My research focuses on understanding the importance of human mitochondrial Hsp70 (Grp75) chaperone machinery for the maintenance of protein quality control inside the mitochondrial matrix. The investigations carried out during this study have been addressed towards gaining better insights into the working of Grp75 chaperone folding machinery in association with its diverse set of co-chaperones residing in human mitochondria. Additionally, the research also focuses on explaining the various modes of Grp75 participation leading to multiple disease conditions. The thesis has been divided into the following sections as follows: Chapter I: An introduction to the mitochondrial import machinery and role of mitochondrial Hsp70 chaperone folding machinery for the maintenance of protein quality control: Mitochondrion is an essential organelle present in the eukaryotic cell and requires more than 1500 proteins for its proper functioning. Although, mitochondria harbour their own genome, it encodes for only 13 proteins in humans. The rest of the entire proteome is encoded by the nuclear genome and requires proper targeting of proteins to different compartments of mitochondria. Remarkably, mitochondrial matrix alone requires more than 60% of the proteome for its suitable functioning. Briefly, the mitochondrial matrix destined polypeptide passes through the outer membrane translocon; the ‘TOM’ complex and then enters the TIM23 translocon present in the inner membrane of mitochondria. The complete translocation of the polypeptide into the mitochondrial matrix side requires the assistance of mtHsp70 based motor system present on the matrix side which pulls the polypeptide into the matrix in an ATP-dependent manner and with the assistance of various co-chaperones. Subsequently, the unfolded polypeptide is to be folded back to its native state, which is ensured again by the mtHsp70 based chaperone folding machinery. Importantly, while 20% of mtHsp70 is involved in protein import, 80% of mtHsp70 is dedicated for protein folding. In addition to mtHsp70, the chaperone folding machinery consists of various soluble co-chaperones such as the J-proteins which stimulate the ATP hydrolysis rate of Hsp70. Furthermore, another co-chaperone termed as a nucleotide exchange factor ensures binding of fresh ATP molecule onto Hsp70 ensuring multiple rounds of folding cycles. To understand the relevance of mitochondrial Hsp70 chaperone folding machine in the maintenance of protein quality control, Chapter I of the thesis has been divided into multiple sections as follows: Briefly, the initial portion of Chapter I provide a glimpse of the translocon components present in mitochondria for targeting of proteins to outer membrane, inner membrane and inter-membrane space. Owing to the vast proteome size of the mitochondrial matrix, the following section describes the detailed mechanism and translocation process of the mitochondrial matrix targeted proteins. Additionally, subsequent sections of Chapter I provide a comprehensive description of each of the mtHsp70 chaperone folding components, which maintain the protein quality control in the matrix. The players that constitute the chaperone folding machines are mitochondrial Hsp70, J-proteins, nucleotide exchange factors and the newly discovered human escort protein. Essentially, the section provides information about the cellular distribution, structure and function of each of these players constituting the mtHsp70 chaperone folding machine. Loss of regulation between these players leads to defects in protein folding. Imbalance in protein homeostasis is one of the primary causes for mitochondrial dysfunction leading to various diseases. Importantly, recent literature has highlighted the involvement of mtHsp70 chaperone folding players in Parkinson’s disease (PD), Myelodysplastic syndrome (MDS) and cancer. In accordance, the last section of the Chapter I has been dedicated to describe the basic cell biology and proposed mechanisms for the above diseased states. Interestingly, in comparison to yeast and bacteria, the composition of mtHsp70 chaperone folding machinery in humans is unique and distinctly different. Owing to a lack of information about the functioning of human mitochondrial Hsp70 chaperone folding machinery and with an emphasis on understanding its role in various disease manifestations, the objectives that were laid for my PhD thesis are as follows: 1) Functional in vitro reconstitution of the human Grp75 chaperone folding machinery by purifying all the Grp75 chaperone folding machinery players namely; Grp75 (human mtHsp70), hTid-1L and hTid-1S (J-proteins), GrpEL1 (nucleotide exchange factor) and Human escort protein (Hep). 2) Dissection of the intrinsic biochemical defects associated with the variants of Grp75 reported in Parkinson’s disease (PD). 3) To understand the correlation between elevated levels of Grp75 and its contribution to malignancy. In conclusion, the current study has highlighted some of the key features of human Grp75 chaperone folding machinery and its regulation in the maintenance of human mitochondrial matrix protein quality control, failure of which leads to pathological conditions. Chapter II: Reconstitution of the human Grp75 chaperone folding machinery to understand the functional interplay between the multiple protein components: The mitochondrial Heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 towards mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5-peptide as compared to the other Hsp70’s. We observed that the two human mitochondrial matrix J-protein splice-variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hep possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C-terminus of mtHsp70 in a full-length context, and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C-terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thereby promoting a similar conformational change that leads to ATPase stimulation. Moreover, we have also dissected out the inter-domain defective nature associated with the point mutant of Grp75 implicated in Myelodysplastic syndrome thus providing an explanation for the loss of function of Grp75 eventually leading to loss of protein quality control in the diseased state. Chapter III: Enhanced J-protein interaction and compromised protein stability of Grp75 variants leads to mitochondrial dysfunction in Parkinson’s disease: Parkinson’s disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multi-factorial in nature, a recent proteomic screen involving PD-patients revealed two mitochondrial Hsp70 variants (P509S and R126W) that are implicated in PD-pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD-variants are centrally involved in PD-progression is totally elusive. In this report, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD-variants. Biochemically, R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, P509S variant exhibits significantly enhanced interaction with J-protein co-chaperones involved in folding and import machinery, thus altering the overall regulation of chaperone mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD-mutations at the cellular level, we have developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD-mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with ‘mitochondrial dysfunction’ such as mitochondrial DNA (mtDNA) loss and increased susceptibility to oxidative stress recapitulating the cellular features of dopaminergic neurons similar to those reported in other PD-models. Together, our observations for both the variants strongly indicate a definite involvement of mtHsp70 as a susceptibility factor in Parkinson’s disease. Chapter IV: To understand the correlation between elevated levels of Grp75 and its contribution to malignancy: Multiple studies carried out by various groups have reported the presence of elevated levels of Grp75 in cancer cells. Furthermore, proteomic screens show a positive correlation with the higher levels of Grp75 and the aggressive or metastatic nature of cancer. Importantly, cancer cells also exhibit altered mitochondrial metabolism and are found to be under constant oxidative stress pressure. Moreover, Grp75 actively participates in maintenance of mitochondrial function and as well is reported to interact with many putative oncoproteins. However, there is little information available on the possible role of Grp75 in modulating the cellular niche which might favor towards increased malignant transformation of cells. To identify pathways for explaining the correlation between Grp75 and cancer, our initial attempts have focused on monitoring the multiple cellular changes influenced by elevated levels of Grp75 in a cell line based system. To our surprise, transient transfection of cells with Grp75 led to a tremendous increase in the reactive oxygen species levels. Furthermore, a strong positive correlation between the extent of increased levels of Grp75 and the amount of ROS generated in these cells was established. As expected, increased ROS levels observed in Grp75 overexpressing cells also resulted in reduced cell viability. Notably, mitochondrial superoxide generation was found to be the major source for the observed increment in ROS levels in Grp75 expressing cells. In addition, the localization profile of the exogenously expressed Grp75 protein highlighted the fact that the protein was found to be predominantly targeted to mitochondria. Strikingly, the elevated Grp75 levels led to an increase in mitochondrial mass and also displayed a higher proportion of circular and fragmented mitochondria in these cells. Together, the above preliminary observations hint towards a strong correlation between the levels of Grp75 and its influence on the redox biology of cells providing an additional and a possible explanation of the mode of participation of Grp75 in generation and progression of malignancy.

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

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

This thesis entitled “Mechanism of Anticancer and Antimalarial Action of a Modulator of Heat Shock Proteins” describes the successful elucidation of the mechanism of anticancer and antimalarial action of 15-Deoxyspergualin (DSG). DSG, a relatively well known immunosuppressant and antitumor molecule has been demonstrated to kill the malaria parasite in vitro and in vivo (Midorikawa et al., 1997; Midorikawa et al., 1998). A highly polar molecule, DSG binds the carboxy terminal “EEVD” motif of heat shock proteins, Hsp70 and Hsp90, enhances the ATPase activity of Hsp70 (Nadler et al., 1992; Nadler et al., 1998), and modulates several seemingly unrelated cellular processes. DSG has also been demonstrated to inhibit protein synthesis and polyamine synthesis in cells (Kawada et al., 2002; Hibasami et al., 1991), and previously speculated to inhibit malaria parasite growth by inhibiting polyamine synthesis. The grim situation with regard to malaria infection and mortality, principally an offshoot of the emergence of chloroquine resistant strains of the causative agent of malaria - Plasmodium falciparum, calls for intense efforts towards developing efficacious antimalarial agents with few side effects. DSG, having been used already in graft rejection cases in man and demonstrated to potently inhibit malaria in mice (Midorikawa et al., 1997), offers promise in this regard. It was, therefore, of interest to solve the mystery of its mechanism of antimalarial action. Chapter 1 surveys literature related to DSG mechanism of action and presents the thesis objective. Chapter 1 also gives an overview of heat shock proteins and their role in cancer, and the biology of the malaria parasite (Plasmodium falciparum), the working of the principal metabolic pathways existing in it, and a description of processes related to the intriguing, relict plastid present in apicomplexans. The metabolic processes previously speculated to be targeted by DSG, and those later found to be involved in DSG mechanism of action – polyamine synthesis and transport, protein synthesis and apicoplast processes are dealt with in more detail. Though DSG has been speculated to kill the malaria parasite by inhibiting polyamine synthesis, that DSG could clear malaria infection in Plasmodium berghei infected mice did not corroborate with the observation that inhibitors of polyamine biosynthesis are incapable of inhibiting the malaria parasite in vivo probably because the parasites make do with polyamines salvaged from the host (Assaraf et al., 1984; Bitonti et al., 1987). On the other hand, DSG is known to bind heat shock proteins, and inhibit protein synthesis, and heat shock proteins are speculated to be involved in the activation of HRI (heme regulated inhibitor), a type of eIF2á kinase that phosphorylates the eukaryotic initiation factor, eIF2á in conditions of heme deficiency or other cellular stress. eIF2á phosphorylation leads to stalling of protein synthesis. It seemed likely that if HRI is activated upon sequestration of heat shock proteins by DSG, it would culminate in protein synthesis inhibition and ultimately, cell death. With the intention to investigate this line of thought, the PlasmodB database was mined for proteins essential to the existence of heme dependent protein synthesis in Plasmodium falciparum. Two Hsp70 proteins from Plasmodium falciparum, one with the carboxy terminal “EEVD” motif implicated in DSG binding, and one without, and an Hsp70 interacting protein were cloned and expressed in their recombinant form in Escherichia coli. The preliminary characterization of these heat shock proteins described in Chapter 2 revealed that they were functionally active. DSG did not inhibit either the chaperone activity of the Hsp70s or the interaction of Hsp70 with Hip, but stimulated their ATPase activity as anticipated. Chapter 3 gives a complete picture of the mechanism of protein synthesis inhibition by DSG in the standard protein synthesis system – reticulocyte lysate. The experiments carried out revealed that DSG inhibits protein synthesis precisely through the mechanism envisaged, i.e. through phosphorylation of HRI following sequestration of Hsp70. Experiments involving exogenous addition of heat shock protein to in vitro translation reactions confirmed this hypothesis. Moreover, DSG inhibited protein synthesis in cancer cells in vivo, too, and HRI knockdown cells were not affected by DSG. Interestingly, the Hsp70 levels in various cancer cell lines inversely correlated with the inhibitory activity of DSG, and modulation of Hsp70 levels through standard methods altered DSG inhibition of protein synthesis in these cells. It was thus confirmed that DSG did indeed inhibit mammalian cells through the pathway envisaged. Its previously reported antitumor property is probably through this outlined mechanism of interference with protein regulation. In the malaria parasite, too, DSG inhibited protein synthesis through eIF2 alpha phosphorylation following Hsp70 sequestration as outlined in Chapter 4. However, while the concentration of DSG required for inhibition of malaria parasite growth was in the nanomolar range, high micromolar concentrations of DSG were required to effect protein synthesis inhibition in the malaria parasite, indicating that yet another target for DSG existed in the malaria parasite. With protein synthesis no longer a candidate target of DSG, I looked into the previously implicated polyamine synthesis pathway. In the event of DSG inhibiting polyamine transport in addition to polyamine biosynthesis, it would be expected to clear malaria infection in vivo contrary to other inhibitors of polyamine biosynthesis. In Chapter 5, evidence for the polyamine synthesis pathway in the malaria parasite is provided. Experiments involving incorporation of radiolabeled precursors in the malaria parasite and in mammalian cells, however, revealed that only high micromolar concentrations of DSG inhibit polyamine synthesis. Polyamine transport was also studied in considerable detail in malaria parasite infected red blood cells. Though infected red blood cells demonstrated different kinetic parameters, implying that new polyamine transporters were employed by the parasite on the red blood cell upon infection, DSG did not potently inhibit polyamine transport, either. The mystery of the target of DSG in the malaria parasite was, however, close to solution, when the growth inhibition of the malaria parasite by DSG was studied carefully. DSG invoked “delayed death” – a phenomenon wherein death is invoked only one cycle after incubation with the inhibitor. “Delayed death” is typical of inhibitors that target apicoplast processes (Fichera and Roos, 1997). DSG did not inhibit either fatty acid synthesis or prokaryotic protein synthesis – processes that occur in the apicoplast, but effected a decrease in the amount of nucleus encoded proteins that are targeted to the apicoplast, suggesting that it inhibited the trafficking of nucleus encoded proteins to the apicoplast. Confocal microscopy of parasites transfected with GFP fusion protein confirmed these findings, and is described in Chapter 6. The thesis ends with a summary of the findings in Chapter 7. Apicoplast processes have always been considered to harbor immense potential in the development of antimalarial agents, thanks to the absence of an equivalent organelle and hence pathways, in the human host. Trafficking of nucleus encoded proteins to the apicoplast has remained unexplored however. The work done in this thesis not only serves to demystify DSG with regard to its mechanism of action, but also paves the way for further studies in this area of intracellular trafficking, which could help in the development of more efficacious antimalarial agents. It also adds a new dimension to previous work conducted with regard to the anticancer action of DSG. Appendix 1 revolves around inhibitors which target various apicoplast processes. Apicoplast processes have been conventionally linked to the intriguing but unfortunate (with respect to clinical application) “delayed death”. Results presented in this section demonstrate that not all apicoplast processes invoke “delayed death”. Inhibition of apicoplast processes such as fatty acid biosynthesis and heme synthesis evoke rapid death. Inhibitors designed to target these processes could, therefore, be highly efficacious.

The Hsp70 family of molecular chaperones is essential for protein folding, re-folding misfolded client proteins, clearance of aberrant client proteins, and can also inhibit programmed cell death. There are two major cytosolic members of this family: the constitutive Hsc70, and the inducible Hsp72. Under stress conditions the Hsp70 family protects the cell from protein related damage by the induction of Hsp72. Hsc70 and Hsp72 are highly homologous with minor differences in substrate binding. In cancers, Hsp72 is commonly induced and this induction is thought to aid in cancer cell survival. In these studies we demonstrate the differential regulation of the prosurvival kinase Akt by Hsc70 and Hsp72. We demonstrate that of the two cytosolic forms, Hsp72 is the primary Akt regulator. Using a phenothiazine class inhibitor of Hsp70-family activity, methylene blue, we demonstrate dose dependent decreases in the levels of Akt; produced breast cancer specific cell death. This cell death could be rescued by the use of an Hsp70 family ATPase stimulating compound, SW02. We also demonstrate a similar phenotype with a rhodacyanine class Hsp70 family inhibitor, YM-1, also capable of reducing Akt and causing cancer specific cytotoxicity. The resulting Akt decreases were sufficient to block a tamoxifen-resistance pathway, allowing previously resistant cells to regain sensitivity to tamoxifen. These results demonstrate the capabilities of Hsp70 family inhibitors as potent compounds for the treatment of breast cancer.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 17, 2009). Includes bibliographical references.

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Agradecemos a compreensão. on 2016-09-14T20:21:24Z (GMT) / Submitted by YADIRA ANSOAR RODRIGUEZ null (yansoar@gmail.com) on 2016-09-14T21:01:11Z No. of bitstreams: 1 Tese VF.pdf: 2830119 bytes, checksum: 3b30a5eef5a71be48f3fcf3950b1dc44 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-09-15T19:20:51Z (GMT) No. of bitstreams: 1 ansoarrodriguez_y_dr_rcla.pdf: 2830119 bytes, checksum: 3b30a5eef5a71be48f3fcf3950b1dc44 (MD5) / Made available in DSpace on 2016-09-15T19:20:51Z (GMT). No. of bitstreams: 1 ansoarrodriguez_y_dr_rcla.pdf: 2830119 bytes, checksum: 3b30a5eef5a71be48f3fcf3950b1dc44 (MD5) Previous issue date: 2016-07-26 / Asociación Universitaria Iberoamericana de Postgrado (AIUP) / A aplicação indiscriminada de agrotóxicos constitui uma das maiores preocupações na atualidade, sendo o Brasil um dos países que mais uso faz destes produtos. O imidacloprido (IMI) é um dos inseticidas mais utilizados no mundo, principalmente nas culturas de cana-de-açúcar, citros, algodão e café. Apesar de seus benefícios, pode apresentar potencial tóxico e genotóxico em organismos não alvo. O uso de bioindicadores permite o estudo dos possíveis riscos destas substâncias nos organismos. Entre estes, plantas superiores e organismos aquáticos são considerados excelentes para avaliar efeitos de agrotóxicos no ambiente. Neste estudo, foram avaliados os efeitos de IMI em organismos não alvos (Allium cepa, Tradescantia pallida e Oreochromis niloticus) expostos a diferentes concentrações, baseadas na aplicação deste inseticida na cultura de cana-de-açúcar, por meio de ensaios celulares e moleculares. Foram testadas concentrações equivalentes à dose do produto recomendada para esta cultura (400 g/ha), a metade (200 g/ha) para simulação da diluição natural e o dobro (800 g/ha) para simulação do uso indiscriminado. O teste de aberrações cromossômicas e de micronúcleos (MN) em A. cepa e T. pallida foram utilizados para avaliar a toxicidade e genotoxicidade. Ensaio do cometa e teste do MN em eritrócitos de O. niloticus, avaliaram danos em nível primário e cromossômico. Análise das alterações histopatológicas no fígado de O. niloticus e localização in situ das proteínas de choque térmico (HSP70) analisadas sob microscopia de luz e imuno-histoquímica, respectivamente, foram empregadas para verificar o potencial tóxico em nível celular. Os resultados no teste de A. cepa e T. pallida, demonstraram que o IMI induziu alterações cromossômicas e o aumento da frequência de MN. Também foi observado indução do dano primário em eritrócitos de O. niloticus nas concentrações testadas e dano em nível cromossômico na maior concentração. Alterações hepáticas também foram observadas em todas as concentrações testadas, entre elas: degeneração hidrópica, núcleos picnóticos e perda do limite celular. A concentração mais alta (250μg/L) induziu um aumento de lípidos ácidos e neutros e dos níveis de marcação da proteína HSP70. Diante dos resultados pode-se concluir que o IMI, nas concentrações testadas, foi genotóxico para os organismos, além de induzir alterações histopatológicas e ativar mecanismos citoprotetores mediados por proteína de choque térmico. Este inseticida apresentou potencial tóxico e genotóxico nos organismos testados, os quais não são alvos de ação deste agrotóxico, fato este que deve ser levado em consideração para sua aplicação. / The indiscriminate application of pesticides is a major concern nowadays, and Brazil is one of the countries which use these products heavily on agriculture. The imidacloprid (IMI) is an insecticide sold worldwide, being widely used on sugar cane, citrus, cotton and coffee crops. Despite its benefits, IMI may have potential for inducing genetic changes in non-target organisms. In this sense, the use of bioindicators like higher plants and fishes allow the assessment of possible effects and risks to the environment derived from the use of this insecticide in agriculture. In this study, we evaluated the effects of IMI on non-target organisms (Allium cepa, Tradescantia pallida and Oreochromis niloticus) exposed to different concentrations, based on the application of this insecticide in the sugarcane culture, through cellular and molecular assays. A concentration equivalent to the recommended dose of the product for this culture (400 g/ha), the half (200 g/ha) for simulation of natural dilution and double (800 g/ha) that simulates the indiscriminate use. Chromosomal aberrations and micronucleus test (MN) in A. cepa and T. pallida were analyzed for toxicity and genotoxicity study. Comet assay and MN test in O. niloticus erythrocytes assess damage to primary and chromosomal level. Analysis of histopathological changes in the O. niloticus liver and in situ localization of heat shock protein (HSP70) were analyzed by light microscopy and immunohistochemistry, respectively, were performed to measure the toxic potential at the cellular level. The results in A. cepa and T. pallida assay, demonstrated that the IMI induced chromosomal alterations and increased frequency of MN. It was also observed induction of primary damage in O. niloticus erythrocytes at all concentrations and damage to the chromosomal level in the highest concentration. Liver changes were also found in all tested concentrations, as: hydropic degeneration, pyknotic nuclei and loss of cell limits. The highest concentration (250μg/L) showed an increase of acid and neutral lipids and labelling levels of HSP70 protein. Given the results it is possible to concluded that the IMI in the tested concentrations was genotoxic in these organisms, besides inducing histopathologic changes and activated cytoprotective mechanisms mediated by heat shock protein. This insecticide has a toxic and genotoxic potential for these organisms, which are not the target of action of this pesticide; fact should be considered for its application.

Orientador: Aureo Tatsumi Yamada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T22:06:48Z (GMT). No. of bitstreams: 1 Lima_PatriciaDanieleAzevedo_M.pdf: 2003445 bytes, checksum: 597310400704b8df5b1e07544bd6caca (MD5) Previous issue date: 2008 / Resumo: Durante a gestação em animais que possuem placentação hemocorial, a hipóxia no primeiro terço da prenhez é um dos fatores cruciais para indução da angiogênese e o adequado desenvolvimento da placenta. Contudo, esta hipóxia se contrapõe à intensa atividade das células que requerem elevado metabolismo, gerando um estresse fisiológico para estas células presentes na interface materno-fetal. Presume-se que estas células necessitem de mecanismos apropriados de citoproteção para sua sobrevida enquanto comprometidos ativamente no suporte funcional do útero gestante. Neste sentido, o presente trabalho teve como objetivo investigar a expressão e a distribuição da proteína de choque térmico 70 (HSP70) na interface materno-fetal durante a gestação normal em camundongos e a sua possível variação em condição de estresse adicional induzido experimentalmente através da lesão embrionária. Sítios de desenvolvimento embrionário/fetal de camundongos prenhes do dia de gestação (dg) 6 ao 17 e, após 30 minutos, 1, 6 e 12 h dos animais submetidos à lesão cirúrgida do embrião (LCE) no dg 9 foram coletados para: - processamento histotécnico convencional de embebição em parafina destinados às análises citoquímicas (lectina DBA e reação de TUNEL) e imunocitoquímicas (anti-HSP72/73, anti-PCNA); - embebição em resina Lowilcryl- K4M para imunocitoquímica ultraestrutural (anti-HSP72/73); - obtenção de homogenados teciduais destinados à SDS-PAGE das frações protéicas e Westernblot (anti-HSP72/73) e, - extração de RNA de homogenados teciduais e de células uNK isoladas para análise de transcritos (HSP72 e 73) com amplificação pelo RTPCR. As análises imunocitoquímicas demonstraram que as células uNK eram as únicas células que expressavam de forma constante as isoformas HSP72/73 ao longo da gestação, sendo confirmada a expressão dos transcritos gênicos das isoformas HSP72/73 nas células uNK isoladas pelo RT-PCR. A imunomicroscopia eletrônica detectou marcação conspícua nas mitocôndrias das células uNK. A análise quantitativa demonstrou que a lesão do embrião reduz o número de células uNK positivas para HSP72/73 e, o SDS/PAGE/Western-blotting identificou as isoformas HSP72 e 73 presente nos homogenados teciduais do útero com uma perceptível redução na intensidade da banda correspondente ao HSP73 nas amostras de pós-lesão, sem afetar significativamente a isoforma HSP72. As análises realizadas com a dupla marcação de TUNEL e PCNA demonstrarm redução de células uNK PCNA positivas no útero submetido a lesão embrionária e aumento de núcleos marcadas positivamente pelo TUNEL. Estes resultados demonstram de forma inédita a expressão de HSP72/73 nas células uNK, sendo inédita também a constatação em leucócitos, sugerindo um papel citoprotetor para estas células importantes na manutenção da gestação. A redução de células uNK HSP72/73 positivas no útero gestante desencadeada pela lesão embrionária, consubstancia a hipótese da atuação da HSP 72/73 como chaperona citoprotetora nas células uNK sendo crítica a atuação da isoforma HSP73 presente na mitocôndria através da regulação negativa das vias de morte celular por apoptose nas células uNK / Resumo: Durante a gestação em animais que possuem placentação hemocorial, a hipóxia no primeiro terço da prenhez é um dos fatores cruciais para indução da angiogênese e o adequado desenvolvimento da placenta. Contudo, esta hipóxia se contrapõe à intensa atividade das células que requerem elevado metabolismo, gerando um estresse fisiológico para estas células presentes na interface materno-fetal. Presume-se que estas células necessitem de mecanismos apropriados de citoproteção para sua sobrevida enquanto comprometidos ativamente no suporte funcional do útero gestante. Neste sentido, o presente trabalho teve como objetivo investigar a expressão e a distribuição da proteína de choque térmico 70 (HSP70) na interface materno-fetal durante a gestação normal em camundongos e a sua possível variação em condição de estresse adicional induzido experimentalmente através da lesão embrionária. Sítios de desenvolvimento embrionário/fetal de camundongos prenhes do dia de gestação (dg) 6 ao 17 e, após 30 minutos, 1, 6 e 12 h dos animais submetidos à lesão cirúrgida do embrião (LCE) no dg 9 foram coletados para: - processamento histotécnico convencional de embebição em parafina destinados às análises citoquímicas (lectina DBA e reação de TUNEL) e imunocitoquímicas (anti-HSP72/73, anti-PCNA); - embebição em resina Lowilcryl- K4M para imunocitoquímica ultraestrutural (anti-HSP72/73); - obtenção de homogenados teciduais destinados à SDS-PAGE das frações protéicas e Westernblot (anti-HSP72/73) e, - extração de RNA de homogenados teciduais e de células uNK isoladas para análise de transcritos (HSP72 e 73) com amplificação pelo RTPCR. As análises imunocitoquímicas demonstraram que as células uNK eram as únicas células que expressavam de forma constante as isoformas HSP72/73 ao longo da gestação, sendo confirmada a expressão dos transcritos gênicos das isoformas HSP72/73 nas células uNK isoladas pelo RT-PCR. A imunomicroscopia eletrônica detectou marcação conspícua nas mitocôndrias das células uNK. A análise quantitativa demonstrou que a lesão do embrião reduz o número de células uNK positivas para HSP72/73 e, o SDS/PAGE/Western-blotting identificou as isoformas HSP72 e 73 presente nos homogenados teciduais do útero com uma perceptível redução na intensidade da banda correspondente ao HSP73 nas amostras de pós-lesão, sem afetar significativamente a isoforma HSP72. As análises realizadas com a dupla marcação de TUNEL e PCNA demonstrarm redução de células uNK PCNA positivas no útero submetido a lesão embrionária e aumento de núcleos marcadas positivamente pelo TUNEL. Estes resultados demonstram de forma inédita a expressão de HSP72/73 nas células uNK, sendo inédita também a constatação em leucócitos, sugerindo um papel citoprotetor para estas células importantes na manutenção da gestação. A redução de células uNK HSP72/73 positivas no útero gestante desencadeada pela lesão embrionária, consubstancia a hipótese da atuação da HSP 72/73 como chaperona citoprotetora nas células uNK sendo crítica a atuação da isoforma HSP73 presente na mitocôndria através da regulação negativa das vias de morte celular por apoptose nas células uNK / Abstract: During the pregnancy of animals developing hemochorial placenta, the hypoxia in the first third of pregnancy is one of the crucial factor for induction of angiogenesis and adequate placental development. However, this hypoxia is contradictory to the great dynamism and metabolism of cells required in the pregnant uterus, conditioning a physiological stress for the cells present at the maternal-fetal interface. It is presumed these cells demand appropriate cytoprotective mechanism for their survival while are committed to actively support the pregnancy. In this way, the present work aimed to investigate the expression and distribution of the chapelone isoforms heat shock protein 72 and 73 (HSP72/73) at the maternal fetal-interface through the pregnancy in mice and its possible variations under additional stressing condition induced experimentally by embryo lesion. Embryo/fetus developing sites of pregnant mice from gestational days (gd) 6 to 17 and, after 30min, 1h and 6h of surgical embryo lesion (SEL) on gd 9 mice, were collected for: - conventional paraffin embedding for cytochemical (DBA lectin and TUNEL reaction) and immunocytochemical (anti-HSP72/73, anti-PCNA) analysis; - LR-white resin embedding for ultrastructural immunocytochemistry (anti- HSP72/73); - uterine tissue homogenates for SDS-PAGE of proteins fractions and Western-blot (anti-HSP7273) and; - RNA extratction form uterine tissue homogenates and isolated uNK cells for transcripts (HSP72 and 73) amplification by RT-PCR. The immunocytcchemical analysis showed the uNK cells as the only cell expressing constantly the HSP72/73 isoforms throughout the gestation, being confirmed the expression of both gene isoforms by RT-PCR in uNK cells. The immunoelectron microscopy detected conspicuous labeling in the mitochondria of uNK cells. The quantitative analysis demonstrated that embryo-lesion reduced the number of HSP72/73 positive uNK cells in the uterus and, SDS/PAGE and Westernblot identified the HSP72 and 73 isoforms present in the tissue homogenates with low reactive intensity of the band corresponding to HSP73 in the after-lesion samples, without affecting significantly the HSP72 isoform. The analysis of TUNEL and PCNA double labelling showed decreasing of PCNA positive-uNK cells in the uterus after embryo-lesion and increasing of TUNEL positive nuclei. These results confirms the expression of HSP72 and HSP73 isoforms in the uNK cells through the gestation and to date, this is also the first report showing HSP70 in leukocytes, suggesting a cytoptotective function to this cell while working actively as important cells supporting the pregnancy. The decreasing of HSP72/73 positive uNK cells in the pregnant uterus triggered by embryo lesion consubstantiate the hypothesis of HSP72/73 working as cytoprotective chaperone in the uNK cells, and the HSP73 isoform in the mitochondria seems to be critical on down-regulation of apoptotic cell depth pathway / Abstract: During the pregnancy of animals developing hemochorial placenta, the hypoxia in the first third of pregnancy is one of the crucial factor for induction of angiogenesis and adequate placental development. However, this hypoxia is contradictory to the great dynamism and metabolism of cells required in the pregnant uterus, conditioning a physiological stress for the cells present at the maternal-fetal interface. It is presumed these cells demand appropriate cytoprotective mechanism for their survival while are committed to actively support the pregnancy. In this way, the present work aimed to investigate the expression and distribution of the chapelone isoforms heat shock protein 72 and 73 (HSP72/73) at the maternal fetal-interface through the pregnancy in mice and its possible variations under additional stressing condition induced experimentally by embryo lesion. Embryo/fetus developing sites of pregnant mice from gestational days (gd) 6 to 17 and, after 30min, 1h and 6h of surgical embryo lesion (SEL) on gd 9 mice, were collected for: - conventional paraffin embedding for cytochemical (DBA lectin and TUNEL reaction) and immunocytochemical (anti-HSP72/73, anti-PCNA) analysis; - LR-white resin embedding for ultrastructural immunocytochemistry (anti- HSP72/73); - uterine tissue homogenates for SDS-PAGE of proteins fractions and Western-blot (anti-HSP7273) and; - RNA extratction form uterine tissue homogenates and isolated uNK cells for transcripts (HSP72 and 73) amplification by RT-PCR. The immunocytcchemical analysis showed the uNK cells as the only cell expressing constantly the HSP72/73 isoforms throughout the gestation, being confirmed the expression of both gene isoforms by RT-PCR in uNK cells. The immunoelectron microscopy detected conspicuous labeling in the mitochondria of uNK cells. The quantitative analysis demonstrated that embryo-lesion reduced the number of HSP72/73 positive uNK cells in the uterus and, SDS/PAGE and Westernblot identified the HSP72 and 73 isoforms present in the tissue homogenates with low reactive intensity of the band corresponding to HSP73 in the after-lesion samples, without affecting significantly the HSP72 isoform. The analysis of TUNEL and PCNA double labelling showed decreasing of PCNA positive-uNK cells in the uterus after embryo-lesion and increasing of TUNEL positive nuclei. These results confirms the expression of HSP72 and HSP73 isoforms in the uNK cells through the gestation and to date, this is also the first report showing HSP70 in leukocytes, suggesting a cytoptotective function to this cell while working actively as important cells supporting the pregnancy. The decreasing of HSP72/73 positive uNK cells in the pregnant uterus triggered by embryo lesion consubstantiate the hypothesis of HSP72/73 working as cytoprotective chaperone in the uNK cells, and the HSP73 isoform in the mitochondria seems to be critical on down-regulation of apoptotic cell depth pathway / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

<p dir="ltr"><b>Background</b></p><p dir="ltr">Heart failure (HF) is a chronic clinical syndrome characterized by symptoms including breathlessness, fatigue, swelling of the ankles, and signs such as edema pulmonary crackles etc. During HF, pathogenic mechanisms including hemodynamic overload, ventricular remodeling, aberrant calcium handling, excessive neurohormonal stimulation contribute to the worsening and progression of the condition. Ventricular arrhythmias are the common cause of sudden cardiac death (SCD) in HF patients.</p><p dir="ltr">Hyperactivation of the sympathetic nervous system (SNS), a characteristic of HF, causes an increase in circulating catecholamines which becomes detrimental to-adrenergic receptors (-AR) leading to signaling dysfunction, and decrease in contractility and the ionotropic reserve. Expression of calcium/calmodulin-dependent protein kinase II (CaMKII), a downstream effector of-AR and a key regulator of calcium homeostasis, has been shown to be enhanced in HF. CaMKII-mediated mechanisms have been demonstrated to contribute to cardiac remodeling, arrhythmias by pathological regulation of ion channels, and contractile dysfunction.</p><p dir="ltr">The human ether-a-go-go related gene (hERG) encodes the pore-forming subunit of the voltage-gated potassium channel that conduct the rapid component of the delayed rectifier potassium current, <i>I</i>_Kr. The gating kinetics of <i>I</i>_Krmakes it a crucial determinant of the duration of the plateau phase of atrial and ventricular action potential (AP). Reduced <i>I</i>_Kr density due to loss-of-function mutations or pharmacological blockage of hERG channels precipitate arrhythmias. Downregulation of <i>I</i>_Kr density and protein have been reported in HF. Recent studies suggest that microRNAs (miRNAs) are involved in pathological downregulation of hERG.</p><p dir="ltr">miRNA are small non-coding RNAs of approximately 22 nucleotides in length that function as gene expression regulatory elements by repression translation. Aberrant miRNA expression has associated with cancer, cardiovascular, autoimmune, and inflammatory disorders.</p><p dir="ltr"><b>Objective</b></p><p dir="ltr">The overarching objective of this study is to investigate the mechanisms of CaMKII-mediated regulation of hERG function, including assessment of an interplay with miR-362-3p during sustained β-AR stimulation. In Specific Aim 1, the effect of CaMKII activation through sustained β-AR stimulation on hERG function and miR-362-3p expression will be assessed. The mechanism of miR-362-3p upregulation will be evaluated in Specific Aim 2, and in Specific Aim 3, the interactome of miR-362-3p and binding sites will be characterized and predicted, respectively.</p><p dir="ltr"><b>Methods</b></p><p dir="ltr">Whole-cell, voltage clamp electrophysiology experiments were performed in HEK 293 cells stably expressing hERG (hERG-HEK) and both hERG and wild-type CaMKIIδ<br>(hERG/CaMKII-HEK) following treatment with isoproterenol for 48 hours, and after transfection with miR-362-3p. The effect of CaMKII activation on miR-362-3p was assessed using real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated 48 hours after isoproterenol treatment and the TaqMan assay was used to reverse transcribe and analyze miR-362-3p expression. Cells were transfected with cJun siRNA and precursor miR-362-3p to assess the role of cJun miR-362-3p upregulation during sustained β-AR stimulation with isoproterenol. The interactome of miR-362-3p was assessed in both cell lines using enhanced crosslinking immunoprecipitation (eCLIP) assay. miR-362-3p binding sites were predicted using RNAStructure Duplexfold after identification of miR-362-3p chimeric molecules from eCLIP experiment. Interaction analysis was performed using GeneMania in Cytoscape to identify genes that were potentially downregulated by miR-362-3p and been reported to interact with hERG.</p><p dir="ltr"><b>Results</b></p><p dir="ltr">In Specific Aim 1, the effect of sustained β-AR stimulation on hERG currents and endogenous miR-362-3p was assessed in hERG-HEK and hERG/CaMKII-HEK cells. Using whole-cell voltage clamp electrophysiology, we demonstrated that 48 hours treatment with 100 nM isoproterenol reduced hERG currents in hERG/CaMKII-HEK cells (p = 0.032) but had no effect on the voltage dependence of activation (p = 0.61) relative to control vehicle. Isoproterenol treatment for 48 hours, however, had no effect on hERG currents (p = 0.58) and the voltage dependence of activation (p = 0.99) in hERG-HEK cells. The effect of sustained isoproterenol treatment on miR-362-3p was also assessed using RT-qPCR. In hERG/CaMKII cells, 48 hours isoproterenol treatment increased miR-362-3p expression (2.3 folds; p = 0.038) relative to control vehicle. hERG/CaMKII-HEK cells were also treated with 500 nM KN-93 or its inactive analogue, KN-92, in an attempt to reverse CaMKII effect on miR-362-3p expression. Treatment with KN-93 decreased miR-362-3p expression (0.5-fold; p = 0.002) relative KN-92 treatment. Isoproterenol treatment had no effect on miR-362-3p expression in hERG-HEK cells (p = 0.38).</p><p dir="ltr">The regulatory mechanism of miR-362-3p expression was evaluated in Specific Aim 2. The role of an activator protein-1 (AP-1)-like sequence located at 98 base pairs upstream of miR-362-3p transcription start site was probed using siRNA inhibition of cJun, a central protein of the AP-1 complex, and deletion of the site sequence. The effect of exogenous miR-362-3p on hERG currents were first assessed. Precursor miR-362-3p decreased hERG currents (p = 0.003) compared to control plasmid. The effect of CaMKII overexpression was also assessed on exogenous miR-363-3p expression. Isoproterenol treatment in hERG/CaMKII-HEK cells transfected with precursor miR-362-3p increased mature miR-362-3p expression (0.029) compared to control vehicle treatment. Inhibition of cJun inhibition with cJun-specific siRNA decreased mature miR-362-3p expression (0.5-fold; p = 0.027) compared to scramble siRNA in hERG-HEK cells. In hERG-HEK cells transfected with mutated precursor miR-362-3p (AP-1-like site deleted), cJun inhibition with siRNA had no effect on miR-362-3p expression (p = 0.40).</p><p dir="ltr">The focus of Specific Aim 3 was to characterize the interactome of miR-362-3p as well as predict the miRNA response element (MRE) of its target mRNAs using enhanced crosslinking immunoprecipitation. A network analysis was also performed to identify miR-362-3p targets that have been reported to interact with hERG. Approximately 23% of miR-362-3p mRNA targets from the eCLIP assay have also been catalogued in miRNA database, TargetScanHuman, as miR-362-3p targets. miR-362-3p chimeric molecules with 853 unique targets, of which 75 were identified to interact with hERG through the network analysis. Four unique chimeric molecules between miR-362-3p and hERG mRNA were identified, but the interactions were non-canonical (located in the coding sequence of hERG and outside the seed region of miR-362-3p). Thirty five of the 75 miR-362-3p targets that were identified to interact had a chimeric read ≥ 3, a cutoff number indicating non-random chimeric formation. Using RNAStructure DuplexFold, miR-362-3p was predicted to form canonical binding with 12 of 35 mRNA targets. HSPA4, a heat shock protein involved in the maturation and trafficking of hERG, was identified in a canonical interaction (8-mer) with miR-362-3p.</p><p dir="ltr"><b>Conclusion</b>:</p><p dir="ltr">Sustained β-AR stimulation increases miR-362-3p expression and decreases hERG currents in CaMKII overexpressing cells. cJun mediates miR-362-3p upregulation by interacting with an AP-1-like sequence upstream of miR-362-3p transcription start site. Pathological regulation of <i>I</i>_Kr by CaMKII mediated by miR-362-3p during sustained-AR may contribute to increased risk of arrhythmias in states of increase catecholaminergic activity, such as HF.</p>

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