Abstract Protein folding of outer membrane and secreted proteins, including receptors, cytokines and antibodies is often linked to disulphide bond formation. Native disulphide bond formation is complex and is usually the rate limiting step in the folding of such proteins. The enzymes which catalyse the slow steps in disulphide bond formation belong to the protein disulphide isomerase (PDI) family. PDI catalyses formation, reduction and isomerization of newly synthesized disulphide bonds. The mechanisms of action of the PDIs are currently poorly understood and this not only inhibits our understanding of the biogenesis of a range of medically important proteins, and hence associated disease states, but also prevents the effective manipulation of the cellular environment by the biotechnology industry for the production of high value therapeutic proteins. Hence, understanding the mechanism of action of these enzymes is vital for a wide range of medically important processes and therapies. In this study the role of a conserved arginine residue in the catalytic activity of PDI was shown. The movement of this residue into and out of the active site locale of PDI was shown to modulate the pKa of the C-terminal active site cysteine of PDI and by that way to allow the enzyme to act efficiently as catalyst both of oxidation and isomerization reactions. The possible role of hydrogen peroxide produced by sulphydryl oxidases during disulphide bond formation was studied in an oxidative protein refolding assay. Analysis showed that hydrogen peroxide can be used productively to make native disulphide bonds in folding proteins with minimal side reactions. In addition, the kinetics of oxidation and reduction of the <b>a</b> domains of PDI and Pdi1p by glutathione was studied in this thesis. The kinetics obtained with stopped-flow and quenched-flow experiments showed the reactions to be more rapid and complex than previously thought. Significant differences exist between the kinetics of PDI and Pdi1p. This implies that the use of yeast systems to predict physiological roles for mammalian PDI family members should be treated cautiously.

This thesis is based on studies, described in four papers, in which the folding energy landscape of MerP was investigated by various techniques. MerP is a water-soluble 72 amino acid protein with a secondary structure consisting of four anti-parallel β-strands and two α-helices on one side of the sheet in the order β1α1β2β3α2β4. The first paper describes the use of CD and fluorescence analysis to examine the folding/unfolding process of MerP. From these experiments it was found that the protein folds according to a two-state model in which only the native and unfolded forms are populated without any visible intermediates. With a rate constant of 1.2 s-1, the folding rate was found to be unusually slow for a protein of this size. The studies presented in the second and third papers were based on measurements of native-state amide proton exchange at different temperatures (Paper II) and GuHCl concentrations (Paper III) in the pre-transitional region. In these studies partially unfolded forms were found for MerP which are essentially unrelated to each other. Thus, in the folding energy landscape of MerP, several intermediates seem to occur on different folding trajectories that are parallel to each other. The slow folding rate of MerP might be coupled to extensive visitation of these conformations. Hydrogen exchange in MerP did also reveal structure-dependent differences in compactness between the denatured states in GuHCl and H2O. In the last paper multivariate data analysis was applied to 2-dimensional NMR data to detect conformational changes in the structure of MerP induced by GuHCl. From this analysis it was suggested that regions involved in the most flexible part of the protein structure are disrupted at rather low denaturant concentrations (&lt; 2.1 M GuHCl) while the native structures of the most stable parts are still not completely ruptured at 2.9 M GuHCl. Finally, the stability, kinetics, contact order and folding nuclei of six proteins with similar topology (MerP, U1A, S6, ADA2h, AcP and HPr) were compared. In this analysis it was found that their folding properties are quite diverse, despite their topological similarities, and no general rules that have been formulated yet can adequately predict their folding behaviour.

Different methods to predict barrier failure in packaging board or paper after converting were investigated. The approach was to compare substrates before and after creasing/folding by applying different barrier tests and to propose a methodology to predict failure in the barrier layer.  Different coatings were used to develop and verify the methodology; a hemicellulose based dispersion barrier coating, a dispersion coated PVOH coating and an extrusion coated PE. Creasing was performed according to standard procedure using recommended creasing geometries. Folding of paper was performed by a gentle creasing with a board backing followed by folding the paper between two metal plates with a well defined distance. The first step in the evaluation was to visually inspect creased/folded substrates by light microscopy to search for coating failures in form of cracks. Both good and bad samples were then tested for grease resistance with a standard test, i.e. TAPPI 454. The TAPPI 454 test showed to be effective to expose barrier failure since oil would penetrate quite fast through the creasing line of cracked samples. Even some samples that appeared to have no cracks in the light microscope showed failure with the grease test. The results showed that only the PE coated samples could sustain a barrier after creasing and folding. This was probably due to a high ductility of the PE-coating combined with a high thickness. The water vapour transmission rate, WVTR, of the samples that passed the TAPPI 454 test was then measured on the samples that endured the grease resistance test. Since PE is a good water vapour barrier, WVTR-measurements were proper for detecting barrier defects. The VWTR of the creased/folded samples was slightly higher for the creased samples than the un-creased references despite the absence of cracks. This was probably due to that the barrier layer got thinner as a result of the strains applied on the coating during the creasing/folding operation.  A methodology to predict barrier failure in barrier coated packaging board and paper after creasing and folding was proposed. Well defined creasing and folding geometries were used in combination with screening for cracks in the barrier layer, first by visual inspection in light microscopy and then by a standard grease resistance test. The samples that passed then screening tests could then be analyzed using more exact but also more time consuming methods such as WVTR. / Olika metoder att förutspå skador i barriärskikt på kartong eller papper efter konvertering undersöktes. Tillvägagångssättet var att jämföra substrat före och efter bigning och vikning genom att tillämpa olika barriärtest och att föreslå en metod för att förutspå defekter i barriärlager. Olika barriärmaterial användes för att utveckla och bekräfta metoden: en hemicellulosa baserad dispersionsbestrykning, en dispersionsbestrykt PVOH barriär och en extruderad PE barriär. Bigandet utfördes enligt standard proceduren och rekomenderade biggeometrier användes. Vikningen av papret utfördes genom varsam bigning med kartong som stöd följt av vikning av pappret genom två metallplattor med ett bestämt avstånd. Utvärderingen började med visuell inspektion av bigade/vikta substrat i ljusmikroskop för att finna barriärdefekter i form av sprickor. Både bra och dåliga prover testades sedan för fettbeständighet med hjälp av ett standardtest, dvs TAPPI 454. TAPPI 454 testet visades sig att vara ett effektivt sätt att identifiera barriärdefekter på grund av att penetration av olja vid biglinjen skedde snabbt på de prov som uppvisade sprickor. Även några av de prov som ej uppvisade sprickor i ljusmikroskop klarade inte av fettbeständighetstest. Resultatet visade att det enda material som kunde bibehålla barriäregenskaper efter bigning och vikning var de PE belagda proven. Detta är antagligen tack vare PE-bestrykningens höga duktulitet och tjocklek. Vattenångspermeabiliteten, WVTR, uppmättes på de prov som uthärdade fettbeständighetstestet. Eftersom PE är en utmärkt vattenångbarriär, var WVTR-mätningar lämpliga för att upptäcka barriärfel. WVTR resultaten för de bigade/vikta proven visade ett något högre värde än de obigade referenserna även om de inte hade sprickor. Det något högre WVTR värdet beror antagligen på att barriärskiktet blev tunnare på grund av töjningen i barriärskiktet under big/vikningen. En metod för att förutspå skador i barriärbestrykt kartong och papper efter bigning och vikning föreslogs. Definierade big- och vikgeometrier användes i kombination av screening av sprickor i barriärskikten, först genom visuell inspektion i ljusmikroskop och sedan ett standarderiserat fettbeständighetstest. Proven som passerar screeningen kan sedan bli analyserade för mer exakta och tidskrävande metoder som WVTR.

Examensarbetet handlar om en jämförelsestudie av beräkningseffektivitet med avseende på energi- och tidsförbrukning mellan grafikkort och processorer i persondatorer och PlayStation 3. Problemet studeras för att göra allmänheten uppmärksam på att det går att lösa en del av energiproblematiken med beräkningar genom att öka energieffektiviteten av beräkningsenheterna. Undersökningen har genomförts på ett explorativt sätt och studerar förhållandet mellan processorer, grafikkort och vilken som presterar bäst i vilket sammanhang. Prestandatest genomförs med molekylberäkningsprogrammet F@H och med filkomprimeringsprogrammet WinRAR. Testerna utförs på MultiCore- och SingleCorePCs och PS3s av olika karaktär. I vissa test mäts effektförbrukning för att kunna räkna ut hur energieffektiva vissa system är. Resultatet visar tydligt hur den genomsnittliga effektförbrukningen och energieffektiviteten för olika testsystem skiljer sig vid belastning, viloläge och olika typer beräkningar. / The thesis is a comparative study of computational efficiency in terms of energy and time consumption of graphics cards and processors in personal computers and Playstation3’s. The problem is studied in order to make the public aware that it is possible to solve some of the energy problems with computations by increasing energy efficiency of the computational units. The audit was conducted in an exploratory way, studying the relationship between the processors, graphics cards and which one performs best in which context. Performance tests are carried out by the molecule calculating F@H-program and the file compression program WinRAR. Tests performed on MultiCore and SingleCore PC’s and PS3’s with different characteristics. In some tests power consumption is measured in order to figure out how energy-efficient certain systems are. The results clearly show how the average power consumption and energy efficiency for various test systems at differ at load, sleep and various calculations.

Epithelial folding is an important morphogenetic process that is essential in transforming simple sheets of cells into complex three-dimensional tissues and organs during animal development (Davidson, 2012). Epithelial folding has been shown to rely on constriction forces generated by the apical actomyosin network (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). However, the contributions of mechanical forces acting along lateral and basal cell surfaces to epithelial folding remain poorly understood. Here we combine live imaging with force measurements of epithelial mechanics to analyze the formation of two epithelial folds in the Drosophila larval wing imaginal disc. We show that these two neighboring folds form via two distinct mechanisms. These two folds are driven either by decrease of basal tension or increase of lateral tension, none of them depends on apical constriction. In the first fold, a local decrease in extracellular matrix (ECM) density in prefold cells results in a reduction of mechanical tension on the basal cell surface, leading to basal expansion and fold formation. Consistent with that, a local reduction of ECM by overexpression of Matrix metalloproteinase II is sufficient to induce ectopic folding. In the second fold a different mechanism is at place. Here basal tension is not different with neighboring cells, but pulsed dynamic F-actin accumulations along the lateral interface of prefold cells lead to increased lateral tension, which drives cell shortening along the apical-basal axis and fold formation. In this thesis I described two distinct mechanisms driving epithelial folding, both basal decrease and lateral increase in tension can generate similar morphological changes and promote epithelial folding in the Drosophila wing discs. / Die Faltung von Epithelien ist ein wichtiger morphogenetischer Prozess, der die Entstehung komplexer, dreidimensionaler Gewebe und Organe aus einfachen Zellschichten ermöglicht (Davidson, 2012). Es ist bekannt, dass Kräfte erzeugt durch das apikale Aktomyosin-Netzwerk wichtig sind für die erfolgreiche Faltung von Epithelien (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). Die Rolle von mechanischen Kräften, die entlang der lateralen und basalen Seite wirken, ist jedoch kaum verstanden. Wir verbinden Lebendmikroskopie mit der Messung von mechanischen Eigenschaften, um die Entstehung von 2 Epithelfalten in den Imaginalscheiben von Drosophila zu verstehen. Wir können dadurch zeigen, dass die beiden Falten durch unterschiedliche Mechanismen entstehen. Sie entstehen entweder durch eine Verringerung der Spannung auf der basalen Seite oder durch eine Erhöhung der Spannung auf der lateralen Seite, aber keine von beiden entsteht durch zusammenziehende Kräfte auf der apikalen Seite. Die erste Falte entsteht durch eine lokale Verringerung der extrazellulären Matrix in den Vorläuferzellen, was zu einer Reduktion der Spannung auf der basalen Seite und zur Ausbildung der Falte führt. Die zweite Falte wird durch einen anderen Mechanismus ausgebildet. Hier ist nicht die Spannung auf der basalen Seite reduziert sondern dynamische Anreicherungen von F-Aktin auf der lateralen Seite resultieren in einer erhöhten lateralen Spannung, die zu einer Verkürzung der Zellen und damit zur Ausbildung einer Falte führt. In meiner Arbeit zeige ich 2 neue Mechanismen zur Entstehung von Epithelfalten auf, durch Absenken der Spannung auf der basalen oder Erhöhen auf der lateralen Seite.

Les riboswitches sont des modules structurés dans les régions 5’ (voire 3’) UTR des ARNm. Chaque riboswitch reconnaît spécifiquement un petit métabolite, ce qui provoque un changement de conformation et permet de moduler au niveau transcriptionnel ou traductionnel (voire par épissage alternatif) l’expression d’un gène impliqué dans la synthèse du métabolite.Ce travail portait sur la cinétique de repliement des domaines aptamères de deux riboswitches homologues liant la thiamine pyrophosphate (TPP), un régulant la transcription du gène thiC (E. coli) et l’autre régulant l’épissage alternatif du gène THIC (A. thaliana).La première approche utilisée (méthode cinétique classique par quenched-flow) consistait à sonder la structure des aptamères par des radicaux hydroxyles au cours du repliement initié par l’addition de TPP. Nous avons également développé (coll. avec A.Griffiths), une approche microfluidique visant à remplacer l’appareillage classique et, à terme, de le dépasser en permettant d’augmenter le nombre d’entrées pour l’étude de systèmes complexes. Nous avons aussi utilisé une méthode (kinITC) récemment développée au laboratoire qui permet d’obtenir des informations thermodynamiques et cinétiques inédites par microcalorimétrie isotherme.Nos résultats ont montré que l’aptamère bactérien se replie beaucoup plus vite que celui d’A. thaliana. Cependant, l’aptamère d’A. thaliana étudié dérivait de la forme sauvage (raccourcissement de l’hélice P3). Par comparaison avec d’autres travaux récents, nos résultats soulignent le rôle fondamental de P3 dans la cinétique de repliement. La méthode kinITC a aussi mis en évidence que le régime cinétique du fonctionnement global du riboswitch de E. coli n’est pas contradictoire avec une première étape de fixation du TPP sous régime thermodynamique.Les résultats obtenus avec la nouvelle méthode de microfluidique sont mitigés. Si nous avons pu reproduire le schéma de coupure de l’ARN lié au TPP obtenu par sondage chimique classique (valide ainsi la première étape de ce développement), la cinétique de repliement observée est plus rapide sans que nous en ayons pour le moment une explication satisfaisante. / Riboswitches are RNA modules found in the 5’-UTR of bacterial mRNA where they control gene expression at the transcriptional or translational level. They are occasionally found in the 3’-UTR where they control alternative splicing. Each riboswitch-controlled gene is involved in the biosynthetic pathway of a metabolite and a feedback loop is ensured by the specific binding of the metabolite.To study the folding kinetics of TPP-binding riboswitch aptamer domains from E.coli (regulating transcription of thiC) and from A. thaliana, (regulating alternative splicing of THIC) two different approaches were used. First, each aptamer structure was probed by hydroxyl radical footprinting during RNA folding triggered by TPP . We also developed (coll. with A. Griffiths) a microfluidic approach aimed at replacing the classical quenched-flow apparatus and, eventually, superseding it by giving access to more entries. We also used a method recently developed in our lab (kinITC), which gives access to rich kinetic and thermodynamic information from isothermal titration calorimetry.Our results showed that the E. coli aptamer folds much faster than its A. thaliana counterpart. However, the form that we used for the latter had a helix P3 shorter than that of the wt and, when compared with recent results, our results highlight the fundamental role of this helix in the kinetics of folding. It was also clear that, globally, the E. coli riboswitch is kinetically controlled but the kinITC method allowed us to show that this is not in contradiction with a thermodynamic control of the first TPP binding step.The results obtained with the microfluidic device are mitigated. We were indeed able to make the proof of concept of hydroxyl RNA probing on a microfluidic chip, but the kinetics of RNA folding appeared to be faster than that observed with the quenched flow. We are not yet able to propose an explanation for this strange fact.

To respond to rapidly-changing stresses in their environment, bacterial cells must be able to sense a variety of chemical cues and respond to them by activating the relevant genes. The lysine riboswitch is a short RNA motif, located just upstream of a gene encoding a lysine biosynthesis protein, that suppresses the expression of that gene when sufficient lysine is present in the cell. It acts by binding a lysine monomer in a region called the aptamer, which in turn rearranges an adjacent domain called the expression platform, sequestering the ‘start' sequence of the gene and preventing it from being transcribed. In this thesis, the lysine riboswitch's ligand-binding transition is studied using single-molecule fluorescence microscopy, optical tweezers, and a hybrid optical force/fluorescence technique. Förster Resonance Energy Transfer (FRET) is used with a fluorescently-labeled aptamer to show that it has a previously-undescribed, partially-folded structural state with enhanced ligand affinity compared to the unfolded structure. The Mg²⁺ dependence of the transition between these states is shown to resolve existing debates in the literature about the sensitivity of the riboswitch. The kinetics of the folding transition are explored using FRET, optical force, and hybrid ‘Fleezers' to map the free energy landscape of ligand binding and show that the ligand itself promotes transitions into the aptamer's folded state, a so-called ‘induced fit' mechanism rare among riboswitches. Finally, high-resolution optical tweezers are used to explore the link between the aptamer's secondary structure (the sequence of paired nucleotides) and its tertiary structure (three-dimensional folding) to illuminate the role of ligand binding in gene regulation, which depends on the equilibrium between competing secondary structures. Hybrid biophysical techniques like optical force/fluorescence microscopy are shown to be indispensable for addressing all the states in the reaction pathways of complex biomolecules like riboswitches and for discriminating between multiple levels of structure formation and interaction with the environment. Not only do the results presented here shed light on the RNA folding problem, particularly the role of tertiary structure in determining the minimum-energy configuration of an RNA sequence, but they could have implications for biomedical research, as the lysine riboswitch has already been shown to be a potential target for next-generation antibiotics.

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.

The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.

Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.