The androgen receptor (AR) fulfils important roles for both sexes. By mediating the biological function of androgens, the AR has remained the target for endocrine therapies treating prostate cancer. The AR also determines the effectiveness of medroxyprogesterone acetate (MPA) in treating AR positive breast cancer. Every man will be affected by prostate cancer if he lives long enough. Prostate cancer continues to be a leading cause of death for males despite research into this cancer covering more than 60 years since Huggins' seminal 1941 study showing that androgens play a key role in this cancer. Unfortunately, significant advances have not been forthcoming and the effect of treatment has remained largely the same over past decades, whereby initial treatment provides temporary remission but eventually advanced cases become refractory to further intervention and the disease recurs in a more aggressive form. A plethora of factors are exquisitely sensitive to minute changes in the AR's structural profile, which can be altered by a single mutation, resulting in aberrant activity. A principal feature of these variant ARs associated with prostate cancer, is enhanced capacity to bind a number of molecules other than its cognate ligand, dihydrotestosterone (DHT). The promiscuous activity of this receptor leads to continued AR signalling and stimulus for the cancer despite low androgen levels induced by treatment regimes. A key question is whether mutations occurring within the AR occur as a result of cancer or contribute to the propagation of the cancer. Recent research has demonstrated that treatments incorporating anti-androgens such as flutamide, which are designed to impede prostate cancer progression by inhibiting AR activity, may actually provide selective pressure favouring somatic mutation of the receptor to take place. The specific changes to the AR which are responsible for gains of function have not been resolved as their crystal structures, which are used to provide conformational analysis of proteins, are tremendously problematic to produce with little success found in literature. Generating representative crystals of the AR protein involves producing soluble recombinant protein. Unfortunately the AR is prone to aggregation and is highly unstable, especially in the presence of antagonistic molecules or absence of a stabilising ligand, preventing the protein from being maintained in the soluble state required for crystallization. In order to produce sufficient quantities of soluble material for crystallization, the androgen receptor's ligand binding domain (LBD) was produced as a recombinant protein in Escherichia coli bacteria strain BL21 (DE3) in the presence of DHT, flutamide, as well as in the absence of ligand. Since soluble unbound AR-LBD has not been produced until now, the bacterial culture containing no ligand was further processed to the stage of cleaving the purification tag from the recombinant protein and represents considerable progress into producing soluble material for crystallizing the troublesome yet considerably important AR in the absence of ligand. Although distinct from prostate cancer in males, AR activity in breast tissue is also a factor determining the action of drugs, such as MPA, included in therapies aimed at breast cancer. The use of MPA has declined primarily due to its adverse effects including unsuccessful generation of a biological response, as well as the advent of other drugs administered for hormonal therapies treating breast cancer. Alternative drugs are needed when breast cancer therapies fail as tumours develop resistance to primary drugs. Although there are a number of drugs on the market, success would be maximised if the determined therapy is matched with the patient, based for example, on their genetic makeup. There is a conundrum whereby some patients with an AR do not respond to MPA, a drug normally recognised by the receptor. In clinical trials it was discovered that an AR with threonine instead of methionine at residue 780 (M780T) fails to activate in response to MPA, but the exact mechanism has remained elusive and needs to be answered at the molecular level. The X-ray crystallographic studies that generate 3D images of macromolecules and wet chemistry, which have traditionally been used to provide insight into science in these dimensions, are incorporated with computer based molecular simulation. This is both complementary and distinct to traditional scientific methodologies, enabling further elucidation of protein-protein interactions, and the influence applied to such inter-relations by natural and drug ligands. This approach has been used, and is continually developed, to understand the binding mechanisms of current drugs as well as designing new drugs. In order to produce a receptor representing the M780T variant, the crystal structure representing the AR-LBD was mutated in silico, into which MPA was then docked. It was found that MPA binds into the M780T AR-LBD with considerably more spatial displacement compared to the position of DHT in the crystal structure, and is predicted to be the primary reason for the inability of MPA to activate this variant AR. The clarification of MPA binding and failure to elicit a response from the variant AR is significant for a cohort of breast cancer patients, as not only does the presence of an AR in the tumour determine the effectiveness of MPA, but correct composition of the AR, specifically, the absence of a M780T mutation. In the absence of this AR mutation, MPA could effectively be used either as an alternative to primary drugs, or in secondary therapies when primary therapies fail. Aberrant activity of variant ARs in response to MPA should also be taken into consideration when analysing drug studies about the effectiveness of MPA. The findings on the loss of response to MPA by the M780T variant AR have been included in the journal article &quotDecreased Androgen Receptor Levels and Receptor Function in Breast Cancer Contribute to the Failure of Response to Medroxyprogesterone Acetate" appearing in the September 2005 issue of Cancer Research journal.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 108-115.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 70-86.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 124-136).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 137-167).

A complex network of protein filaments collectively known as the cytoskeleton carries out several crucial cellular processes. These functions include, but are not limited to, motility, cell shape, mitosis and organelle trafficking. The cytoskeleton is also highly responsive, allowing the cell to alter its shape in response to its immediate needs and environment. One of the major components of the cytoskeleton is the microtubule network. To refer to the array of micro tubules in the cell as a skeleton is a misnomer. Microtubules, by virtue of their structure and nature, are highly dynamic, continuously growing and shrinking. They also bind a variety of accessory molecules that aid in regulating and directing their dynamic activity. In this way they provide a structural basis for integral cell functions that require rapid assembly and disassembly. In some cases, perturbations of the microtubule network results in structural anomalies that lead to undesirable outcomes for the cell, namely chromosomal missegregation events and instability. The accumulation of these events may induce aneuploidy, which has been a fundamental component of tumorigenesis. This dissertation examines the role of the microtubule cytoskeleton within three distinct contexts. The first chapter investigates the association of the anti-apoptotic protein survivin with the microtubule network and its potential impact upon the cell from interphase to cytokinesis. The second chapter of this dissertation explores a little-studied, microtubule-dense organelle, referred to as the midbody, and the highly orchestrated events that take place within it during cytokinesis. The third and final chapter describes a unique experimental condition that may further our understanding of the interaction between the tumor suppressor p53 and the centrosome in cell cycle regulation and tumorigenesis.

Circadian clocks are biological timekeepers that help maintain an organism’s behavior and physiological state optimally timed to the Earth’s day/night cycle. To do this, these internal pacemakers must accurately keep track of time. Equally importantly, they must be able to adjust their oscillations in response to external time cues to remain properly synchronized with the environment, and correctly anticipate environmental changes. When the internal clock is offset from its surrounding day/night cycle, clinically relevant disruptions develop, ranging from inconveniences such as jet-lag to more severe problems such as sleep disorders or mood disorders. In this work, I have used the fruit fly, Drosophila melanogaster, as a model organism to investigate how light and temperature can synchronize circadian systems. My initial studies centered on an intracellular photoreceptor, CRYPTOCHROME (CRY). CRY is a blue light photoreceptor previously identified as a major component of the primary light-input pathway into the Drosophila circadian clock. We used molecular techniques to show that after light-activation, CRY binds to the key circadian molecule TIMELESS (TIM). This interaction irreversibly targets TIM, but not CRY, for degradation. Further studies characterizing a newly isolated cry mutant, crym, showed that the carboxyl-terminus of CRY is not necessary for CRY’s ability to impart photic information to the molecular clock. Instead, the C-terminus appears to be necessary for normal CRY stability and protein-protein interactions. Thus, we conclude that in contrast to previous reports on CRYs of other species, where the C-terminal domain was required for transduction of photic information, the C-terminus of DrosophilaCRY has a purely modulatory function. During the second part of my dissertation work, I focused my studies on circadian thermoreception. While the effects of light in synchronization of the Drosophilaclock to environmental cycles have been extensively characterized, significantly less is known about temperature input pathways into the circadian pacemaker. I have used two approaches to look at how temperature affects the circadian system. First, I conducted a series of behavioral analyses looking at how locomotor rhythms can be phase-shifted in response to temperature cycles. By examining the behavior of genetically ablated flies, we determined that the well-characterized neurons controlling morning and evening surges of activity during light/dark cycles are also implicated in morning and evening behaviors under temperature cycles. However, we also find evidence of cells that contribute to modulating afternoon and evening behavior specifically under temperature cycles. These data contribute to a growing number of studies in the field suggesting that pacemaker cells may play different roles under various environmental conditions. Additionally, we provide data showing that intercellular communication plays an important role in regulating circadian response to temperature cycles. When the morning oscillator is absent or attenuated, the evening cells respond abnormally quickly to temperature cycles. My work thus provides information on the roles of different cell groups during temperature cycles, and suggests that beyond simply synchronizing individual oscillating cells, intercellular network activity may also have a role in modulating proper response to environmental time cues. Finally, I present some preliminary work looking at effects of temperature on known circadian molecules. Using a combination of in vivo and cell culture techniques, I have found that TIM protein levels decrease at higher temperatures. My cell culture data suggest that this is a proteasome-independent degradation event. As TIM is also a key molecule in the light-input pathway, the stability of TIM proteins may be a key point of integration for light and temperature input pathways. While additional research needs to be conducted to confirm these effects in vivoin wild-type flies, these preliminary results identify a possible avenue for further study. Taken together, my work has contributed new data on both molecular and neuronal substrates involved in processing light and temperature inputs into the Drosophila circadian clock.

Made available in DSpace on 2015-03-26T13:54:42Z (GMT). No. of bitstreams: 1 texto completo.pdf: 309281 bytes, checksum: 0de79a6a62424dcb2d046099dc929615 (MD5) Previous issue date: 2008-07-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Two experiments were conducted, one for performance and another for metabolism, with objectives of evaluating the effect of different digestible threonine: digestible lysine relations, supplemented or not with glycine on the performance, the threonine aldolase, threonine dehydrogenase and threonine dehydratase enzyme activity and the uric acid excretion of chicks cutting in the period of 8 to 21 days of age. For both experiments, 560 chicks cutting were used, all male, Coob lineage, distributed in completely randomized design, in a 3 x 2 factorial arrangement, being the digestible threonine: digestible lysine relations (55; 65 e 75 %), supplemented or not with glycine. An additional treatment containing meat flour was used with a 65% digestible threonine: digestible lysine relation. All treatments had eight repetitions and 10 birds per experimental unit, being lodged e metabolic batteries. A full collection of excrete proceeded during the experimental phase. For the analysis of the threonine aldolase, threonine dehydrogenase and threonine dehydratase enzyme activities, two birds per experimental unit were slaughtered and the liver and the pancreas were removed. The glycine supplementation did not influence (P>0,05) the nitrogen retention by the birds, however, it was essential to improve (P<0,05) the chicks cutting performance. The activity of the three examined enzymes in the liver and pancreas was influenced by the threonine levels of the diet. The birds fed with the diet that contained the meat flour had a more intense threonine aldolase activity in the liver and in the pancreas; the threonine dehydrogenase and the threonine dehydratase activity was smaller in the liver and higher in the pancreas in comparison to the birds fed with vegetable diet. The threonine levels of the diet and the supplementation of glycine did not influence the uric acid excretion from the birds. The glycine supplementation reduced the evaluated enzyme activity in the liver and in the pâncreas, providing a higher threonine amount for the protein deposition. Although, the threonine catabolism has been high in birds fed with the digestible threonine relation: digestible lisina of 55%, the feed conversion was not affected by the levels of threonine, therefore, the 55% threonine: lysine relation (0,631% digestible threonine), supplemented with glycine is enough to attend the demands of chicks cutting in the period of 08 to 21 days of age. / Foram conduzidos dois experimentos, um de desempenho e outro de metabolismo, com objetivos de avaliarem o efeito de diferentes relações treonina digestível: lisina digestível, suplementados ou não com glicina sobre o desempenho, a atividade das enzimas treonina aldolase, treonina desidrogenase e treonina desidratase e a excreção de ácido úrico, de pintos de corte no período de 08 a 21 dias de idade. Utilizaram-se, para ambos os experimentos, 560 pintos de corte, macho, da linhagem Coob, distribuídos em delineamento inteiramente casualisado, em um arranjo fatorial 3 x 2, sendo três relações de treonina digestível: lisina digestível (55; 65 e 75 %), suplementadas ou não com glicina. Foi utilizado um tratamento adicional contendo farinha de carne, com relação treonina digestível: lisina digestível de 65 %%. Todos os tratamentos tiveram oito repetições e 10 aves por unidade experimental, sendo alojadas em baterias metálicas. Procedeu-se a coleta total de excreta durante toda a fase experimental. Para análise da atividade das enzimas treonina aldolase, treonina desidrogenase e treonina desidratase, foram abatidas, ao final do período experimental, duas aves por unidade experimental e retirados o fígado e o pâncreas. A suplementação de glicina não influenciou (P>0,05) a retenção de nitrogênio nas aves, porém, foi essencial para melhorar (P<0,05) o desempenho de pintos de corte. A atividade das três enzimas analisadas no fígado e no pâncreas foi influenciada pelos níveis de treonina da dieta. As aves alimentadas com dieta contendo a farinha de carne tiveram a atividade da treonina aldolase maior no fígado e no pâncreas; a atividade da treonina desidrogenase e da treonina desidratase foi menor no fígado e maior no pâncreas, em relação às aves alimentadas com dieta vegetal. Os níveis de treonina da dieta e a suplementação de glicina não influenciaram a excreção de ácido úrico das aves. A suplementação de glicina reduziu a atividade no fígado e no pâncreas de todas as enzimas avaliadas, disponibilizando maior quantidade de treonina para a deposição protéica. Embora, o catabolismo da treonina tenha sido alto nas aves alimentadas com a relação treonina digestível: lisina digestível de 55,0%, a conversão alimentar não foi afetada pelos níveis de treonina, portanto, a relação de 55,0% treonina: lisina (0,631% de treonina digestível), suplementada com glicina é suficiente para atender as exigências de pintos de corte no período de 08 a 21 dias de idade.

Made available in DSpace on 2015-03-26T13:54:55Z (GMT). No. of bitstreams: 1 texto completo.pdf: 105984 bytes, checksum: eac5b7c639b49b694da3dfe59f2995f8 (MD5) Previous issue date: 2006-02-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The experiment was carried out to estimate the level of digestible threonine for laying japanese quail in posture. It was used four hundred quails, with 57 days old and with averaging 75.50% of the rate egg production, during 63 days. A complete randomized experimental design was used with five treatments, eight replicates and ten quail per experimental unit. The basal diet was deficient in threonine, containing 20% of crude protein, 2.910 kcal of ME/kg and supplied with five levels of L-threonine 98% (0.018; 0.074; 0.128; 0.183 and 0.239%), corresponding of digestible threonine: digestible lysine ratios 0.65; 0.70; 0.75; 0.80 and 0.85, respectively to compose the experimental treatments. The following parameters were evaluated: feed intake (g/quail/day), threonine intake (mg/quail/day), egg production (%), production of commercial eggs (%), egg weight (g), egg mass (g/quail/day), feed conversion (kg feed/kg egg), feed conversion (kg feed /egg dozen), weight gain (g), egg quality (yolk (g and %), albumen (g and %) and shell (g and %)), egg length and width (mm) and specific gravity (g/cm3). Linear effect (P<0.05) was only observed in the threonine intake. It is ended that, the japanese quail doesn't demand more than 0.65% of digestible threonine to provided the best productive performance and quality of eggs, for a consumption of 149.2 mg of digestible threonine/day or a daily consumption of 14.34 mg of digestible threonine/g egg, corresponding to the digestible threonine: digestible lysine ratio of 0.65. / O experimento foi conduzido a fim de se estimar o nível de treonina digestível para codorna japonesa em postura. Foram utilizadas 400 codornas, com idade inicial de 57 dias e com taxa de postura média de 75,50%, durante 63 dias. Utilizou-se delineamento inteiramente casualizado com 5 tratamentos, 8 repetições e 10 aves por unidade experimental.A dieta basal foi deficiente em treonina, contendo 20% de proteína bruta, 2910 Kcal de energia metabolizável/kg, sendo suplementada com cinco níveis de L- treonina 98% (0,018; 0,074; 0,128; 0,183 e 0,239%), correspondendo à relação treonina digestível:lisina digestível de 0,65; 0,70; 0,75; 0,80 e 0,85, respectivamente para compor os tratamentos experimentais. As variáveis estudadas foram: consumo de ração (g/ave/dia), consumo de treonina (mg/ave/dia), produção de ovos por ave dia (%), produção de ovos comercializáveis (%), peso do ovo (g), massa de ovos (g/ave/dia), conversão alimentar por massa de ovos (kg de ração/kg de ovos), conversão alimentar por dúzia de ovos (kg de ração/dz de ovos), mudança de peso (g), componentes dos ovos (gema (g e %), albúmen (g e %) e casca (g e %)), altura e diâmetro dos ovos (mm) e gravidade específica (g/cm3). Observou-se efeito linear (P<0,05) somente no parâmetro de consumo de treonina. Conclui-se que para proporcionar os melhores resultados de desempenho e de qualidade de ovos, a codorna japonesa não exige mais que 0,65% de treonina digestível, para um consumo de 149,2 mg de treonina digestível/dia ou um consumo diário de 14,34 mg de treonina digestível/g de ovo, correspondendo à relação treonina digestível: lisina digestível de 0,65.