Ets-1 est un facteur de transcription qui régule des gènes impliqués dans divers processus physiologiques et pathologiques. Il n’agit pas seul au niveau de ses promoteurs cibles mais en coopération avec une variété de partenaires protéiques. L’identification de nouvelles protéines interagissant avec Ets-1 devrait nous permettre de mieux appréhender certaines de leurs fonctions intrinsèques. Dans ce but, nous avons mis au point une stratégie de purification par affinité des partenaires protéiques d’Ets-1. Ceci nous a permis d’identifier et de confirmer comme partenaires d’interaction d’Ets-1, les trois protéines du complexe DNA-PK : Ku70, Ku86 et DNA-PKcs. Nous avons ensuite déterminé les conséquences fonctionnelles des interactions. Ces résultats ont permis de définir une nouvelle régulation de son activité qui pourrait fournir de nouveaux indices pour comprendre les diverses fonctions d’Ets-1.Dans une autre étude, nous avons démontré que l’isoforme majoritaire Ets-1 p51 est un nouveau substrat de la caspase-3, protéase effectrice de la machinerie apoptotique. En effet, Ets-1 p51, mais pas le variant d’épissage Ets-1 p42, est clivé in vitro et dans des cellules apoptotiques par la caspase-3. Ce clivage génère trois fragments C-terminaux : Cp20, Cp17 et Cp14 et un fragment N-terminal Np36. le fragment Cp17, le principal produit de clivage, est capable de bloquer l’activation des promoteurs cibles induites par Ets-1 p51, démontrant ainsi ses propriétés de dominant négatif naturel. Ces données suggèrent un nouveau mécanisme de régulation d'Ets-1 via son clivage par la caspase-3 en générant un fragment dominant négatif qui peut jouer un rôle actif durant l'apoptose / Ets-1 is a transcription factor that regulates genes involved in various physiological and pathological processes. Ets-1 proteins do not act alone on their target promoters but with a wide range of protein partners. Identifying novel proteins that interact with Ets-1 should permit a better understanding of the intrinsic functions of the Ets-1 proteins. For this purpose, we develop an affinity purification strategy of Ets-1 interaction partners using streptavidin pull-down assay. We thereby identified new potentials interaction partners consisting for a heterotrimeric complex of DNA-PK, made up of Ku70, Ku86 and DNA-PKcs. Then, we characterized the functional consequences of the interactions. These results have permit to identify a new regulation of its activity that could provide new clues to understand the diverse functions of Ets-1.In another study, we demonstrated that the majority isoform Ets-1 p51 is a novel cleavage substrate of caspase-3, an effector protease of apoptosis. Indeed, Ets-1 p51, but not the spliced variant Ets-1 p42, is processed in vitro and in cells undergoing apoptosis by caspase-3. These cleavages lead to the generation of three C-terminal fragments Cp20, Cp17 and Cp14 and a N-terminal fragment, Np36. The Cp17 fragment, the major cleavage product generated during apoptosis, inhibits Ets-1 p51-mediated transactivation of target genes, acting thus as a natural dominant-negative of the full-length Ets-1 p51 protein. These data suggest a novel mechanism of Ets-1 p51 regulation through its caspase-mediated cleavages and generation of a dominant-negative fragment, which may play active role during apoptosis

Diss. (sammanfattning) Umeå : Univ., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 116-138).

Orientador: Sara T. O. Saad / Deissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T23:58:25Z (GMT). No. of bitstreams: 1 Souza_DeniseSilviade_M.pdf: 5043387 bytes, checksum: 45f6a641dda3292cdb3759ed8468aae3 (MD5) Previous issue date: 2005 / Resumo: Para que a terapia gênica seja aplicada à terapêutica clínica, é necessário o desenvolvimento de um sistema que permita a eficácia biológica e segurança no caso de efeitos tóxicos. O gene da eritropoetina (EPO) pode ser utilizado para o tratamento de anemias crônicas, porém estudos in vivo têm demonstrado que efeitos tóxicos, como trombose e policitemia fatal, são freqüentes devido ao descontrole da expressão da EPO e alta elevação do hematócrito (Hct). O objetivo deste estudo foi buscar um sistema capaz de induzir a apoptose de células transformadas com o gene da EPO, baseado na ativação artificial da caspase9, através de indutores químicos de dimerização (CIO), comparando este sistema ao do Tet-Off. Duas construções foram utilizadas: a primeira contendo o cDNA da EPO murina (pBS-mEpoD) sob o controle do sistema Tet-off (ptetTA) e o segundo contendo o cDNA da caspase9 ligado a duas moléculas de FKBP em tandem (iCasp9). As moléculas de FKBP contêm uma mutação F36V que permite a ligação específica do indutor químico de dimerização (CIO) AP20187, gentilmente cedido pela ARIAD Pharmaceutics (Cambridge, MA). Estudos in vitro mostraram que, em células murinas C2C12 transfectadas estavelmente, o vetor icasp9 apresentou expressão adequada e 90% das células entraram em apoptose após a administração do AP 20187. Os ensaios in vivo foram realizados, inicialmente, com 3 grupos de camundongos (11/grupo), que receberam '5mu¿p pBS-mEpoD , '0,5mu¿g ptet-tTA e diferentes concentrações de iCasp9: grupo 1: zero, grupo 2= '6mu¿g e grupo 3= '10mu¿ g. As injeções intramusculares foram seguidas de eletroporação (8 pulsos, 30mseg , 200V/cm, 1Hz). Após cinco semanas, os animais apresentaram aumento no Hct de 33% (Student T test p=0.0013), 36.8% (p=0.0004) e 12.8% (p=0.0374) nos grupos 1,2 e 3 respectivamente, sugerindo que a administração de '6mu¿g de icasp9 não altera a expressão do vetor pBSmEpoD, enquanto que '10mu¿g de iCasp9 reduz sua eficiência. Novo experimento foi realizado, onde 49 animais participaram do estudo, sendo que treze destes receberam as concentrações administradas ao grupo 1, acima descrito, e 36 receberam as concentrações do grupo 2. Através deste experimento, foi possível verificar a eficiência do sistema na elevação e sustentação do hematócrito, assim como comparar os sistemas iCasp9/AP20187 e Tet-Off, no controle da expressão da eritropoetina. ... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: In order to apply gene therapy to clinical practice, it is necessary to develop a system establishing biological efficiency and safety in case of toxic effects.The erythropoietin gene (EPO) can be used in the treatment of chronic anemia, however in vivo studies have revealed that toxic effects, such as thrombosis and fatal polycythemia, are frequent due to uncontrolled EPO expression and a high increase in the hematocrit (Hct) levels.The aim of this study was to search for a system capable of inducing apoptosis in cells transformed by the EPO gene, based on the artificial activation of caspase-9, using chemical inducers of dimerization (CIO), comparing this system to Tet-Off. Two constructions were used: the first one containing the cONA of murine EPO (pBSmEpoO) under Tet-Off system (ptet-tTA) control and the second containing the cONA of caspase-9 connected to two FKBP molecules in tandem (iCasp9). The FKBP molecules contain a F36V mutation that permits a specific connection to the chemical inducer of dimerization (CIO) AP20187, kindly provided by ARIAO Pharmaceutics (Cambridge, MA). Studies in vitro demonstrated that, in stably transfected murine cells C2C12,the icasp9 vector presented and adequate expression and 90% of the cells entered apoptosis after AP20187 administration. The in vivo assays were performed, initially, with 3 groups of mice (11/group) that received '0,5mu¿g pBS-mEpoO, '0,5mu¿g ptet-tTA and different concentrations of iCasp9: group 1: zero, group 2='6mu¿g, and group 3='10mu¿g.The intramuscular injections were followed by electroporation (8 pulses, 30mseg, 200V/cm, 1Hz). After five weeks, the animais demonstrated a 33% increase in Hct (Student T test p=0.0013), 36.8% (p=0.OO04)and 12.8% (p=0.0374) in groups 1, 2, and 3 respectively, suggesting that the administration of '6mu¿g of icasp9 does not alter the pBS-mEpoO vector expression, although '10mu¿g of iCasp9 reduces its efficiency. A new assay was performed, the study involved 49 animais, thirteen of which received the same concentrations administered in group 1, described above, and 36 received the same concentrations as group 2. ... Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

As glândulas salivares são estruturas essenciais para a manutenção da homeostase da cavidade oral pela síntese e secreção do fluido salivar. A disfunção ou perda permanente das glândulas salivares causadas por radioterapia, doenças inflamatórias ou desordens congênitas elevam principalmente o risco de infecções da mucosa oral e de estruturas dentárias, além de potencialmente prejudicar funções fisiológicas como fala, mastigação e paladar, diretamente interferindo na qualidade de vida dos indivíduos afetados. Os tratamentos atualmente disponíveis são apenas paliativos, ressaltando a necessidade de se compreender melhor os processos embriogênicos a fim de desenvolver novas estratégias terapêuticas capazes de regenerar as glândulas salivares. O princípio da formação das glândulas salivares baseia-se na coordenação de diversos processos morfogenéticos, e este trabalho foca particularmente em investigar a formação do espaço luminal do sistema de ductos, uma vez que a adequada abertura dos lumens é um processo essencial para a secreção salivar. Relata-se que a remoção das células centrais dos cordões sólidos epiteliais por morte celular apoptótica é o principal mecanismo de abertura do espaço luminal dos futuros ductos glandulares em camundongos. Porém, pouco se sabe sobre o controle temporal da apoptose durante o desenvolvimento glandular e sobre seu comportamento em glândulas salivares humanas. Neste trabalho, o perfil de expressão de diversas proteínas envolvidas na cascata apoptótica em glândulas salivares fetais humanas foi analisado de acordo com cada estágio morfogenético por imunoistoquímica (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x, Bcl-xL, caspase-3 clivada, caspases-6, -7 e -9, apaf-1, survivina e citocromo c). As análises semi-qualitativas resultaram em negatividade apenas para as proteínas Bcl-2, Bad, Bid e caspase-3 clivada em todas as fases de desenvolvimento. A expressão nuclear de Bax e Bak foi identificada em presumidos espaços luminais em estágios precoces, enquanto Bcl-xL foi o fator antiapoptótico da família Bcl-2 que exibiu expressão nuclear mais importante. Caspases-6, -7 e -9 foram positivas em todas as fases, e a ausência de caspase-3 clivada sugere caspase-7 como principal caspase efetora da apoptose em desenvolvimento de glândulas salivares humanas. Ambos os componentes do complexo apoptossomo foram positivos durante o desenvolvimento glandular, e o inibidor survivina demonstrou mais positividade nuclear em estágios mais avançados. Ao observar a expressão de reguladores apoptóticos durante o desenvolvimento glandular humano, foram realizados experimentos funcionais com culturas de tecido glandular de camundongos para avaliar o papel das caspases durante a formação desta estrutura. Inicialmente detectou-se a atividade apoptótica em glândulas salivares de camundongos albinos no centro dos cordões epiteliais primários a partir de estágios precoces de desenvolvimento através de TUNEL e caspase-3 clivada. A partir disso, foi realizada a inibição apoptótica funcional in vitro durante o mesmo período, que resultou em ductos significativamente mais amplos e em defeitos morfológicos importantes nas estruturas luminal e acinar. Este trabalho evidenciou portanto atividade apoptótica durante a formação de glândulas salivares humanas e de camundongo, expressando-se em fases mais precoces do que reportadas anteriormente. Além disso, a ausência de Bad e Bid indica que a via intrínseca está mais ativa que a extrínseca, e distintos perfis de expressão da maioria das moléculas sugere adicionais funções não-apoptóticas durante a morfogênese glandular. / Salivary glands are essential structures for the maintenance of homeostasis of the oral cavity by synthesizing and secreting saliva. Permanent dysfunction or loss of salivary glands caused by radiotherapies, inflammatory diseases or congenital disorders increase mainly the risk of infections of the oral mucosa and tooth surface, also impairing physiological functions as speech, mastication and taste, directly interfering in quality of life. Current treatments are only palliative-based, which highlights the need of having a better understanding of embryonic processes to develop therefore new therapeutic strategies able to regenerate salivary glands. The development of glandular secretory units and ductal system involves the coordination of several morphogenetic processes, and this study particularly focuses in investigating the formation of the lumenal space of the ductal system, as the proper lumen opening is an essential step for the salivary secretion. The clearance of the central cells of developing solid epithelial stalks by apoptotic cell death is the main mechanism of lumen space opening within presumptive ducts in mouse salivary glands. However little is known about its temporal regulation and its function in human salivary glands. Here we analysed the profile expression of several apoptosis-related proteins during human salivary gland development in correlation to each morphogenetic stage by immunohistochemistry (Bax, Bak, Bad, Bid, Bcl-2, Bclx, Bcl-xL, cleaved caspase-3, caspases-6, -7 e -9, apaf-1, survivin e citocromo c). Immunohistochemical results were analysed semi-qualitatively, and proteins Bcl-2, Bad, Bid and cleaved caspase-3 were considered completely negative at all stages of development. The nuclear expression of Bax and Bak were observed within the presumptive luminal spaces at early stages, while Bcl-xL was the antiapoptotic factor of Bcl-2 family that showed more prominent nuclear expression. Caspases-6, -7 and -9 were positive at all stages, and the absence of cleaved caspase-3 suggests caspase-7 as the main effector caspase during human salivary gland development. Both components of the apoptosome complex were also positive through all development, and the inhibitor of apoptosis survivin has shown more nuclear positivity at later stages. As the expression of apoptotic regulators was observed during human salivary gland development, functional experiments were then performed in mouse salivary gland cultures to determine the apoptotic activity of during the glandular formation. Initially, the apoptotic activity was detected in mouse salivary glands within the centre of primary epithelial stalks from early stages of development by TUNEL and cleaved caspase-3. Thus the in vitro apoptotic inhibition was performed at the same stages, which resulted in significant wider ducts and important morphological defects within luminal and acinar structures. This work has therefore evidenced the existence of apoptotic role in salivary gland lumen formation of both human and mouse models, having an earlier start point as reported before. Moreover, the absence of Bad and Bid indicates that the intrinsic pathway is more active than the extrinsic during human development, and the distinct subcellular expression of most molecules suggests additional non-apoptotic functions.

As glândulas salivares são estruturas essenciais para a manutenção da homeostase da cavidade oral pela síntese e secreção do fluido salivar. A disfunção ou perda permanente das glândulas salivares causadas por radioterapia, doenças inflamatórias ou desordens congênitas elevam principalmente o risco de infecções da mucosa oral e de estruturas dentárias, além de potencialmente prejudicar funções fisiológicas como fala, mastigação e paladar, diretamente interferindo na qualidade de vida dos indivíduos afetados. Os tratamentos atualmente disponíveis são apenas paliativos, ressaltando a necessidade de se compreender melhor os processos embriogênicos a fim de desenvolver novas estratégias terapêuticas capazes de regenerar as glândulas salivares. O princípio da formação das glândulas salivares baseia-se na coordenação de diversos processos morfogenéticos, e este trabalho foca particularmente em investigar a formação do espaço luminal do sistema de ductos, uma vez que a adequada abertura dos lumens é um processo essencial para a secreção salivar. Relata-se que a remoção das células centrais dos cordões sólidos epiteliais por morte celular apoptótica é o principal mecanismo de abertura do espaço luminal dos futuros ductos glandulares em camundongos. Porém, pouco se sabe sobre o controle temporal da apoptose durante o desenvolvimento glandular e sobre seu comportamento em glândulas salivares humanas. Neste trabalho, o perfil de expressão de diversas proteínas envolvidas na cascata apoptótica em glândulas salivares fetais humanas foi analisado de acordo com cada estágio morfogenético por imunoistoquímica (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x, Bcl-xL, caspase-3 clivada, caspases-6, -7 e -9, apaf-1, survivina e citocromo c). As análises semi-qualitativas resultaram em negatividade apenas para as proteínas Bcl-2, Bad, Bid e caspase-3 clivada em todas as fases de desenvolvimento. A expressão nuclear de Bax e Bak foi identificada em presumidos espaços luminais em estágios precoces, enquanto Bcl-xL foi o fator antiapoptótico da família Bcl-2 que exibiu expressão nuclear mais importante. Caspases-6, -7 e -9 foram positivas em todas as fases, e a ausência de caspase-3 clivada sugere caspase-7 como principal caspase efetora da apoptose em desenvolvimento de glândulas salivares humanas. Ambos os componentes do complexo apoptossomo foram positivos durante o desenvolvimento glandular, e o inibidor survivina demonstrou mais positividade nuclear em estágios mais avançados. Ao observar a expressão de reguladores apoptóticos durante o desenvolvimento glandular humano, foram realizados experimentos funcionais com culturas de tecido glandular de camundongos para avaliar o papel das caspases durante a formação desta estrutura. Inicialmente detectou-se a atividade apoptótica em glândulas salivares de camundongos albinos no centro dos cordões epiteliais primários a partir de estágios precoces de desenvolvimento através de TUNEL e caspase-3 clivada. A partir disso, foi realizada a inibição apoptótica funcional in vitro durante o mesmo período, que resultou em ductos significativamente mais amplos e em defeitos morfológicos importantes nas estruturas luminal e acinar. Este trabalho evidenciou portanto atividade apoptótica durante a formação de glândulas salivares humanas e de camundongo, expressando-se em fases mais precoces do que reportadas anteriormente. Além disso, a ausência de Bad e Bid indica que a via intrínseca está mais ativa que a extrínseca, e distintos perfis de expressão da maioria das moléculas sugere adicionais funções não-apoptóticas durante a morfogênese glandular. / Salivary glands are essential structures for the maintenance of homeostasis of the oral cavity by synthesizing and secreting saliva. Permanent dysfunction or loss of salivary glands caused by radiotherapies, inflammatory diseases or congenital disorders increase mainly the risk of infections of the oral mucosa and tooth surface, also impairing physiological functions as speech, mastication and taste, directly interfering in quality of life. Current treatments are only palliative-based, which highlights the need of having a better understanding of embryonic processes to develop therefore new therapeutic strategies able to regenerate salivary glands. The development of glandular secretory units and ductal system involves the coordination of several morphogenetic processes, and this study particularly focuses in investigating the formation of the lumenal space of the ductal system, as the proper lumen opening is an essential step for the salivary secretion. The clearance of the central cells of developing solid epithelial stalks by apoptotic cell death is the main mechanism of lumen space opening within presumptive ducts in mouse salivary glands. However little is known about its temporal regulation and its function in human salivary glands. Here we analysed the profile expression of several apoptosis-related proteins during human salivary gland development in correlation to each morphogenetic stage by immunohistochemistry (Bax, Bak, Bad, Bid, Bcl-2, Bclx, Bcl-xL, cleaved caspase-3, caspases-6, -7 e -9, apaf-1, survivin e citocromo c). Immunohistochemical results were analysed semi-qualitatively, and proteins Bcl-2, Bad, Bid and cleaved caspase-3 were considered completely negative at all stages of development. The nuclear expression of Bax and Bak were observed within the presumptive luminal spaces at early stages, while Bcl-xL was the antiapoptotic factor of Bcl-2 family that showed more prominent nuclear expression. Caspases-6, -7 and -9 were positive at all stages, and the absence of cleaved caspase-3 suggests caspase-7 as the main effector caspase during human salivary gland development. Both components of the apoptosome complex were also positive through all development, and the inhibitor of apoptosis survivin has shown more nuclear positivity at later stages. As the expression of apoptotic regulators was observed during human salivary gland development, functional experiments were then performed in mouse salivary gland cultures to determine the apoptotic activity of during the glandular formation. Initially, the apoptotic activity was detected in mouse salivary glands within the centre of primary epithelial stalks from early stages of development by TUNEL and cleaved caspase-3. Thus the in vitro apoptotic inhibition was performed at the same stages, which resulted in significant wider ducts and important morphological defects within luminal and acinar structures. This work has therefore evidenced the existence of apoptotic role in salivary gland lumen formation of both human and mouse models, having an earlier start point as reported before. Moreover, the absence of Bad and Bid indicates that the intrinsic pathway is more active than the extrinsic during human development, and the distinct subcellular expression of most molecules suggests additional non-apoptotic functions.

Multiple lines of experimental evidence demonstrate that chronic alcoholconsumption causes mitochondrial injury, as well as acinar cell oxidative and metabolicstress. Alcoholics are more susceptible to acute and chronic pancreatitis. Despite alcoholrelatedacinar cell injury, apoptosis, or programmed cell death, appears to be reduced inacinar cells rather than increased, as is seen in the liver.This work describes the possible mechanisms through which alcohol affects theacinar cell apoptosis pathways in the rat pancreas. Two apoptotic pathways wereinvestigated: (1) receptor-mediated apoptosis via Fas/FasL and caspase-8, and (2)mitochondrial-mediated apoptosis via Bcl-2/Bax and caspase-9. Both pathways canactivate the final apoptosis executer caspase-3. Using the Lieber-DeCarli alcohol/controldiet, rats fed alcohol for 14 weeks had a significant decrease of key mediators of theFas/Fas ligand receptor-mediated pathway, while the mitochondria-associated apoptoticpathway is inappropriately deactivated. In addition, this study describes the mRNAexpression of inflammatory cytokines, such as IL-1??, IL-6, TNF??, IL-18 and TGF??,which are reported to influence inflammation and apoptosis. The anti-inflammatoryeffects of alcohol were confirmed with decreased expression of regulatory cytokinesincluding IL-1??, IL-18, TGF?? and IL-6 in alcohol-fed rats.Alcohol appears to block apoptosis in the pancreas through multiple mechanisms.Activity of the Fas/Fas ligand receptor-mediated pathway appears to be suppressed at thelevel of caspase-8, with further inhibition by down-regulation of caspase-3. Despiteknown acinar cell stress and mitochondria injury, the mitochondria-mediated apoptoticpathway was not activated. This data suggest that alcohol consumption suppresses theremoval of mitochondria injured acinar cells, promoting apoptosis resistance, and mayincrease the susceptibility to pancreatitis. The increased susceptibility to pancreatic injurywas further investigated by using lipopolysaccharide (LPS). Alcohol exacerbates LPSinducedpancreatoxicity by enhanced pancreatic apoptosis. The attenuation of apoptosisby ethanol increased the threshold of apoptosis in response to LPS and acceleratesapoptosis. Here it is hypothesized that alcoholics are more susceptible to endotoxinmediatedacute pancreatitis and the response is more severe than in non-alcoholics.

Stroke induces multiple pathological sequelae directly affecting neuronal survival and eliciting short and long-term deficits in behavioral outcome. Most of stroke models utilized to investigate these pathological consequences are based on pure cerebral ischemia models. However, human thromboembolic stroke is characterized by a complex multifactorial response that involves the activation of the cerebral microcirculation by the occluding thrombus. Here, we have characterized a novel mouse model of tromboembolic stroke that mimics most of the clinical aspects of the human pathology. The common carotid artery thrombosis (CCAT) model produces consistent and reproducible infarcts and triggers an inflammatory response comparable to other well established models of stroke. Several of the pathological consequences of cerebral ischemia are triggered by focal inflammatory processes that occur early after the ischemic event. Cerebral inflammation is initiated by an early release of pro-inflammatory cytokines. These active cytokines promote the recruitment of inflammatory cells from the blood cerebral circulation into the brain parenchyma and subsequent release of additional amounts of inflammatory cytokines. This exacerbated cytokine response result in further irreversible neuronal and histopathological damage. Cytokines interleukyne-1 beta (IL-1 beta) and interleukyne-18 (IL-18) maturation requires the presence of active caspase-1. Activation of caspase-1 in the peripheral immune response involves the recruitment of several caspase-1 molecules into a macromolecular complex termed the inflammasome. Cerebral ischemia triggers the synthesis and activation of caspase-1. However, the cellular mechanisms associated to the activation of caspase-1 in the ischemic brain remain to be elucidated. In this study, we demonstrate that the NLRP1-inflammasome composed by capase-1, ASC (apoptosis-associated speck-like protein containing a caspase-activating recruitment domain) and NLRP1 (NLR (nucleotide binding, leucine-rich repeat) is assembled following the ischemic event. Moreover, we have characterized the cellular distribution of the inflammasome proteins in the normal and the ischemic brain. Data from this investigation suggest that six to twenty four hours following CCAT the inflammasome complex is assembled in neurons while microglia, macrophages and astrocytes form this complex at 7 days following cerebral ischemia. On the basis of these findings we next investigated whether inhibition of the inflammasome complex reduces the inflammatory response after ischemia. Neutralization of NLRP1 utilizing a specific antibody, revealed decreased activation of caspase-1 and IL-1 beta and reduced histopathological damage within the ischemic brain. Thus, the inflammasome complex is a major contributor of the inflammatory response following cerebral ischemia and inhibition of this complex may be a novel therapeutic target for reducing the pathological consequences of stroke.

The inducible heat shock protein Hsp72 inhibits apoptosis and promotes long term survival after a number of stresses but the mechanism by which this is achieved remains unclear. A role for Hsp72 in modulating apoptosis mediated through members of the TNF-receptor super family other than TNF-R1 has not been clearly established. Given the observations of high levels of Hsp72 in tumours of poor prognosis, we set out to determine whether Hsp72 could specifically modulate apoptosis induced through the death receptor pathways mediated by Fas and the TRAIL receptors. Both these pathways are of relevance in tumour surveillance. (For complete abstract open document)

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: Alzheimer's; Ependymins; Caspases; SOD. Includes bibliographical references (p. 55-59).