenoxy)phenylalkanoates, and (3- (benzyloxy)phenoxy)-6-(trifluoromethyl)benzonitriles were synthesized and evaluated in vitro for CB1 affinity. The olivetol diaryl ether analogue was the most potent ligand of the alkyl series, but the diaryl ester analogues exhibited modest affinity for CB1 receptors. The most potent compound of the series was the 2-(3-(benzyloxy)phenoxy)-6- (trifluoromethyl)benzonitrile.

abstract: Emerging infectious diseases (EIDs) in vulnerable populations are a proposed cause of reduced global biodiversity due to local and regional extinctions. Chytridiomycosis, a fungal disease caused by Batrachochytrium dendrobatidis (Bd), is affecting amphibian populations worldwide. Chapter 1 of this thesis reports using lab-raised larval tiger salamanders (Ambystoma tigrinum nebulosum), collected as eggs, to test if Bd infects them. Bd infects metamorphosed tiger salamanders; however, it is currently unknown if larvae can be infected by Bd. Adult frogs tend to host Bd on ventral surfaces and hind legs while tadpoles host Bd in keratinized mouthparts. No research has considered differences in infection between life stages of salamanders. It was hypothesized that Bd can colonize larvae in the same manner as metamorphosed animals. Larval salamanders were inoculated to test if Bd concentrations differ among body regions in larvae compared to metamorphosed salamanders. Larvae can carry Bd with the concentration of Bd varying between body region. Chapter 2 report using native tiger salamanders (Ambystoma tigrinum nebulosum), from northern Arizona and Bd as a study system to test if Bd is native or introduced to Arizona. It was hypothesized that Bd is not endemic to Arizona, but is introduced. There are multiple hypotheses regarding potential routes Bd may have traveled through Arizona and into Mexico. These hypotheses were tested using the Kaibab Plateau in Coconino County, Arizona, as a study site. The plateau is isolated from surrounding areas by the Grand Canyon to the south and the Vermillion Cliffs to the north serving as major biogeographical barriers. It is hypothesized that tiger salamanders are not dispersing into or out of the Kaibab Plateau due to geological restrictions. Bd, therefore, should not be present on salamanders on the Kaibab Plateau due to geological restriction. Tiger salamanders in stock tanks located on the Kaibab as well as preserved museum specimens housed in the Arizona State University Natural History Collection were sampled. The results indicate that Bd occurs at low levels on Kaibab Plateau tiger salamanders. / Dissertation/Thesis / Masters Thesis Biology 2019

Thesis (B.A.)-Oregon State University, 1997. / Includes bibliographical referenes (leaves 20-22). Also available via the Internet.

Nearly every species on the planet has at least one parasite, which, by definition, incurs a cost in the host. Therefore, organisms must resist parasites - preventing or reducing infections - or tolerate parasites - reducing the costs of infection - in order to maintain their fitness despite the presence of parasites. Here, I investigated: 1) whether parasitic, larval trematodes (cercariae) can detect the least resistant tadpole host species, 2) a hypothetical framework for how host life history impacts the utilization of inflammation and thus, resistance and tolerance, 3) whether a common anesthesia technique used in experimental infections immunocompromises tadpoles, 4) the relationship between tadpole host life history, tolerance, and behavioral resistance to cercarial infection, 5) how tadpole behavior affects trematode infection location, and 6) how host life history impacts trematode infection location and the implications of this for host tolerance. In the first chapter, I investigated whether trematode cercariae could discriminate among several tadpole host species to identify the most susceptible hosts. Cercariae were consistently more attracted to Bufo terrestris tadpoles, which was the most susceptible host species. Other tadpole species varied in attractiveness in an order similar to their susceptibility to infection. Furthermore, there was consistent and significant variation among individual attractiveness and susceptibility within host species. If susceptibility to infection is heritable, chemical cues used by cercariae to identify susceptible hosts could represent a substrate on which natural selection acts, setting up a "Red Queen" arms race between host cues and parasite detection of those cues. In the second chapter, I proposed a framework which outlined the cost-benefit relationship between host life history and immune responses. Because inflammatory immune responses are known to cause self-damage to hosts, anti-inflammatory immune responses should vi be used for long-lived, slowly-developing ("slow-paced") hosts, those infected with relatively less virulent parasites, and/or ongoing but ineffective inflammatory responses. Conversely, the cost of inflammation might be less expensive than the cost of infection among shortlived, rapidly-developing ("fast-paced") hosts, those infected with virulent parasites, and/or those undergoing protracted but ineffective anti-inflammatory immune responses. In the third research chapter, I investigated whether two common anesthesia agents, benzocaine and tricaine mesylate (MS-222), immunocompromise tadpoles. These chemicals are used extensively to study the behavioral resistance of tadpoles to cercariae; if treatment increases infections not only by removing these behaviors but also by suppressing the immune system, behavior might appear artificially effective at preventing trematode infection. I found that neither benzocaine nor MS-222 affected the abundance of circulating white blood cells relative to waterexposed control tadpoles. Furthermore, there was no difference in trematode infection success when tadpoles were anesthetized, allowed to recover from anesthesia, and subsequently experimentally infected. The results of this experiment indicate that benzocaine and MS-222 are both practical, non-immunosuppressive anesthesia agents to use when studying trematode infections in amphibians. In the fourth research chapter, I quantified tadpole hosts' use of behavioral resistance (parasite-induced behaviors) and tolerance of exposure to cercariae. Across seven host species, parasite-induced behaviors were negatively correlated with pace-of-life, with rapidly-developing ("fast-paced") tadpoles exhibiting significantly more behavior than slowly-developing ("slowpaced") tadpoles. The opposite pattern was true of tolerance, where fast-paced species had poorer tolerance of cercarial exposure than slow-paced species. Given that slow-paced species are more likely to be exposed to cercariae because they 1) occur in water more likely to harbor cercariae and 2) have longer developmental times, tolerance to trematode exposure might be an vii evolutionary adaptation that circumvents the costs of behavioral - and, possibly, immunological - resistance to infection. In the fifth research chapter, I investigated whether parasite-induced behaviors were capable of affecting encystment location of trematode cercariae in Hyla femoralis tadpoles and whether the resulting encystment location affected tolerance of infection. Benzocaineanesthetized and control tadpoles had similar infection intensities. However, among benzocaineanesthetized tadpoles, the majority of cercarial infections occurred in tadpoles' heads, but unanesthetized control tadpoles were predominantly tail-infected. Furthermore, the number of head infections were negatively associated with mass change (poor tolerance), whereas the number of tail infections was positively associated with mass change (good tolerance). These results suggest that parasite-induced behavior is not only an important mechanism of resistance to trematodes, as other researchers have described, but also a mechanism of tolerance, whereby tadpoles can prevent the deleterious effects of trematode infection by controlling infection location. The sixth research chapter extends the work of the fifth and investigates whether host life history predicts encystment location of cercariae and whether encystment location affects tolerance of infection. Among seven host species, fast-paced species had significantly more infections in the head and body, whereas slow-paced species had the majority of infections in their tails. Slow-paced species were also less resistant to trematode infections in any body location than fast-paced species. These patterns were partially explained by surface area, with a slow-paced species having more surface area (making them more likely to be contacted and infected by cercariae) and a larger proportion of slow-paced species' surface area was comprised by tail than heady and body. Tail infections were less expensive than head and body infections; slow-paced species were more tolerant of infection and across species, tail infections had no effect on tolerance (mass change) whereas head infections were negatively associated with tolerance. These results suggest that slow-paced tadpoles, which are relatively more likely to viii become infected by cercariae and probably have more evolutionary history with these parasites, have invested in a morphology that improves their tolerance to parasites. The body of work that I have produced demonstrates that variation in resistance and tolerance to trematode parasites is ubiquitous among tadpole hosts. Furthermore, this variation is predictable based on host life history. Because tadpole life history can dramatically impact the likelihood of exposure to cercariae and encounters are necessary for host-parasite selective pressure to occur, life history can predict the adaptations of hosts and parasites. Given amphibians' status as the most rapidly declining taxon on the planet and the ubiquity of emerging infectious diseases for amphibians and other organisms, these findings should inform future research on host- and parasite-mediated mechanisms of disease.

No description available.

No description available.

Current municipal wastewater treatment plants (MWWTP) technologies are insufficiently removing emerging contaminants of concern. These emerging contaminants are an issue as many are known endocrine disrupting compounds (EDCs). EDCs are contaminants that can have severe and irreversible impacts on highly conserved endocrine systems that are critical during developmental periods in vertebrates as well as during adult life. Many EDCs have non-monotonic dose-response curves yet they are not often tested at low, environmentally relevant concentrations. EDC research to date has focused heavily on xenoestrogenic compounds whereas thyroid hormone (TH) disruption has been largely overlooked. TH is conserved in all vertebrates and plays crucial roles in neural development, basal metabolism, and thermoregulation. TH is comprised of thyroxine (T4), often known as the transport form of TH, and triiodothyronine (T3), the more bioactive form of TH. A TH spike occurs in the perinatal period of humans, and when disrupted, this spike can cause severe developmental defects. An analogous, but perhaps more overt, TH spike occurs in amphibians. TH is the sole hormone that drives amphibian metamorphosis, thus providing an excellent model for TH action. Our lab has previously developed the cultured tailfin (C-fin) assay, which uses biopsies from premetamorphic Rana catesbeiana tadpole tailfins cultured in the presence of an exogenous chemical of concern to assess perturbations to TH- and stress-responsive gene transcript levels by QPCR. This thesis uses the C-fin assay to assess the efficacy of removal of biological TH- and stress-altering activity in conventional municipal wastewater treatment systems. We first assess the successive levels of a full-scale conventional activated sludge (CAS) MWWTP in its ability to reduce perturbations of mRNA transcript levels of the critical TH receptors alpha (thra) and beta (thrb), and stress responsive gene transcripts superoxide dismutase (sod), catalase (cat) and heat shock protein 30 (hsp30). Secondary treatment of wastewater effluents removes cellular stress perturbations when compared to influents, but thr disruptions remain after conventional secondary wastewater treatment. We then assess three pilot-sized conventional secondary MWWTP configurations run at two operational conditions. The C-fin assay results suggest that the current understanding of operational conditions and the efficiency of complex MWWTP configurations is not clear-cut when assessed by biological endpoints such as the transcript abundance perturbations in the C-fin assay. Finally, the C-fin assay is used to investigate transcript profiles of genes of interest when the tissues are treated with the endogenous hormones T3, T4, and estradiol (E2). Our results indicate that T4 acts as more than solely a T3-prohormone and that gene expression levels in response to the two different forms of TH can be T3 or T4 specific. E2 effects, although implicated in altering TH-mediated responses in other contexts, do not affect TH-responsive gene transcripts in the C-fin. The data presented use the novel C-fin assay to challenge and advance the currently accepted views of TH-action, as well as develop necessary yet practical biological knowledge for management of emerging contaminant release from MWWTPs. / Graduate / 0383 / 0768 / 0307 / polaw@uvic.ca

Identifying amphibian habitats within a landscape provides a tool for managing their populations. I identified if and how amphibians used small ephemeral wetlands (≤ 0.1ha) and streams within the mixedwood forest area managed by Daishowa Marubeni International Ltd. near Peace River, north-western Alberta. Twenty-seven wetlands and their riparian zones were sampled for all life stages of amphibians in 2008 using timed visual encounter surveys. The riparian zones of 11 small streams were sampled with pitfall traps within 120 m of their beds from 2006 to 2008. Habitat features were also measured. Lithobates sylvaticus, Anaxyrus boreas and Pseudacris maculata used small ephemeral wetlands and the riparian zones of ephemeral, intermittent and permanent streams at different life stages. Water temperature and canopy cover influenced amphibian presence and abundance in wetlands. Coniferous and deciduous tree density were associated with L. sylvaticus abundance at the stream sites. I conclude that small waterbodies are amphibian habitats in the mixedwood forest of boreal Alberta. / Ecology

To date, the majority of studies into sex determination and sexual development have focused on the mammalian system due to the mouse model being an excellent tool for developmental biology as well as obvious implications to human health and development. However, the focus on the mammalian system has caused a large fraction of other vertebrate groups to be overlooked. The last decade has seen an array of studies into various non-mammalian organisms like fishes, birds, reptiles, and amphibians. These studies have unveiled a remarkably conserved molecular background utilized for sexual differentiation amongst all vertebrates, ranging from the alligator which employs temperature dependent sex determination to the mouse, which employs genotypic sex determination In this project, I implemented molecular methods traditionally used to study model organisms, to investigate an amphibian species, the cane toad (Bufo marinus). The cane toad was chosen due to its invasive status in Australia, as well as being a representative of one of the most successful and specious families of vertebrates, Bufonidae. Since, its introduction, this species has rapidly spread across the continent, adversely effecting native species throughout its introduced range. Recent studies have identified the disruption of the sex-determination pathway as a realistic goal in an otherwise, seemingly futile, effort to curtail their expansion. We decided to approach the study of cane toad sex determination and development on three levels, in order to have a broader understanding of the similarities between toads and other groups, and additionally, to better assess the feasibility of utilizing molecular means to control cane toads in non-native habitats. Firstly, I used a candidate gene approach to clone and characterize five genes which were previously known to be involved in both mammalian as well as amphibian sex determination: Sox9, Dmrt1, p450arom, Sf1, and Dax1. I chose Sox9 and Dmrt1 due to their known involvement in the male-specific pathway of mammals. Dmrt1 was also known to be male specific in other groups, including amphibians. Conversely, I also decided to investigate p450arom and Dax1, due to their affiliation with the female pathway, while Sf1 has a role in both sexes. All of these genes were expressed in the gonads of both sexes of cane toads. However, Sox9 exhibited strong transcriptional up-regulation in testes at the time of sexual differentiation, similar to mammals. Thus, using the candidate gene approach, I was successful in identifying a sex-specific marker which could be utilized in the manipulation of the sex determination pathway (e.g. female to male sex reversal). VIIThe second approach involved the study of sex determination on a cytogenetic level. Traditionally, Bufonids have been assumed to utilize a ZZ/ZW (male homogametic/female heterogametic) sex chromosome system, yet only one recent study has been successful in the identification of sex chromosomes in a toad species. Moreover, the authors identified the sex chromosomes in only one population (of 18 studied), highlighting the scarcity of discernible sex chromosomes in toads. When we began to investigate the karyotype of cane toads, we were able to identify a female-specific length polymorphism in the nucleolus organizer region (NOR) of chromosome 7, making this chromosome pair a strong candidates for the Z and W sex chromosomes. In order to verify our hypothesis, we also performed chromomycin A3 staining to reveal a differential signal between the chromosome pair, indicative of heterochromatin accumulation on the brighter NOR. This study lent strong support to the observed difference between the chromosomes, yet we required further investigation in order to identify the W and the Z chromosomes, specifically. By utilizing comparative genomic hybridization (CGH), I was able to identify a female specific region on the chromosome with the larger NOR, identifying it as the W chromosome. This study was successful in identifying the sex chromosomes in the cane toad. Moreover, it allows us to make a more accurate prediction of the possible sex determination method utilized: either a dosage dependent male determination system, or a female-specific gene in a female determination system. The third approach focused on the Bidder’s organ and its role in sexual development of cane toads. The Bidder’s organ is a gonadal structure comprised of ovary-like tissue, found in both males and females, with no known function. Since our study was focused on female to male sex-reversal of cane toads, we decided to investigate the potential of the Bidder’s organ to interrupt this process. By utilizing the candidate genes, which I had previously cloned, I was able to identify the Bidder’s organ as having an entirely distinct transcriptional pattern in comparison to the gonads of either sex. Additionally, the Bidder’s organ showed significantly higher levels of p450arom expression than the gonads, identifying it as a possible key player in the production of aromatase enzyme for oestrogen production. Moreover, gene expression patterns in the Bidder’s organ strongly correlated with the significant developmental time points in the sexual development of the toads, implying a possible function in the development process of toads. In conclusion, I have performed the first study of the molecular, cytogenetic and anatomical aspects of sexual development in a toad. I was able to verify that cane toads utilize a ZZ/ZW chromosome system and furthermore, likely utilize the mammalian male-linked gene, Sox9, in male development. I was also able to show that the Bidder’s organ is transcriptionally active at key time points, likely indicative of a functional role during development.

The decline of amphibian populations around the world is a well documented phenomenon. The Baw Baw Frog Philoria frosti belongs to a group of high-elevation, mountain-top amphibians in Australia that have undergone recent population declines, but an understanding of the responsible agents is deficient or absent for most species. The inability to diagnose agents of decline has mostly been attributed to a paucity of knowledge on the natural history of these species. / The discipline of conservation biology provided a scientific basis for commencing investigation into the decline of P. frosti. This thesis examines the pattern and extent of decline, and the autoecology and demography of the species, in order to provide a basis for evaluating conceivable decline-agents, and to establish a platform to commence diagnosis of the decline. (For complete abstract open document)