Diante do quadro atual de previsões de mudanças climáticas, estudos a respeito das possíveis respostas dos organismos a estas alterações são importantes. Com a finalidade de prever e verificar se estas serão de fato deletérias ou se os organismos são capazes de lidar com elas sem alterações na sua fisiologia, e consequentemente na estrutura do ambiente, E. brasiliensis foi utilizada como modelo para estudar possíveis impactos do aumento da temperatura e acidificação dos oceanos na sua fisiologia. Para isso, espécimes foram expostos a 9 possíveis combinações de temperatura (24ºC, 28ºC e 30ºC) e pH (8.0, 7.7 e 7.3) em diferentes intervalos de tempo (1, 3, 12, 24 e 48 h). Amostras de gônadas e fluido celomático foram coletadas para avaliar a expressão das proteínas de estresse HSP70, AIF-1 e p38-MAPK, e a variação no número e viabilidade dos celomócitos. Nossos resultados mostram que o modelo é sensibilizado pelas mudanças no ambiente, através da hiper-regulação das proteínas de estresse. O cenário considerado mais extremo (30°C + pH7.3) ocasionou a morte de 100% dos organismos após 24horas. E o segundo cenário mais severo (30°C + pH7.7) desencadeou o desenvolvimento de ulceração de pele. Os efeitos são mais pronunciados nos celomócitos e a acidificação da água parece ter efeitos antagônicos com a temperatura nos celomócitos e sinérgicos nas gônadas. Embora a resposta tenha sido sistêmica, o grau e a dinâmica foram distintos em relação às diferentes amostras e estresses. Podendo causar modificações na resposta imune dos organismos e consequentemente na sobrevivência da espécie a longo prazo. / Under the current Climate Change context, studies about the potential responses of the organisms to their changing environment are of extreme importance. Recent studies point out the synergy of temperature and ocean acidification altogether. In this study, we used the sea star E. brasiliensis to assess the physiological effects of rising temperature, seawater acidification and the interaction of both factors. Independent individuals (N=225) were exposed to 9 possible combinations of temperature (24ºC, 28ºC and 30ºC) and pH (8.0, 7.7 and 7.3), for 1, 3, 12, 24 and 48 h. We compared the stress produced by these treatments measuring the expression of heat shock proteins (HSP70), the production of the allograft inflammatory factor (AIF−1) and the activation of mitogen kinases (MAPKs) at both gonad and celomic fluid. Furthermore, we assessed the quantity and quality of coelomocytes. Our results demonstrated that E. brasiliensis is vulnerable to the interaction of temperature and acidification. All the stress proteins evaluated were upregulated. The extreme scenario (30°C + pH7.3) caused the death of 100% of organisms after 24 hours, while the second most severe scenario (30°C + pH7.7) triggered skin ulceration. Nevertheless, we found that water acidification produces antagonistic effects to the temperature in coelomocytes and synergistic effects in gonad cells. Furthermore, these effects were more pronounced in the coelomocytes than in the gonads. The systemic response found in this study suggest that the interactive effects of elevated temperatures in conjunction with ocean acidification may endanger the survival of this species, and it could compromise the ecosystem functioning at long term.

A leishmaniose é uma enfermidade infecciosa causada por várias espécies de parasitas do gênero Leishmania e representa um dos principais problemas de saúde pública nos países subdesenvolvidos. No hospedeiro, a sobrevivência do protozoário causador dessa doença depende de uma classe especial de proteínas, as chaperonas moleculares ou proteínas de choque térmico como também são conhecidas. A função dessas proteínas é auxiliar no processo de enovelamento protéico, no transporte de proteínas entre as membranas e em muitas outras importantes funções celulares. Neste processo, as chaperonas moleculares são auxiliadas pelas suas co-chaperonas que desempenham função de destaque. Dentre as principais famílias de chaperonas moleculares temos as Hsp70 e as Hsp90 com suas respectivas co-chaperonas, as Hsp40 e a Aha1. O presente trabalho pretendeu inicialmente expressar e purificar as co-chaperonas moleculares Hsp40I e Hsp40II de L. braziliensis para realizar estudos de caracterização estrutural por meio das técnicas de dicroísmo circular e fluorescência. Contudo, a insolubilidade dessas proteínas, que pode ter sido causada pela presença de mutações nas sequências de DNA, motivou a caracterização de outra co-chaperona, a Aha1 de L. braziliensis (LbAha1). A LbAha1 foi expressa no sobrenadante celular e purificada por três etapas cromatográficas (troca aniônica, afinidade por íons cálcio e gel filtração). A análise da sequência de aminoácidos dessa proteína mostra que ela possui 9 resíduos de triptofano distribuídos nos dois domínios característicos da LbAha1. Estudos de desnaturação química por uréia, monitorados pelas técnicas de dicroísmo circular e fluorescência, mostram que os dois domínios da LbAha1 apresentam estabilidades diferentes. Os estudos estruturais realizados permitiram identificar as transições com o respectivo domínio. / Leishmaniasis is an infectious disease caused by several species of Leishmania species and represents major public health problems in developing countries. In the harborer, the survival of the parasite that cause this disease depends on a special class of proteins, molecular chaperones or heat shock proteins as they are also known. The function of these proteins is to assist in protein folding, transport of proteins and many other important cellular functions. In this process the molecular chaperones are helped by their co-chaperones that play a prominent role. Among the main families of molecular chaperones, there are Hsp70 and Hsp90 with their respective co-chaperones, Hsp40 and the Aha1. The present work, initially pretended to express and purify the molecular co-chaperones Hsp40I and Hsp40II of the L. braziliensis for structural characterization by spectroscopic techniques like fluorescence and circular dichroism. However, the insolubility of these proteins, possibly caused by the presence of mutations in their DNA sequences, led to the characterization of another co-chaperone, the Aha1 of the L. braziliensis. These proteins were expressed in the cell supernatant and purified by three chromatographic steps (anion exchange, affinity for calcium ions and gel filtration). The analysis of the DNA sequence of this protein shows that it has nine Trp residues distributed between the two domains and by urea denaturation studies monitored by fluorescence techniques and circular dichroism show that they have different stabilities.

Diante do quadro atual de previsões de mudanças climáticas, estudos a respeito das possíveis respostas dos organismos a estas alterações são importantes. Com a finalidade de prever e verificar se estas serão de fato deletérias ou se os organismos são capazes de lidar com elas sem alterações na sua fisiologia, e consequentemente na estrutura do ambiente, E. brasiliensis foi utilizada como modelo para estudar possíveis impactos do aumento da temperatura e acidificação dos oceanos na sua fisiologia. Para isso, espécimes foram expostos a 9 possíveis combinações de temperatura (24ºC, 28ºC e 30ºC) e pH (8.0, 7.7 e 7.3) em diferentes intervalos de tempo (1, 3, 12, 24 e 48 h). Amostras de gônadas e fluido celomático foram coletadas para avaliar a expressão das proteínas de estresse HSP70, AIF-1 e p38-MAPK, e a variação no número e viabilidade dos celomócitos. Nossos resultados mostram que o modelo é sensibilizado pelas mudanças no ambiente, através da hiper-regulação das proteínas de estresse. O cenário considerado mais extremo (30°C + pH7.3) ocasionou a morte de 100% dos organismos após 24horas. E o segundo cenário mais severo (30°C + pH7.7) desencadeou o desenvolvimento de ulceração de pele. Os efeitos são mais pronunciados nos celomócitos e a acidificação da água parece ter efeitos antagônicos com a temperatura nos celomócitos e sinérgicos nas gônadas. Embora a resposta tenha sido sistêmica, o grau e a dinâmica foram distintos em relação às diferentes amostras e estresses. Podendo causar modificações na resposta imune dos organismos e consequentemente na sobrevivência da espécie a longo prazo. / Under the current Climate Change context, studies about the potential responses of the organisms to their changing environment are of extreme importance. Recent studies point out the synergy of temperature and ocean acidification altogether. In this study, we used the sea star E. brasiliensis to assess the physiological effects of rising temperature, seawater acidification and the interaction of both factors. Independent individuals (N=225) were exposed to 9 possible combinations of temperature (24ºC, 28ºC and 30ºC) and pH (8.0, 7.7 and 7.3), for 1, 3, 12, 24 and 48 h. We compared the stress produced by these treatments measuring the expression of heat shock proteins (HSP70), the production of the allograft inflammatory factor (AIF−1) and the activation of mitogen kinases (MAPKs) at both gonad and celomic fluid. Furthermore, we assessed the quantity and quality of coelomocytes. Our results demonstrated that E. brasiliensis is vulnerable to the interaction of temperature and acidification. All the stress proteins evaluated were upregulated. The extreme scenario (30°C + pH7.3) caused the death of 100% of organisms after 24 hours, while the second most severe scenario (30°C + pH7.7) triggered skin ulceration. Nevertheless, we found that water acidification produces antagonistic effects to the temperature in coelomocytes and synergistic effects in gonad cells. Furthermore, these effects were more pronounced in the coelomocytes than in the gonads. The systemic response found in this study suggest that the interactive effects of elevated temperatures in conjunction with ocean acidification may endanger the survival of this species, and it could compromise the ecosystem functioning at long term.

Orientadores: Selma Giorgio, Fernanda Ramos Gadelha / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:07:59Z (GMT). No. of bitstreams: 1 Codonho_BarbaraSantoni_M.pdf: 2501497 bytes, checksum: d7ceeef1653b2df4a7d52b28ebd16543 (MD5) Previous issue date: 2014 / Resumo: As leishmanioses são um conjunto de doenças causadas pelo protozoário do genêro Leishmania, que atingem milhões de pessoas por ano. O tratamento é realizado primeiramente com antimoniais pentavalentes e, em casos de resistência, são indicadas a pentamidina ou anfotericina B. Todos estes fármacos são tóxicos e induzem efeitos colaterais nos pacientes. Devido a dificuldades no tratamento, o estudo de moléculas presentes no parasita se torna importante. Dentre essas, as heat shock proteins 70 (HSP70) são proteínas essenciais para o ciclo de vida da Leishmania. Durante a passagem do vetor para o hospedeiro vertebrado, o parasita encontra vários tipos de estresses que induzem a uma maior expressão da HSP70. Nesse projeto avaliou-se os efeitos da superexpressão da HSP70 em Leishmania (Leishmania) amazonensis, comparando-se parasitas que superexpressam a proteína HSP70 (pTEX-HSP70) com parasitas contendo somente o vetor (pTEX). Os resultados mostraram que os promastigotas transfectados pTEX e pTEX-HSP70 apresentaram vários aspectos ultraestruturais semelhantes aos não transfectados (WT), porém mostraram ser maiores e com o tamanho da área nuclear maior. A superexpressão da proteína HSP70 conferiu aos parasitas uma fase estacionária de proliferação mais estendida do que a observada em parasitas pTEX. Uma maior resistência e capacidade proliferativa foram observadas nos parasitas pTEX-HSP70 quando submetidos a diferentes condições de estresses (tratamentos com H2O2, choque térmico e ambiente hiperbárico), em relação a parasitas pTEX. Os resultados também mostraram que parasitas pTEX e pTEX-HSP70 infectam culturas de macrófagos peritoneais e macrófagos humanos derivados de sangue periférico, em taxas (% de infecção e número de amastigotas/macrófago) semelhantes a de parasitas WT. O processo de infecção em camundongos BALB/c mostrou que o tamanho da lesão induzida pelos parasitas pTEX e pTEX-HSP70 na pata foi diferente nas primeiras semanas, mas semelhante no curso final da infecção. Adicionalmente, as cargas parasitárias nas lesões dos camundongos BALB/c infectados com os parasitas pTEX e pTEX-HSP70 foram semelhantes, mas maiores que as cargas parasitárias nas lesões induzidas por WT. Além disso, os baços dos camundongos infectados com os parasitas pTEX e pTEX-HSP70 apresentaram visceralização. Ensaios da bioenergética destes promastigotas mostraram que parasitas pTEX-HSP70 apresentam maiores taxas de consumo de O2 do que parasitas pTEX, apesar de apresentarem produção de ATP semelhante. A produção de superóxido nos parasitas pTEX-HSP70 e pTEX foram similares, apesar da liberação de H2O2 ser bem inferior nos de parasitas pTEX-HSP70. Os resultados obtidos indicam que a superexpressão da proteína HSP70 protege a L.(L.) amazonensis de situações de estresse imediato, mas não interfere com a sua capacidade infectiva / Abstract: Leishmaniasis are a group of diseases caused by the protozoan genus Leishmania, which affect millions of people each year. The treatment is performed primarily with pentavalent antimony and resistance cases are indicated pentamidine or amphotericin B. All these drugs are toxic and induce side effects in patients. Due to difficulties in treatment, the study of molecules present in the parasite becomes important. Among these, the heat shock protein 70 (HSP70) proteins are essential for the life cycle of Leishmania. During the transition from vector to vertebrate host, the parasite finds various types of stresses that induce a higher expression of HSP70. In this project was evaluated the effects of overexpression of HSP70 in Leishmania (Leishmania) amazonensis, comparing parasites that overexpressing HSP70 (pTEX-HSP70) protein with parasites containing the empty vector (pTEX). The results showed that transfected promastigotes pTEX and pTEX-HSP70 showed several similar ultrastructural aspects similar to promastigotes of L.(L.) amazonensis untransfected (WT), but proved to be larger and the size of the largest nuclear area. Overexpression of HSP70 protein gave the parasites a stationary phase of proliferation more extended than that observed in parasites pTEX. Higher strength and better proliferative capacity were observed in parasites pTEX-HSP70 when submitted to different stress conditions (hydrogen peroxide, heat shock treatments and hyperbaric environment), in relation to parasites pTEX. The results also showed that pTEX and pTEX-HSP70 parasites infect cultures of peritoneal macrophages and human peripheral blood-derived macrophages in rate (% infection and the number of amastigotes / macrophage) similar to WT parasites. The process of infection in BALB/c mice showed that the size of the induced parasitic pTEX and pTEX-HSP70 foot injury was different in the first few weeks but similar in the final course of infection. Additionally, parasitic loads on the lesions of BALB/c mice infected with pTEX and pTEX-HSP70 parasites were similar, but larger than the parasitic loads in lesions induced by WT. Moreover, spleens from infected with pTEX and pTEX-HSP70 parasites mice showed visceralization. Assays of bioenergetics promastigotes showed that these pTEX-HSP70 parasites consume more O2 than pTEX parasites, despite showing similar ATP production. Superoxide production in parasites pTEX and pTEX-HSP70 were similar, despite the release of hydrogen peroxide is considerably lower than pTEX-HSP70 parasites. The results indicate that overexpression of HSP70 protein protect L.(L.) amazonensis in immediate situations of stress, but does not interfere with its infective capacity / Mestrado / Imunologia / Mestra em Genética e Biologia Molecular

Background Cisplatin is used for treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC) but treatment with cisplatin often leads to acquired resistance to cisplatin, resulting in poor patient survival. Globotriaosylceramide (Gb3) and multidrug resistance protein 1 (MDR1) have been associated with cisplatin resistance. Gb3 serves as a receptor for verotoxin-1 (VT-1), which induces apoptosis, and has been shown to have a functional dependency to MDR1 and heat shock protein 70 (HSP7o). The Bcl-2 family of proteins and inhibitors of apoptosis (IAPs) are key regulators of apoptosis. BH3-mimetics mimic pro-apoptotic BH3-only proteins, while Smac mimetics mimic the IAP-binding protein Smac/Diablo. These drugs have shown great promise in reversing cisplatin resistance. Exosomes are small bio-nanoparticles secreted and taken up by both cancer cells and normal cells. They have the ability to transfer properties between cells and have been shown to confer resistance to cisplatin. Methods In this thesis, NSCLC cell line H1299 and MPM cell line P31 were studied using western blot, flow cytometry, proteome profilers, confocal microscopy and gene expression arrays to investigate changes in protein and gene expression after acquisition of cisplatin resistance (P31res and H1299res) or after incubation with exosomes or drugs that target these. The cytotoxic and apoptotic effects were studied using fluorometric cytotoxicity assay (FMCA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results This thesis confirms that Gb3 is a potential target for cisplatin resistance reversal. Incubation with glycosphingolipid production inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and VT-1 led to reduced Gb3 cell surface expression and increased cytotoxic effect of cisplatin in all cell lines. Gb3 and MDR1 was not co-localized in any studied cell line, but Gb3 and HSP70 were co-localized on the cell surface and PPMP and VT-1 led to a decrease of both Gb3 and HSP70. Both BH3-mimetic obatoclax and Smac mimetic AT-406 had an additive effect on cisplatin-induced cytotoxicity and apoptosis in P31 and a synergistic effect in P31res. Results indicate that exosomes from cisplatin-resistant cell lines can transfer HSP70 to the surface of cells. Conclusion Cell surface Gb3 and HSP70, the Bcl-2/IAP-family proteins and exosomal transfer of cisplatin resistance characteristics are potential targets in combatting cisplatin resistance that show therapeutic promise and warrant further research.

Situações de estresse metabólico e térmico deflagram a expressão celular e liberação para o plasma, de proteínas de choque térmico da família de 70 kDa (HSP70) para desempenhar função de defesa-chave em um processo conhecido como resposta ao choque térmico ou ao estresse, que as caracterizam como proteína citoprotetora. Estudos indicam a ocorrência de HSP70 extracelular (eHSP70) na circulação por até 24 h após desafios como o exercício físico, cuja liberação dá-se por tecidos do território hepatoesplâncnico, em uma resposta mediada por receptores α-adrenérgicos. Esta liberação de HSP70 para o plasma é atenuada pela ingestão de glicose. Estas evidências levaram-nos a supor que a liberação de eHSP70 em resposta ao estresse possa ter um papel fisiológico na regulação da glicemia, a qual é afetada por estímulo simpático. Neste trabalho, os resultados dos experimentos in silico indicaram a possibilidade de ancoragem e interação estável entre a HSP70 e a molécula de insulina. Ensaios de imunoprecipitação de insulina com amostras de plasma de animais submetidos ao tratamento de insulina Regular e Detemir revelaram HSP70 coimunoprecipitada especialmente em situação de jejum severo. Parte destas amostras foi submetida à quantificação de glicemia, insulinemia e HSP72 circulante, cujos resultados evidenciam elevada concentração desta proteína de choque térmico por ocasião de hipoglicemia induzida por insulina. Parte dos estudos in vivo deste trabalho foram desenvolvidos com ratos submetidos ao choque térmico agudo (uma sessão de 15 minutos) com hipertermia induzida (41,5 °C) ou com injeção de HSP70 (HSPA1A) exógena, com grupos experimentais organizados em grupos Controle (C) e Choque Térmico (HS) e, em tempos experimentais: imediatamente (tempo zero), 6, 12 e 24 h pós-choque. Foi observada a ocorrência das formas constitutiva e induzível de eHSP70 no plasma de animais submetidos ao choque térmico com valor maior nos períodos de 12 e 24 h pós-choque. Nossos estudos mostram que, in vivo, a eHSP70 plasmática produz alteração na tolerância à glicose, com maior hiperglicemia, quando o teste é realizado 12 h após o choque térmico. Este efeito foi bloqueado pela administração (i.p.) de anticorpo anti-HSP70 e se mostrou independente da síntese de novo de HSP70. Ainda, a presença de eHSP70 plasmática (induzida por choque térmico ou administrada via endovenosa) causa menor captação de glicose pelo músculo esquelético. Ensaios com músculo isolado indicam que o efeito inibidor da eHSP70 sobre a captação de glicose é dependente de insulina. Apesar das evidências de que a ligação HSP70-insulina no plasma tenha implicação sobre a oferta de glicose, não se pode descartar também a possibilidade de que as ações da eHSP70 estejam ocorrendo pela sua ligação a receptores específicos, e/ou de que sejam parte de uma resposta ao estresse na qual estejam implicados os hormônios contrarregulatórios. Em suma, os resultados indicam que a eHSP70 plasmática é capaz de se ligar- à insulina, provocar tolerância alterada à glicose e diminuir a captação de glicose pelo músculo esquelético, aumentando a disponibilidade de glicose circulante. / Metabolically stressful situations trigger the expression and release of the 70 kDa (HSP70) as an essential protective response, in a process known as heat shock (HS) response or stress response. HSP70 is characterized as cytoprotector entities. Previous studies indicate the occurrence of extracellular HSP70 (eHSP70) in the circulation up to 24 h after stressful challenges, such as physical exercise, and that such eHSP70 liberation comes from hepatosplancnic tissues, in an 1-adrenergic dependent response, which is attenuated by glucose ingestion. The above findings led us to suppose that stress-elicited eHSP70 release could play a physiological role on glycemic control, which are affected by sympathetic stimuli. In this work, in silico experiments indicated the possibility of docking and stable interaction between HSP70 and the insulin molecule. Immunoprecipitation of plasma samples revealed that HSP70 co-immunoprecipitates with insulin, especially under severe fast. Part of these samples was subjected to quantification of glucose, insulin and circulating HSP72, whose results show high concentration of heat shock protein during severe hypoglycemia induced by insulin. Part of the in vivo studies from this work were also carried out in healthy rats submitted to an acute (15 min) HS (41.5 °C) session or in HSP70 (HSPA1A)-injected rats, assigned into control (C) and HS groups which were observed immediately and after 6, 12 and 24 h post-shock. Both the constitutive (predominantly) and the inducible forms of eHSP70 were observed in the plasma of heat shocked animals, being the times 12 and 24 h those in which the highest concentrations were noted. Our studies suggest that in vivo produces eHSP70 change in plasma glucose tolerance, greater hyperglycemia when glucose tolerance test is performed 12 h after heat shock, and this effect was abrogated by anti-HSP70 antibody administration (i.p.) and independent of de novo synthesis of HSP70. Moreover, the presence of eHSP70 (HS-induced or injected) in the plasma or in soleus muscle incubations elicits lower glucose uptake from the skeletal muscle. Isolated muscle studies indicate that eHSP70 inhibiting effect over glucose uptake is insulin-dependent. However, although evidence suggests a role for HSP70 binding to insulin over glucose offering, it cannot be discarded the possibility that eHSP70 actions are due to their binding to specific HSP70 membrane receptors within the muscle cell, and/or that are part of a stress response in which the counter-regulatory hormone are involved. In short, the results indicate that eHSP70 plasma is able to bind to insulin, causing impaired glucose tolerance and decrease glucose uptake by skeletal muscle, increasing blood glucose availability.

La production de globules rouges dépend du taux apoptose des précurseurs érythroïdes et est principalement régulé par l'érythropoïétine (Epo). La privation en Epo aboutit à l'activation de la caspase-3 (casp-3) qui clive GATA-1 ce qui entraîne l'apoptose des érythroblastes immatures. L'activation de la casp-3 est également indispensable aux modifications morphologiques caractéristiques observées au cours de la différenciation érythroïde terminale humaine, sans qu'il n'y ait ni d'apoptose ni de clivage de GATA-1. L'objectif de cette thèse était de mettre en évidence si Hsp70 inductible, dont un des rôles principaux est la régulation de l'apoptose, est impliquée dans la protection sélective des substrats de la casp-3 activée au cours de la différenciation érythroïde terminale humaine. Nous avons mis en évidence que lors de la différentiation érythroïde terminale pendant la phase d'activation des caspases, Hsp70 a une expression nucléo-cytoplasmique constitutive et co-localise avec GATA-1 dans le noyau. La localisation nucléaire d'Hsp70 est régulée par l'Epo : après privation des cellules en Epo, il y a une importante diminution de la localisation nucléaire d'Hsp70 et GATA-1 est clivée. L'inhibition de l'expression d'Hsp70 par une approche siRNA a comme conséquence le clivage de GATA-1 lors de l'activation de la casp-3 avec un arrêt de différenciation et une augmentation de la mort cellulaire. Hsp70 est une nouvelle protéine anti-apoptotique de la différenciation érythroïde terminale. Nous proposons un modèle dans lequel l'Epo détermine le destin des érythroblastes (apoptose vs différenciation) en aval de la casp-3 en régulant la localisation nucléaire d'Hsp70.

Hsp104 and ClpB are hexameric ATPases that resolubilize aggregated proteins in collaboration with the Hsp70 chaperone system. Hsp104/ClpB functionally interact only with their respective Hsp70 system and this specificity is mapped to the Hsp104/ClpB coiled-coil domain (CCD). We hypothesize that the interaction between Hsp70 and Hsp104/ClpB CCD stimulates nucleotide exchange and release of substrate from Hsp70. In the current study, the CCDs of E. coli ClpB and S. cerevisiae Hsp104 have been purified. Isolated domains are monomeric and well folded. They inhibit refolding of aggregated firefly luciferase in a species-specific manner. We found that the ATPase activity of E. coli DnaK is stimulated at low concentrations of the E. coli ClpB CCD but not by yeast Hsp104 CCD. However, in another bacterial system (Thermus thermophilus) we found that the ClpB CCD inhibits The ATPase activity of DnaK suggesting that the interaction may have different consequences in distinct chaperone networks.

Two studies were conducted to determine if Hsp70 is able to protect human skeletal muscle from muscle mechanical damage and alterations in SERCA activity associated with prolonged concentric exercise. In the first study, one-legged isometric knee extension exercise at 40% MVC and a duty cycle of 50% (5 sec contraction followed by 5 sec of relaxation) was used to induce a heat shock response in one leg only. Participants were followed over six recovery days to determine the time course of Hsp70 induction and decay. Results showed fiber type specific increases in Hsp70 that persisted in one leg only throughout six days of recovery. These increases in Hsp70 occurred with only transient changes in Ca2+ uptake and muscular force. With the exception of minor decreases in low frequency force, there were no apparent reductions in muscular force or SERCA activity by the third recovery day. Therefore an exercise protocol was established which was able to induce a heat shock response with only minor alterations in muscle mechanical function and SERCA activity. In the second study, the same isometric exercise was employed, however, on the day corresponding to recovery day 3 in the first study, participants were asked to complete a one hour cycling protocol at 70% VO2 max. The goal was to cause similar one-legged increases in Hsp70 as the first study and to then challenge SERCA activity and muscular force in the presence of elevated Hsp70 by using cycling exercise. Results showed cycling induced reductions in maximal Ca2+ ATPase activity, muscular force, rates of muscle relaxation, and rates of muscle force development were attenuated by the preconditioning (isometric) exercise. These studies confirm the idea that preconditioning exercise is able to attenuate subsequent exercise induced insults to SERCA activity and muscular force, likely through an Hsp70 mediated mechanism.

Two studies were conducted to determine if Hsp70 is able to protect human skeletal muscle from muscle mechanical damage and alterations in SERCA activity associated with prolonged concentric exercise. In the first study, one-legged isometric knee extension exercise at 40% MVC and a duty cycle of 50% (5 sec contraction followed by 5 sec of relaxation) was used to induce a heat shock response in one leg only. Participants were followed over six recovery days to determine the time course of Hsp70 induction and decay. Results showed fiber type specific increases in Hsp70 that persisted in one leg only throughout six days of recovery. These increases in Hsp70 occurred with only transient changes in Ca2+ uptake and muscular force. With the exception of minor decreases in low frequency force, there were no apparent reductions in muscular force or SERCA activity by the third recovery day. Therefore an exercise protocol was established which was able to induce a heat shock response with only minor alterations in muscle mechanical function and SERCA activity. In the second study, the same isometric exercise was employed, however, on the day corresponding to recovery day 3 in the first study, participants were asked to complete a one hour cycling protocol at 70% VO2 max. The goal was to cause similar one-legged increases in Hsp70 as the first study and to then challenge SERCA activity and muscular force in the presence of elevated Hsp70 by using cycling exercise. Results showed cycling induced reductions in maximal Ca2+ ATPase activity, muscular force, rates of muscle relaxation, and rates of muscle force development were attenuated by the preconditioning (isometric) exercise. These studies confirm the idea that preconditioning exercise is able to attenuate subsequent exercise induced insults to SERCA activity and muscular force, likely through an Hsp70 mediated mechanism.