Rozprawy doktorskie na temat „Hsp31”

Orientador: Agnaldo Lopes da Silva Filho
Resumo: Introdução: As proteínas de choque térmico (“Heat Shock Proteins”) são produzidas em resposta ao estresse patofisiológico nas células animais e não só fazem parte de várias etapas da carcinogênese, atuando principalmente como agentes antiapoptóticos, como também estão implicadas em mecanismos de resistência à quimioterapia em vários tipos de tumores. Objetivo: O presente estudo visa comparar a expressão dos genes TRAP1, HSPB1, HSPD1, HSPA1L e HSPA1A nas amostras de CEO (no tumor primário ou na metástase) com a expressão dos mesmos em amostras de tumores ovarianos benignos e tecido ovariano normal e correlacionar a expressão gênica com o prognóstico das pacientes e com a resistência ao tratamento com platina. Métodos: Foram avaliadas amostras de 51 pacientes operadas no Hospital Vera Cruz, entre os anos de 2008 a 2011, divididas em quatro grupos: CEO primário (n = 14), CEO metastático (n = 11), cistoadenoma seroso ovariano (n = 07) e ovário normal (n = 19). Utilizou-se a técnica de qRT-PCR para determinar o perfil de expressão dos genes. Resultados: As pacientes incluídas neste estudo apresentavam idade média de 56,75 anos. Não houve diferença significativa (valor-P> 0,050) na comparação entre a expressão dos genes e os grupos estudados. Os genes HSPA1A, HSPA1L e TRAP1 foram subexpressos e se diferiram significativamente dos genes em indivíduos com ovário normal. A expressão dos genes analisados não correlacionou se com as variáveis quantitativas, como idade, menarca, e tempo ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Heat Shock Proteins are produced in response to pathophysiological stress and take part in several stages of carcinogenesis, acting primarily as anti-apoptotic agents. They are also implicated in resistance to chemotherapy in several types of tumors. Herein we correlated the expression of genes encoding these proteins and the clinical and pathological aspects of patients with ovarian cancer (OC). METHODS: 51 patients included in the study were divided into four groups: those with primary EOC (n = 14), metastatic EOC (n = 11), ovarian serous cystadenoma (n = 7), with no evidence of ovarian malignancy or control (n = 11). The 57 tumor samples obtained were submitted to RNA extraction and reverse transcription. qRT-PCR was performed to compare the expression of TRAP1, HSPB1, HSPD1, HSPA1A and HSPA1L in primary and HSP60, HSP70, HSPA1L genes did not differ among the groups (p-value> 0.050) .HSPA1A, HSPA1L and TRAP1 we underexpressed in the primary and metastatic EOC groups with HSPA1L showing the lowest expression with compare with normal ovary tissue. TRAP1 expression was higher in tumors at stage I/II than at stages III/IV. Grade II subjects showed higher HSPB1 expression. There was no correlation between HSPs expression and age, menarche, parity, period after menopause initiation and CA-125. HSPA1A gene was negatively correlated with the risk of dying of OC. There was no differences between HSP expression gene evaluated and overall and disease-free survival. In conclusion, we ... (Complete abstract click electronic access below)
Mestre

HSPC1 (Hsp90), a member of the anti-apoptotic Heat Shock Protein (HSP) family appears to play a pivotal role in the development and maintenance of several tumour cell characteristics and as a result has become a target for novel anti-cancer therapies. HSPC1 inhibitors have been tested in clinical trials on a wide variety of cancer types with moderate success. However, despite recent advantages in HSPC1 inhibitor development, the effects of these drugs are not consistent. A number of factors may play a role in determining cell sensitivity to these inhibitors. As Chronic Lymphocytic Leukaemia (CLL) is such a heterogeneous disease with great variation in baseline HSP levels and other proteins amongst the patient cohort, it would not be unreasonable to assume that HSPC1 inhibitors may have varying success as a treatment strategy for this disease. The present study examined the effects of four HSPC1 inhibitors on primary CLL cells, as well as cells from healthy control subjects, and analysed a number of HSPC1 client proteins to assess the efficacy of these inhibitors. Great variation in cellular response to these drugs was observed in both CLL and healthy control subjects. Analysis of HSPC1 client proteins in these cells including ZAP-70, Akt, NF-kB and HSPA1A, revealed that HSPC1 inhibitors do not effect client protein levels in all samples. The results suggest that these inhibitors should not be considered as a universal treatment strategy for CLL and provide a basis for further study into elucidating the mechanisms behind HSPC1 inhibitor resistance. The final aim of this work was to investigate the role of the microenvironment in CLL progression, where a co-culture system was used as an in-vitro tool. Whilst consistent data was obtained using cell lines, and showed that microenvironmental factors promoted resistance to HSPC1 inhibitors, use of primary CLL cells in this model produced inconsistent data, again highlighting the heterogeneity of the disease.

O diabetes mellitus tipo 1 é uma doença metabólica, caracterizada pela desregulação glicêmica, que ocorre devido a um ataque autoimune. A insulinoterapia é o tratamento clássico para o DM1. Contudo, alguns pacientes que apresentam essa doença não respondem de forma eficiente a este tratamento e apresentam episódios frequentes de hipoglicemia severa e despercebida (pacientes hiperlábeis). Essas complicações comprometem de forma significativa a qualidade de vida dessas pessoas. O transplante de ilhotas é uma importante alternativa para o tratamento de pacientes hiperlábeis com DM1. No entanto, essa terapia apresenta restrições como a necessidade de mais de um doador por transplante e significativa morte das ilhotas devido ao estresse provocado pelo procedimento de isolamento, além da morte promovida pelo sistema imune do paciente nos primeiros momentos pós-transplante. A autofagia é um mecanismo de reciclagem de componentes citoplasmáticos que é fundamental para a homeostase celular. Em condições de estresse, este mecanismo é ativado acima do seu nível basal, promovendo a degradação de agregados proteicos e organelas defeituosas, evitando assim, danos celulares que comprometam a viabilidade da célula. Trabalhos realizados por nosso grupo têm mostrado a citoproteção que PRL promove em células-beta, reduzindo a apoptose induzida por citocinas pró-inflamatórias. Também demonstramos o papel essencial de HSPB1 na inibição de apoptose induzida por PRL após o tratamento com citocinas. Além disso, resultados recentes de nosso laboratório mostraram um aumento nos níveis de autofagia em células-beta após sua exposição a citocinas, bem como uma restauração a níveis normais na presença de PRL. Visando um melhor entendimento do papel da PRL na modulação da autofagia em células-beta, o objetivo desse projeto foi estudar se HSPB1 também é essencial no mecanismo de regulação da autofagia induzido por PRL.Para tal, fizemos experimentos em modelos de células-beta MIN6, MIN6 silenciadas para HSPB1 (MIN6-shHSPB1) e MIN6 com sequencia short hairpin aleatória (MIN6- SsC), medindo a morte celular através de ensaios de viabilidade, e ensaios de western blot para avaliar os níveis de marcadores de autofagia e fluxo autofágico (degradação de autofagossomos), tratando as células com citocinas, prolactina e indutores ou inibidores de autofagia. Os resultados mostraram que a modulação da autofagia ocasionada pela prolactina em células-beta se dá, em parte, através de HSPB1. O tratamento com prolactina foi capaz de inibir a morte celular induzida por citocinas, mesmo na presença de cloroquina, um bloqueador de autofagia, o que nos levou a concluir que a autofagia não é uma via envolvida na citoproteção de células beta induzida por PRL. Os resultados gerados nesse estudo contribuíram para uma melhor compreensão dos eventos moleculares induzidos por PRL em células-beta, e poderão permitir a inferência de novas abordagens que melhorem a citoproteção, cultura e transplante dessas células em pacientes com diabetes tipo 1.
Type 1 diabetes mellitus is a metabolic disease characterized by glycemic dysregulation, which occurs due to an autoimmune destruction of beta-cells. Insulin therapy is the gold standard treatment for DM1. However, some DM1 patients do not respond efficiently to this treatment and suffer frequent episodes of severe hypoglycemia unawareness. Since this complication jeopardizes the quality of life of these people, Islet transplantation is a therapeutic alternative indicated to treat these patients. However, besides the lack of enough organ donors, the loss of beta cells during both the isolation as well as the infusion of islets into the recipient induce a great estresse and thus a significant cell death is one of the drawbacks of this procedure. Autophagy is a mechanism of recycling cytoplasmic components and is essential for cellular homeostasis. Under estresse conditions, this mechanism is activated above basal levels, promoting the degradation of protein aggregates and defective organelles, thus avoiding cell damage that could compromise cell viability. Studies carried out by our group have shown not only that PRL promotes cytoprotection in beta-cells, reducing pro-inflammatory cytokines-induced apoptosis, but also that HSPB1 plays an essential role in this inhibition of apoptosis mediated by PRL after treatment with cytokines. Moreover, recent results from our laboratory showed an increase in autophagy levels in beta-cells after exposure to cytokines, as well as a restauration to normal levels in the presence of PRL. In order to better understand the role of PRL in the modulation of autophagy in these cells, the aim of this project is to study whether HSPB1 is also essential in the mechanism of autophagy regulation induced by PRL. Using MIN6 beta cell models where HSPB1 was silenced (MIN6-shHSPB1) or not (MIN6-SsC), we studied cell death by viability assays. Moreover, western blot assays were performed in order to assess levels of autophagy and autophagic flux markers in the cells.Our results showed that HSPB1 in one of the mediators of PRL-induced modulation of autophagy. Nevertheless, since hormonal treatment was still able to inhibit cytokinesinduced cell death even in the presence of chloroquin, an autophagy blocker, we conclude that autophagy is not a signaling pathway involved in PRl-induced beta-cell cytoprotection. Altogether, the results shown in this study may help to increase the knowledge of the molecular events induced by PRL in beta-cells, and may allow to infer new approaches to improve cytoprotection, culture and transplantation of these cells into type 1 diabetic patients.

SPC1/furine est une protéase à sérine active dépendante du Ca[indice supérieur ++] qui est membre de la famille des convertases de mammifères et dont l'homologue bactérien est la subtilisine. Elle est impliquée dans la maturation de plusieurs précurseurs protéiques tels que le prorécepteur à l'insuline, le pro-[bêta]-NGF, le pro-BMP-1 et la pro-fibrilline, les deux derniers étant des substrats de la matrice extracellulaire. Nous avons voulu identifier de nouveaux déterminants du pro-domaine de hSPC1 impliqués dans son auto-activation ainsi que d'autres déterminants moléculaires responsables et nécessaires au largage de l'enzyme dont la forme soluble pourrait cliver des substrats matriciels. Les études ont donc été divisées en deux parties. (1) Une étude d'homologie de séquence entre les pro-domaines des convertases a permis d'identifier des résidus conservés. (2) SPC1 est une protéine transmembranaire de type I dont une forme soluble est générée suite à son largage."--Résumé abrégé par UMI.

Heat shock proteins (HSPs) are a ubiquitous family of protein chaperones, which prevent irreversible protein aggregation. HSPs help confer thermotolerance to organisms and can protect against other environmental stresses, such as oxidative stress. Expression levels of HSPs increase during exposure to these stresses. Small heat shock proteins (sHSPs) bind to denaturing protein to prevent aggregation. Since sHSPs are ATP-independent, ATP-dependent chaperones are involved in actively refolding proteins after release from sHSPs. Plants, including the model organism Arabidopsis thaliana, contain multiple HSPs, which are localized to the cytosol, chloroplasts, mitochondria, the nucleus, peroxisomes, and ER. This project focuses on the function of chloroplast-localized Hsp21, which is encoded by a single nuclear gene, making it amenable to genetic analysis. One goal is to identify the phenotypic effects of elevated temperatures and other stresses on Arabidopsis plants null for Hsp21. An Hsp21 null mutant line was identified in which a point mutation in the conserved intron base at the 3' splice site prevents the accumulation of Hsp21 after heat stress. Mutants unable to synthesize Hsp21 during stress showed a mild heat-sensitive phenotype. Additional assays to study chlorophyll levels and the effects of oxidative stress will be performed. Another goal is to identify possible in vivo substrates of Hsp21. This is being approached by introducing an affinity tag (strep-tag II encoding WSHPQFEK) to the 3' end of the Hsp21 gene. The DNA encoding Hsp21-strep has been inserted into an entry vector and will be recombined with a plant transformation plasmid, which is transformed into Agrobacterium tumefaciens. This bacterium is used to insert the tagged protein gene into plants null for Hsp21. Hsp21 and any associated proteins can then be isolated by separating cell lysates from these plants via streptactin affinity chromatography. Mass spectrometry will be used to determine the identity of the in vivo substrates of Hsp21. It is hypothesized that heat-labile proteins crucial for chloroplast function are most likely to be protected by Hsp21.

Orientador: Valéria Marçal Felix de Lima
Coorientador: Paulo César Ciarlini
Banca:Luiz Daniel de Barros
Banca:Flavia Lombardi Lopes
Banca:Suely Regina Mogami Bomfim
Banca; Rosimeri de Oliveira Vasconcelos
Resumo: A leishmaniose visceral canina (LVC) é uma doença crônica que causa imunossupressão nos animais doentes, principalmente por prejudicar a resposta imunológica celular, diminuindo a proliferação linfocitária e a capacidade fagocítica das células de defesa. Recentemente, a enzima heme oxigenase-1 (HO-1) vem ganhando destaque por estar envolvida na regulação da resposta imune celular em algumas condições patológicas, sendo uma enzima induzível por condições de estresse, como o estresse oxidativo que sabidamente ocorre na LVC. Nesse contexto, esse trabalho teve por objetivo determinar o papel da HO-1 na LVC, determinando sua concentração e expressão em cães infectados e saudáveis, correlacionando-a com o estresse oxidativo, carga parasitária e IL-10. Objetivou-se também avaliar o efeito da indução e inibição da enzima sobre a resposta linfoproliferativa de células de linfonodo de cães doentes e sobre a taxa de infecção macrofágica por promastigotas de Leishmania infantum, determinando as citocinas envolvidas. Os cães com LVC apresentaram marcante estresse oxidativo e aumento da concentração e expressão de HO-1, obtendo-se correlação positiva entre HO-1e estresse oxidativo e IL-10 de acordo com o tecido analisado. A inibição de HO-1 aumentou a taxa de proliferação celular na presença de antígeno solúvel de L. infantum, enquanto a indução de HO-1 diminuiu a taxa de proliferação antígeno-específica e aumentou a taxa de infecção macrofágica e o número de amastigotas por macrófago. Con... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Canine visceral leishmaniasis (CVL) is a chronic disease that causes immunosuppression by reducing the cellular response of infected animals, impairing the cell proliferation and the phagocytic ability of defense cells. Recently, heme oxygenase-1 (HO-1) has been highlighted for being involved in regulation of cell response in certain pathological conditions, and for being an enzyme that can be induced by stress conditions, such as oxidative stress, that is known to occur in CVL. In this context, this study aimed to determine the role of HO-1 in CVL, determining its levels and expression in infected and healthy dogs, correlating these findings with oxidative stress, parasite load and IL-10. The effect of induction and inhibition of HO-1 on lymphoproliferative response by lymph node cells of infected dogs and macrophage infection rate by promastigotes of Leishmania infantum were also evaluated. Dogs with CVL showed marked oxidative stress and increased levels and expression of HO-1, obtaining a positive correlation between HO-1 and oxidative stress and IL-10 in a tissue-dependent way. Inhibition of HO-1 increased proliferation rate in the presence of L. infantum soluble antigen, while induction of HO-1 decreased antigen-specific proliferation and increased macrophage infection rate and number of amastigotes per macrophage. The increase in HO-1 metabolism observed in CVL is associated to oxidative stress present in these dogs and could be one of the mechanisms involved in the in... (Complete abstract click electronic access below)
Doutor

Hsp27 appartient à la famille des protéines dites de survie comme Bcl2 ou la survivine. C'est une protéine anti-apoptotique qui subit une dérégulation de son expression dans de nombreux types tumoraux. Elle est caractérisée comme étant une cible thérapeutique majeure. Au cours de ma thèse, j'ai isolé des peptides stabilisés, dit aptamères, capables d'inhiber fonctionnellement les activités anti-apoptotiques et tumorigènes d'Hsp27. Ces aptamères perturbent la biochimie structurale de Hsp27 et induisent le blocage du cycle cellulaire in vivo. Parallèlement à cette étude, j'ai caractérisé les effets de la déplétion de Hsp27 sur la formation de métastases et de tumeurs osseuses. J'ai aussi montré que la modification du taux de Hsp27 induisait la dégradation de différentes protéines, dites clientes, comme la caspase3, HDAC6 et STAT2.

O transplante de ilhotas pancreáticas é uma terapia promissora para o tratamento da diabetes mellitus tipo 1 (DM1). No entanto, ilhotas transplantadas estão sujeitas à rejeição pelo sistema imune dos pacientes receptores, portanto faz-se necessário o desenvolvimento de mecanismos moleculares que protejam essas células. Estudos mostraram que o hormônio prolactina (PRL) é capaz de inibir a apoptose desencadeada por citocinas pró-inflamatórias sobre células beta pancreáticas e que este processo citoprotetor depende da presença da chaperona HSPB1. Foi observado que durante o desenvolvimento do DM1, as células beta pancreáticas sofrem estresse de retículo endoplasmático e que isso contribui para desencadear apoptose. O estresse de retículo endoplasmático é caracterizado pelo acúmulo de proteínas mal dobradas nessa organela resultando na ativação da resposta a proteínas mal dobradas (UPR) que tem como finalidade recuperar a homeostase celular. No presente estudo mostramos, pela primeira vez, que PRL foi capaz de proteger células beta pancreáticas contra estresse de retículo endoplasmático promovido tanto por citocinas pró-inflamatórias (TNFα, IFNγ e IL1β) quanto pelos estressores de retículo endoplasmático: tunicamicina e tapsigargina; e que HSPB1 é essencial nesse mecanismo de citoproteção. No contexto do DM1, esse hormônio parece ter um efeito modulador da UPR aumentando os níveis de BiP, antecipando a ativação de ATF6 e PERK, mantendo a via de PERK ativa por mais tempo, inibindo a via de IRE1α, e diminuindo os níveis de CHOP em tempos maiores. Coletivamente, os resultados aqui apresentados aprofundam os conhecimentos sobre a função de HSPB1, conduzindo para o desenvolvimento de estratégias que visam à atenuação da morte de células beta por meio da modulação de uma via de proteção endógena, a qual é independente da modulação do sistema imunológico.
The islet transplantation is a promising therapy for the treatment of type 1 diabetes mellitus (T1DM). However, transplanted islets are subject to rejection by the immune system of the recipient patients, therefore the development of molecular mechanisms that protect these cells is necessary. Studies have shown that the hormone prolactin (PRL) is capable of inhibiting apoptosis triggered by pro-inflammatory cytokines on pancreatic beta cells and that this cytoprotective process depends on the presence of the chaperone HSPB1. It was observed that during the development of type 1 diabetes, pancreatic beta cells undergo endoplasmic reticulum stress and that this contributes to trigger apoptosis. The endoplasmic reticulum stress is characterized by accumulation of misfolded proteins in this organelle resulting in the activation of unfolded protein response (UPR) that aims to restore cellular homeostasis. In the present study, we show for the first time that PRL was able to protect pancreatic beta cells against endoplasmic reticulum stress promoted by both pro-inflammatory cytokines (TNFα, IFNγ and IL1β) as the endoplasmic reticulum stressors: tunicamycin and thapsigargin; and HSPB1 is essential that cytoprotective mechanism. In the context of T1DM, PRL appears to have a modulating effect of the UPR by increasing the levels of BiP, anticipating the activation of ATF6 and PERK, keeping the PERK pathway active for longer, inhibiting the pathway IRE1α, and decreasing the levels of CHOP for longer times. Collectively, the results presented here deepen the knowledge of the HSPB1 function, leading to the development of strategies inducing attenuation of beta cells death through modulation of endogenous protection means, which are independent of the modulation of the immune system.

Bibliography: leaves 59-64.
One of the contributors of the widespread interest the yeast Saccharomyces cerevisiae has received is its ability to yield and tolerate high levels of ethanol. S. cerevisiae is able to grow and remain viable in growth media containing ethanol concentrations as high as 19.8% (w/v), a level that is toxic to many other microorganisms. Since production of ethanol is a normal event in the growth cycyle of S. cerevisiae, this organisms has evolved a number of mechanisms to cope with deleterious effects of ethanol. These include induction of heat shock proteins (HSPs). Among these, HSP30 is particularly interesting in that it is the only stress-induced protein known to be instrinsically bound to the yeast plasma membrane. Another ethanol induced HSP; HSP12 has previously been shown to have a peripheral plasma membrane localisation. It has further been shown that HSP12 protects liposomes against damage by ethanol. This study was initially aimed at investigating whether there is co-operation between HSP30 and HSP12 in this membrane protection role.

La recherche des marqueurs biologiques de la tendreté a fait l’objet de nombreux travaux chez les animaux producteurs de viande et en particulier les bovins. A l’issue de ces études, une expression différentielle de la protéine Hsp27 entre des groupes de tendreté extrême a été mise en évidence. Cette protéine est présente à un « carrefour » biologique de l’interactome lié à la tendreté. Comprendre les mécanismes d’action de la protéine Hsp27 dans la tendreté de la viande bovine est l'un des défis de recherche dans le domaine de la production de viande. Dans cette optique, mon travail de thèse (2010-2013) avait pour objectif d’analyser le rôle de Hsp27 dans le développement du muscle et son implication dans le déterminisme des caractéristiques des tissus liés à la qualité de la viande. La première étape de ce travail a consisté à produire un modèle de souris présentant une inactivation du gène de la protéine Hsp27 (KO HspB1) et d’analyser leur phénotype comparativement à des témoins. Les souris KO HspB1 sont viables, fertiles et ne présentent aucune anomalie majeure, mais ont un format plus petit que celui de leurs témoins. L’analyse de leurs caractéristiques musculaires par une technique immunohistoligique mise au point spécifiquement (Publication 1) n’a pas révélé de différences. Au niveau ultrastructural, l'observation du muscle des souris par microscopie électronique à transmission a révélé des différences ultrastructurales entre les deux génotypes à T0 post-mortem avec des écarts entre les myofibrilles très espacées chez les souris KO HspB1 et un appareil contractile musculaire moins organisé. Ces différences sont encore plus marquées à T72 heures post-mortem. Ainsi le phénotype musculaire fin des souris KO HspB1 est plus altéré (Publication 2). Une analyse bio-informatique a été réalisée dans l'objectif de compléter la liste des interacteurs de la protéine Hsp27 et des gènes cibles de l’invalidation d’HspB1 susceptibles de participer à des différences de structure du muscle et de la tendreté. Les partenaires ou cibles prédits de Hsp27 sont des protéines impliquées dans différentes fonctions, comme des Heat shock proteines, des régulateurs de l'apoptose, des facteurs de traduction, des protéines du cytosquelette et des antioxydants. Les abondances de 15 protéines ont été quantifiées par Western-bloting dans deux muscles (m. Soleus, m. Tibialis). Elles sont modifiées chez les souris dépourvues de Hsp27 principalement dans le muscle le plus oxydatif. Cette étude démontre l'existence de liens fonctionnels entre Hsp27 et ses cibles prédites qui pourraient participer au phénotype fin des souris (Publication 3). Pour compléter cette étude, une analyse protéomique du muscle Tibialis anterior a été menée en utilisant la technique d’électrophorèse bidimensionnelle couplée à la spectrométrie de masse. La comparaison des protéomes spécifiques de ces deux génotypes a permis de mettre en évidence des profils d’expression différents pour plusieurs protéines. Elle confirme l’effet muscle spécifique du KO et révèle un lien avec le métabolisme du calcium et des Hsps différentes de celles mises en évidence dans le muscle oxydatif (Publication 4). L'ensemble des données issues de cette étude réalisée dans une espèce modèle apporte des connaissances nouvelles susceptibles d’éclairer sur les mécanismes moléculaires impliqués dans l’établissement de la tendreté de la viande bovine. Elle suggère que le statut en Hsp, les processus apoptotiques et la protection contre le stress oxydatif contribuent à l'évolution de l'ultrastructure post-mortem des muscles et à la tendreté de la viande. Ces nouvelles connaissances seront validées ultérieurement sur muscle bovin
Thanks to genomics, we have previously identified markers of beef tenderness, and computed a bioinformatic analysis that enabled us to build an interactome in which we found Hsp27 at a crucial node. Understanding the role of Hsp27 in the development of muscle and in the determinism of beef tenderness is one of the research challenges in meat production. In this context, my pHDthesis (2010-2013) aimed to analyze the role of Hsp27 in muscle development and its involvement in the determination of the characteristics related to the quality of the meat tissue. In this study, we generated mice devoid of Hsp27 protein by homologous recombination of the HspB1 gene as an animal model. The HspB1-/ - mice were viable and fertile, showing no apparent abnormality but a smaller than their control format. The muscle structure of animals was examined by optical microscopy and transmission electron microscopy. The first approach, made by a developed immunohistochemical classification (Publication 1), did not reveal any differences in the characteristics of muscle fibers (contractile and metabolic type, shape, perimeter, cross-sectional area) but a trend for a higher proportion of small fibers. Different myosin heavy chains electrophoretic profiles were also observed in HspB1-/- mice. At the ultrastructural level, examination of the myofibrillar material showed destructured myofibrils and higher gaps between myofibrils in HspB1-/-, and a greater disintegration of myofibrils at 72h postmortem (Publication 2). We have used a network-based approach for understanding the contribution of Hsp27 to tenderness through the prediction of its interactors related to tenderness. We have revealed the direct interactors of Hsp27. The predicted partners of Hsp27 included proteins involved in different functions e.g. members of Hsp families, regulators of apoptosis, translation factors, cytoskeletal proteins and antioxidants. The abundances of 15 proteins were quantified by Western blotting in two muscles of HspB1-null mice and their controls. We observed changes in the amount of most of the Hsp27 predicted targets in mice devoid of Hsp27 mainly in the most oxidative muscle (Soleus. Our study demonstrates the functional links between Hsp27 and its predicted targets. It suggests that Hsp status, apoptotic processes and protection against oxidative stress are crucial for post-mortem muscle metabolism, subsequent proteolysis, and therefore for beef tenderness (Publication 3). To complete this study, we performed a proteomic analysis of m. Tibialis anterior (glycolytic muscle), using 2D gel electrophoresis, to detect changes in protein abundance subsequent to the invalidation of HspB1 gene. This study confirms the muscle specific effect of HspB1 invalidation and reveals a new list of Heat shock proteins different from those highlighted in oxidative muscle and relationships with calcium (Publication 4). All together, these results provided from a model species showed the very important role of Hsp27 for muscle ultrastructure and revealed its implication in different muscle biological pathways. This provided new elements for understanding the crucial role for Hsp27 in the modulation of the tenderizing process of muscle during meat ageing that will be further examined in beef

Exposure of cells to environmental or chemical stressors will initiate the heat shock response, which is mediated by heat shock proteins. Heat shock proteins are molecular chaperones which are classified by size into six main families: HSP100, HSP90, HSP70, HSP60, HSP40 and the small heat shock proteins (sHsps). The sHsp family members bind to denatured proteins and maintain them in a folding competent state such that they may be refolded by other molecular chaperones.

The present study examined the expression and function of two amphibian sHsps, namely, Rana catesbeiana HSP30 and Xenopus laevis HSP27. Initially, an antisense riboprobe was produced to study the mRNA accumulation of Rana hsp30 in cultured tongue fibroblast (FT) cells. Results showed that Rana hsp30 mRNA was optimally induced when maintained at 35°C for 2 h. An antibody to the recombinant Rana HSP30 protein was also produced in order to study HSP30 protein accumulation in Rana FT cells. Analysis showed that Rana HSP30 was heat-inducible and accumulated maximally at 4 h when maintained at 35°C and then allowed to recover at 22°C for 2 h. Immunocytochemical analysis indicated that Rana HSP30 protein was present primarily in the nucleus, with diffuse localization in the cytoplasm. Additional immunocytochemical analysis showed that Rana HSP30 remained in the nucleus following heat stress and extended periods of recovery.

The molecular chaperone function of Rana HSP30 was also studied. Recombinant Rana HSP30 was found to inhibit the heat induced aggregation of various target proteins including citrate synthase, luciferase and malate dehydrogenase. Also, no major difference was detected between Rana HSP30 and Xenopus HSP30C in the inhibition of heat-induced aggregation of target proteins.

This study also examined the expression and function of Xenopus laevis HSP27. Analysis of the putative amino acid sequence of the Xenopus hsp27 cDNA revealed that it had an identity of 71% with chicken, 65% with zebrafish, 63% with human and 53% with topminnow. Most of the identity was located within the α-crystallin domain of the protein. Interestingly, Xenopus HSP27 shared only a 19% identity with 2 other Xenopus sHsps, HSP30C and HSP30D.

Western blot analysis using an anti-Xenopus HSP27 antibody revealed that HSP27 was not detectable in cultured kidney epithelial cells. However, examination of early Xenopus embryos revealed that HSP27 was first detected in tadpole embryos (stage 44). Heat-inducible HSP27 was also first detected at this stage. The accumulation pattern of Xenopus HSP27 protein was distinct from Xenopus HSP30, which was heat-inducible at midtailbud stage 26, approximately two and a half days earlier in development.

Analysis of recombinant HSP27 by native pore exclusion limit electrophoresis showed that it formed high molecular weight, multimeric complexes. The molecular chaperone function of HSP27 was assessed by means of thermal aggregation assays employing citrate synthase, luciferase and malate dehydrogenase. Xenopus HSP27 inhibited the heat-induced aggregation of all of these target proteins. This study has revealed that Xenopus HSP27 is a member of the HSP27 subfamily of small heat shock proteins in Xenopus and distinct from the HSP30 family. The accumulation of HSP27 under constitutive and stress-inducible conditions is developmentally regulated. Finally, this sHsp appears to function as a molecular chaperone.

Le repliement intracellulaire des protéines nouvellement synthétisées est assisté par des réseaux cellulaires de protéines chaperons. Chez Escherichia coli, la coopération entre les protéines chaperons Trigger Factor (TF) et DnaK est prédominante dans ce processus. En accord avec ceci, la délétion simultanée des gènes codants pour ces deux protéines chaperons conduit à une croissance bactérienne très réduite et à l'accumulation d'un grand nombre de protéines cytoplasmiques sous forme d'agrégats. Au cours de cette étude, nous avons utilisé ces phénotypes afin de mettre en évidence des interactions potentielles au sein du réseau de protéines chaperons in vivo. Nous avons montré que la perte des protéines chaperons TF et DnaK, et donc des voies de repliements dans lesquelles elles sont impliquées, pouvait être secourue de façon efficace par la surexpression du chaperon Hsp33, connu pour être activable en réponse à un stress oxydatif sévère. En outre, la délétion du gène hslO, codant pour Hsp33, n'était plus tolérée en l'absence de TF et DnaK. Cependant, en comparaison avec d'autres protéines chaperons comme GroESL ou SecB, la suppression de ces phénotypes par Hsp33 n'a pas pu être attribuée à un éventuel chevauchement de fonctions avec DnaK et TF. Au contraire, nos résultats montraient qu' Hsp33 surexprimée fixait de façon spécifique le facteur d'élongation-Tu (EF-Tu) et favorisait sa dégradation par la protéase Lon. Cette action synergétique entre Hsp33 et Lon était responsable du rétablissement de la croissance bactérienne en l'absence de TF et DnaK, possiblement via le rétablissement du couplage entre la vitesse de traduction et les capacités de repliement des protéines nouvellement synthétisées du double mutant. Afin de soutenir cette hypothèse, nous avons ensuite montré que la surexpression de la toxine HipA qui inhibe EF-Tu, était aussi capable de supprimer le phénotype de thermosensibilité et de réduire significativement l'agrégation des protéines en l'absence de TF et DnaK
Intracellular de novo protein folding is assisted by cellular networks of molecular chaperones. In Escherichia coli, cooperation between the chaperones Trigger Factor (TF) and DnaK is central to this process. Accordingly, the simultaneous deletion of both chaperone-encoding genes leads to severe growth and protein folding defects. Herein, we took advantage of such defective phenotypes to further elucidate the interactions of chaperone networks in vivo. We show that disruption of the TF/DnaK chaperone pathway is efficiently rescued by over-expression of the redox-regulated chaperone Hsp33. Consistent with this observation, the deletion of hslO, the Hsp33 structural gene, is no longer tolerated in the absence of the TF/DnaK pathway. However, in contrast with other chaperones like GroESL orSecB, suppression by Hsp33 was not attributed to its potential overlapping general chaperone function(s). Instead, we show that over-expressed Hsp33 specifically binds to elongation factor-Tu (EF-Tu) and targets it for degradation by the protease Lon. This synergistic action of Hsp33 and Lon was responsible for the rescue of bacterial growth in the absence of TF and DnaK, by presumably restoring the coupling between translation and the downstream folding capacity of the cell. In support of this hypothesis, we show that over-expression of the stress-responsive toxin HipA, which inhibits EF-Tu, also rescues bacterial growth and protein folding in the absence of TF and DnaK

La carcinose péritonéale est une atteinte néoplasique métastatique de la séreuse péritonéale caractérisée par la diffusion de multiples nodules tumoraux. Son pronostic est sombre, marqué par une chimiorésistance. Les traitements intrapéritonéaux, développés pour délivrer des drogues de chimiothérapie anti-cancéreuse directement au contact de ces nodules, ont permis d’améliorer en partie les résultats oncologiques de cette pathologie. Le principe est de mettre à profit la barrière péritonéo-plasmatique pour administrer des posologies plus élevées de drogues, directement au contact des nodules, et ainsi majorer leur cytotoxicité. En stratégie curative, la chimiothérapie intrapéritonéale est associée à une chirurgie de cytoréduction (CRS) complète et son efficacité est majorée par l’adjonction d’une hyperthermie (ChimioHyperthermie IntraPéritonéale - CHIP). Si ce traitement combiné a transformé le pronostic de patients sélectionnés, les résultats restent insatisfaisants. Par exemple les patients atteints de carcinose d’origine colorectale présentent un taux de survie globale à 5 ans de 40% lorsqu’ils sont éligibles à la CRS-CHIP et une médiane de survie de l’ordre de 16 mois quand le traitement se cantonne à de la chimiothérapie systémique.Une meilleure compréhension des mécanismes cellulaires impliqués dans cette chimiorésistance est donc nécessaire pour déterminer de nouvelles cibles thérapeutiques. Les protéines de choc thermique jouent un rôle fondamental dans l’homéostasie protéique intracellulaire en agissant comme protéines chaperonnes et en régulant l’architecture du cytosquelette. L’Hsp27 (ou HspB1) en particulier est impliquée dans la réponse à différents stress cellulaires comme le choc thermique, le stress oxydatif et l’exposition aux drogues de chimiothérapie. Via des mécanismes finement régulés, Hsp27 exerce une protection garantissant la survie cellulaire, en adaptant ses niveaux d’expression, d’oligomérisation et de phosphorylation. Le taux d’Hsp27 est dès lors augmenté dans la plupart des cancers et apparaît comme marqueur fort de mauvais pronostic. Cela en fait un acteur clé de la chimiorésistance et une cible thérapeutique potentielle.Parmi les thérapeutiques ciblées basées sur l’ARN, les oligonucléotides antisens (ASO) sont des molécules issues du génie génétique capables de bloquer spécifiquement la traduction d’un ARN messager cible en protéine. L’apatorsen, un ASO anti-Hsp27 de deuxième génération, a été développé pour bloquer la synthèse d’Hsp27 au sein de la cellule cancéreuse et ainsi rétablir la chimiosensibilité. Après avoir mis en place un modèle de carcinose péritonéale colorectale traitée par CRS et CHIP chez le rat, nous avons étudié in vitro et in vivo, l’effet de l’adjonction de l’apatorsen au traitement standard de cette maladie. Nos résultats ne montrent pas de gain significatif de survie et donnent lieu à une discussion sur cette stratégie de traitement
Peritoneal carcinomatosis is a neoplasic metastatic process of the peritoneal serous lining characterized by the spread of multiple tumoral nodules. The prognosis of such attempt is very poor, characterized by a global chemoresistance. Intraperitoneal treatments were developed to improve drug’s cytoxicity by delivering them directly on nodules. The principle is to take advantage of the peritoneal-plasma barrier that allows to deliver higher drug’s concentration directly onto nodules and so to improve cytotoxicity. In curative intent strategy intraperitoneal chemotherapy is combined to a complete surgical cytoreduction (CRS) and to hyperthermia to enhance efficiency (Hyperthermic Intraperitoneal Chemotherapy - HIPEC). Thanks to this strategy overall survival improved in selected patients but still be flawed. For example, patients with colorectale peritoneal carcinomatosis present a 40% five-year overall survival, whereas those not eligible to that aggressive treatment present a 16 months median survival. So a better understanding of cellular molecular mechanisms responsible for this chemoresistance that will allow identifying new therapeutic targets is needed. Heat shock proteins play a fundamental role in intracellular protein homeostasis by acting as chaperone and regulating cytoskeleton architecture. In particular, Hsp27 acts as a regulator of the cellular response to various stress, such as thermic choc, oxidative stress, exposition to antineoplasic drugs. Through finely regulated process, Hsp27 exerts a cytoprotective role to guaranty cell survival, by adapting its level of expression, oligomerization and phosphorylation. As so Hsp27 is a key actor of chemoresistance and a designated therapeutic target.Antisens oligonucleotides are a new class of molecular targeted treatment able to specifically block the traduction into protein of a messenger RNA. Apatorsen, a second generation anti-Hsp27 ASO, has been developed to decrease Hsp27 levels in neoplastic cells and so restore chemosensitivity.After establishing a colorectal peritoneal carcinomatosis rat model with CRS and HIPEC, we studied in vitro and in vivo the effect of the apatorsen adjunction to this standard treatment. Our results did not show a significant survival improvement and give rise to a discussion upon this treatment strategy

Mestrado em Biologia Molecular e Celular
A síntese proteica é um processo essencial para que todos os organismos mantenham a homeostasia celular. Os tRNAs são elementos cruciais na síntese proteica, uma vez que codificam a informação genética presente no mRNA. A linha celular HeLa, utilizada neste estudo, foi primeiramente isolada de uma mulher com cancro do colo do útero e desde então tem sido bastante usada na investigação, sendo muito importante no estudo das bases moleculares de muitas doenças. De modo a monitorizar a agregação proteica nesta linha celular, um sistema repórter foi desenvolvido utilizando uma fusão entre HspB1 (Hsp27) e a GFP. HspB1 é um chaperone molecular com capacidade de recrutar outros chaperones e restabelecer a conformação ideal das proteínas em situações de stress. A GFP é uma proteína fluorescente que marca certas condições biológicas de interesse. Para perceber o impacto dos erros da tradução na agregação de proteínas e no surgimento das doenças, o principal objetivo deste estudo foi desenvolver uma linha celular estável (HeLa) expressando um sistema repórter HspB1-GFP, de modo a monitorizar os erros no enovelamento das proteínas em resposta ao stress proteotóxico. Ao longo deste estudo o sistema repórter expressando HspB1-GFP foi desenvolvido com sucesso, permitindo assim a sua utilização para identificar situações fisiológicas e patológicas em que a agregação de proteínas ocorre em células de mamífero.
Protein synthesis is essential for all organisms to maintain cell homeostasis. tRNAs are crucial elements in protein synthesis as they decode the genetic information organized in the mRNA codons. A HeLa cell line, used in this study, was first isolated from a woman with cervical cancer and since then was highly used in biological studies, being extremely important in the study of the molecular basis of several diseases. In order to monitor protein aggregation in this cell line, a reporter system was developed using an HspB1 (Hsp27) and a GFP fusion. HspB1 is a small heat shock protein that, in stress situations, recruits other proteins in order to restore the conformation of the proteins. GFP is a biosensor that reports several cellular conditions of interest. To understand the impact of translation errors on protein aggregation and on the disease arising, the main goal of this study was to develop a stable cell line (HeLa) expressing a reporter system HspB1-GFP to monitor the protein misfolding in response to proteotoxic stress. During this study, the reporter system expressing HspB1-GFP was developed successfully, allowing the identification of physiological and pathological situations where protein aggregation occurs in mammalian cells.

In a healthy cell, the ROS levels are stringently regulated by the action of various enzymatic or non-enzymatic antioxidant systems. Imbalance in the ROS homeostasis generates oxidative stress resulting in damage to cellular macromolecules. Besides, pro-oxidants, glyoxals which are normally generated as an intermediate compound in the glycolytic pathway and other metabolic activity are known to cause oxidative stress in the cell. Elevated oxidative stress is one of the prominent cellular aetiologies associated with premature aging, cardiovascular and retinal disorders, atherosclerosis, and several neurological disorders. Parkinson disease (PD) is one of well-known neurodegenerative diseases whose pathogenicity is correlated to mitochondrial dysfunction due to elevated oxidative stress in the neuronal cells. Several proteins which are associated with development of familial form of PD, DJ-1, a member of ThiJ/DJ-1/PfpI super family, is known to act as an oxidative sensor in humans. Interestingly, heat shock protein (Hsp)31 from S. cerevisiae which belongs to DJ-1 family was shown to provide a similar oxidative stress resistance in yeast. However, the mechanistic aspects how these family members functions as an oxidative stress sensor are not clearly defined. The main focus of my investigation is to understand the involvement of these DJ-1 proteins in regulation of redox homeostasis and mitochondrial health, which are major hallmarks in the pathogenesis of PD. My major findings demonstrate the importance of Hsp31 family proteins in protecting cells against oxidative stress, which is induced by methylglyoxal (MG). The deletion of Hsp31 leads to a compromised growth phenotype in yeast upon MG induced stress. Moreover, Hsp31 exhibited robust GSH-independent glyoxalase activity both in vivo and in vitro. Besides, the glyoxalase activity is critical for glyoxal detoxification as well as suppression of ROS levels in cells. On the other hand, in agreement with the observed growth phenotypes, Hsp34 protein possesses a very mild glyoxalase activity as compared to Hsp31. Furthermore, active site mutational analysis reveals that methylglyoxalase activity of Hsp31 protein is critical for providing protection against oxidative stress in yeast. Importantly, endogenous expression of human DJ-1 could complement the growth of yeast under oxidative and glyoxal stress conditions signifying its functional conservation across species. Mechanistically, my findings highlight that Hsp31 regulates cellular GSH and NADPH homeostasis thereby protecting cells against oxidative stress. In addition, cellular localization experiment reveals that though Hsp31 is a cytosolic protein, it predominantly localizes into mitochondria under oxidative stress conditions and protects the organelle from severe oxidative damages. Lastly, my findings uncover the role of Hsp31 paralogs in the maintenance of mitochondrial health integrity and other stress related pathways. To test their role in the mitochondrial health, I have analysed several parameters such as mass, dynamics and functionality. Interestingly, though the single deletions of these paralogs do not have significant effects over the mitochondrial phenotypes, the deletion of DJ-1 homologs in combination of hsp31 and hsp34 in yeast led to enhanced total as well as functional mitochondrial mass in cells. To address how mitochondrial mass enhancement occurs in the cells, the organelle turnover (mitophagy) was assessed. The microscopic and western analysis indicates, there was no alteration in mitophagy among the ∆hsp31∆hsp34 compared to WT. On the contrary, an enhancement in the basal levels of ROS stimulated increased biogenesis of mitochondria in ∆hsp31∆hsp34 cells was observed. Strikingly, ∆hsp31∆hsp34 cells also exhibit upregulation of mitochondrial fusion proteins resulting hyperfusion of mitochondria. Additionally, our results demonstrates that ∆hsp31∆hsp34 cells exhibited a long-term G2/M cell cycle arrest, which was rescued upon overexpression of mitochondrial fission protein, Dnm1. Lastly, absence of these paralogs in yeast, resulted in induction of apoptotic-like features in the cells and decreased lifespan in Saccharomyces cerevisiae. Altogether, my studies highlight the importance of DJ-1 class of proteins in maintaining the cellular redox status, mitochondrial integrity and cellular health in yeast. In conclusion, overall my studies highlight that Hsp31 is a robust methylglyoxalase and regulates cellular NADPH and GSH pool thereby helps in the maintenance of redox homeostasis. Hsp31 predominantly translocate into mitochondria upon oxidative stress to protect the organelle from oxidative damages. Furthermore, my findings provide the first evidence over the involvement of DJ-1 family proteins in the regulation of mitochondrial health and dynamics, cell-cycle arres and reduced lifespan in yeast.

碩士
國立陽明大學
傳統醫藥學研究所
97
Hsp32 is a stress-inducible protein with prominent cardio-protective functions. The aim of this study was to investigate the effect of multimodality stimulations at acupoint PC 6 on myocardial Hsp32 expression in rats. Male Sprague-Dawley rat, weighing 260~320g, were divided into six groups, namely, lectroacupuncture group (EA), laser acupuncture group (LA), hydroacupuncture group (HA), local somatothermal stimulation group (LSTS), sham group (for EA, LA, LSTS) and normal group (for HA), under adequate anesthesia, multimodalities, such as EA, LA, HA and LSTS were separately applied at left acupoint Neiguan (PC 6). The parameters were 2/15 Hz, alternatively, 1-2 mA and 20min for EA; semiconductor laser, emission wavelengths of 660 nm, 60 mW and 15 min for LA; a dose of 3mg/100g body weight of chloralose and urethane mixture for HA; fluctuating temperature, 27min/per dose and three doses for LSTS treatment. The pathological examinations, myocardial Hsp32 protein and mRNA expression were detected by using immunostaining, Western blotting, and real time PCR, respectively. Serum CK-MB and Troponin-I were determined to evaluate the possibility of myocardium injury after HA treatment at PC 6 acupoint . Electrocardiography was recorded to detect the incidence of arrhythmia in ischemia-reperfusion (I/R) model. Besides, primary endocardium and myocardium cultures were used to investigate the direct effect of drug mixture , which was used for in vivo HA treatment, on cardiac Hsp32 expression. The results showed that there was a significant increase of Hsp32 and Nrf2 expression in HA group and an increase of Hsp70 in LSTS group. Hsp32 was time-dependently and dose-dependently increased by HA treatment. However, there was no significant difference, in terms of IκB expression, in all experimental groups. Results from immunostaining showed that the Hsp32 expression was located in myocardium and endocardium in the heart, but mainly in smooth muscle layer of ascending aorta. By using primary heart cultures, Nrf2 protein expression was increased in both endocardium and myocardium. Besides, preconditioning HA at left PC 6 significantly decreased the duration of arrhythmia (VPB, VF and total account) and incidence of stony heart in subsequent I/R model. We concluded that LSTS at left PC 6 increased Hsp70 protein expression while HA at left PC 6 increased hsp32 gene expression and Nrf2 protein expression, which might provide a new strategy for treatment of ischemic heart disease.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.

Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. The present study examined the pattern of accumulation of HSP30 and HSP70 in cells recovering from heat shock as well as the effect of proteasome inhibition on cytoplasmic/nuclear and endoplasmic reticulum (ER) molecular chaperone accumulation, large multimeric HSP30 complexes, stress granule and aggresome formation in Xenopus laevis A6 kidney epithelial cells. Initial immunoblot analysis revealed the presence of elevated levels of HSP30 after 72 h of recovery. However, the relative levels of HSP70 declined to near control levels after 24 h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24 h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. To verify if proteasome inhibition in A6 cells induced the formation of similar HSP30 granules, immunoblot and immunocytochemical analyses were performed. MG132, celastrol and withaferin A enhanced ubiquitinated proteins, inhibited chymotrypsin-like activity of the proteasome and induced the accumulation of cytoplasmic/nuclear HSPs, HSP30 and HSP70 as well as ER chaperones, BiP and GRP94 and heme oxygenase-1. Northern blot experiments determined that proteasome inhibitors induced an accumulation in hsp30, hsp70 and bip mRNA but not eIF1α. The final part of this study demonstrated that treatment of A6 cells with proteasome inhibitors or sodium arsenite or cadmium chloride induced HSP30 multimeric complex formation primarily in the cytoplasm. Moreover, these stressors also induced the formation of RNA stress granules, pre-stalled translational complexes, which were detected via TIA1 and polyA binding protein (PABP), which are known stress granule markers. These stress granules, however, did not co-localize with large HSP30 multimeric complexes. In comparison, proteasome inhibition or treatment with sodium arsenite or cadmium chloride also induced the formation of aggresome-like structures, which are proteinaceous inclusion bodies formed as a result of an abundance of aggregated protein. Aggresome formation was identified by monitoring the presence of vimentin and γ-tubulin, both of which are cytoskeletal proteins and serve as markers of aggresome detection. Aggresome formation, which was also verified using the ProteoStat assay, co-localized with large HSP30 multimeric complexes. Co-immunoprecipitation experiments revealed that HSP30 associated with γ-tubulin and β-actin in cells treated with proteasome inhibitors or sodium arsenite or cadmium chloride suggesting a possible role in aggresome formation. In conclusion, this study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation. Finally, proteasome inhibition, sodium arsenite and cadmium chloride treatment of A6 cells induced cytoplasmic/nuclear and ER chaperones as well as resulting in the formation stress granules and aggresome-like structures which associated with large HSP30 multimeric complexes.

Research Doctorate - Doctor of Philosophy (PhD)
The remarkable cellular communication events that characterise the highly species specific interactions observed during the ontogeny of mammalian fertilization, represent some of the most intriguing in all of biology. Given the 60 years or so of research conducted to elucidate the precise mechanisms that underpin these interactions, it is surprising that they still remain largely unknown. This can be mostly attributed to the unique luminal environment in which the sperm reside following insemination and the direct effects that these fluids have on their functionality. Although immense controversy surrounds the precise ligand responsible for the spermatozoas binding to the oocyte’s zona pellucida, considerable contention is also afforded to the mechanism by which they bind. A number of landmark papers have recently emerged to suggest that these initial binding events may be facilitated by the formation and presentation of multimeric zona pellucida receptor complexes on the sperm surface during their terminal maturation, rather than the widely held paradigm that the zona pellucida receptor is a single molecular entity. During these studies the use of blue native polyacrylamide gel electrophoresis, for the first time in mammalian sperm, has provided direct evidence that a number of multimeric zona receptor complexes indeed reside on the apical plasma membrane of capacitated sperm and that two of these complexes have the ability to interact with the zona pellucida. Proteomic analysis of these two complexes has indicated that molecular chaperones (CCT/TRiC complex and HSPD1) are responsible for the formation of each complex, and individually, these complexes contain a number of receptor proteins (ZPBP2, ZP3R and ADAMTS10) that potentially provide the zona pellucida affinity. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize.

Toxoplasma gondii is an intracellular parasite that causes a persistent infection in 10- 80% of the world's population, depending on geographic location. Infection with this parasite causes changes in behavior and physiological functions of an infected host, including warm- blooded animals including humans. Recent studies also show influence of infection with T.gondii on male reproductive fitness in mice and rat and to investigate this issue further became a task of my diploma thesis. In this study, detailed histometric screening of testicular function along with levels of a pituitary luteinizing hormone (LH) and screening of epigenetic modification of testicular DNA were analysed in infected and control mice. Testicular function and sperm production was significantly decreased in T. gondii positive group after a 30-day infection. The level of LH in urine of T. gondii infected mice was decreased compares to control and the number of leptotene primary spermatocytes and spermatids was lowered, but also number of Sertoli cells and tubule diameter were elevated. In epigenetic study the testicular epigenom was tested to measure global methylation and methylation of specific genes Hspa1, Crem and Creb1 which are essential for successfully ongoing spermatogenesis. The global level of methylation of...

Breast cancer accounts as the most prevalent cancer and the leading cause of cancer death worldwide among women. Estrogen is one main factor responsible for tumour growth in breast cancer patients through stimulation of estrogen receptor (ER) signalling. ER positive (ER+) breast cancer patients are eligible for anti-estrogenic drugs. Fulvestrant (Faslodex®) represents a second-line therapy for the treatment of postmenopausal women with ER+ advanced breast cancer. Unfortunately, a significant number of ER+ patients will develop resistance to second-line fulvestrant treatment. It is therefore important to understand the molecular mechanisms of resistance and to identify biomarkers capable of predicting response to this treatment. The aim of this project is to establish a genome-wide shRNA functional screening to identify key proteins central in resistance mechanisms and potentially predictive biomarkers capable of identifying ER+ patients that are responsive or resistant to fulvestrant treatment. To do so, a MCF-7-based fulvestrant resistant breast cancer cell line was used. MCF-7/LCC1 and MCF-7/LCC9 were transduced with shRNA libraries covering over 15,000 mRNAs and treated with fulvestrant. This led to depletion and/or enrichment of shRNAs targeting genes evaluated by next generation sequencing (NGS). Deconvolution of NGS data from genomic DNA of LCC1 and LCC9 cells transduced by shRNA libraries led to identification of 206 genes that may have functional significance in fulvestrant resistance. Ingenuity Pathway Analysis of the candidate genes identified HSD17B10 and HSPE1 as key-molecules in networks related to cell proliferation and death. We have found that these genes are upregulated in different fulvestrant-resistant cell lines when compared to their fulvestrant-sensitive parental cell line at gene and protein expression levels using RT-qPCR and Western blotting. This expression is enhanced in fulvestrant presence, suggesting that these proteins may have critical importance in the resistance phenotype. Further studies on these proteins may elucidate on how to overcome fulvestrant resistance.