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A pathway for the biosynthesis of tropic acid from tryptophan in Datura Stramonium has been proposed by Goodeve, and was supported by Hamon. The purpose of this investigation was to determine quantitatively the percentage of incorporation of tryptophan into tropic acid. This was attempted using vacuum infiltration and sterile root culture techniques with autoradiography being utilized to identify the radioactive metabolites. Tryptophan was not found to be converted to tropic acid under the experimental conditions used in this investigation. The purpose of this investigation was also to extract crude enzyme preparations which would convert certain postulated intermediate compounds into tropic acid. The conversion of a-phenyl-3-aminopropionic acid to atropic acid and the conversion of atropic acid to tropic acid was attempted. The enzyme extracts of Datura Stramonium root tissues were prepared from acetone powders, from fresh tissue, and from freeze-dried tissues. These extracts did not show any activity in vitro. It is concluded that some differences existed between the tissues used in the present investigation and those used in previous work. / Pharmaceutical Sciences, Faculty of / Graduate

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 244-258).

No description available.

Bone metastases are a major cause of morbidity in patients of advanced breast cancer. Development of osteolytic bone metastasis depends on the interaction between malignant tumour cells and bone microenvironment. Receptor Activator of Nuclear Factor Kappa B (RANK) is a member of tumour necrosis factor (TNF) superfamily that is expressed by osteoclasts (the bone resorbing cells) and primary breast tumour cells. Previous studies demonstrated that RANK receptor and its ligand (RANKL) play an important role in bone remodelling, mammary gland development and immune system. RANKL was also found to serve as a chemotactic factor that facilitates breast tumour metastasis to bone. However, the role of the RANK receptor in breast cancer cell metastatic behaviour in bone is not fully understood. Therefore, the aim of this thesis was to explore the role of the RANK receptor in parental and bone-tropic breast cancer cell growth, motility and invasion, and assess these cells influence on breast cancer cell induced osteoclastogenesis. Functional studies in breast cancer cells showed that RANKL (100 - 300 ng/ ml) significantly enhanced parental human MDA-231 (MDA-231P) and mouse 4T1 breast cancer cell spreading within minutes. RANKL induced chemotactic cell migration of MDA-231P cells in vitro. I also found that RANKL significantly stimulated random and directional 2D and 3D cell migration of parental MDA-231P and bone-tropic (MDA-231BT) breast cancer cells in vitro. These effects were observed at concentrations (100 – 300 ng/ml) that were sufficient to induce osteoclast formation in the presence and absence of breast cancer cells in vitro. In contrast, high concentrations of RANKL (1000 ng/ ml) dramatically suppressed human MDA-231P breast cancer cell invasion in vitro. These data indicate that the RANK receptor in the breast cancer cell lines tested influences cancer cell spreading, migration and invasion in vitro. Thus, targeting RANK in tumour cells may be of value in the prevention of tumour burden associated with breast cancer bone metastasis. Mechanistic studies revealed that RANKL stimulated the phosphorylation of p38 kinase in human and mouse breast cancer cells. Interestingly, RANKL had no effect on NFᴋB, JNK and AKT pathways in parental human MDA-231 and mouse 4T1 breast cancer cells at concentrations up to 300 ng/ ml. These data implies that the RANK receptor modulates human and mouse breast cancer cell metastatic behaviour via p38 activation and independently of the NFᴋB and PI3K/AKT pathways. Silencing of the RANK receptor in the bone-tropic human breast cancer cells MDA- 231BT2 reduced directional cell migration without affecting cell viability and growth. Functional studies in osteoclast and breast cancer cell revealed that knockdown of RANK expression in both parental and bone-tropic human breast cancer cells significantly inhibited the ability of these cells to stimulate osteoclast formation. Although, I cannot exclude the possibility of the involvement of other signalling pathways downstream of the RANK receptor, these studies suggest that the RANK/P38 signalling in osteoclast and breast cancer cells contributes significantly to breast cancer cell behaviour in bone. Genetic analysis of the RANK gene in human parental and bone-tropic MDA-231 breast cancer cells showed a number of polymorphisms. One variant detected was found to be deleterious for the RANK protein. This variant changes the amino acid sequence from alanine to threonine (Ala ˃ Thr) and only appeared in the RANK gene in the parental human MDA-231P breast cancer cells. Moreover, of the four known RANK isoforms that were detected in the parental and bone-tropic breast cancer cells tested, two lacked the TRAF6 binding motifs associated with NFκB activation. All RANK isoforms detected on the bone-tropic MDA-231BT breast cancer cells expressed the P38 binding motifs. Altogether, these findings support the role of the RANK/P38 signalling pathway in breast cancer cell behaviour in bone. Epigenetic analysis in parental human MDA-231P breast cancer cells showed that continuous and long-term exposure to RANKL (10 and 100 ng/ ml) for up to 50 passages (approximately 120 days) did not induce epigenetic changes, particularly DNA methylation, in the RANK gene. However, I found DNA methylation changes in a set of genes that are known to be involved in cell development and regulation. The methylation status of the altered CpG loci either hypermethylated or hypomethylated are located at different parts in the CpG islands. Whole genome DNA methylation pattern of the bone-tropic breast cancer cells showed a number of genes that appeared in both bone-tropic variants are correlated with different biological function of the cells. I also found that long-term exposure of human MDA-231P to RANKL (100 ng/ ml) enhanced the ability of these cells to stimulate osteoclastogenesis in vitro. These data together indicate that long-term exposure to RANKL induces “boney” epigenetic changes in a set of genes that enhances breast cancer cell behaviour in bone. Overall, this thesis illustrated that the RANK receptor on human parental and bone-tropic breast cancer cells plays an important role in cell motility and ability of these cells to influence osteoclastogenesis and ultimately osteolysis. Therefore, agents that selectively target the RANK receptor may be of value in the treatment of both tumour burden and osteolytic bone disease associated with breast cancer. However, the role of the RANK receptor in bone metastasis will require further in vivo investigation.

La Cuenca del Madre de Dios, es una región que está plenamente inserta en el trópico húmedo y abarca una considerable superficie del oriente peruanoboliviano. Esta región con vastas áreas naturales, empieza a mostrar cambios fundamentales en la organización de su espacio. Su ocupación si bien se reconoce es desde muy antiguo, por parte de grupos etnicos; ha visto en el presente siglo, principalmente en las tres últimas décadas, el acontecer de diferentes fases en las relaciones del hombre con su medio. El estudio para el desarrollo integral de espacios definidos por cuencas hidrográficas multinacionales como la del Madre de Dios, supone problemas técnicos y políticos. El diseño del estudio, con un enfoque amplio, debe considerar factores de tipo físico, socio-económico, político e institucional.Estas notas, resultado de la experiencia de trabajo en la región, presentan una visión sintética de la realidad de la Cuenca y ponen el acento en: La Oferta Ambiental, la Ocupación del Territorio, la Caracterización de sus Unidades Político-Administrativas y la Estrategia para el Desarrollo Integral. El ecodesarrollo, la ordenación territorial y la integración son elementos claves de una estrategia de actuación que considere a la Cuenca como marco de la planificación regional que define un contexto espacial y ambiental adecuado para las tareas del desarrollo. Por todo lo anterior, el Ecodesarrollo como estilo y la Integración Territorial Multinacional como política-, son consideradas de suma importancia para el desarrollo integral y sostenido de espacios fronterizos en las regiones tropicales como lo es la Cuenca del Madre de Dios.  The Madre de Dios Basin is a region totally inserted in the humid tropic and covers a large section of the Peruvian and Bolivian Oriente. This region with extensive natural areas has already started to show changes in its spatial organisation. Though it was settled by different ethnic groups since ancient times, it has been in the last three decades that sorne important changes have occurred in the man-environment relationships. To study binational basins such as the one of Madre de Dios, brings about many technical and political difficulties. This is because the research project design must be broadly oriented to cover topics such as physical, socioeconomic, political, and institutional. In this paper 1 present the results of a fieldwork experience in the region. They give us a synthetic view of the Madre de Dios reality, placing special attention to the environmental realm, its characterization of the political and administrative units, and a strategy to integral development. Land management, ecodevelopment and integration are key elements toward a decision-making strategy that takes the basin as a framework to regional planning.

Yes / ABSTRACT Entry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant’s phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable. IMPORTANCE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug-resistant virus is particularly interesting because it tends to be rare, with lack of sensitivity usually associated with the presence of CXCR4-using virus (CXCR4 is the main alternative coreceptor HIV-1 uses, in addition to CD4). We analyzed envelope sequences from HIV-1, obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure, without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We found that in these individuals, resistant viruses form a distinct population that evolved once and was successful as a result of drug pressure. Further evolutionary analysis placed the complex network of interdependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance. / X.J. was supported by Medical Research Council (G1001806/1) and Wellcome Trust (097820/Z/11/A) funding and F.F. by a Biotechnology and Biological Sciences Research Council studentship to D.L.R.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Interactions between nesting birds and invertebrates are a common, yet poorly understood ecological phenomenon. Many of these types of interactions are close and prolonged, and therefore potentially critical to one or both of the species involved in the interaction. However it is unusual for the nature of the interaction to be evaluated in a manner that reveals the impact of the relationship on both parties to the interaction. This study examines two relationships between nesting birds and invertebrates, both of which involve the hooded parrot (Psephotus dissimilis), a small grass parrot that inhabits the tropical savannas of northern Australia. ¶ The field-work for this project was conducted over two parrot breeding seasons in 2006 and 2007 near Katherine, Northern Territory, in the Australian dry tropics. In chapter II, I present data on the breeding biology of the hooded parrot as background for the study that follows. Nest building commenced in January, with peak activity in February and the last chicks fledged in April. Fifty three active nests were located. The mean number of eggs laid per nest was 4.5 (s.d. ± 0.9), of which 3.0 (± 1.79) hatched and 2.0 (± 2.0) fledged. Clutches were laid asynchronously over a period of a week and chicks remained in eggs for 18.6 (± 1.95 days). Chicks were 29.2 (± 2.9) days old when they fledged from the nest. These data are typical for Australian parrots. ¶ In chapter III, the unusual nature of the parrot’s nest site is examined. Many species of bird nest in natural cavities or those they excavate. Whilst cavity nesters as a whole experience increased nesting success, the greatest success is experienced by species that can excavate their own nests. Certain arboreal cavity nesters, such as woodpeckers, require extensive morphological adaptation for this behaviour, but this has not occurred in Australia, despite competition among birds and a suite of arboreal mammals for naturally occurring cavities. Some species, however, have adapted their behaviour to make use of substrates that are not as hard as wood. Hooded parrots excavate nests in terrestrial termitaria, releasing them from competition for limited arboreal cavities. However, I show that only termitaria with a high level of termite activity, and which are more than two metres tall, are suitable, and that the parrots exhibit a strong preference for the cathedral mounds of Nasutitermes triodiae. Nests placed in highly active mounds had a significantly higher success rate than those in mounds where activity was somewhat lower, suggesting that the behaviour is adaptive. ¶ The thesis then shifts focus from the parrot to its nest symbiont, first describing the species involved in the interaction in chapter IV, and then its behaviour in the nests of hooded parrots in chapter V. Trisyntopa neossophila sp. n. (Lepidoptera: Oecophoridae) was reared from the nest of the hooded parrot and described using morphological characters. Aspects of its biology are reported and similarities to the biology of Trisyntopa scatophaga found in the nests of the golden-shouldered parrot (Psephotus chrysopterygius) are discussed. The possibility that a moth was associated with the extinct paradise parrot (Psephotus pulcherrimus) is considered in the light of the phylogenetic relationships between the parrots. ¶ Trisyntopa neossophila is an unusual moth whose breeding cycle is shown to be closely synchronised with the hooded parrot. T. neossophila is one of three coprophagous, nest dwelling moths in the genus Trisyntopa. True coprophagy is rare in the Lepidoptera, although some species occasionally consume faeces to gain rare nutrients. T. neossophila lays its eggs in the nest of hooded parrots so that larvae hatch in synchrony with the hatching of the parrot’s eggs. The larvae spend their larval period in the nest and exclusively consume the excrement of the nestling parrots. When the parrot chicks fledge, the larvae move to the walls of the nest cavity to pupate, emerging the following wet season to repeat the process during the next parrot breeding season. ¶ With a description of the ecology of both species involved in the nesting symbiosis, chapter VI reviews the literature surrounding other interactions between nesting birds and invertebrates. A large number of birds are shown to nest in, or in close proximity to, structures made by invertebrates and avian nesting material provides a reliable shelter for many invertebrate species. However, the nature of such relationships has rarely been experimentally demonstrated. I propose that in order to understand the nature of these relationships they need to be explored within the theoretical framework of community ecology. Putative commensal and parasitic relationships have all been documented in the bird/invertebrate nesting literature, yet researchers, with few exceptions, repeatedly overlook the impact that these relationships are having on the invertebrate, at best assuming the nature of its impact, but more often ignoring its impact entirely. Here I present a framework for formulating hypotheses to ensure that the nature of the relationship can be identified. Only by explicitly stating the level of organisation at which the experiment is to occur (individual or population), identifying the net cost or benefit of the interaction, the range of conditions under which such costs or benefits would apply and the spatial and temporal context in which they apply, can an investigator expect to recognise and describe the often complex nature of these relationships. ¶ While parasitic and commensal relationships between nesting birds and invertebrates are commonly reported, mutualisms between birds and invertebrates have not been reported. Despite this, candidates for this type of relationship exist. Chapter VII uses the framework outlined in the literature review (chapter VI) to experimentally examine the relationship between the hooded parrot and Trisyntopa neossophila. By manipulating the populations of moth larvae in a sample of hooded parrot nests, we sought to establish the impact of the relationship on each species. The moth depends on the parrot for provision of shelter and a reliable food source. The parrot however, was neither benefited nor harmed by the interaction in terms of short term reproductive output or chick growth, although differences between the experimental and control nests were noted. The relationship between the hooded parrot and T. neossophila, at least during the study period, is therefore concluded to be commensal. ¶ Collectively, the chapters of this thesis explore the complicated interactions between species. The dependence of the moth on the parrot and the parrot on the termite, demonstrate the importance of understanding interactions between species in a manner that reveals the impacts of the interactions, the range of conditions under which they would apply and the level of organisation at which they apply, as outlined in chapter VI. The dependence of the animals in this study on each other makes them more vulnerable to extinction than previously thought. Whilst this may not be immediately significant for the hooded parrot/T. neossophila system, which is thought to be secure, the ecologically similar system on the Cape York Peninsula, involving the golden-shouldered parrot and its nest attendant moth Trysintopa scatophaga, is vulnerable to extinction and subject to intensive management to ensure its persistence. This study brings new information to the management of the golden-shouldered parrots and urgently recommends increased protection for Trysintopa scatophaga.