L’inflammasome NLRP3 est un complexe multi-protéique responsable de la production d’IL-1β en réponse à des signaux de danger. Certaines mutations de NLRP3 étant responsables de maladies inflammatoires l’activation de ce complexe se doit d’être finement régulée. Dans cette étude je me suis intéressé à l’importance de la protéine de choc thermique HSP70 dans l’activation de l’inflammasome NLRP3. J’ai dans un premier temps mis en évidence que l’absence d’HSP70 entraine une amplification des symptômes de la péritonite chez la souris. Le manque d’HSP70 augmente également l’activation de la caspase-1 et la production d’IL-1β par les macrophages issus de la moelle osseuse de souris (BMDMs) à la suite d’un traitement par différents activateurs de NLRP3 in vitro. Ces phénomènes sont associés à une augmentation du nombre et de la taille des complexes ASC/NLRP3 dans la cellule. De manière correspondante, la surexpression d’HSP70 dans les BMDMs diminue l’activation de la caspase-1 et la production d’IL-1β après traitement par des activateurs de NLRP3. Une des explications possibles de l’effet inhibiteur d’HSP70 est son interaction avec NLRP3 que j’ai observé par PLA (Proximity Ligation Assay). J’ai également utilisé un choc thermique pour surexprimer HSP70 et observé une inhibition de l’inflammasome NLRP3 in vitro. Finalement, une hyperthermie in vivo inhibe également les symptômes de la péritonite chez la souris, soulignant la relevance physiologique de ces observations. Cette étude fournit donc des preuves de l’effet inhibiteur d’HSP70 sur l’inflammasome NLRP3 et met en lumière un possible nouvel outil de traitement des maladies inflammatoires. / NLRP3 inflammasome is a multi-protein complex aimed at producing IL-1β in response to danger signals. Gain of function mutations of NLRP3 are responsible for inflammatory diseases, so NLRP3-dependent inflammation required tight regulation. Here we investigated the importance of the stress sensor, Heat Shock Protein 70 (HSP70) on the NLRP3 inflammasome activation. First, the lack of HSP70 leads to a worsening of NLRP3-dependent peritonitis in mice. HSP70 deficiency also enhances caspase-1 activation and IL-1β production by murine Bone Marrow-Derived Macrophages (BMDMs) under NLRP3 activators treatment in vitro. These phenomena are associated with an increase in the number and size of ASC/NLRP3 specks. At the opposite side, the overexpression of HSP70 in BMDMs decreases caspase-1 activation and IL-1β production under NLRP3 activators treatment in vitro. One possible explanation of the inhibitory effect of HSP70 is its interaction with NLRP3. A heat shock, used as a way to induce the expression of HSP70 also inhibits the NLRP3 inflammasome activation in vitro. Finally, in vivo hyperthermia also inhibits peritonitis features in mice, highlighting the physiological relevance of our observations. This study provides evidences on the inhibitory role of HSP70 on the NLRP3 inflammasome and on the possibility to treat inflammatory diseases by inducing its expression, mainly by hyperthermia.

Les HSP ou protéines de stress ont été découvertes chez la drosophile par Ritossa en 1962. Hautement conservées entre les espèces, elles sont essentielles à l’homéostasie cellulaire et plus encore à la survie lors de stress d’origine diverse (chimique, physique, métabolique etc…). Actuellement, chez les mammifères, il existe cinq principales familles d'HSPs en fonction de leur poids moléculaires : HSP110, HSP90, HSP70 et HSP60 et les petites HSPs à laquelle appartient HSP27 (Kampinga et al., 2009). Parmi les HSPs, HSP70 est la plus fortement induite que ce soit par des agressions telles que le stress oxydatif, les agents anticancéreux ou les radiations ionisantes. HSP70 est, contrairement aux cellules « normales », fortement exprimée dans les cellules cancéreuses et confère à ces cellules une résistance aux drogues anticancéreuses (Goloudina, Demidov & Garrido, 2012) favorisant ainsi le développement tumoral. Les propriétés cyto-protectrices de HSP70 ont été jusqu’à présent attribuées par ces fonctions intracellulaires, principalement en inhibant le processus apoptotique à différentes étapes clés de la signalisation cellulaire (Ravagnan et al., 2001). Cependant une forme membranaire de HSP70 a été détectée à la surface des exosomes dérivant des cellules tumorales (Kuppner et al., 2001). HSP70 membranaire participerait au processus de tumorigenèse en inhibant l’activation des cellules myéloïdes suppressives (MDSCs) et en conséquence, la réponse immune anti-tumorale (Pfister et al., 2007; Schmitt, Gehrmann, Brunet, Multhoff, & Garrido, 2007). Par cette double facette, HSP70 représente donc une cible thérapeutique de choix pour la thérapie anticancéreuse. Compte tenu du rôle clé de HSP70, à la fois par ses fonctions intracellulaires et extracellulaires dans le développement tumoral, l’un des projets phare de notre groupe a été de développer des inhibiteurs spécifiques de HSP70, notamment des aptamères peptidiques et des peptides. Dans un premier travail, nous avons démontré in vitro que deux aptamères A8 et A17 (et son dérivé P17), interagissent avec HSP70 sur des domaines différents et sensibilisent les cellules cancéreuses à la mort induite par des agents chimiothérapeutiques. Des études in vivo réalisées chez la souris et le rat confirment ces résultats et mettent en évidence une réduction significative de la croissance tumorale par ces aptamères peptidiques. Dans un deuxième travail, nous avons généré un dérivé de l’aptamère A8, le peptide P8.1. Nous avons démontré que ce peptide est capable de neutraliser la forme membranaire de HSP70, présente à la surface des exosomes, bloquant ainsi l’activation des MDSCs et restaurant la réponse immunitaire anti-tumorale. A plus long terme, ce travail vise à mettre au point et à valider en clinique une thérapie anticancéreuse plus efficace, en associant aux traitements anticancéreux actuels, les inhibiteurs de HSP70. / Heat shock proteins (HSPs) were first discovered in Drosophila by Ritossa in 1962. As stress proteins, HSPs are induced in response to a wide variety of physiological and environmental insults. HSPs have a cyto-protective function and act as molecular chaperones by assisting the folding of nascent or misfolded proteins and by preventing their aggregation. Mammalian HSPs have been classified into 5 families according to their molecular weight: HSP110, HSP90, HSP70, HSP60 and the family of small HSPs such as HSP27 (Kampinga et al., 2009). The most well-known inducible stress chaperone HSP70 is hardly detectable at basal level in normal “non-stressed” cells, but in cancer cells HSP70 is constitutively highly expressed. In that respect, this HSP play a key role in oncogenesis and in resistance to chemotherapeutic drugs (Goloudina et al., 2012).Until now, the cytoprotective properties of HSP70 were attributed to its intracellular functions mainly via its ability to block the apoptotic process at key points of the signal (Ravagnan et al., 2001). More recently, a membrane bound form of HSP70 was detected but also at the surface of exosomes derived from tumor cells but not non-cancerous cells (Kuppner et al., 2001). Moreover, growing evidence support the critical role of this membrane-bound HSP70 in the process of tumorigenesis (Pfister et al., 2007; Schmitt et al., 2007) via the activation of myeloid suppressor cells (MDSCs), which inhibit the anti-tumor immune response (Chalmin et al., 2010). Thereby, HSP70 by this dual action represents an attractive target for new anti-cancer therapy.In that aim, we developed specific inhibitors of HSP70, including peptide aptamers and peptides. In this work, we demonstrated that two aptamers A8, A17 (and the peptide P17), interact with different domains of HSP70 and, significantly sensitized cancer cells to apoptosis induced by chemotherapeutic drugs. Accordingly, in vivo studies in mice and rats showed a significant reduction of tumor growth by these inhibitors. Finally, we generate an A8 derived peptide called P8.1 that specifically neutralized the extracellular region of HSP70 at the surface of exosomes. Our results demonstrated that this peptide P8.1 inhibits MDSC activation and restored the antitumor immune response in vitro and in vivo, respectively.Overall, our work will help to develop and validate more effective cancer therapy based on the association of conventional chemotherapy with HSP70 inhibitors.

Neste trabalho foi avaliado como caranguejos Ucides cordatus, provenientes de ambientes preservados e impactados pela ação antrópica, respondem a exposição ao cádmio através da regulação da expressão da HSP70 (Heat Shock Proteins). O caranguejo de manguezal Ucides cordatus é uma espécie típica de ambiente estuarino, amplamente utilizado como recurso pesqueiro pelas comunidades locais. Vários estudos já foram desenvolvidos, tratando a maioria deles da sua dinâmica populacional ou aspectos de manejo. Por isso, a avaliação do seu estado fisiológico se faz importante para a complementação destes estudos ecológicos bem como na identificação dos impactos causados à sobrevivência desses organismos pela ação antrópica. Para avaliar a influência dos poluentes foram escolhidas duas áreas para coleta no litoral de São Paulo. Uma área impactada, Cubatão, e outra bem preservada, Juréia. Os animais foram expostos a 5mg/L de cloreto de cádmio, diluído na água dos aquários por até 24 horas, em intervalos de tempo de 30 minutos, 1, 2, 6 e 12 horas. A expressão de HSP70 foi medida no hepatopâncreas e brânquias através de imunoensaios utilizando o método ELISA. Foi observado que a sobrevivência dos organismos é influenciada pela manutenção em laboratório durante aclimatização bem como pela exposição ao cádmio, entretanto, os organismos provenientes de áreas preservadas se mostram mais responsivos ao estresse. A regulação de HSP70 foi pouco alterada em ambos os grupos e nos diferentes órgãos, não sendo considerada como um bom biomarcador para este tipo de estresse no organismo modelo utilizado / This study evaluated how the crab Ucides cordatus from impacted and preserved environments responds to cadmium exposure through the expression of HSP70 (Heat Shock Proteins). The mangrove crab Ucides cordatus is an estuarine specimen, tipically used as fishing resource by local communities. Several studies have been developed, most of them dealing with population dynamics and management aspects. Therefore, it is important to comprehend their physiological status to complement these ecological studies and to understand the impact caused by human action in the survival of these organisms. The species was collected in two different areas of São Paulo coast, polluted and conserved areas, Cubatão and Juréia, respectively. These two sites were chosen to evaluate the influence of pollutants in the organism physiology. At the laboratory the animals were exposed to 5mg/L of cadmium chloride, diluted directly in the water. After an interval of 30 minutes, 1, 2, 6 and 12 hours, HSP70 expression was measured in the hepatopancreas and gills using immunoassays through ELISA method. It was observed that the survival of the organisms was influenced by laboratory management as well as by exposure to cadmium, however, the organism from preserved areas were more responsive to stress. The regulation of HSP70 was little changed in both groups and in different organs, so maybe it should not be considered as a good biomarker for this type of stress

Oilseed and oilseed meal extracted from members of <i>Brassicaceae</i> release broadspectrum biocidal isothiocyanate when ground and exposed to moisture. The compounds are released when the seed enzyme myrosinase catalyzes the hydrolysis of glucosinolates producing glucose, sulfate, and pesticidal isothiocyanates. Allylisothiocyanate (AITC), the predominant isothiocyanate of <i>Brassica juncea</i>, has broad-spectrum biological activities against plants, animals and fungi. Knowledge of the concentration of AITC arising from a treatment with mustard and AITC toxicity to many target and non-target species is not known. Therefore, factors affecting AITC release and assays of mustard toxicity were conducted. The rate of AITC release from mustard meal was affected by temperature and pH. Current isothiocyanate extraction and quantification methods measure a change in the concentration of glucose (a predominant product of myrosinase-catalysed glucosinolate hydrolysis) to determine myrosinase activity. The objectives of this work were to study: 1) factors affecting myrosinase activity in mustard (<i>Brassica juncea</i>), 2) the effects of AITC on seed germination and 3) the toxicity of AITC and mustard meal.<p> Attempts were made to improve the Herb and Spice Method, the only available industrial method to measure total isothiocyanate production in mustard meal. The effects of a wide range of reaction temperatures (7 to 97°C) and incubation times (0 min to 2 h) on myrosinase-catalyzed conversion of sinigrin (a glucosinolate) to allyl isothiocynate (AITC) were studied. Significant inhibition of enzyme activity was observed at all temperatures over 57°C, and at 97°C no myrosinase activity was found. It was concluded that myrosinase-catalyzed conversion of sinigrin to AITC was a rapid process and detectable amounts of AITC could be found in samples in two min, and that higher temperatures inhibited myrosinase activity. The pH of the reaction mixture significantly affected myrosinase-catalyzed conversion of sinigrin to AITC. A change in pH did not affect the substrate, but severely affected the activity of myrosinase. Furthermore, other compounds viz., boric acid (H3BO3), succinic acid (C2H4(COOH)2),calcium chloride (CaCl2) and ethanol (C2H5OH), were explored for their ability to inhibit myrosinase activity. Calcium chloride and ethanol were particularly effective.<p> It was hypothesized that AITC might act as a plant growth promoter/regulator based on the fact that AITC and ethylene, a plant growth regulator, exhibit structural similarity (R-CH=CH2, where R is -CH2SCN and -H in AITC and ethylene, respectively). Therefore, AITC might act as an ethylene analogue. Ethylene is known to promote seed germination and overcome seed dormancy in a dose- and species-dependent manner.Flax and tomato seeds were used as model systems to test the germination enhancing properties of AITC. It was concluded that AITC promoted flax and tomato seed germination and thus might be used for this application in agricultural practice.<p> An assay was developed for testing AITC toxicity in ground seed by exploring HSP70 expression in <i>Caenorhabditis elegans</i> as a marker of toxicity. <i>C.elegans</i> strain N2 was exposed to different concentrations (0 to 10 ìM) of AITC for 2 h at room temperature. Western blotting with anti-<i>HSP70</i> antibody showed a marked increase in the expression of <i>HSP70</i> protein in a dose-dependent manner. Assays of the expression of <i>HSP70A</i> mRNA by quantitative real time reverse transcriptase (RT) PCR revealed no significant change in the expression of <i>HSP70A</i> mRNA at low concentrations of AITC (< 0.1 ìM). However, treatment with higher concentrations (>1ìM) resulted in four- to five - fold increase in expression of <i>HSP70A</i> mRNA over the control. To understand if mustard toxicity was due to AITC alone, or if other compounds in mustard ground seed affected <i>HSP70</i> transcript production, <i>C. elegans</i> was exposed to AITC or <i>Brassica juncea</i> cv. Arrid ground seed (Arrid is a mustard variety with a lower level of sinigrin (<3 ìM per gram of seed), or both. ELISA revealed increased expression of HSP70 protein in C. elegans treated with AITC + ground seed, but the level of protein was less than that observed with AITC alone. These results indicated that mustard ground seed toxicity was contributed primarily by AITC, and that some ground seed components antagonized AITC toxicity in <i>C. elegans</i>.

Oilseed and oilseed meal extracted from members of <i>Brassicaceae</i> release broadspectrum biocidal isothiocyanate when ground and exposed to moisture. The compounds are released when the seed enzyme myrosinase catalyzes the hydrolysis of glucosinolates producing glucose, sulfate, and pesticidal isothiocyanates. Allylisothiocyanate (AITC), the predominant isothiocyanate of <i>Brassica juncea</i>, has broad-spectrum biological activities against plants, animals and fungi. Knowledge of the concentration of AITC arising from a treatment with mustard and AITC toxicity to many target and non-target species is not known. Therefore, factors affecting AITC release and assays of mustard toxicity were conducted. The rate of AITC release from mustard meal was affected by temperature and pH. Current isothiocyanate extraction and quantification methods measure a change in the concentration of glucose (a predominant product of myrosinase-catalysed glucosinolate hydrolysis) to determine myrosinase activity. The objectives of this work were to study: 1) factors affecting myrosinase activity in mustard (<i>Brassica juncea</i>), 2) the effects of AITC on seed germination and 3) the toxicity of AITC and mustard meal.<p> Attempts were made to improve the Herb and Spice Method, the only available industrial method to measure total isothiocyanate production in mustard meal. The effects of a wide range of reaction temperatures (7 to 97°C) and incubation times (0 min to 2 h) on myrosinase-catalyzed conversion of sinigrin (a glucosinolate) to allyl isothiocynate (AITC) were studied. Significant inhibition of enzyme activity was observed at all temperatures over 57°C, and at 97°C no myrosinase activity was found. It was concluded that myrosinase-catalyzed conversion of sinigrin to AITC was a rapid process and detectable amounts of AITC could be found in samples in two min, and that higher temperatures inhibited myrosinase activity. The pH of the reaction mixture significantly affected myrosinase-catalyzed conversion of sinigrin to AITC. A change in pH did not affect the substrate, but severely affected the activity of myrosinase. Furthermore, other compounds viz., boric acid (H3BO3), succinic acid (C2H4(COOH)2),calcium chloride (CaCl2) and ethanol (C2H5OH), were explored for their ability to inhibit myrosinase activity. Calcium chloride and ethanol were particularly effective.<p> It was hypothesized that AITC might act as a plant growth promoter/regulator based on the fact that AITC and ethylene, a plant growth regulator, exhibit structural similarity (R-CH=CH2, where R is -CH2SCN and -H in AITC and ethylene, respectively). Therefore, AITC might act as an ethylene analogue. Ethylene is known to promote seed germination and overcome seed dormancy in a dose- and species-dependent manner.Flax and tomato seeds were used as model systems to test the germination enhancing properties of AITC. It was concluded that AITC promoted flax and tomato seed germination and thus might be used for this application in agricultural practice.<p> An assay was developed for testing AITC toxicity in ground seed by exploring HSP70 expression in <i>Caenorhabditis elegans</i> as a marker of toxicity. <i>C.elegans</i> strain N2 was exposed to different concentrations (0 to 10 ìM) of AITC for 2 h at room temperature. Western blotting with anti-<i>HSP70</i> antibody showed a marked increase in the expression of <i>HSP70</i> protein in a dose-dependent manner. Assays of the expression of <i>HSP70A</i> mRNA by quantitative real time reverse transcriptase (RT) PCR revealed no significant change in the expression of <i>HSP70A</i> mRNA at low concentrations of AITC (< 0.1 ìM). However, treatment with higher concentrations (>1ìM) resulted in four- to five - fold increase in expression of <i>HSP70A</i> mRNA over the control. To understand if mustard toxicity was due to AITC alone, or if other compounds in mustard ground seed affected <i>HSP70</i> transcript production, <i>C. elegans</i> was exposed to AITC or <i>Brassica juncea</i> cv. Arrid ground seed (Arrid is a mustard variety with a lower level of sinigrin (<3 ìM per gram of seed), or both. ELISA revealed increased expression of HSP70 protein in C. elegans treated with AITC + ground seed, but the level of protein was less than that observed with AITC alone. These results indicated that mustard ground seed toxicity was contributed primarily by AITC, and that some ground seed components antagonized AITC toxicity in <i>C. elegans</i>.

Le réseau de protéines chaperons est hautement conservé dans l'ensemble du vivant. Il régule l'homéostasie des protéines au sein de la cellule en condition de croissance normale ainsi qu'en réponse à des stress environnementaux. Les chaperons membres de la famille HSP70 (Heat Shock Protein 70 kDa), famille particulièrement conservée, agissent tout au long de la biogénèse des protéines et orchestrent une pléthore de processus cellulaires liés au repliement et/ou au remodelage de protéines. Le cycle ATP-dépendant du chaperon HSP70 repose sur une étroite collaboration avec ses partenaires co-chaperons. Parmi ces co-chaperons, on distingue les membres de la famille DnaJ/HSP40 qui transfèrent les substrats vers HSP70 et stimulent son activité ATPasique, et les facteurs d'échange de nucléotides qui assurent la réinitialisation du cycle d'HSP70 permettant ainsi la libération du substrat. Au sein de la bactérie E. coli, la protéine HSP70 est appelée DnaK. Elle agit de concert avec les deux co-chaperons DnaJ et GrpE (ensemble nommés DnaKJE) afin d'assister les protéines dans leur repliement au cours de la synthèse de novo, de désagréger des protéines mal repliées, de faciliter l'adressage et le passage de protéines à travers les membranes biologiques, et de remodeler certains complexes protéiques impliqués dans des processus cellulaires variés. DnaKJE coopère efficacement avec d'autres systèmes chaperons majeurs, tels que la protéine Trigger Factor (TF) associée aux ribosomes et le complexe chaperonine GroESL, notamment pour le repliement de protéines nouvellement synthétisées dans le cytosol. De plus, une des fonctions cellulaires majeure du système DnaKJE est son implication dans la réponse au stress thermique (Heat Shock Response - HSR). DnaKJE contrôle la HSR en interagissant directement avec le facteur de transcription s32, sous-unité de l'ARN polymérase. Cette interaction facilite la dégradation de s32 par la protéase FtsH. En condition de stress, l'accumulation de protéines mal repliées au sein de la cellule entraine le recrutement de DnaKJE et par conséquent, la stabilisation de s32. Suite à cette stabilisation, une induction de la transcription de plus d'une centaine de gènes codant entre autres, pour des protéines chaperons et des protéases se met en place dans la cellule pour lutter contre le stress environnant. De ce fait, DnaK et ses co-chaperons sont considérés comme des éléments clés de la réponse cellulaire contre le collapse de l'homéostasie protéique par action directe sur des protéines mal repliées et indirecte en modulant la synthèse de nombreuses HSPs, via s32. L'étude récente de l'intéractome de DnaK révèle qu'au moins 50% des enzymes impliquées au sein du métabolisme central (MC) de la cellule interagissent avec DnaK à température physiologique. A travers l'analyse d'une banque de suppresseurs multi-copie, nous avons identifié six gènes associés au MC : ackA, ldhA, lpd, pykF, talB et csrC qui lorsqu'ils sont surexprimés, permettent de restaurer partiellement le défaut de croissance d'une souche mutante n'exprimant pas les chaperons DnaK et Trigger Factor (deltatig deltadnaKJ). Remarquablement, la surexpression d'ackA, talB et csrC supprime également le défaut de croissance d'un mutant dnaK à haute température, ce qui suggère une implication importante de DnaK au niveau du MC. Dans ce projet, l'implication de DnaK dans le fonctionnement du métabolisme carboné a été établi par une analyse métabolique combinant analyses macro-cinétiques (suivi de croissance, analyse de la consommation des substrats et de la production de produits du métabolisme) sur différentes sources de carbones seules ou en mélange et analyses micro-cinétiques (flux métaboliques par marquage 13C). Finalement, ces travaux apportent différentes hypothèses quant au rôle de DnaK dans le contrôle du MC, directement ou indirectement via la régulation de la HSR, en réponse à une défaillance de l'homéostasie protéique ou d'une carence nutritionnelle. / Intricate networks of highly conserved molecular chaperone machines govern cellular protein homeostasis, both under lenient and more stressful growth conditions. Members of the highly conserved HSP70 family of molecular chaperones are key players in this process, acting at nearly every step in protein biogenesis. The ATP-dependent chaperone cycle of HSP70 chaperones relies upon the cooperation with a cohort of essential cochaperones, including DnaJ/HSP40 family members that recruit the chaperone to specific substrate and/or cellular localization and stimulate its ATPase activity, and nucleotide exchange factors, which insure proper resetting of the chaperone cycle and the resulting substrate release. In the bacterium Escherichia coli, the multifunctional HSP70 chaperone, named DnaK, acts in concert with its cochaperones DnaJ and GrpE (all together referred as DnaKJE) to efficiently, assist de novo protein folding, protein disaggregation, protein targeting and translocation through biological membranes, and protein complexes remodeling leading to multiple cellular activities. Remarkably, previous works also showed that DnaKJE can efficiently cooperate with other major cytosolic chaperones, including the ribosome-bound Trigger Factor (TF) and the chaperonin GroESL, especially during the folding of newly-synthesized cytosolic proteins. In addition, one of the key cellular functions of DnaKJE in E. coli is the regulation of the heat shock response (HSR). In this case, DnaKJE controls the HSR by interacting directly with the heat shock sigma factor s32 subunit of the RNA polymerase to facilitate it degradation by the FtsH protease. Under stress condition, DnaKJE is recruited to accumulating misfolded proteins, leading to an increased stability of s32 and the subsequent induction of more than hundred heat shock proteins. Therefore, DnaK, and its cochaperones are central components of the cellular response to proteostasis collapse, both by acting directly on misfolded proteins and by modulating the synthesis a plethora of heat shock chaperones and proteases. The recently described in vivo interactome of DnaK in E. coli revealed that at least 50% of the central metabolism enzymes interact with DnaK at physiological temperature. Remarkably, through a multicopy suppression analysis we have now identified six genes of the central metabolism (CM), namely ackA, ldhA, lpd, pykF, talB and csrC, which when overexpressed partially suppress the growth defect of the sensitive double mutant lacking DnaK and Trigger Factor (deltatig deltadnaKJ ), with half of them, namely ackA, talB and csrC, additionally suppressing the growth defect of the single ?dnaKJ mutation at high temperature, thus strongly suggesting a major role of DnaK in this process. Using a combination of growth assays on specific carbon sources entering the CM at various metabolic nodes with NMR analyses for characterizing the carbon source assimilation, identifying and quantifying the metabolism by-products and determining metabolic flux rearrangements, we show that DnaKJE impacts the responsiveness of the central metabolism by acting either directly at the level of the CM or along the first step of substrate assimilation. How does the multifunctional DnaK chaperone modulate the CM, either directly or indirectly via the control of the HSR, in response to proteostasis failure or nutrient starvation is discussed.

HSP70 plays an important role in cancer development. However, the molecular role of HSP70 in cancer is poorly understood. Previous work from our laboratory demonstrated that HSP70 is essential for initiation of Her2-positive breast cancer by controlling oncogene-induced senescence. Here we demonstrate that HSP70 is critical for both initiation and progression of mammary cancer. Interestingly, the role of HSP70 in cancer development did not involve its canonical function as a molecular chaperone. Instead, HSP70 had multiple effects on signaling pathway components related to tumor initiation, growth, and metastasis, such as FOXM1, HIF1, NF-𝜅B, and SRC. HSP70 regulated signaling networks via association with the co-chaperone, BAG3, a scaffold protein with capacity to interact with multiple key regulators of cell signaling. Using SRC as a model, we demonstrated that association with HSP70 attenuates BAG3's interaction with the SH3 domain of SRC. We also show that an HSP70-interacting small molecule, YM-1, can specifically inhibit the HSP70-BAG3 signaling axis, leading to selective inhibition of tumor growth in vivo and in vitro. This compound mimicked the effects seen with depletion of HSP70 in a dose dependent manner, providing a proof of principle that the association of HSP70 and BAG3 is needed for regulation of these pathways. Additionally, a second generation of YM-1 analogs, JG-98 and JG-84, were shown to be more potent than YM-1 while acting in a similar fashion on signaling pathways. A less potent analog, JG-36, was not able to modulate these pathways as effectively. These studies demonstrate that the HSP70-BAG3 axis is a major regulator of cancer signaling and suggest that targeting the interface between HSP70 and BAG3 is a novel therapeutic approach.

Cadmium (Cd) is a toxic metal known to accumulate in and have adverse effects on the olfactory systems of fish. The objective of this project was to investigate the effect of cadmium on zebrafish larvae, specifically the effects on the olfactory system at cellular and functional levels. Zebrafish larvae (72 hours post fertilization) were exposed to sublethal concentrations of cadmium (0.5, 1, 5, and 10 µM) for 96 h. Whole-body cadmium accumulation during this exposure period as quantified using GFAAS (graphite furnace atomic absorption spectroscopy) was found to increase with both exposure length and concentration. Using a transgenic strain of hsp70/eGFP (heat shock protein 70/enhanced green fluorescent protein reporter gene) zebrafish, dose-dependent induction of the heat shock response was observed in the olfactory epithelium. Expression of hsp70/eGFP in the olfactory epithelium was a highly sensitive biomarker for the effects of cadmium in the olfactory system with a lowest observed effects concentration (LOEC) of 0.5 µM Cd. Strong induction of the transgenic reporter gene correlated closely with cell death (LOEC of 5 µM Cd) and histological alterations (LOEC of 1 µM Cd) in the olfactory epithelium of zebrafish larvae following cadmium exposure. Additionally, loss of sensory cilia from the surface of the olfactory epithelium was observed in larvae exposed to 5 and 10 µM Cd. Furthermore, behaviour tests to assess olfactory function revealed sensory deficits, likely due in part to the cadmium-induced degeneration of the olfactory epithelium (p<0.05 for 1 µM; p<0.001 for 5 and 10 µM Cd). <p>To determine if cadmium was entering the cells of the olfactory epithelium by acting as a calcium (Ca) antagonist, zebrafish larvae were co-exposed to 1, 5, or 10 µM Cd with 1 or 5 mM Ca for 96 h. Whole-body cadmium accumulation as quantified using ICP-MS (inductively coupled plasma mass spectrometry) was decreased in larvae co-exposed to cadmium and calcium. Additionally, induction of the heat shock response was reduced in the presence of increasing calcium co-treatment. These ameliorating effects of calcium were further revealed in cell death and histological analyses of the olfactory epithelium. Also, larvae co-exposed to cadmium and calcium exhibited greater olfactory sensory function compared to larvae exposed to cadmium only. Significant increases in aversion response were observed in larvae exposed to 5 µM Cd with 1 and 5 mM Ca (p<0.05). These results indicate that cadmium gains entry to the olfactory epithelium via calcium uptake systems, wherein it causes damage to the olfactory system and can lead to sensory impairment.

Cadmium (Cd) is a toxic metal known to accumulate in and have adverse effects on the olfactory systems of fish. The objective of this project was to investigate the effect of cadmium on zebrafish larvae, specifically the effects on the olfactory system at cellular and functional levels. Zebrafish larvae (72 hours post fertilization) were exposed to sublethal concentrations of cadmium (0.5, 1, 5, and 10 µM) for 96 h. Whole-body cadmium accumulation during this exposure period as quantified using GFAAS (graphite furnace atomic absorption spectroscopy) was found to increase with both exposure length and concentration. Using a transgenic strain of hsp70/eGFP (heat shock protein 70/enhanced green fluorescent protein reporter gene) zebrafish, dose-dependent induction of the heat shock response was observed in the olfactory epithelium. Expression of hsp70/eGFP in the olfactory epithelium was a highly sensitive biomarker for the effects of cadmium in the olfactory system with a lowest observed effects concentration (LOEC) of 0.5 µM Cd. Strong induction of the transgenic reporter gene correlated closely with cell death (LOEC of 5 µM Cd) and histological alterations (LOEC of 1 µM Cd) in the olfactory epithelium of zebrafish larvae following cadmium exposure. Additionally, loss of sensory cilia from the surface of the olfactory epithelium was observed in larvae exposed to 5 and 10 µM Cd. Furthermore, behaviour tests to assess olfactory function revealed sensory deficits, likely due in part to the cadmium-induced degeneration of the olfactory epithelium (p<0.05 for 1 µM; p<0.001 for 5 and 10 µM Cd). <p>To determine if cadmium was entering the cells of the olfactory epithelium by acting as a calcium (Ca) antagonist, zebrafish larvae were co-exposed to 1, 5, or 10 µM Cd with 1 or 5 mM Ca for 96 h. Whole-body cadmium accumulation as quantified using ICP-MS (inductively coupled plasma mass spectrometry) was decreased in larvae co-exposed to cadmium and calcium. Additionally, induction of the heat shock response was reduced in the presence of increasing calcium co-treatment. These ameliorating effects of calcium were further revealed in cell death and histological analyses of the olfactory epithelium. Also, larvae co-exposed to cadmium and calcium exhibited greater olfactory sensory function compared to larvae exposed to cadmium only. Significant increases in aversion response were observed in larvae exposed to 5 µM Cd with 1 and 5 mM Ca (p<0.05). These results indicate that cadmium gains entry to the olfactory epithelium via calcium uptake systems, wherein it causes damage to the olfactory system and can lead to sensory impairment.

Submitted by Andre Moraes Queiroz (andre.moraesqueiroz@ufpe.br) on 2015-04-14T14:38:12Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Evilis da Silva Monte.pdf: 1778277 bytes, checksum: 32ce58e371b16f1861c19808c2c73f9f (MD5) / Made available in DSpace on 2015-04-14T14:38:12Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Evilis da Silva Monte.pdf: 1778277 bytes, checksum: 32ce58e371b16f1861c19808c2c73f9f (MD5) Previous issue date: 2013 / CNPq / A disponibilidade de 12 genomas sequenciados de espécies do gênero Drosophila enfatizou a importância da citogenética, incluindo a construção de fotomapas dos cromossomos politênicos de outras espécies do gênero. Esta ferramenta é relevante para a localização de genes, detecção de diversidade genética entre populações e para a verificação de inferências evolutivas. A proposta deste trabalho foi de ampliar os estudos citogenéticos no gênero Drosophila, através da construção do fotomapa dos cromossomos politênicos e mapeamento gênico em Drosophila sturtevanti, devido à escassez de dados citogenéticos do grupo saltans. Para a preparação do fotomapa foi utilizada a linhagem DIR (Dois Irmãos, Recife) coletada no Campus da UFRPE em 2009. O fotomapa mostrou que o complemento cromossômico é constituído por cinco braços eucromáticos. O par I é submetacêntrico, representado pelos braços XL e XR, o par II é metacêntrico representado pelos braços IIL e IIR e o cromossomo III é acrocêntrico. Foram observadas duas diferentes inversões paracêntricas, uma no braço XL e outra no cromossomo III. Para os genes Hsp70 e Hsp83 foram identificadas apenas uma marcação relevante, no cromossomo III (seção 86) e no braço XR (seção 32), respectivamente. Os resultados encontrados possibilitaram a comparação do genoma de D. sturtevanti com o genoma de outras espécies e, ainda, sugerem a homologia cromossômica entre os grupos saltans, willistoni e melanogaster.