A soja geneticamente modificada tolerante ao herbicida glifosato foi testada em ensaio nutricional. A qualidade protéica da soja foi avaliada durante 14 dias de experimento com ratos machos tipo Wistar recém desmamados. Além de um grupo controle aproteico, quatro dietas testadas continham cerca de 10% de proteínas de diferentes fontes: caseína, soja comercial, soja parental e soja GM. Resultados similares entre os grupos demonstraram o baixo aproveitamento da proteína ingerida, conforme esperado para todas as dietas com soja não suplementadas com metionina e expressos pelos valores de PDCAAS. As análises hematológicas realizadas demonstraram a síntese comprometida de células eritrócitárias e imunológicas nos mesmos grupos experimentais. Este comportamento fisiológico dos animais indica que a ingestão da variedade GM não causou diferença significativa no desenvolvimento dos animais entre as três amostras de soja ensaiadas e tampouco foram observados efeitos adversos em órgãos dos animais e nos parâmetros químicos analisados. / A glyphosate tolerant soybean obtained by genetic modification was tested on a nutritional essay. The quality of the soy protein was assessed by a 14-day long experiment with Wistar male rats, three weeks old. Besides the control free protein group, four different diet groups containing about 10 % protein were pooled out: casein, commercial, parental and GM soybeans. Similar results showed the regular low biological value of the consumed soy proteins not supplemented by methionine displayed by PDCAAS values. The hematological analysis pointed to a commitment of the synthesis of erythrocytic and immunologic cells at the experimental soy groups. The overall behavior of the animals indicate the ingestion of the GM variety of soybean did not cause significant differences for the rat development when compared to the other soybean groups, neither side effects on inner organs and chemical analyzed parameters.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The common bean (Phaseolus vulgaris L.) have high content of dietary fiber and protein of high quality, so it can be considered a functional food. The objectives of this work were: (1) to verify if occurs maternal effect for the insoluble fiber, soluble fiber, methionine and cysteine content in grains common bean; (2) to estimate the heritability and gain from selection for these traits; (3) to assess the composition of amino acids of early generations of common bean obtained from controlled crosses with genitor of high cysteine content. Six generations were obtained (F1, F1 reciprocal, F2, F2 reciprocal, retrocruzamento 1 - RCP1 and retrocruzamento 2 - RCP2) from crosses between genitors contrasting to the insoluble fiber (Guateian 6662 x Guapo Brilhante), soluble fiber (Guapo Brilhante x Pérola), methionine (BRS Valente x Iapar 44) and cysteine (TPS Nobre x Minuano) content. The soluble and insoluble fiber contents were determined by enzyme-gravimetric method and the amino acids content were determined by high performance liquid chromatography (HPLC). The experimental design was a completely randomized, with variable number of replications for generations obtained. The results showed presence of genetic variability for the insoluble fiber, methionine and cysteine contents. No was observed effect maternal significant for insoluble fiber, soluble fiber, methionine and cysteine contents in grains of common bean. Estimates of heritability moderate in the broad sense of 45.22% and gain from selection predicto of 10.15% was obtained for the insoluble fiber. Unable to estimate the heritability in the broad sense and in the narrow sense for soluble fiber, because of lack of genetic variability in different generations, and for the methionine and cysteine content, because genetic negative variances were obtained. The selection of plants F2 obtained from crosses between Guateian 6662 x Guapo Brilhante and between BRS Valente x Iapar 44 may result in superior genotypes for the insoluble fiber and methionine content, respectively. The genitors TPS Nobre and Minuano and F1 and F1r generations showed essential amino acids and nonessential contents suitable for use in food. For the cysteine content, it is not possible to select any plant in the F2 generation due to the low contents of this amino acid sulfur. / O feijão (Phaseolus vulgaris L.) apresenta alto teor de fibra alimentar e proteína de alta qualidade, por isso pode ser considerado um alimento funcional. Os objetivos desse trabalho foram: (1) investigar a ocorrência de efeito materno para os teores de fibra insolúvel, fibra solúvel, metionina e cisteína em grãos de feijão; (2) obter estimativas de herdabilidade e de ganho por seleção para esses caracteres; (3) avaliar a composição de aminoácidos de gerações precoces de feijão obtidas a partir de cruzamentos controlados com genitor de alto teor de cisteína. Sendo assim, foram obtidas seis gerações (F1, F1 recíproco, F2, F2 recíproco, retrocruzamento 1 - RCP1 e retrocruzamento 2 - RCP2), a partir de cruzamentos entre genitores contrastantes para os teores de fibra insolúvel (Guateian 6662 x Guapo Brilhante), fibra solúvel (Guapo Brilhante x Pérola), metionina (BRS Valente x Iapar 44) e cisteína (TPS Nobre x Minuano). Os teores de fibra insolúvel e solúvel foram determinados pelo método enzimático-gravimétrico e os teores dos aminoácidos foram quantificados por cromatografia líquida de alta performance (HPLC). O delineamento experimental utilizado foi o inteiramente casualizado, com número de repetições variável para as diferentes gerações. Os resultados obtidos revelaram presença de variabilidade genética para a fibra insolúvel, metionina e cisteína. Não foi verificado efeito materno significativo para os teores de fibra insolúvel, fibra solúvel, metionina e cisteína em grãos de feijão. Estimativa moderada de herdabilidade, em sentido amplo, de 45,22% e ganho por seleção predicto de 10,15% foram obtidos para a fibra insolúvel. Não foi possível estimar a herdabilidade, em sentido amplo, e em sentido restrito para a fibra solúvel, devido à ausência de variabilidade genética nas diferentes gerações, e para os teores de metionina e de cisteína, pois variâncias genéticas negativas foram obtidas. A seleção de plantas F2 obtidas a partir do cruzamento entre Guateian 6662 x Guapo Brilhante e entre BRS Valente x Iapar 44 poderá resultar em genótipos superiores quanto aos teores de fibra insolúvel e de metionina, respectivamente. Os genitores TPS Nobre e Minuano e as gerações F1 e F1r apresentaram teores de aminoácidos essenciais e não-essenciais adequados para uso na alimentação. Para o teor de cisteína, não é possível selecionar nenhuma planta na geração F2 devido aos baixos teores desse aminoácido sulfurado.

Made available in DSpace on 2014-07-29T15:23:42Z (GMT). No. of bitstreams: 1 Dissertacao Amanda Goulart de Oliveira Sousa.pdf: 1097784 bytes, checksum: efe44a45c995339f0d2e83886704fc64 (MD5) Previous issue date: 2011-04-01 / The aim of this study was to determine the nutritional quality and protein value of the baru almond, pequi almond, and cerrado cashew nut, native fruits from the Brazilian Savanna, compared to the peanut. Standardized methods were used to determine centesimal composition, amino acid profile, fatty acids and mineral content. The experiment was carried out with 42 male weanling Wistar rats. The animals were randomly assigned into seven groups. The experiment lasted fourteen days. The diets were formulated according to AIN-93G, six diets with 10% protein: CAS7 (7% lipid casein), CAS15 (15% lipid casein), AMB (baru almond), AMP (pequi almond), CJC (cerrado cashew nut), AMD (peanut) and a protein-free diet. A biological assay was carried out to assess the protein value, by Net Protein Ratio (NPR), Relative Net Protein Ratio (RNPR), and Protein Digestibility-Corrected Amino Acid Score (PDCAAS) methods. We found that the exotic almonds and the nut are rich in proteins (22.7 29.9 g/100 g), lipids (41.9 50.0 g/100 g), fibres (baru and pequi almonds, around 10.0 g/100 g), iron and zinc (4.3 7.4 mg/100 g). Baru almond s protein did not show deficiency in essential amino acids and lysine was the first limiting amino acid in the proteins of the pequi almond and cerrado cashew nut. The baru almond showed a RNPR of 86%, similar to that of the cerrado cashew nut (78%), but higher than that of the peanut (72%) and of the pequi almond (54%). The PDCAAS value of the baru almond (91%) was the highest and cerrado cashew nut and peanut presented similar values of this index (82%), which were higher than that of the pequi almond (55%). The baru almond has the highest protein quality, but the cerrado cashew nut and peanut are sources of good quality protein, too. We recommend the inclusion of these exotic foods in healthy diets and in food industry, and the baru almond and cerrado cashew nut as sources of complementary protein. / O objetivo deste estudo foi determinar a qualidade nutricional e o valor protéico das amêndoas de baru e de pequi e da castanha-de-caju-do-cerrado, frutas nativas do Cerrado brasileiro, e comparar com o amendoim. Determinou-se a composição química centesimal, teor de minerais e perfil de aminoácidos, conforme métodos padronizados. Foi realizado um experimento com 42 ratos Wistar, machos, recém-desmamados, distribuídos em sete grupos segundo delineamento por blocos casualizados, durante catorze dias. As dietas foram formuladas segundo AIN-93G, sendo seis dietas com 10% de proteína: CAS7 (caseína com 7% de lipídios); CAS15 (caseína com 15% de lipídios); amêndoas de baru (AMB); amêndoa de pequi (AMP); castanha-de-caju-do-cerrado (CJC) e amendoim (AMD), e uma dieta aprotéica (APO). O valor protéico foi estimado por meio dos métodos Net Protein Ratio (NPR), Relative Net Protein Ratio (RNPR) e Protein Digestibility-Corrected Amino Acid Score (PDCAAS). As amêndoas de baru, de pequi e a castanha-de-caju-do-cerrado são ricas em proteínas (22,7-29,9 g/100 g), lipídios (41,9-50,0 g/100 g), fibras (amêndoas de baru e de pequi, em torno de 10,0 g/100 g), ferro e zinco (4,3-7,4 mg/100 g). A proteína da amêndoa de baru não apresentou deficiência em aminoácidos essenciais, e a lisina foi o primeiro aminoácido limitante nas proteínas da amêndoa de pequi e da castanha-de-caju-do-cerrado, e o segundo limitante na proteína do amendoim. A amêndoa de baru apresentou RNPR de 86%, estatisticamente similar ao da castanha-de-caju-do-cerrado (78%), mas superior ao do amendoim (72%) e da amêndoa do pequi (54%). A amêndoa de baru apresentou maior valor de PDCAAS (91%), e a castanha-de-caju-do-cerrado e o amendoim apresentaram valores semelhantes para este índice (82%), seguidos pela amêndoa de pequi (55%). A amêndoa de baru possui maior qualidade protéica, porém a castanha-de-caju-do-cerrado e o amendoim também são fontes de proteína de boa qualidade. Recomendamos a inclusão destes alimentos nativos em dietas saudáveis e na indústria alimentícia, e a amêndoa de baru e a castanha-de-caju-do-cerado como fontes de proteínas complementares.

A soja geneticamente modificada tolerante ao herbicida glifosato foi testada em ensaio nutricional. A qualidade protéica da soja foi avaliada durante 14 dias de experimento com ratos machos tipo Wistar recém desmamados. Além de um grupo controle aproteico, quatro dietas testadas continham cerca de 10% de proteínas de diferentes fontes: caseína, soja comercial, soja parental e soja GM. Resultados similares entre os grupos demonstraram o baixo aproveitamento da proteína ingerida, conforme esperado para todas as dietas com soja não suplementadas com metionina e expressos pelos valores de PDCAAS. As análises hematológicas realizadas demonstraram a síntese comprometida de células eritrócitárias e imunológicas nos mesmos grupos experimentais. Este comportamento fisiológico dos animais indica que a ingestão da variedade GM não causou diferença significativa no desenvolvimento dos animais entre as três amostras de soja ensaiadas e tampouco foram observados efeitos adversos em órgãos dos animais e nos parâmetros químicos analisados. / A glyphosate tolerant soybean obtained by genetic modification was tested on a nutritional essay. The quality of the soy protein was assessed by a 14-day long experiment with Wistar male rats, three weeks old. Besides the control free protein group, four different diet groups containing about 10 % protein were pooled out: casein, commercial, parental and GM soybeans. Similar results showed the regular low biological value of the consumed soy proteins not supplemented by methionine displayed by PDCAAS values. The hematological analysis pointed to a commitment of the synthesis of erythrocytic and immunologic cells at the experimental soy groups. The overall behavior of the animals indicate the ingestion of the GM variety of soybean did not cause significant differences for the rat development when compared to the other soybean groups, neither side effects on inner organs and chemical analyzed parameters.

No description available.

Die Interaktion von RING-finger-Ubiquitin (Ub)-Ligasen (E3-Enzyme) mit Ub-konjugierenden Enzymen (E2-Enzyme) bestimmt wie schnell ein Zielprotein mit einer Ub-Modifikation versehen wird. In dieser Arbeit wird die Stimulation der E2-Enzyme Ubc6 und Ubc7 durch die E3-Enzyme Hrd1 und Doa10 untersucht. Es wird gezeigt, dass Ubc6~Ub-Konjugate bereitwilliger sogenannte "closed conformations" annehmen als Ubc7~Ub-Konjugate, was wiederum die Tendenz, Ub zu übertragen, steigert. Die katalytische Aktivität von Ubc7 kann durch RING-Domänen stimuliert werden. Durch einen allosterischen Mechanismus, der linchpin allostery, werden Ubc7~Ub-Intermediate in "closed conformations" gedrängt. Zusätzlich werden spezifische Kontakte zwischen RING-finger-Domänen und der Ub-Einheit in einem E2~Ub-Konjugat identifiziert. Diese schränken die Flexibilität des Konjugates weiter ein und begünstigen dadurch die Reaktivität des E2~Ub-Intermediates. Dieser Mechanismus scheint weit verbreitet zu sein und wurde schon bei anderen Ub-Ligasen beobachtet. Poly-Ub-Signale werden in mehreren Schritten generiert. In einer Priming genannten Reaktion wird die erste Ub-Einheit auf das Zielprotein übertragen. Dieser Vorgang erfordert sehr flexible Enzyme, die in diversem Umfeld Akzeptorstellen finden und mit Ub modifizieren. Die zweite Reaktion, die elongation, umfasst das schrittweise Anheften weiterer Ub-Moleküle an die erste Einheit. Im Gegensatz zum Priming, beruht die Bildung einheitlicher Ketten auf der wiederholten und robusten Konjugation von Ub-Molekülen in gleichbleibendem Milieu. Ub-Ligasen verwenden verschiedene Strategien, um die unterschiedlichen Herausforderungen dieser Reaktionen zu bewältigen. Während Doa10 je ein E2-Enzym pro Reaktion nutzt, kann Hrd1 ein einzelnes E2-Enzym durch linchpin allostery ausreichend stimulieren, um beide Prozesse durchzuführen, wie diese Arbeit zeigt. / The interaction of RING-finger ubiquitin (Ub) ligases (E3 enzymes) with Ub conjugating enzymes (E2 enzymes) dictates how fast a Ub modification is synthesized on a client protein. This thesis addresses the catalytic stimulation of the E2 enzymes Ubc6 and Ubc7 by their cognate E3 enzymes Hrd1 and Doa10. Results show that Ubc6~Ub conjugates adopt closed conformations more readily than Ubc7~Ub conjugates, indicative for an inherently higher propensity to transfer Ub. The catalytic activity of Ubc7 can be stimulated by a RING domain which relies on so-called linchpin allostery. This drives Ubc7~Ub intermediates into a closed conformation. In addition, specific contacts of the RING-finger domain and the Ub moiety in an E2~Ub conjugate were identified which further restrict the flexibility of the conjugate and thereby increase the reactivity of the E2~Ub intermediate. This seems to represent a common mechanism for the stimulation of E2 enzymes because similar contacts of RING-finger proteins with Ub have been observed for other Ub ligases. Poly-Ub signals on proteins are generated in successive steps. The first reaction, called "priming", comprises the attachment of an initial Ub moiety to the target. This requires high flexibility of the involved enzymes to modify acceptor sites in a versatile environment. The second step is the sequential addition of Ub to previously attached Ub molecules in a process termed elongation. In contrast to priming, the formation of uniform Ub chains relies on the repeated and robust conjugation of Ub moieties in a mostly invariant setting. Ub ligases employ different strategies to meet the divergent requirements of these reactions. Doa10 uses separate E2 enzymes for priming and elongation. This thesis shows that Hrd1 efficiently stimulates a single E2 enzyme for the catalysis of both steps via linchpin allostery.

Proteins are the center of all biochemical reactions in living organisms. Proteins need to be present at the right time, in the right place, with the correct concentration and have the right shape to carry their designated function. Protein homeostasis is when all proteins in the proteome are in functional balance, and such balance is maintained by synthesis, folding, and degradation machinery. When protein homeostasis is lost, organisms start to age and develop diseases. To truly unveil disease mechanisms and provide more efficient means for treatment and prevention, we need a holistic understanding of the mechanism of protein homeostasis. Currently, most biomarker studies focus on the quantity aspect of the proteome. The quality aspect has been neglected because of the difficulties in measuring quality in vivo with cellular context retained. This work first proposes a kinetic model of protein homeostasis, which can provide a holistic view, including both quantity and quality aspects, as well as monitor the complex protein interactions. Using mass spectrometry, the model quantifies the quality of proteome by linking the concentration of protein, mRNA, and the rate protein synthesis, folding, unfolding, misfolding, refolding, degradation of the correctly folded protein, and degradation of protein aggregation. We then applied the ideas within the kinetic model of protein homeostasis to study several proteins in human blood serum. We reviewed the current known mechanism of transthyretin mediated amyloidosis and proposed a study approach that can measure the quality difference between different transthyretin's mutation stages, as well as monitor if the transthyretin amyloidosis has been developed at the early stage. We also used mass-spectrometry to quantify the surface accessibility differences in human serum albumin (HSA) between patients with and without rheumatoid arthritis (RA). We found certain residues are less reactive in the RA group, indicating a structural change in HSA. Such structural changes, possibly caused by ligand binding, stabilized HSA and explained the heat denature curve shift we observed. In the end, we introduced a novel assay, Iodination Protein Stability Assay (IPSA). IPSA is used to quantify protein quality by measuring protein folding stability. We applied IPSA to human serum, and it is the first in situ study, to our best knowledge, that measure the protein folding stability of proteins from human serum. We confirmed that IPSA is sensitive to measuring the differences in protein folding stability between transferrin's different iron-binding states. Together, this dissertation conveys the importance of adding quality aspects to current quantity-focused research in curing diseases and improving the quality of human life.

No description available.

No description available.

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.