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Orientador: Ilda de Godoy / Resumo: O tabagismo é responsável pelo maior número de mortes evitáveis ​​no mundo e está relacionado ao desenvolvimento de várias doenças, principalmente a doença pulmonar obstrutiva crônica (DPOC). Assim, a busca por biomarcadores precoces torna-se relevante para sua identificação e para o sucesso terapêutico. Os objetivos do nosso estudo foram avaliar a concentração da proteína de choque térmico 70 (HSP70), expressão do gene HSP70, anticorpos anti-HSP70 auto, marcador inflamatório sistêmico através da citocina interleucina-8 (IL-8) e proteína C reativa (PCR), alterações imunológicas e danos no DNA no sangue periférico de fumantes crônicos assintomáticos e não fumantes. Nossos resultados mostraram concentrações séricas aumentadas de HSP70, anti-HSP70, IL-8, PCR e neutrófilos, e danos no DNA de células sanguíneas de fumantes em comparação ao não-fumantes. Portanto, o tabagismo foi responsável por levar a alteração nos parâmetros fisiológicos e moleculares associados ao risco de desenvolver DPOC e outras doenças pulmonares. Com base nos dados, sugerimos que a HSP70 pode ser responsável pelo aumento dos níveis de citocinas inflamatórias e, consequentemente, o aumento do influxo de neutrófilos para os pulmões e aumento dos danos ao DNA e auto-anticorpos anti-HSP70. / Abstract: Smoking is responsible for the largest number of preventable deaths in the world, and is related to the development of several diseases, mainly chronic obstructive pulmonary disease (COPD). Thus, the search for early biomarkers of such diseases becomes relevant for their identification and for successful therapy. The objectives of our study were to evaluate the concentration of heat shock protein 70 (HSP70), expression of the HSP70 gene, anti-HSP70 auto antibodies, the systemic inflammatory marker through cytokine interleukin-8 (IL-8) and CPR, immunological changes and DNA damage in peripheral blood of chronic asymptomatic smokers and non-smokers. Our results showed increased serum concentrations of HSP70, anti-HSP70, IL-8, CPR and neutrophils, and DNA damage in blood cells of smokers than in non-smokers. Therefore, cigarette smoking was confirmed as a noxious agent on physiological and molecular parameters associated with the risk for developing COPD and other lung diseases. Based on the data we suggest that HSP70 can be responsible for the increased levels of inflammatory cytokines, and consequently, the increased influx of neutrophils into the lungs and increased DNA damages e anti-HSP70 auto antibodies. / Doutor

The centromeres of eukaryotes have a region on which the kinetochore is assembled, flanked by heterochromatin which provides cohesion between the sister chromatids during cell division. When centromeric heterochromatin is lost chromosomes no longer segregate evenly into the daughter cells during cell division. In the fission yeast Schizosaccharomyces pombe (S. pombe) RNA interference (RNAi) is responsible for maintaining this heterochromatin. The pathway is part of a feedback loop whereby siRNAs generated from non-coding centromere transcripts are loaded into an Argonaute complex. The siRNAs guide the complex to the homologous centromere repeats in order to recruit Clr4 which modifies histone H3 with the heterochromatin mark H3K9me. A previous screen to find factors affecting centromere silencing isolated 13 loci termed centromere: suppressor of position-effect (csp) 1-13. Several csp mutants have been identified to be RNAi components. In this investigation the csp6 locus has been identified to be the Hsp70 gene ssa2+. It has been demonstrated that Argonaute proteins from plants and flies require Hsp70/90 chaperone activity for loading of siRNA. It therefore seems likely that Hsp70 may play a similar role in fission yeast. Genetic and biochemical techniques have been used in this study to investigate if the csp6 alleles are affecting siRNA loading in S. pombe. RNA Polymerase II (RNAPII) transcribes the pre-siRNA transcripts from the centromere repeats. csp3 was identified to be an allele of the RNAPII subunit rpb7+. rpb7-G150D was found to cause a silencing defect in the centromeric heterochromatin through a defect in transcription. Another RNAPII mutation, rpb2-m203, was found to have strong silencing defects caused by an unidentified non-transcriptional role in RNAi-mediated heterochromatin formation at the centromere. In order to gain more insight into the role of RNAPII in heterochromatin assembly I performed a screen in which the subunits rpb3 and rpb11 were subjected to random mutagenesis. Several mutants were isolated and characterisation of phenotypes regarding heterochromatin at the centromere has been carried out for nine of the mutants. As a result a novel phenomenon of RNAi-independent silencing at the centromere has been discovered.

At the cellular level, the stress response involves the synthesis of a highly conserved family of heat shock proteins (Hsps). These proteins are essential for maintenance of cellular homeostasis, both in times of stress and in normal cell functioning. Some of the most abundant forms of Hsps in the cell are members of the 70 kDa family. Intracellular heat shock protein 70 (Hsp70) expression in response to proteotoxicity is a highly conserved cellular stress response, but little is known about the role of extracellular Hsp70 (eHsp70) in fish. In order to begin characterizing eHsp70 in fish, the hypothesis that an acute stressor will elevate plasma Hsp70 levels in rainbow trout (Oncorhynchus mykiss) was tested. Subsequent in vitro studies examined whether eHsp70 level was modulated by cortisol and if this involved the action of the glucocorticoid receptor (GR), a ligand-activated transcription factor. The effect of cortisol on the eHsp70 response is important to consider because this steroid is elevated as a result of stressor exposure to allow for short-term allocation of energy stores to cope with stress. Cortisol is the primary corticosteroid in fish and exerts its main effects by binding to either GR or mineralocorticoid receptors (MR). Furthermore, eHsp70 has been previously implicated as having important immunoregulatory roles in mammalian models, but nothing has yet been reported in fish. To this end, a hypothesis tested here was that eHsp70 levels will increase after exposure to the bacterial endotoxin lipopolysaccharide (LPS), and that this response is modulated by cortisol. Finally, research on the effects of exogenous Hsp70 has not been reported in lower vertebrates; however, the relevance of this protein in intercellular signaling, especially in regards to immune regulation, is gaining increasing importance in mammalian models. Therefore, an experiment to determine whether Hsp70 would elicit upregulation of key immunoregulatory cytokines was also conducted. To accurately measure the low levels of Hsp70 in the plasma, a competitive antibody-capture enzyme-linked immunosorbent assay (ELISA) was developed. In the in vivo study, fish exposed to an acute heat shock (1h at 10°C above ambient temperature) exhibited a significant elevation in red blood cell Hsp70 levels over a 24 h period. There was also a significant increase in plasma Hsp70 levels at 4 h, but not at 24 h post-heat shock. To more specifically determine how cortisol affected the release of Hsp70, in vitro studies using primary cultures of hepatocytes demonstrated that cortisol significantly decreased eHsp70 levels in the medium at 24 h when compared with untreated controls, and this response was abolished in the presence of a GR antagonist, mifepristone (RU486). This result for the first time established a link between cortisol signaling and eHsp70 release in any animal model. When hepatocytes were exposed to LPS in vitro, eHsp70 levels were significantly lower in the LPS (30 µg/ml) group; however, heat shock abolished this effect at 24 h. Though eHsp70 levels in the heat shocked hepatocytes treated with low-dose LPS (10 µg/ml) was similar to untreated control levels, high-dose LPS treated hepatocytes showed significant elevation of eHsp70 levels above the low dose group. The ability of LPS to modulate eHsp70 release was not observed to be further regulated by cortisol. While this work suggests the modulation of eHsp70 by LPS, the physiological role remains to be elucidated. Finally when hepatocytes were exposed to exogenous Hsp70, there was no effect on key immunoregulatory genes (IL-1β and IL-8) transcript levels; however, the effect of this protein remains to be tested using other cell systems, including immune cells in fish. Overall, eHsp70 concentration was measured in trout plasma using a competitive ELISA and demonstrates for the first time that stressor exposure affects plasma eHsp70 levels in fish. Furthermore, cortisol, the primary corticosteroid in teleosts, modulates eHsp70 release in trout hepatocytes and this is action is mediated by GR signaling. Also, while trout hepatocytes secrete eHsp70 in response to endotoxin shock, a role for eHsp70 in eliciting an immune response is not clear in lower vertebrates. Taken together the results from this study suggest a role for eHsp70 in acute stress adaptation in fish, but the target tissues involved and the physiological responses remain to be elucidated. Further work on the effects of eHsp70 on target tissues effects, and the mechanisms involved, may have important implications in our understanding of the role of this stress protein in cell signaling and stress adaptation in fish.

Purpose: Excessive day time sleepiness, sleep disorders and neurobehavior changes are common clinical observations in the patients with epilepsy. From literature review, they were highly related with epilepsy risk characteristics (age of onset, types or numbers of drugs, seizure frequency), co-morbidities or neuronal network changes. The serological biomarkers have been reported to reflect the phenomenon of seizure, while their correlations with neurobehavior changes were still not concluded. There were two purposes of this thesis. (1) To understand the relationship between sleep disturbance with day time performances (2) To understand the relationships between serological biomarkers, neurobehavior performances and neuronal networks in patients with temporal lobe epilepsy. Material and Methods: The study enrolled patients from epilepsy outpatient clinic. By using self-appreciated questionnaire (The Pittsburgh Sleep Quality Index, The Epworth Sleepiness Scale, Euroqol Quality of Life Scale-5D), we collected the characteristics of sleep related behavior and life quality changes and explored the relationship with epilepsy risk characteristics. In patients with temporal lobe epilepsy, we assessed the neurobehavior performances, measured the serological biomarkers (heat shock protein 70, S100£]protein, neuron specific enolase, brain derived neurotrophic factor, plasma and mitochondrial DNA) and brain magnetic resonance imaging. In statistical analysis, we compared the differences with age matched controls or performed correlation analysis among the parameters Result: One hundred and seventeen patients with epilepsy completed the sleep quality questionnaires. The results showed that 20 percent of patients had day time sleepiness, while the sleep disorder was prolong sleep latency and impaired sleep efficiency. In epilepsy characteristics, patients with complex partial seizure, intractable seizure or with multi-pharmacy were related with poor sleep quality. A total of 34 patients completed the serological, neurobehavior and brain magnetic resonance analysis. The results showed that patients with temporal lobe epilepsy had higher heat shock protein 70 and S100£]protein levels, while those with attacks more than twice per month had significant higher heat shock protein 70, S100£]protein and neuron specific enolase levels. Compared with the matched controls, the regions showing atrophy included hippocampus and parahippocampus, putamen, thalamus and supplementary motor areas. In correlation study, only heat shock protein 70 showed an inverse correlation with hippocampal volume (R square = 0.22, p = 0.007) after controlling for the effect of age. Conclusion: The study suggested that epilepsy risk characteristics, serological biomarkers, brain atrophic regions were important factors for day time sleepiness, sleep disturbances and neurobehavior changes in patients with epilepsy.

In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. Ubiquitin is best known for its role in targeting proteins for degradation by the proteasome. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is found specifically in central and peripheral neurons, and is responsible for the removal of small peptide fragments from the ubiquitin chain and for co-translational processing of ubiquitin gene products to generate free monomeric ubiquitin. In response to extreme conditions, cells exhibit an up-regulation of heat shock protein (HSP) expression, which contributes to repair and protective mechanisms. Within the HSP family, HSP70 is the major inducible member that protects against cell death. Based on the pharmacologic property of organophosphates as an inhibitor of cholinesterase, it is generally contended that manifestations of organophosphate poisoning, including secretion and muscle fasciculation, stupor, cardiopulmonary collapse, respiratory failure, coma or death, result from accumulation of, and over-stimulation by acetylcholine at peripheral of central synapses. One approach in furthering our understanding on organophosphate poisoning is delineation of its potential protective mechanisms. In this regard, the information on the cellular and molecular mechanisms that underlie organophosphate poisoning has received attention. Our laboratory demonstrated previously that a crucial brain site via which mevinphos (Mev), an organophosphate insecticide of the P=O type, acts is the rostral ventrolateral medulla (RVLM), the medullary origin of premotor sympathetic neurons that are responsible for the maintenance of vasomotor tone. The phasic changes in cardiovascular events over the course of acute Mev intoxication also parallel fluctuations of the ¡§life-and-death¡¨ signals that emanate form the RVLM. Based on a rat model of organophosphate poisoning that provides continuous information on cellular and molecular mechanisms in the RVLM, the present study was undertaken to evaluate whether changes in protein level of UCH-L1 or HSP70 are associated with death arising from Mev intoxication. We also evaluated the efficacy of both of them in the neuroprotection against fatality during Mev intoxication. The first part of this study investigated whether UCH-L1 plays a neuroprotective role at the RVLM, where Mev acts to elicit cardiovascular toxicity. In Sprague-Dawley rats maintained under propofol anesthesia, Mev (960 µg/kg, i.v.) induced a parallel and progressive augmentation in UCH-L1 or ubiquitin expression at the ventrolateral medulla during the course of Mev intoxication. The increase in UCH-L1 level was significantly blunted on pretreatment with microinjection bilaterally into the RVLM of a transcription inhibitor, actinomycin D (5 nmol) or a translation inhibitor, cycloheximide (20 nmol). Compared to artificial cerebrospinal fluid (aCSF) or sense uch-L1 oligonucleotide (100 pmol) pretreatment, microinjection of an antisense uch-L1 oligonucleotide (100 pmol) bilaterally into the RVLM significantly increased mortality, reduced the duration of the phase I (¡§pro- life¡¨ phase), blunted the increase in ubiquitin expression in ventrolateral medulla, and augmented the induced hypotension in rats that received Mev. The second part of this study investigated whether HSP70 plays a neuroprotective role at the RVLM. Intravenous administration of Mev (960

(理学療法基礎系7, 第42回日本理学療法学術大会)

<p>Inducible Heat shock protein (Hsp70i) is a protein chaperone that is utilized during tumorigenesis and viral infections for efficient propagation. Overexpression of Hsp70i is observed in a wide spectrum of human tumors, and this overexpression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Hsp70i aids in cancer cell propagation through regulation of anti-apoptotic and cell survival pathways. Furthermore, Hsp70i is induced following infection for several viruses and aids viral propagation, in part through regulation of anti-apoptotic pathways as well as promoting the folding of newly synthesized proteins. Due to the parallel role of Hsp70i in both cancer and viral pathogenesis, identification of small-molecule inhibitors selective for Hsp70i could provide tools for the development of novel therapeutics and further elucidate the role of Hsp70i in both cancer and viral infections.</p><p>To date, few Hsp70 inhibitors have been identified and characterized, and their efficacy in clinical settings is unknown. Through the fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, an allosteric inhibitor selective for Hsp70i was identified, called HS-72. We show that HS-72 is highly selective for Hsp70i, over the broader purinome and other Hsp70 family members, in particular the closely related constitutively active Hsp70 family member, Hsc70. Additionally, HS-72 acts as an allosteric inhibitor to induce a conformational change and inhibit Hsp70i activity. HS-72 displays hallmarks of Hsp70i inhibition in vitro by promoting Hsp70i substrate protein degradation, protein aggregation, and selective growth inhibition of cancer cells. In wild type mice HS-72 is well tolerated and a limited PK study shows HS-72 is bioavailable. Furthermore, in a MMTV-neu breast cancer mouse model, HS-72 shows efficacy to inhibit tumor growth and promote survival.</p><p>Due to the similar utilization of Hsp70i in cancer and viral pathogenesis, this suggests the potential for HS-72 as an antiviral agent. Dengue virus (DENV) is of great public health importance due to estimates of up to 400 million infections per year, coupled with the geographic distribution of the virus, which is now endemic in over 100 countries worldwide. There is also a pressing need for DENV interventions, owing to the lack of approved vaccines or antiviral therapies. DENV is reliant on host factors throughout the viral life cycle and Hsp70i has been implicated as a host factor in DENV pathogenesis. Additionally, the complete role of Hsp70i in DENV pathogenesis remains to be elucidated, highlighting a unique opportunity to use HS-72 as a tool to specifically probe Hsp70i function. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Furthermore, inducing Hsp70i expression prior to infection, correlates with an increase in DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained, through inhibiting the entry stage of the viral life cycle. Following infection, Hsp70i localizes to the cell surface and interacts with the DENV receptor complex to mediate viral entry. While, HS-72 treatment results in a disruption of the interaction of Hsp70i with the DENV receptor complex, yielding a reduction in infected cells. </p><p>Collectively this work further supports Hsp70i as an anticancer and anti-dengue virus target, and identifies HS-72, a chemical scaffold that is amenable to resynthesis and iteration, as an ideal starting point for a new generation of therapeutics targeting Hsp70i.</p> / Dissertation

At the cellular level, the stress response involves the synthesis of a highly conserved family of heat shock proteins (Hsps). These proteins are essential for maintenance of cellular homeostasis, both in times of stress and in normal cell functioning. Some of the most abundant forms of Hsps in the cell are members of the 70 kDa family. Intracellular heat shock protein 70 (Hsp70) expression in response to proteotoxicity is a highly conserved cellular stress response, but little is known about the role of extracellular Hsp70 (eHsp70) in fish. In order to begin characterizing eHsp70 in fish, the hypothesis that an acute stressor will elevate plasma Hsp70 levels in rainbow trout (Oncorhynchus mykiss) was tested. Subsequent in vitro studies examined whether eHsp70 level was modulated by cortisol and if this involved the action of the glucocorticoid receptor (GR), a ligand-activated transcription factor. The effect of cortisol on the eHsp70 response is important to consider because this steroid is elevated as a result of stressor exposure to allow for short-term allocation of energy stores to cope with stress. Cortisol is the primary corticosteroid in fish and exerts its main effects by binding to either GR or mineralocorticoid receptors (MR). Furthermore, eHsp70 has been previously implicated as having important immunoregulatory roles in mammalian models, but nothing has yet been reported in fish. To this end, a hypothesis tested here was that eHsp70 levels will increase after exposure to the bacterial endotoxin lipopolysaccharide (LPS), and that this response is modulated by cortisol. Finally, research on the effects of exogenous Hsp70 has not been reported in lower vertebrates; however, the relevance of this protein in intercellular signaling, especially in regards to immune regulation, is gaining increasing importance in mammalian models. Therefore, an experiment to determine whether Hsp70 would elicit upregulation of key immunoregulatory cytokines was also conducted. To accurately measure the low levels of Hsp70 in the plasma, a competitive antibody-capture enzyme-linked immunosorbent assay (ELISA) was developed. In the in vivo study, fish exposed to an acute heat shock (1h at 10°C above ambient temperature) exhibited a significant elevation in red blood cell Hsp70 levels over a 24 h period. There was also a significant increase in plasma Hsp70 levels at 4 h, but not at 24 h post-heat shock. To more specifically determine how cortisol affected the release of Hsp70, in vitro studies using primary cultures of hepatocytes demonstrated that cortisol significantly decreased eHsp70 levels in the medium at 24 h when compared with untreated controls, and this response was abolished in the presence of a GR antagonist, mifepristone (RU486). This result for the first time established a link between cortisol signaling and eHsp70 release in any animal model. When hepatocytes were exposed to LPS in vitro, eHsp70 levels were significantly lower in the LPS (30 µg/ml) group; however, heat shock abolished this effect at 24 h. Though eHsp70 levels in the heat shocked hepatocytes treated with low-dose LPS (10 µg/ml) was similar to untreated control levels, high-dose LPS treated hepatocytes showed significant elevation of eHsp70 levels above the low dose group. The ability of LPS to modulate eHsp70 release was not observed to be further regulated by cortisol. While this work suggests the modulation of eHsp70 by LPS, the physiological role remains to be elucidated. Finally when hepatocytes were exposed to exogenous Hsp70, there was no effect on key immunoregulatory genes (IL-1β and IL-8) transcript levels; however, the effect of this protein remains to be tested using other cell systems, including immune cells in fish. Overall, eHsp70 concentration was measured in trout plasma using a competitive ELISA and demonstrates for the first time that stressor exposure affects plasma eHsp70 levels in fish. Furthermore, cortisol, the primary corticosteroid in teleosts, modulates eHsp70 release in trout hepatocytes and this is action is mediated by GR signaling. Also, while trout hepatocytes secrete eHsp70 in response to endotoxin shock, a role for eHsp70 in eliciting an immune response is not clear in lower vertebrates. Taken together the results from this study suggest a role for eHsp70 in acute stress adaptation in fish, but the target tissues involved and the physiological responses remain to be elucidated. Further work on the effects of eHsp70 on target tissues effects, and the mechanisms involved, may have important implications in our understanding of the role of this stress protein in cell signaling and stress adaptation in fish.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21339号 / 医博第4397号 / 新制||医||1031(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 宮本 享, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM