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.

Orientador: Jose Barreto Campello Carvalheira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T02:56:20Z (GMT). No. of bitstreams: 1 Mendes_MariaCarolinaSantos_D.pdf: 2658779 bytes, checksum: 153ed5344815e7e59a41c04c4a965670 (MD5) Previous issue date: 2014 / Resumo: A proteína mTOR é um proteína reguladora chave de vários processos celulares, dentre eles proliferação, crescimento e sobrevivência celular. Fatores de crescimento, oxigênio, status energético e a presença de aminoácidos são fundamentais para que todos esses processos ocorram normalmente. Descobertas realizadas nas últimas décadas mostraram que a via da mTOR encontra-se ativada em vários processos celulares, incluindo formação tumoral e angiogênese. A leucina é um aminoácido de cadeia ramificada que tem o maior potencial em ativar a via da mTOR. Devido sua capacidade de promover a síntese proteica e ganho de massa muscular, seu uso é constantemente estimulado em pacientes com câncer. No entanto, seus efeitos no crescimento tumoral não está claro. Dessa forma, realizamos um estudo cujo objetivo principal foi investigar os efeitos da dieta suplementada com leucina na modulação do crescimento tumoral em diferentes linhagens de células tumorais que se diferenciem em relação à ativação constitutiva da via IRS1/Akt/mTOR. Estudos in vivo e in vitro realizados demonstraram que as células que se diferenciam em relação à ativação da via IRS1/AKT/mTOR respondem de maneira distinta à suplementação com leucina. Linhagens de células tumorais que possuem a via da mTOR constitutivamente ativada, PC-3 e MCF-7, quando suplementadas com doses elevadas de leucina in vitro reduziram a proliferação celular e causaram retenção das células na fase G1 do ciclo celular. Já o xenoenxerto tumoral da PC-3 reduziu sua proliferação e aumentou a morte celular quando os animais foram suplementados com leucina na dieta. Nós também observamos aumento da atividade da mTOR e da p70S6K em todas as linhagens celulares quando suplementadas com leucina. O aumento da atividade da proteína mTOR foi acompanhado de redução na fosforilação de AKTser473 nas células que possuíam a via da PI3K hiperativada (PC-3 e MCF-7). Esse fato pode estar ocorrendo devido a ativação das alças de contraregulação ocasionadas pela estimulação excessiva provocada pela suplementação com leucina, naquelas linhagens celulares que já possuem a via hiperativada. Fato este comprovado pelo aumento da fosforilação em serina 307 da proteína IRS1. Dessa forma, nossos resultados sugerem que a ativação da via da mTOR é central para determinar a sensibilidade de tumores à dieta suplementada com leucina, podendo modular o desenvolvimento tumoral naquelas células que já possuem a via IRS1/AKT/mTOR constitutivamente ativada. O mecanismo pelo qual a leucina pode retardar o desenvolvimento tumoral em células que possuem a via da mTOR hiperativada parece estar relacionado com o eixo de regulação negativa p70S6K-PI3K, com consequente redução da fosforilação de AKT e liberação das vias apoptóticas nos tecidos tumorais / Abstract: mTOR is a key regulatory protein in various cellular processes including proliferation, cell growth and survival. Growth factors, oxygen, energy status and amino acids are all essential to these processes. New findings in the last few decades have shown that the mTOR pathway is activated in many cellular processes, including tumorigenesis and angiogenesis. The branched chain amino acid leucine has the greatest potential to activate the mTOR pathway. Due to its ability to promote protein synthesis and muscle mass gain, use of leucine is frequently utilized in patients with cancer. However, the effect of leucine on tumor growth is not clear. The aim of this study is therefore to investigate the effect of diet-supplemented leucine on the modulation of tumor growth in several tumor cell lines that differ in the constitutive activation status of the insulin receptor substrate 1 (IRS1)/AKT/mTOR pathway. Both in vitro and in vivo experiments demonstrated different cell proliferation responses when cells were exposed to high doses of leucine. Tumor cell lines PC-3 and MCF-7, which have a constitutively activated mTOR signaling, displayed reduced cell proliferation and G1 phase cell cycle arrest when supplemented with high doses of leucine in vitro. Likewise, leucine-supplemented PC-3 cell tumor xenografts displayed reduced proliferation and increased cell death. We also observed increased activity of mTOR and its downstream substrate p70S6K in all cell lines supplemented with leucine. Increased mTOR activity was accompanied by a reduction in AKT serine 473 (ser473) phosphorylation in cell lines with a hyperactivated PI3K pathway (PC-3 and MCF-7). This most likely occurred because leucine supplementation further increased mTOR and p70S6K activity, triggering the inhibitory p70S6K/IRS1 axis. In fact, we found increased IRS1 ser307 phosphorylation in hyperactivated cell lines (PC-3 and MCF-7) supplemented with high doses of leucine. Therefore, our results suggest that mTOR pathway activation is central to determining the sensitivity of tumors to leucine supplementation. Furthermore, this could affect the response to leucine-supplemented therapies of those tumors in which the PI3K pathway is constitutively activated. The mechanism for this appears to be related to the negative p70S6K/IRS1 regulation axis, with consequent reduction of AKT phosphorylation and the release of apoptotic pathways in tumor tissues / Doutorado / Fisiopatologia Médica / Doutora em Ciências

L’Hypertension Hyperkaliémique Familiale (HHF) est une forme rare d’hypertension associée à une hyperkaliémie et une acidose métabolique hyperchlorémique, très sensible aux diurétiques thiazidiques. Les premières analyses génétiques ont permis d’identifier deux gènes responsables, WNK1 et WNK4, mais qui n’expliquaient que 8 % de notre cohorte. L’objectif de ma thèse était de rechercher de nouveaux gènes ou variants à l’origine de l’HHF. Notre stratégie initiale a été de combiner une analyse de liaison à un séquençage d’exome entier sur trois grandes familles atteintes. Nous avons ainsi identifié un nouveau gène responsable de la maladie codant pour un acteur jusque là insoupçonné, KLHL3 (Kelch-like 3), responsable de 39% des cas de notre cohorte. La majorité des mutations sont présentes à l’état hétérozygote entrainant un phénotype modéré, alors que les rares patients porteurs d’une mutation homozygote, tous issus de familles consanguines, présentent un phénotype plus marqué. Le spectre des mutations a montré l’importance des structures en boucles de cette protéine qui joue un rôle d’adaptateur de substrat dans un complexe d’ubiquitination (publié dans Nature Genetics, 2012). La découverte d’un type unique de mutations sur le gène CUL-3 par une équipe concurrente a été confirmée dans notre cohorte, entrainant un phénotype plus précoce et plus sévère. Ces mutations ont mis en lumière l’importance de ces deux protéines dans la constitution du complexe E3 ubiquitine-ligase et la dégradation des WNKs dans le néphron, par le protéasome après ubiquitination. Nous avons aussi identifié des mutations faux-sens dans le domaine acide de WNK1 très conservé chez des patients ayant un phénotype HHF mais sans hypertension artérielle. Ce motif, similaire à celui porteur de mutations sur WNK4 est responsable de la liaison à l’adaptateur de substrat KLHL3. Les sujets atteints présentent un âge plus précoce d’apparition de la maladie avec des valeurs de pression artérielle normales. La comparaison phénotypique avec les cas porteurs d’une mutation WNK4 et d’une délétion de l’intron 1 de WNK1 a montré des différences de pression artérielle significatives. La transfection d’ARNc mutés dans les œufs de Xénope, effectuées en collaboration, a permis de démontrer que ces nouvelles mutations faux-sens de WNK1 entrainent une accumulation de son isoforme rénale KS-WNK1 (soumis à J Am Soc Nephrol). L’ensemble de ces résultats ouvre une nouvelle voie de compréhension moléculaire de la régulation du transport des ions sodium, potassium et chlore au niveau du rein et par conséquence de la pression artérielle. / Familial Hyperkalemia Hypertension (FHHt), also known as Gordon syndrome is a rare form of hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis, very sensitive to thiazide diuretics. In 2001, the first genetic analysis identified two genes, coding for two serine/threonine kinases WNK1 and WNK4, which explained only 8% of our cohort. The aim of my thesis was to search new genes or variants responsible for FHHt. We decided to combine a linkage analysis and a whole exome sequencing in three affected families. We identified a new gene responsible for the disease coding for an unsuspected actor KLHL3 (Kelch-like 3), responsible for 39% of our cohort. The majority of the mutations are present at a heterozygous state leading to a moderate phenotype, whereas patients with homozygous mutation, all from consanguineous families, displayed a stronger phenotype. The spectrum of mutations showed the importance of the loop structures of this protein playing an adaptor role in an ubiquitination complex (published in Nature Genetics, 2012). The discovery of a particular type of mutations in CUL-3 by another team was confirmed in our cohort, leading to an earlier and more severe phenotype. These changes have highlighted the importance of these two proteins in the formation of the E3 ubiquitin-ligase-complex and in the WNKs degradation in the nephron by the proteasome after ubiquitination. We have also identified missense mutations in the acidic motif of WNK1, highly conserved in patients with FHHt without hypertension. This pattern is similar to the WNK4 mutations and is responsible for the binding of the substrate adaptor KLHL3. Affected individuals have an earlier age of onset with normal blood pressure values for most of them. Phenotypic comparison with cases carrying WNK4 mutations and deletion of the intron 1 of WNK1 showed significant differences in blood pressure values. Transfection of mutated cRNA in Xenopus laevis oocyte demonstrated that these new WNK1 missense mutations result in the accumulation of the renal isoform KS-WNK1 (submitted to J Am Soc Nephrol). Taken together, these results open a new pathway for understanding the molecular regulation of ion transport and WNK kinases in the kidney and consequently the regulation of blood pressure.

Pim (Provirus Integration site for Moloney murine leukemia virus) kinases are a family of three serine/threonine kinases involved in cell cycle, survival and metabolism. These kinases were first identified in malignant cells and are most often associated with their role in cancer. Their role in immunity and lymphocytes is less well known. To date, it has been shown that Pim 1 and/or Pim 2 are important for T lymphocyte survival and activation when the Akt signaling pathway is inhibited by rapamycin. In addition, our laboratory has shown that Pim 2 is critical for BLyS-mediated naive B lymphocyte survival in the presence of rapamycin. This thesis extends the role(s) for Pim 1 and/or 2 to include functions during B cell activation and the generation of immune responses. We found that during in vitro activation of purified resting splenic B cells from wild type mice with a variety of activators that use multiple signaling pathways, including the BCR, TLR and CD40 receptors, both Pim 1 and 2 kinases were induced by 48 hours post-activation, suggesting that they could play a role in B cell activation and differentiation to antibody secreting or memory B cells. Immunization of Pim 1-/-2-/- knockout mice with T cell dependent antigens showed impairment in antibody and antibody secreting cell generation as well as lack of germinal center formation clearly demonstrating an involvement of Pim 1 and/or 2 in the immune response. FACS examination of B cell populations from naive Pim 1-/-2-/- knockout mice revealed normal levels of splenic marginal zone and follicular B cells and T cells, however, decreased numbers of all peritoneal B cell populations and decreased B cells in Peyer's Patches was seen. An examination of serum antibody found in naive Pim 1-/-2-/- knockout mice showed decreased levels of natural antibody, which is likely due to loss of the peritoneal B1 cells but does not explain the significantly decreased TD immune response. To determine whether the defect was B cell intrinsic or a more complex interaction between B and T cells, we determined whether Pim 1-/-2-/- mice would respond to T cell independent, TI-1 and TI-2, antigens. Antibody production and antibody secreting cell formation were also significantly decreased in these mice supporting our notion of a B cell intrinsic defect. To further examine the B cell response problem, we attempted to establish chimeric mice using either bone marrow derived cells or fetal liver cells from WT or Pim 1-/-2-/- donors so that the B cells were derived from Pim 1-/-2-/- mice and the T cells would be WT. Unfortunately, we were not able to consistently engraft and develop mature Pim 1-/-2-/- B cells, which indicate that there is a stem cell defect in these knockout mice that requires further investigation. Because one of the major failures in activated Pim 1-/-2-/- B cells is the generation of antibody secreting cells, an analysis of the expression of transcription factors IRF-4 and BLIMP-1, known to play a role in this process was carried out. Although IRF-4 induction was not affected by the loss of Pim 1 and 2, the number of cells able to increase BLIMP-1 expression was significantly decreased, revealing a partial block in the generation of ASCs. Taken together the data presented in this thesis reveals a new and critical role for Pim 1 and 2 kinases in the humoral immune response.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.