A subfamília p21 RAS de pequenas GTPases, incluindo KRAS, HRAS e NRAS, participa de muitas redes de sinalização, incluindo proliferação celular, organização do citoesqueleto e apoptose, e é o alvo mais freqüente de mutações ativadoras em câncer. Mutações germinativas em KRAS e HRAS causam graves anormalidades desenvolvimentais levando às síndromes de Noonan, cárdio-facial-cutânea e Costello, porem mutações ativadoras germinativas em NRAS não foram descritas até hoje. A síndrome autoimune linfoproliferativa (ALPS) é o mais comum defeito genético de apoptose linfocitária, cursando com autoimunidade e acúmulo excessivo de linfócitos, particularmente do tipo T + CD4- CD8-. As mutações causadoras de ALPS descritas até hoje afetam a apoptose mediada por Fas, uma das vias extrínsecas de apoptose. Nós demonstramos aqui que os principais achados clínicos de ALPS, bem como uma predisposição para tumores hematológicos, podem ser causados por uma mutação heterozigota ativadora G13D no oncogene NRAS, sem causar prejuízo na apoptose mediada por Fas. O aumento na quantidade intracelular de NRAS ativo, ligado a GTP, induziu a um aumento da sinalização na via RAF/MEK/ERK, o que suprimiu a expressão da proteína pró-apoptótica BIM, e atenuou a apoptose intrínseca mitocondrial. Desta forma, uma mutação germinativa ativadora em NRAS causou um fenótipo clinico diferente do visto em pacientes com mutações em outros membros da família p21 RAS, cursando com um defeito imunológico seletivo, sem distúrbios generalizados do desenvolvimento / The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4-, CD8- ab T cells. Mutations in ALPS typically affect Fas-mediated apoptosis, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair Fas-mediated apoptosis. The increase in active, GTP-bound NRAS augmented RAF/MEK/ERK signaling which markedly decreased the pro-apoptotic protein BIM and attenuated intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects

A infecção persistente por HPV-16 está fortemente associada ao risco de desenvolvimento de neoplasias do colo do útero, vagina, vulva, pênis, canal anal e orofaringe. O estudo detalhado da variabilidade nucleotídica intra-típica de HPV-16 resultou em importantes achados no que concerne à filogenia e evolução viral, e à história natural das infecções. Variantes Asiático-Americanas (AA) e E-350G de HPV-16 foram associadas com maior risco de persistência da infecção viral e desenvolvimento de câncer de colo de útero quando comparadas à variante Européia protótipo (E-P ou E-350T), embora esta ainda apresente alto risco quando comparada aos outros tipos virais. Mais recentemente, diferenças funcionais entre as proteínas E6/E7 das distintas variantes moleculares de HPV- 16 estão sendo descritas, a fim de explicar as diferenças nas associações epidemiológicas observadas. Dados do nosso grupo apontaram para a transcrição aumentada do gene MEK2 especificamente em queratinócitos humanos primários (PHKs) transduzidos com E6/E7 da variante E-350G. Pelo exposto, objetivou-se: (1) Analisar os níveis de ativação de proteínas efetoras das vias de transdução de sinal mediadas por MAPK e PI3K/AKT em queratinócitos imortalizados por E6/E7 de três variantes moleculares de HPV-16 (AA, E-P, E-350G); (2) Analisar os efeitos das proteínas E6/E7 dessas variantes sob as vias de MAPK quanto à indução de fatores de transcrição; (3) Analisar o potencial transformante de PHKs imortalizados pelas diferentes variantes, e em cooperação com a proteína celular c-MYC; (4) Analisar o potencial de migração e invasão em PHKs imortalizados pelas diferentes variantes de HPV-16, e em cooperação com a proteína celular c-MYC. Neste estudo observou-se que a variante AA de HPV-16 induziu a maior ativação das vias de sinalização estudadas (MAPK, e PI3K/AKT). Ademais, PHKs imortalizados por esta variante apresentaram maior capacidade de migração, de invasão através de uma matriz de colágeno, além de maior potencial transformante. Adicionalmente, as células imortalizadas pela variante AA apresentaram maior expressão da proteína mesenquimal vimentina e diminuição dos níveis da proteína epitelial E-caderina, sugerindo ativação parcial de Transição Epitélio Mesênquima (EMT) nestes queratinócitos. Ademais, quando o oncogene c-MYC foi co-transduzido nas diferentes linhagens infectadas por E6/E7 de HPV-16, foi observado que em PHKs imortalizados pela variante AA também houve maior ativação da via de MAPK-ERK, maior migração, e um potencial transformante semelhante, em relação às células co-transduzidas pela variante E-350G e c-MYC. Em conjunto, estes dados sugerem que a variante AA de HPV-16 possui vantagem seletiva sob as outras variantes em promover transformação celular, migração e invasão, e isto poderia explicar, ao menos em parte, a maior prevalência desta variante no câncer cervical. Os resultados gerados neste estudo são de extrema relevância para avaliar o impacto da variabilidade intra-típica de HPV-16 sobre o potencial oncogênico observado em estudos epidemiológicos / Persistent infection with HPV-16 is strongly associated with risk of developing neoplasia in the uterine cervix, vagina, vulva, penis, anal canal and oropharynx. The detailed study of HPV-16 intra-typical nucleotide variability resulted in important findings regarding phylogeny and viral evolution, and the natural history of infections. Asian-American (AA) and E-350G variants of HPV-16 were associated with increased risk of persistent viral infection and development of cervical cancer compared to the European prototype (E-P or E-350T), although this variant still presents higher risk when compared to other viral types. More recently, functional differences between the E6/E7 proteins of distinct molecular variants of HPV-16 are being described, in order to explain the differences in the epidemiological associations observed. Data from our group pointed to increased transcription of the MEK2 gene specifically in primary human keratinocytes (PHKs) transducing E6/E7 of the E-350G variant. Consequently, the aims of this study were: 1) To examine the activation levels of effector proteins of the signal transduction pathways mediated by MAPK and PI3K/AKT in PHKs immortalized by E6/E7 of three different molecular variants of HPV-16 (AA, E-P, E-350G); (2) To analyze the effects of E6/E7 of different molecular variants of HPV-16 upon MAPK pathways concerning the induction of transcription factors; (3) To analyze the transforming potential of PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC; (4) To analyze the potential of migration and invasion in PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC. In this study we observed that the AA variant of HPV-16 induced higher activation of both signaling pathways studied (MAPK, and PI3K/AKT). Furthermore, this variant presented increased migration capacity, higher invasion through a collagen matrix, and greater transforming potential. Moreover, cells immortalized by the AA variant showed higher expression of the mesenchymal protein vimentin and a decrease of the epithelial protein E-cadherin, suggesting partial activation of Epithelial Mesenchymal Transition (EMT). In addition, when the c-MYC oncogene was co-transduced in the different cells lines infected with HPV-16 E6/E7, we observed that in PHKs immortalized by the AA variant there was also an enhanced activation of the MAPK-ERK pathway, a higher ability to migrate, and similar transformation potential in comparison with cells co-transduced with the E-350G variant and c-MYC. Taken together, this data suggest that the AA molecular variant of the HPV-16 has a selective advantage over the other variants to promote cell transformation, migration and invasion, and this could partly explain the higher prevalence of this variant in cervical cancer. The results generated in this study are very important to assess the impact of intra-typical variability of HPV-16 on the oncogenic potential observed in epidemiological studies

L'action de la dopamine dans les cellules est transmise par sa fixation sur des récepteurs qui appartiennent à deux classes, D1 et D2. Quatre sous-types du récepteur D1 (D1A, D1B/D5, D1C et D1D) ont été clonés jusqu'à présent chez les vertébrés. L'analyse évolutive montre que les sous-types D1A et D1B sont les plus conservés alors que les sous-types D1C et D1D sont absents chez les mammifères. Malgré cette diversité, les fonctions de la dopamine dans l'organisme ne peuvent pas être rapportées à l'action d'un sous-type précis. C'est pourquoi au cours de ce travail nous avons identifié des caractères fonctionnels capables de distinguer chacun des sous-types et de comprendre pour quelle raison ils ont été conservés chez les vertébrés. La désensibilisation est un des paramètres fonctionnels les plus important. Le récepteur D1A se caractérise par une baisse d'activité forte et biphasique, le récepteur D1B par un profil proche avec une amplitude plus faible conséquence de son activité constitutive. Enfin, le récepteur D1C ne semble pas être capable de se désensibiliser. La construction de chimères entre chacun des sous-types du récepteur D1 et la protéine GFP ont permis la visualisation des récepteurs au cours de la désensibilisation. Elles montrent que l'internalisation ne semble pas, pour les récepteurs D1 de la dopamine, intervenir dans le processus de désensibilisation fonctionnelle. L'activation simultanée des récepteurs A1 de l'adénosine bloque l'activité des récepteurs D1. L'analyse des voies de signalisation MAPK a montré que l'activation de la voie ERK était rapide et forte, et différente selon les sous-types. La voie p38 n'est que faiblement activée et la voie JNK semble ne pas l'être du tout. Il semble donc que les mécanismes d'activation et de régulation des voies de signalisation différencient les sous-types du récepteur D1 chez les vertébrés. Ces paramètres participent de façon majeure à la transmission régulée des fonctions de la dopamine dans l'organisme.

<p>Transforming growth factor-β (TGF-β) is a member of a large superfamily of cytokines which participate in many different types of cellular processes, such as growth inhibition, cell migration, differentiation, cell adhesion, wound healing and immunosuppression. Alterations of TGF-β superfamily signalling results in several different disorders, including bone disease, vascular disease and cancer. The TGF-β signalling pathways involve several different proteins, such as the Smad proteins, which upon receptor activation are translocated to the nucleus, where they affect transcriptional responses. </p><p>The actin cytoskeleton is an organised network of filaments with a highly dynamic structure, which is under a continuous reconstruction to control the morphology, survival, growth and motility of eukaryotic cells. The members of the family of small GTP-binding proteins have been shown to be important regulators of the actin cytoskeleton.</p><p>TGF-β was found to induce short term as well as long term actin reorganisation in prostate cancer cells. The short term response included membrane ruffling, and required signalling by the small GTPases Cdc42 and Rho as well as, the involvement of the mitogen-activated protein kinases p38 (p38 MAPK). The long term response included formation of stress fibers and required a cooperation between Smad and Rho GTPase signalling pathways involving the Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). </p><p>The TGF-β-induced activation of Cdc42 was, furthermore, shown to require the inhibitory Smad7 and p38 MAP kinase, via a PI3K-dependent pathway. Mixed lineage kinase 3 (MLK3), a mediator downstream of Cdc42, was necessary for the Cdc42-dependent actin filament reorganisation.</p><p>Apoptosis is an important and carefully regulated process in human development and disease, which allows the multicellular organisms to remove cells that are in excess or potentially dangerous. TGF-β family members can induce apoptosis in many different cell types, in the presence or absence of other growth factors. Smad7 had previously been shown to be necessary for TGF-β-induced apoptosis of epithelial cells. We could show that Smad7 is required for TGF-β-induced activation of the TGF-β activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK pathway, which subsequently leads to apoptosis in prostate cancer cells.</p><p>Members of the lymphoid enhancer factor-1/T-cell factor (LEF1/TCF) family of transcription factors have, together with β-catenin, been shown to be nuclear effectors in the Wnt-signalling pathway. We investigated a possible cross-talk between the TGF-β and Wnt signalling pathways. We found that TGF-β, in a Smad7-dependent manner induced a nuclear accumulation of β-catenin and enhanced the transcriptional activity of β-catenin and the induction of the downstream target gene <i>c-myc</i>. Since β-catenin and c-Myc has been shown to promote apoptosis, our results suggests the possibility that β-catenin contributes to TGF-β-induced apoptosis</p>

<p>Diabetes is a major risk factor for ischemic stroke and is associated with increased mortality. Additionally, hyperglycemia, a common complication in acute stroke, is associated with poor outcome.</p><p>In order to identify the correlation between blood glucose and early mortality, multiple logistic regression analyses were used and odds ratios calculated in a retrospective study of 447 stroke patients. Eighty-one patients (18%) had diabetes. The odds ratios for 30-day case-fatality and blood glucose were 1.9 and 1.6 in diabetic and non-diabetic patients respectively. Optimal blood glucose concentrations in respective group were 10.3 and 6.3 mmol/L, as determined by receiver operator characteristic (ROC) curves.</p><p>Cerebral ischemia triggers different signaling pathways including mitogen-activated protein kinases (MAPK) which regulate fundamental cell functions. In an experimental rat model of combined hyperglycemia and transient middle cerebral artery occlusion (MCAO), the activation pattern of one such MAPK, extracellular signal-regulated kinase (ERK) was studied along with infarct volumes and neurological function. Hyperglycemia resulted in markedly increased ERK activation and approximately three-fold increase of infarcts compared with controls. </p><p>Based on the increased ERK activation, further experiments were conducted to limit the hyperglycemic-ischemic damage by interfering with ERK and supposedly related mechanisms. Consequently, rats were given U0126 (inhibiting ERK activation), PBN (anti-oxidative), PP2 (inhibiting src-family kinases), or vehicle. PBN reduced infarcts and improved neurological function compared with controls while no statistically significant effects were observed for U0126 or PP2. However, when the dose was doubled, U0126 significantly reduced infarcts and improved neurological function after 1 day in hyperglycemic rats. Post-ischemic ERK activation was completely inhibited by U0126 as demonstrated with Western immunoblotting. The findings suggest that ERK is an important mediator of hyperglycemic-ischemic brain injury and possible target for future interventions.</p>

Transforming growth factor-β (TGF-β) is a member of a large superfamily of cytokines which participate in many different types of cellular processes, such as growth inhibition, cell migration, differentiation, cell adhesion, wound healing and immunosuppression. Alterations of TGF-β superfamily signalling results in several different disorders, including bone disease, vascular disease and cancer. The TGF-β signalling pathways involve several different proteins, such as the Smad proteins, which upon receptor activation are translocated to the nucleus, where they affect transcriptional responses. The actin cytoskeleton is an organised network of filaments with a highly dynamic structure, which is under a continuous reconstruction to control the morphology, survival, growth and motility of eukaryotic cells. The members of the family of small GTP-binding proteins have been shown to be important regulators of the actin cytoskeleton. TGF-β was found to induce short term as well as long term actin reorganisation in prostate cancer cells. The short term response included membrane ruffling, and required signalling by the small GTPases Cdc42 and Rho as well as, the involvement of the mitogen-activated protein kinases p38 (p38 MAPK). The long term response included formation of stress fibers and required a cooperation between Smad and Rho GTPase signalling pathways involving the Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). The TGF-β-induced activation of Cdc42 was, furthermore, shown to require the inhibitory Smad7 and p38 MAP kinase, via a PI3K-dependent pathway. Mixed lineage kinase 3 (MLK3), a mediator downstream of Cdc42, was necessary for the Cdc42-dependent actin filament reorganisation. Apoptosis is an important and carefully regulated process in human development and disease, which allows the multicellular organisms to remove cells that are in excess or potentially dangerous. TGF-β family members can induce apoptosis in many different cell types, in the presence or absence of other growth factors. Smad7 had previously been shown to be necessary for TGF-β-induced apoptosis of epithelial cells. We could show that Smad7 is required for TGF-β-induced activation of the TGF-β activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK pathway, which subsequently leads to apoptosis in prostate cancer cells. Members of the lymphoid enhancer factor-1/T-cell factor (LEF1/TCF) family of transcription factors have, together with β-catenin, been shown to be nuclear effectors in the Wnt-signalling pathway. We investigated a possible cross-talk between the TGF-β and Wnt signalling pathways. We found that TGF-β, in a Smad7-dependent manner induced a nuclear accumulation of β-catenin and enhanced the transcriptional activity of β-catenin and the induction of the downstream target gene c-myc. Since β-catenin and c-Myc has been shown to promote apoptosis, our results suggests the possibility that β-catenin contributes to TGF-β-induced apoptosis

Diabetes is a major risk factor for ischemic stroke and is associated with increased mortality. Additionally, hyperglycemia, a common complication in acute stroke, is associated with poor outcome. In order to identify the correlation between blood glucose and early mortality, multiple logistic regression analyses were used and odds ratios calculated in a retrospective study of 447 stroke patients. Eighty-one patients (18%) had diabetes. The odds ratios for 30-day case-fatality and blood glucose were 1.9 and 1.6 in diabetic and non-diabetic patients respectively. Optimal blood glucose concentrations in respective group were 10.3 and 6.3 mmol/L, as determined by receiver operator characteristic (ROC) curves. Cerebral ischemia triggers different signaling pathways including mitogen-activated protein kinases (MAPK) which regulate fundamental cell functions. In an experimental rat model of combined hyperglycemia and transient middle cerebral artery occlusion (MCAO), the activation pattern of one such MAPK, extracellular signal-regulated kinase (ERK) was studied along with infarct volumes and neurological function. Hyperglycemia resulted in markedly increased ERK activation and approximately three-fold increase of infarcts compared with controls. Based on the increased ERK activation, further experiments were conducted to limit the hyperglycemic-ischemic damage by interfering with ERK and supposedly related mechanisms. Consequently, rats were given U0126 (inhibiting ERK activation), PBN (anti-oxidative), PP2 (inhibiting src-family kinases), or vehicle. PBN reduced infarcts and improved neurological function compared with controls while no statistically significant effects were observed for U0126 or PP2. However, when the dose was doubled, U0126 significantly reduced infarcts and improved neurological function after 1 day in hyperglycemic rats. Post-ischemic ERK activation was completely inhibited by U0126 as demonstrated with Western immunoblotting. The findings suggest that ERK is an important mediator of hyperglycemic-ischemic brain injury and possible target for future interventions.

An injured vascular system has a substantial impact on an individuals overall health, and an understanding of the mechanisms that underlie blood vessel pathophysiology is required for the development of rational and effective treatment strategies. The phenotypic modulation of smooth muscle cells (SMC) during vascular injury, characterized by altered adhesion, migration and synthetic behavior, plays an important role in the eventual outcome. Specifically, the ability of SMCs to adhere to and remodel their extracellular environment via regulation of the syndecan class of cell adhesion molecules dictates the response of the vascular wall to local injury. The effect of in vitro syndecan-4 regulation on SMC adhesion was investigated through the use of a glass microsphere centrifugation assay, and an antisense-mediated reduction in gene expression was found to correlate with decreased adhesive strength. Regulation of syndecan-1, syndecan-2, and syndecan-4 gene expression was observed experimentally by mechanical strain of SMCs. Using real-time polymerase chain reaction (PCR), the kinetics of both static and cyclic mechanical strain were found to modify the gene expression in a time and strain magnitude-dependent manner unique to each syndecan. In particular, the responses of syndecan-4 were acute, but transient, while the evolution of syndecan-1 and syndecan-2 regulation was delayed by comparison. Mechanical strain also modulated syndecan-4 protein expression and ectodomain shedding, as measured by Western immunoblotting, and this effect was found, through selective inhibition, to be at least in part dependent on mitogen-activated protein (MAP) kinase signaling. In particular, intact extracellular signal-regulated MAP kinase (ERK) 1/2 and c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK/SAPK) signaling pathways were found to be required for the observed strain-induced shedding. These findings offer a better understanding of syndecan function in response to mechanical strain and suggest potential new mechanisms by which physical forces may modulate vascular SMC behavior and regulation during normal physiology, pathologic conditions, and engineered arterial substitute development.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Non-Latin script record Includes bibliographical references (leaves 112-128). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 199-250.