A transcrição pela RNA polimerase II (RNA polimerase B) e a sínteses de proteínas são os mais importantes processos metabólicos em células eucarióticas, e estão envolvidas no controle da expressão gênica. Trichoderma reesei foi utilizado como modelo de estudo para o desenvolvimento deste estudo. Este fungo filamentoso é um microrganismo que vem sendo utilizado por vários laboratórios no mundo para o estudo dos diversos processos biológicos básicos devido à sua grande importância biotecnológica. Foi estabelecido por nosso grupo de pesquisa um banco de dados ESTs (\"Expressed Sequence Tags\") para este microrganismo e, por meio da técnica de \"microarrays\" de cDNAs, determinou-se a reposta transcricional de T. reesei em função da disponibilidade de oxigênio e glicose, assim como a alguns estresses ambientais. Com base nesses resultados foram escolhidos alguns transcritos afetados pela limitação de oxigênio, tais como o gene btf3. Este gene codifica para a proteína reguladora BTF3 (Fator Transcricional 3 da RNA polimerase B), muito conservada em eucariotos, envolvida na transcrição de vários promotores da classe II e que faz parte do complexo que se liga aos polipeptídios nascentes (NAC). Com o intuito de estudar a funcionalidade do gene btf3 em condições normais e em choque térmico foi realizada uma análise transcricional comparativa em larga escala da cepa selvagem de T. reesei QM9414 e do mutante nocaute do gene btf3. A expressão de aproximadamente 2.000 genes foi analisada, por meio de \"microarrays\", em células submetidas ao estresse por choque térmico produzido pelo incremento da temperatura de cultura a 40°C. O nocaute do gene btf3 produziu o incremento da expressão dos genes das vias metabólicas primárias (ND4 e FBA), da defesa celular (DnaJ, HSP70 e RCI) e da síntese de proteínas (eIF2); enquanto que reprimiu genes da estrutura celular (HFBII) e da síntese de RNA (ATF21). No choque térmico, genes que codificam para proteínas associadas com a defesa celular, como as chaperonas Hsp70 e DnaJ, tiveram sua expressão induzida enquanto que proteínas associadas à divisão celular, como histonas e septina B, e à síntese de proteínas, como as proteínas ribossomais, foram reprimidas em ambas as cepas como resultado do estresse. Os resultados obtidos na análise por \"microarray\" foram validados através de PCR quantitativo em tempo real (qPCR). Estes resultados sugerem que BTF3 possa atuar como repressor de alguns genes transcritos pela RNA polimerase II. / Transcription by RNA polymerase II (RNA polymerase B) and protein synthesis are the most important metabolic processes in eukaryotic cells, and they are involved in the control of gene expression. Trichoderma reesei was used as model of study for the development of this study. This filamentous fungus is a microorganism that has been used by some laboratories around the world for the study of diverse basic biological questions due to its great biotechnological importance. We had established a data base of ESTs (Expressed Sequence Tags) for this microorganism and, through the technique of cDNA microarray, we had determined the transcriptional response of T. reesei to oxygen and glucose availability, as well as some environmental stresses. Based on such studies we chose some transcripts affected by oxygen limitation such as the btf3 gene, for more detailed investigations. This gene codes for the BTF3 regulatory protein (RNA Polymerase B Transcription Factor 3), a conserved transcriptional factor among eukaryotes that is involved in the transcription of several class II promoters and is part of the nascent polypeptide-associated complex (NAC). In order to study the functionality of the btf3 gene in normal and heat shock conditions, a large-scale transcriptional comparative analysis between T. reesei wildtype strain QM9414 and the btf3 knockout mutant was executed. The expression of approximately 2,000 genes was analyzed using microarrays in cells submitted to heat stress produced by culture temperature increment to 40°C. The knockout of btf3 produces the increment of the expression of genes involved with the primary metabolism pathways (ND4 and FBA), cellular defense (DnaJ, HSP70 and RCI) and protein synthesis (eIF2); whereas it repressed genes related to the structure cell (HFBII) and RNA synthesis (ATF21). In heat shock, genes that encode Hsp70 and DnaJ proteins, associated to cellular defense, as chaperones, had their expression induced. On the other hand, genes for proteins associated to cellular division, such as histones and septin B, and those related to protein synthesis, such as ribossomal proteins were transcriptionally repressed in both strains as a result of the stress. The results obtained in the microarray analysis were validated through quantitative real-time PCR (qPCR). These results suggest that BTF3 can act as a repressor for some genes transcribed by RNA polymerase B.

Ng Ho Yuen Andus. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 105-131). / Abstracts in English and Chinese. / Chapter I --- Title page --- p.i / Chapter II --- Thesis committee --- p.ii / Chapter III --- Acknowledgement --- p.iii / Chapter IV --- Abstract --- p.v / Chapter V --- Abstract (Chinese version) --- p.vii / Chapter V --- Table of contents --- p.ix / Chapter VI --- List of abbreviations --- p.xv / Chapter VII --- List of figures --- p.xviii / General introduction --- p.1 / Chapter Chapter 1: --- Literature review --- p.5 / Chapter 1.1. --- Heat shock proteins (HSPs) --- p.6 / Chapter 1.1.1. --- Introduction --- p.6 / Chapter 1.1.2. --- The various heat shock proteins --- p.8 / Chapter 1.1.2.1. --- HSP100s --- p.8 / Chapter 1.1.2.2. --- HSP90s --- p.9 / Chapter 1.1.2.3. --- HSP70s --- p.12 / Chapter 1.1.2.3.1. --- ATPase reaction cycle of HSP70 and protein folding --- p.13 / Chapter 1.1.2.3.2. --- Protein translocation --- p.14 / Chapter 1.1.2.3.3. --- Selective lysosomal proteolysis --- p.16 / Chapter 1.1.2.4. --- HSP60s --- p.16 / Chapter 1.1.2.5. --- Small HSPs --- p.17 / Chapter 1.1.2.6. --- Ubiquitin --- p.19 / Chapter 1.1.3. --- HSP studies in fish --- p.21 / Chapter 1.1.3.1. --- In vivo works --- p.21 / Chapter 1.1.3.2. --- In vitro works --- p.23 / Chapter 1.2. --- Growth hormone / prolactin family in teleostean fishes --- p.26 / Chapter 1.2.1. --- Introduction --- p.26 / Chapter 1.2.2. --- Growth hormone (GH; somatotropin) --- p.29 / Chapter 1.2.2.1. --- Structure --- p.29 / Chapter 1.2.2.2. --- Actions --- p.29 / Chapter 1.2.2.3. --- Insulin-like Growth Factors (IGFs; somatomedins) --- p.31 / Chapter 1.2.3. --- Prolactin (PRL) --- p.34 / Chapter 1.2.3.1. --- Structure --- p.34 / Chapter 1.2.3.2. --- Actions --- p.35 / Chapter 1.2.4. --- Somatolactin (SL) --- p.37 / Chapter 1.2.4.1. --- Structure --- p.37 / Chapter 1.2.4.2. --- Actions --- p.38 / Chapter 1.2.5. --- Growth hormone receptor (GH-R) and prolactin receptor (PRL-R) --- p.39 / Chapter 1.3. --- Cortisol in teleostean fishes --- p.41 / Chapter 1.4. --- Salinity adaptation in teleosts --- p.44 / Chapter Chapter 2: --- Effect of in vitro thermal shock on HSP70 expression in whole blood of Sparus sarba --- p.46 / Chapter 2.1. --- Introduction --- p.47 / Chapter 2.2. --- Materials and methods --- p.49 / Chapter 2.2.1. --- Overall experimental design --- p.49 / Chapter 2.2.2. --- Experimental fish --- p.49 / Chapter 2.2.3. --- Blood sampling and preparation --- p.49 / Chapter 2.2.4. --- Thermal stress regimes --- p.50 / Chapter 2.2.5. --- Protein extraction --- p.51 / Chapter 2.2.6. --- Protein quantification --- p.51 / Chapter 2.2.7. --- Indirect enzyme-linked immunosorbent assay (ELISA) --- p.52 / Chapter 2.2.8. --- Protein gel electrophoresis and immunoblotting (Western blotting) --- p.54 / Chapter 2.2.9. --- Statistical analysis --- p.55 / Chapter 2.3. --- Results --- p.56 / Chapter 2.3.1. --- Validation of indirect ELISA --- p.56 / Chapter 2.3.2. --- Effect of in vitro thermal shock on HSP70 expression in whole blood of Sparus sarba --- p.56 / Chapter 2.4. --- Discussion --- p.60 / Chapter 2.5. --- Conclusion --- p.64 / Chapter Chapter 3: --- Effects of hormones on HSP70 expression in whole blood of Sparus sarba in vitro --- p.65 / Chapter 3.1. --- Introduction --- p.66 / Chapter 3.2. --- Materials and methods --- p.68 / Chapter 3.2.1. --- Overall experimental design and experimental fish --- p.68 / Chapter 3.2.2. --- Hormone treatments --- p.59 / Chapter 3.2.3. --- "Protein extraction and quantification, indirect ELISA，gel electrophoresis, and immunoblotting (Western blotting)" --- p.70 / Chapter 3.2.4. --- Statistical analysis --- p.70 / Chapter 3.3. --- Results --- p.71 / Chapter 3.3.1. --- Effect of Cortisol on HSP70 levels in whole Blood --- p.71 / Chapter 3.3.2. --- Effect of recombinant bream growth hormone on HSP70 levels in whole blood --- p.71 / Chapter 3.3.3. --- Effect of recombinant bream insulin-like growth factor-I on HSP70 levels in whole blood --- p.71 / Chapter 3.3.4. --- Effect of ovine prolactin on HSP70 levels in whole blood --- p.72 / Chapter 3.4. --- Discussion --- p.81 / Chapter 3.4.1. --- Effect of Cortisol on HSP70 levels in whole Blood --- p.81 / Chapter 3.4.2. --- Effect of recombinant bream growth hormone on HSP70 levels in whole blood --- p.83 / Chapter 3.4.3. --- Effect of recombinant bream insulin-like growth factor-I on HSP70 levels in whole blood --- p.85 / Chapter 3.4.4. --- Effect of ovine prolactin on HSP70 levels in whole blood --- p.86 / Chapter 3.5. --- Conclusion --- p.88 / Chapter Chapter 4: --- Effect on HSP70 expression in whole blood of Sparus sarba acclimated to various salinities --- p.89 / Chapter 4.1. --- Introduction --- p.90 / Chapter 4.2. --- Materials and methods --- p.92 / Chapter 4.2.1. --- Overall experimental design and experimental fish --- p.92 / Chapter 4.2.2. --- "Protein extraction and quantification, indirect ELISA, gel electrophoresis, and immunoblotting (Western blotting)" --- p.92 / Chapter 4.2.3. --- Statistical analysis --- p.93 / Chapter 4.3. --- Results --- p.94 / Chapter 4.4. --- Discussion --- p.97 / Chapter 4.5. --- Conclusion --- p.100 / Chapter Chapter 5: --- General discussion and conclusion --- p.101 / References --- p.105

As proteínas de choque térmico (HSP) são expressas em todas as células humanas, sendo superexpressas em condições de estresse celular, tais como hiper ou hipotermia, isquemia, inflamação e reparação. Dentre as várias famílias de HSP, encontram-se a Hsp27 e a Hsp47, cuja superexpressão relaciona-se à inibição da apoptose e à manutenção dos filamentos de actina do citoesqueleto (Hsp27) e à manutenção do sistema de produção de procolágeno e do colágeno (Hsp47). O laser de diodo (LD) tem atualmente amplas aplicações clínicas com vantagens terapêuticas comprovadas, porém os efeitos térmicos que pode acarretar aos tecidos ainda são controversos. Neste trabalho, avaliou-se a influência do LD sobre a expressão imunoistoquímica das Hsp27 e Hsp47 sobre o tecido dentário submetido a gengivoplastia com LD e com bisturi convencional, bem como relacionou-se essa influência com as variações de temperatura provocadas pela irradiação laser. Vinte e quatro ratos adultos foram divididos em dois grupos: grupo 1 12 animais submetidos a gengivoplastia no incisivo central inferior esquerdo utilizando laser de diodo (ZAP soft lase, 810 nm, 0,3 W, densidade de energia de 113,63 J/cm2); grupo 2 12 animais submetidos a gengivoplastia no mesmo local utilizando bisturi convencional. Os animais sofreram eutanásia nos períodos de 0h, 24h, 72h e 120h, sendo os incisivos centrais retirados e descalcificados. Posteriormente foram submetidos a análise imunoistoquímica utilizando anticorpos monoclonais contra Hsp27 e Hsp47. Teste in vitro foi realizado utilizando-se incisivos centrais de ratos extirpados e mantidos resfriados até o momento do teste. Este foi realizado com a instalação de 4 termopares, os quais registraram a variação da temperatura durante gengivoplastia realizada com o LD calibrado com os mesmos parâmetros do teste in vivo. Observou-se haver variação máxima de temperatura entre 1C e 6C, dependendo da região analisada. A expressão da Hsp27 mostrou-se aumentada no grupo irradiado com laser em relação ao bisturi em praticamente todos os períodos experimentais. A Hsp47 também expressou-se mais intensivamente no grupo laser, porém mais tardiamente. A expressão de ambas as proteínas foi mais intensa na região irradiada, porém nas demais regiões medidas não foi possível relacionar as variações imunoistoquímicas com as de temperatura. Concluiu-se que o LD provoca modificações na expressão das Hsp27 e Hsp47, as quais podem estar relacionadas ao aumento da temperatura provocado pelo laser. / Heat shock proteins (HSP) are expressed in all human cells, and are overexpressed under conditions of cellular stress, such as hyper- or hypothermia, ischemia, inflammation and repair. Among the various families of HSP, there are Hsp27 and Hsp47, whose overexpression is related to the inhibition of apoptosis, maintenance of cytoskeletal actin filaments (Hsp27) and maintenance of the procollagen and collagen production system (Hsp47). At present, diode laser (DL) has broad clinical applications with proven therapeutic advantages; however, there is still controversy about the thermal effects it may have on tissues. In this study, the influence of DL on the immunohistochemical expression of Hsp27 and Hsp47 on dental tissue submitted to gingivoplasty with DL and a conventional scalpel was evaluated, as well as how this influence was related to the variations in temperature caused by laser irradiation. Twenty-four adult rats were divided into two groups: Group 1 12 animals submitted to gingivoplasty in the mandibular left central incisor using diode laser (ZAP soft lase, 810 nm, 0.3 W, energy density 113.63 J/cm2); Group 2 12 animals submitted to gingivoplasty in the same site using a conventional scalpel. The animals were euthanized in the periods of 0h, 24h, 72h and 120h, and the central incisors were removed and decalcified. Afterwards they were submitted to immunohistochemical analysis using monoclonal antibodies against Hsp27 and Hsp47. The in vitro test was performed, using the extirpated central incisors of rats, which were kept chilled until the time of the test. This was done by installing 4 thermocouples that recorded the variation in temperature during the gingivoplasty performed with DL calibrated with the same parameters as those of the in vivo test. It was observed that there was a maximum variation in temperature of between 1C and 6C, depending on the region analyzed. Hsp27 expression was shown to be increased in the laser-irradiated group when compared with the group treated by scalpel in practically all the experimental periods. Hsp47 was also more intensely expressed in the laser group, however, much later. The expression of both proteins was more intense in the irradiated region, but in the other regions measured, it was not possible to relate the immunohistochemical variations with those of temperature. It was concluded that DL causes changes in the expression of Hsp27 and Hsp47, which may be related to the temperature increase caused by laser.

The ability to accurately predict danger and implement appropriate protective responses is critical for survival. Environmental fluctuations can cause damage at the cellular level, leading to the misfolding and aggregation of proteins. Such damage is toxic to cells: in age-related neurodegenerative diseases like ALS, Parkinson’s, Alzheimer’s and Huntington’s Diseases, the accumulation of damaged proteins in the brain ultimately leads to neuronal cell death and disease onset. To date, there is still no cure to combat the progressive degeneration and cell death seen in the brains of patients. Cells within an animal possess defense programs to minimize protein damage. One such defense mechanism is the activation of a program called the Heat Shock Response, which increases production of protective proteins known as heat shock proteins (HSPs). These HSPs act as molecular chaperones to assist with the clearing out of damaged proteins. This program is implemented by a conserved transcription factor, Heat Shock Factor 1 (HSF-1). However, in brains of patients with degenerative diseases, this protective mechanism, for reasons yet unknown, is not constantly activated. My thesis has involved the discovery of innate mechanisms that exist in organisms to activate this cellular protective mechanism against protein misfolding. My research, using the model organism Caenorhabditis elegans, has shown that the protective heat shock response in the cells of the animal can be triggered through neurohormonal signaling. The neurohormonal signaling that I am studying is one that is highly conserved across all organisms from plants to insects to mammals – serotonergic signaling. The stimulation of serotonergic signaling appears sufficient to activate the Heat Shock Response, even in the absence of real damage. In fact, the neuronal release of serotonin facilitates a pre-emptive upregulation of protective genes in the animal, which we have observed to be able to reduce the accumulation of damaged proteins in a C. elegans model of Huntington’s Disease. Additionally, I have seen that anticipating danger can enhance the animal’s stress response in a serotonin-dependent manner, thus facilitating better survival against a subsequent insult that can cause protein damage. Together, these studies present the novel possibility of protection against neurodegenerative disease via modulation of neurotransmission and/or neurosecretion. They also allow for understanding how sensory inputs are coupled to gene expression under stressful conditions. I hope to understand the mechanism by which animals adapt to changes in their environment by coordinating their sensory input with changes in behavior and gene expression.

The exposure to heat can adversely affect animal performance and productivity, particularly when associated with pregnancy. The comprehension of the physiological and cellular responses during heat stress assists the decisions to improve the productivity of goats in a tropical environment. In this context, this study evaluated the physiological and cellular responses of Saanen goats in acute and chronic stress conditions. Healthy Saanen goats were randomly assigned to heat stress treatment, short-term, under solar radiation and in the long-term, in climatic chamber. Data were analyzed using the Statistical Analysis System (SAS). The normality was confirmed using the Shapiro-Wilk test. In acute heat stress, solar radiation caused the increase of cortisol release, respiratory rate and reduced T3 and T4 to restore homeothermia. The expression of p53 (pro-apoptotic protein), Bcl-2 (anti-apoptotic protein), HSP60, HSP70 and HSP90 was higher in leukocyte cells for heat stressed goats. In chronic heat stress at the end of gestation, goats significantly mobilized the increase of respiratory rate to lose heat, with cortisol elevation on the 15 days previous to parturition (P < 0.05), which was even greater at day 15 postparturition. At the cellular level, HSP70 was the most expressed during and after heat challenge, with more transcripts at day 15 postparturition for the heat stressed goats. In mammary epithelial cells, there were an increase in apoptosis-related transcripts, p53 and Bax, for the group undergoing heat stress before parturition. The expression of HSP27 was higher before and after parturition for the same group when compared to the animals in thermal neutrality. During lactation, the stressed goats presented lower milk yield. The heat challenge increased the percentage of fat and decreased lactose. The somatic cell count was higher for stressed goats during all lactation. During lactation, the gene expression of prolactin receptor (PRLr) was lower for heat stress goats, which may explain the reduction in milk production at subsequent lactation. In conclusion, our findings suggest that respiratory rate and HSP70 are reliable biomarkers for assessing the thermal comfort and thermotolerance of Saanen goats under acute and chronic heat stress. In addition, chronic thermal stress at final gestation impaired milk production and milk composition from subsequent lactation. / A exposição ao calor pode afetar negativamente o desempenho e a produtividade dos animais, particularmente quando associados à gestação. Compreender as relações entre as respostas fisiológicas e celulares, quando em estresse por calor, auxilia na tomada de decisões para melhorar a produtividade de caprinos em ambiente tropical. Neste contexto, este estudo avaliou as respostas fisiológicas e celulares de cabras Saanen em condição de estresse agudo e crônico. Cabras da raça Saanen saudáveis foram aleatoriamente expostas ao estresse por calor, de curto prazo sob radiação solar e a longo prazo em câmara climática. Os dados foram analizados utilizando o software estatístico \"Statistical Analysis System\" (SAS, 2008). A normalidade foi confirmada com o teste de Shapiro-Wilk. Em estresse agudo, a radiação solar promoveu o aumento da liberação de cortisol, elevou a frequência respiratória e reduziu T3 e T4, para reestabelecer a homeotermia. Observou-se nas células leucocitárias das cabras sob estresse, aumento da expressão da p53 (proteína pró-apoptótica), Bcl-2 (proteína anti-apoptótica), HSP60, HSP70 e HSP90. Sob estresse crônico no final da gestação, as cabras aumentaram a frequência respiratória para perder calor, com elevação do cortisol no dia 15 pré-parto (P <0,05), que foi ainda maior no dia 15 pós-parto. A nível celular, a HSP70 foi a mais expressa durante e após o desafio com calor, com mais transcritos no dia 15 pós-parto para as cabras sob estresse. Nas células epiteliais mamárias, houve aumento dos transcritos relacionados à apoptose, p53 e Bax, para o grupo sob estresse por calor antes do parto. A expressão da HSP27 foi maior antes e após o parto para o mesmo grupo quando comparado aos animais em termoneutralidade. Durante a lactação, as cabras sob estresse apresentaram menor produção de leite. O desafio de calor aumentou o percentual de gordura e diminuiu a lactose. A contagem de células somáticas foi maior para as cabras sob estresse durante toda a lactação. A expressão gênica do receptor de prolactina (PRLr) foi menor para as cabras sob estresse, 15 dias após o parto, o que pode explicar a redução na produção de leite na lactação subsequente. Em conclusão, nossos achados sugerem que a frequência respiratória e a HSP70 são biomarcadores confiáveis para avaliar o conforto térmico e a termotolerância de cabras Saanen sob estresse por calor agudo e crônico. Além disso, o estresse por calor crônico pré-parto prejudicou a produção e a composição do leite da lactação subsequente.

Heat shock protein (HSP)27 is traditionally described as an intracellular chaperone and signaling molecule, but growing evidence suggests it is released from immune cells where it plays an anti-inflammatory role during atherogenesis. Previously, the O’Brien lab found that overexpression of HSP27 led to augmented HSP27 serum levels in female apolipoprotein E knockout (ApoE-/-) mice, attenuated atherogenesis, and inhibited macrophage foam cell formation via physical binding with scavenger receptor (SR)-A. However, the precise mechanism of atheroprotection remained elusive. This thesis sought to ascertain the mechanism(s) by which HSP27 prevents foam cell formation, and determine if SR-A, a key receptor involved in the uptake of lipid into macrophages, plays an important role in HSP27-mediated atheroprotection. Pre-treatment of human macrophages with recombinant HSP27 (rHSP27) inhibited acytelated low density lipoprotein (acLDL) binding and uptake independent from receptor competition effect. Reduction in uptake was associated with attenuation of expression of SR-A mRNA, total protein, and cell surface expression. To explore the signaling mechanism by which HSP27 modulated SR-A expression it was hypothesized that nuclear factor-kappa B (NF-kB), a major regulator of many atherosclerosis gene programs, is altered by extracellular HSP27. Indeed, rHSP27 markedly activated NF-kB signaling in macrophages. Using an inhibitor of NF-kBsignaling there was an attenuation of rHSP27-induced inhibition of SR-A gene and protein expression, as well as lipid uptake, suggesting that SR-A expression is regulated by NF-kB activation. Lastly, to investigate if SR-A is required for HSP27-mediated atheroprotection in vivo, ApoE-/- and ApoE-/-SR-A-/- mice fed a high fat diet were treated with rHSP25, the mouse orthologue of HSP27, or PBS for 3 weeks. While rHSP25 therapy equally reduced serum cholesterol levels in the mouse cohorts, aortic atherogenesis, assessed using en face and sinus cross-sectional analyses, was attenuated in ApoE-/- mice but not ApoE-/-SR-A-/- mice. In conclusion, rHSP27 inhibits foam cell formation by downregulating SR-A expression. This effect may be associated with NF-kB activation. Reductions in atherosclerotic burden by rHSP27 require SR-A, and are independent of changes in serum cholesterol levels, highlighting the importance of macrophage lipid uptake in atherogenesis. Results presented in this thesis demonstrate that SR-A is a major target for HSP27 atheroprotection in the vessel wall, and provide an impetus for further studies that investigate the potential therapeutic value of HSP27.

Les recherches exposées dans ce document portent sur l'étude du rôle de HSF2 au cours du développement du système nerveux central. Les Heat Shock Factors (HSF) sont impliqués dans la réponse au choc thermique et également au cours du développement embryonnaire. Mes travaux ont démontré que HSF2 est requis au cours de la formation du cortex cérébral pour la migration de certains neurones en régulant directement l'expression de p35, sous unité activatrice de CDK5. D'autres cibles ont été identifiées NudE, Dclk, Dab1 nécessaires à la migration des neurones en participant à la dynamique du cytosquelette. De plus, ces travaux montrent que HSF2 module la prolifération et la différenciation des cellules souches neurales (NSC) et des progéniteurs (NP) car i) par électroporation in ovo chez le poulet, la surexpression de HSF2 provoque une augmentation de la prolifération des NP; ii) les NSC Hsf2−/− en culture présentent un retard de prolifération, de survie et de différenciation. Ainsi, HSF2 pourrait assister la décision cellulaire des NSC/NP vers la prolifération ou la différenciation et la migration, tel un aiguilleur de destin cellulaire

Human Epidermal Growth Factor Receptor 2 (HER-2/neu) is a breast tumor antigen (Ag) commonly overexpressed in 30% of breast cancer cases. Both HER-2/neu-targeted DNA-based and transgene modified dendritic cell (DC)-based vaccines are potent elements in eliciting HER-2/neu specific antitumor immune responses; however, there has been no side-by-side comparison of these two different immunization methods. We utilized an in vivo murine tumor model expressing the rat neu Ag to compare the immunization efficacy between DC transduced with replication-deficient adenovirus containing neu (AdVneu), to form DCneu, and plasmid DNA (pcDNA) vaccine. DCneu displayed an upregulation of immunologically important molecules and inflammatory cytokines expression such as IL-6 that partially mediated conversion of the regulatory T (Tr) cell suppression. Wildtype FVB/N mice immunized with DCneu induced stronger HER-2/neu-specific humoral and cellular immune responses compared to plasmid DNA immunized mice. Furthermore, mice immunized with DCneu remained completely protected from tumor challenge compared to partial or no protection observed in DNA immunized mice in two tumor animal models. In FVBneuN transgenic mice, which develop spontaneous breast tumors at 4-8 months of age, DCneu significantly delayed tumor onset when immunization conducted in mice at a younger age. Taken together, we demonstrated that a HER-2/neu-gene modified DC vaccine is more potent than a plasmid DNA vaccine in inducing neu specific immune responses resulting in greater protective and preventative effects in the tumor models examined. <p>In another study, we examined the use of a DC-based cancer vaccine involving the phagocytosis of apoptotic tumor cells expressing heat shock protein 70 (HSP70). The dual role of HSP70, as an antigenic peptide chaperone and danger signal, makes it especially important in DC-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T cell stimulation and overall vaccine efficacy. We found that DC with phagocytosis of MCA/HSP in the early phase of apoptosis expressed more peptide-major histocompatibility class (pMHC) I complexes, stimulated stronger cytotoxic T lymphocytes (CTL) responses and induced greater immune protection against MCA tumor cell challenge, compared to mice immunized with DC that phagocytosed MCA/HSP cells in the late phase of apoptosis. Taken together, our data demonstrated that HSP70 expression on apoptotic tumor cells stimulated DC maturation and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DC with phagocytosis of apoptotic tumor cells in late phase of apoptosis. <p>Overall, we have examined variations in designing DC-based cancer vaccines in two completely different model systems. Taken together, our results may have an important impact in designing DC-based antitumor vaccines.

Human Epidermal Growth Factor Receptor 2 (HER-2/neu) is a breast tumor antigen (Ag) commonly overexpressed in 30% of breast cancer cases. Both HER-2/neu-targeted DNA-based and transgene modified dendritic cell (DC)-based vaccines are potent elements in eliciting HER-2/neu specific antitumor immune responses; however, there has been no side-by-side comparison of these two different immunization methods. We utilized an in vivo murine tumor model expressing the rat neu Ag to compare the immunization efficacy between DC transduced with replication-deficient adenovirus containing neu (AdVneu), to form DCneu, and plasmid DNA (pcDNA) vaccine. DCneu displayed an upregulation of immunologically important molecules and inflammatory cytokines expression such as IL-6 that partially mediated conversion of the regulatory T (Tr) cell suppression. Wildtype FVB/N mice immunized with DCneu induced stronger HER-2/neu-specific humoral and cellular immune responses compared to plasmid DNA immunized mice. Furthermore, mice immunized with DCneu remained completely protected from tumor challenge compared to partial or no protection observed in DNA immunized mice in two tumor animal models. In FVBneuN transgenic mice, which develop spontaneous breast tumors at 4-8 months of age, DCneu significantly delayed tumor onset when immunization conducted in mice at a younger age. Taken together, we demonstrated that a HER-2/neu-gene modified DC vaccine is more potent than a plasmid DNA vaccine in inducing neu specific immune responses resulting in greater protective and preventative effects in the tumor models examined. <p>In another study, we examined the use of a DC-based cancer vaccine involving the phagocytosis of apoptotic tumor cells expressing heat shock protein 70 (HSP70). The dual role of HSP70, as an antigenic peptide chaperone and danger signal, makes it especially important in DC-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T cell stimulation and overall vaccine efficacy. We found that DC with phagocytosis of MCA/HSP in the early phase of apoptosis expressed more peptide-major histocompatibility class (pMHC) I complexes, stimulated stronger cytotoxic T lymphocytes (CTL) responses and induced greater immune protection against MCA tumor cell challenge, compared to mice immunized with DC that phagocytosed MCA/HSP cells in the late phase of apoptosis. Taken together, our data demonstrated that HSP70 expression on apoptotic tumor cells stimulated DC maturation and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DC with phagocytosis of apoptotic tumor cells in late phase of apoptosis. <p>Overall, we have examined variations in designing DC-based cancer vaccines in two completely different model systems. Taken together, our results may have an important impact in designing DC-based antitumor vaccines.

The purpose of this study was to establish whether curcumin protects renal proximal tubule cells against ischemic injury, determine whether this postulated cytoprotective effect is mediated through the upregulation of HSP70, and investigate whether the mechanism by which curcumin induces HSP70 expression and confers its protective effect is through activation of the Unfolded Protein Response. LLC-PK1 cells were cultured on collagen-coated filters to mimic conditions of in vivo renal proximal tubule cells and induce cell polarization. Injury with and without curcumin treatment was studied by using chemically-induced ATP-depletion which mimics renal ischemic injury. Cell injury was assessed using a TUNEL assay in order to evaluate DNA cleavage associated with ischemia-induced apoptosis and actin staining used to assess cytoskeletal disruption. Renal ischemic damage was further investigated by determining detachment of the Na-K ATPase from the basolateral membrane, which represents loss of cell polarity. Cells were incubated with curcumin in a dose- and time-response fashion and subsequent levels of HSP70 expression were assessed. Cells were then incubated with AEBSF, an inhibitor of the Unfolded Protein Response (UPR) and HSP70 and BiP/GRP78 (an ER resident chaperone that is upregulated by the UPR) expression levels were evaluated. Results demonstrated that treatment with curcumin during two hours of injury results in significantly less injury-related apoptosis and cytoskeletal disruption compared to control injured cells. It was demonstrated that curcumin induces HSP70 in both a dose- and time-response fashion. Moreover, curcumin treatment resulted in profound stabilization of Na-K ATPase on the basolateral membranes as there was significantly less Na-K ATPase detachment in cells treated with curcumin during two hours of injury compared to control injured cells. Finally, treatment with AEBSF inhibited HSP70 upregulation in curcumin-treated cells as well as inhibiting the GRP78 over-expression otherwise demonstrated in curcumin-treated cells. Protection of proximal tubule cells against renal ischemic injury by curcumin was therefore indicated to be mediated by the activation of the UPR through which HSP70 is upregulated. Curcumins activation of the UPR and induction of HSP70 explains the stabilization of Na-K ATPase on the cytoskeleton and also provides a potential mechanism explaining many of curcumins therapeutic and protective qualities.