<i>Brucella abortus</i> is capable of resisting the microbicidal mechanisms of phagocytic cells and growing within phagocytic cells, usually macrophages. <I>B. abortus</i>, like several other intracellular bacteria responds to the hostile environment in macrophages by producing heat shock proteins (HSPs) which are induced by environmental stresses. Bacterial HSPs are very immunogenic, eliciting both cellular and humoral immune responses in the infected host. The significance of host cellular and protective immune responses directed against these proteins is currently unresolved. Baculovirus recombinants were generated in <i>Sf9</i> insect cells for <i>B. abortus</i> HSPs and the protein expression was optimized. Humoral (Western blot), cell mediated (CMI, IFN-g- release by splenocytes, and CD3+CD4+, CD3+CD8+ T cell/ total splenocytes ratios) and protective immune responses of BALB/c mice (challenge with virulent <i>B. abortus</i> 2308) against these recombinants, against <i>B. abortus</i> superoxide dismutase (SOD) and ribosomal L7/L12 proteins, inoculated alone or in various combinations with complete Freund's, Ribi and recombinant IL-12 as adjuvants, were analyzed. Vaccinia virus-GroEL recombinant as priming immunogen, followed by baculovirus-GroEL-Ribi booster, was explored. Androstenediol, an immune up-regulator, was tested for its ability to induce resistance against challenge. None of the mice inoculated with individual, divalent or trivalent HSP-expressing <i>Sf9</i> cells combined with Freund's were protected against challenge and the <i>Sf9</i> cell-induced response masked the recombinant protein-specific CMI responses. Recombinant HSPs were purified and combined with Ribi. Although significant IFN-g release was induced by immunization with the HtrA-Ribi combination, no mice were protected against challenge. Priming with vaccinia virus-GroEl recombinant and boosting with purified baculovirus-GroEL protein-Ribi combination did not induce protection. Androstenediol did not enhance in vivo resistance to challenge. IL-12 alone did not activate splenocytes but induced significant IFN-g release in mice when combined with killed <i>B. abortu</i>s RB51 vaccine, purified recombinant HtrA or purified SOD proteins, or L7/L12 expressing <i>Escherichia coli</i> cells. Significant protection was induced by SOD combined with IL-12. No correlation was seen between IFN-g release by splenocytes and protection against challenge in the SOD/IL-12-immunized mice. The results suggest that <i>B. abortus</i> HSPs are not highly immunogenic in mice and though various immune responses may be induced by one or another HSPs, protective immune response, unfortunately, is not among them. The results of this study reflect the difficulties in experimenting with immune responses against single or a limited number of recombinant <i>B. abortus</i> proteins. This is particularly true when the task includes induction of a protective immune response and finding significant correlation between different types of immune response assays. / Ph. D.

Creeping bentgrass (Agrostis stolonifera L.) is a major low-cut cool-season turfgrass used worldwide. The objectives of this research were to: 1) to gain insight into the diurnal fluctuation of N metabolism and effects of cytokinin (CK) and nitrate; 2) to characterize the impacts of N and CK on creeping bentgrass under heat stress; 3) to investigate the simultaneous effects of CK and N on the antioxidant responses of heat stressed creeping bentgrass; and 4) to examine the expression pattern of the major heat shock proteins (HSPs) in creeping bentgrass during different heat stress periods, and then to study the influence of N on the expression pattern of HSPs. The transcript abundance of nitrate reductase (NR), nitrite reductase (NIR), plastidic glutamine synthetase (GS2), ferredoxin-dependent glutamate synthase (Fd-GOGAT), and glutamate dehydrogenase (GDH) and N metabolites in shoots were monitored during the day/night cycle (14/8 h). All the measured parameters exhibited clear diurnal changes, except GS2 expression and total protein. Both NR expression and nitrate content in shoots showed a peak after 8.5 h in dark, indicating a coordinated oscillation. Nitrate nutrition increased diurnal variation of nitrate content compared to control and CKHowever, CK shifted the diurnal in vivo NR activity pattern during this period. Grass grown at high N had better turf quality (TQ), higher Fv/Fm, normalized difference vegetation index (NDVI), and chlorophyll concentration at both 15 d and 28 d of heat stress than at low N, except for TQ at 15 d. Shoot NO3-, NH4+, and amino acids increased due to the high N treatment, but not water soluble proteins. High N also induced maximum shoot nitrate reductase activity (NRmax) at 1 d. CK increased NDVI at 15 d and Fv/Fm at 28 d. In addition, grass under 100 µM CK had greatest NRmax at both 1 d and 28 d. Under high N with 100 µM CK, root tZR and iPA were 160% and 97% higher than under low N without CK, respectively. Higher O2- production, H2O2 concentration, and higher malonydialdehyde (MDA) content in roots were observed in grass grown at high N. The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) in roots were enhanced by high N at 19, 22, and 24% levels, respectively, relative to low N. Twenty-eight days of heat stress resulted in either the development of new isoforms or enhanced isoform intensities of SOD, APX, and POD in roots compared to the grass responses prior to heat stress. However, no apparent differences were observed among treatments. No CK effects on these antioxidant parameters were found in this experiment. At week seven, grass at medium N had better TQ, NDVI, and Fv/Fm accompanied by lower shoot electrolyte leakage (ShEL) and higher root viability (RV), suggesting better heat resistance. All the investigated HSPs (HSP101, HSP90, HSP70, and sHSPs) were up-regulated by heat stress. Their expression patterns indicated cooperation between different HSPs and that their roles in creeping bentgrass thermotolerance were affected by N level. / Ph. D.

Heat shock protein 90 (HSP90) is a molecular chaperone protein that protects proteins from degradation, repairs damaged proteins, and assists proteins in carrying out their functions. HSP90 has hundreds of clients, many of which are inflammatory signaling kinases. The mechanism by which HSP90 enables inflammatory pathways is an active area of investigation. The HSP90 inhibitors such as geldanamycin (GA) and its derivative 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) have been shown to reduce inflammation. It was hypothesized that inhibiting HSP90 would reduce inflammatory signal cascade levels. To test this, J774 mouse macrophage cells were treated with 17-DMAG and immune-stimulated with lipopolysaccharide (LPS). 17-DMAG treatment reduced nitric oxide (NO) production and the expression of pro-inflammatory cytokines interleukin (IL)-6, IL-12, and TNF-α. Inhibition of HSP90 also prevented nuclear translocation of NF-κB. To investigate the anti-inflammatory effects of HSP90 inhibition in vivo, MRL/lpr lupus mice were administered 5 mg/kg 17-DMAG for six weeks via intraperitoneal injection. Mice treated with 17-DMAG were found to have reduced proteinuria and reduced splenomegaly. Flow cytometric analysis of splenocytes showed that 17-DMAG decreased double negative T (DNT) cells. Renal expression of HSP90 was also measured and found to be increased in MRL/lpr mice that did not receive 17-DMAG. The mechanistic interactions between HSP90 and the pro-inflammatory nuclear factor-κB (NF-κB) pathway were studied and a computational model was developed. The model predicts cellular response of inhibitor of κB kinase (IKK) activation and NF-κB activation to LPS stimulation. Model parameters were fit to IKK activation data. Parameter sensitivity was assessed through simulation studies and showed a strong dependence on IKK-HSP90 binding. The model also accounts for the effect of a general HSP90 inhibitor to disrupt the IKK-HSP90 interaction for reduced activation of NF-κB. Model simulations were validated with experimental data. In conclusion, HSP90 facilitates inflammation through multiple signal pathways including Akt and IKK. Inhibition of HSP90 by 17-DMAG reduced disease in the MRL/lpr lupus mouse model. A computational model supported the hypothesis that HSP90 is required for IKK to activate the NF-κB pathway. Taken together, HSP90 is a prime target for therapeutic regulation of many inflammatory processes and warrants further study to understand its mechanism of regulating cell signaling cascades. / Ph. D.

External stresses surrounding bone can stimulate heat shock proteins (HSPs), which are involved in anti-apoptosis, cell proliferation, and differentiation. In vitro stress modulation and HSP induction may be critical factors for enhancing bone regeneration. We investigated whether applying individual or combinatorial stress conditioning (thermal, tensile, and biochemical) and effective HSP modulation could induce in vitro responses in preosteoblasts indicating mitogenic/osteogenic/angiogenic/anti-osteoclastic effects. A preosteoblast cell line (MC3T3-E1) was exposed to conditioning protocols utilizing thermal stress applied with a water bath, tensile stress using a Flexcellâ„¢ bioreactor, and biochemical stress with the addition of growth factors (GFs) (i.e. transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2)). Furthermore, the role of HSP70 in osteogenesis under normal conditions and in response to heat was investigated by transfecting preosteoblasts with HSP70 small interfering RNA alone or in combination with thermal stress and measuring cellular response. Heating at 44°C (for 8 minutes) rapidly induced osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), vascular endothelial growth factors (VEGF), and cyclooxygenase 2 (COX-2) mRNA at 8 hour post-heating (PH). The addition of GFs with heating induced OPG and VEGF genes more than heating or GF addition alone. OPN, OCN, and OPG secretions increased with the addition of GFs. However, matrix metalloproteinase-9 (MMP-9) secretion was inhibited by heating, with more significant declines associates with GF inclusion. Equibiaxial tension (5%, 0.2 Hz, 10 seconds tension/10 seconds rest, 6 days) with GFs enhanced proliferation than tension or GF addition alone. MMP-9 secretion decreased in response to tension alone or more with GFs. Tension (1-5%, 24 hours) with GFs induced prostaglandin E synthase 2 (PGES-2), OPG, and VEGF genes more than tension or GFs alone. Combinatorial conditioning with thermal stress (44°C, 8 minutes) and tension (3%, 0.2 Hz, 10 seconds tension/10 seconds rest, 4 hours for HSP gene and 24 hours for VEGF secretion and MMP-9 gene) induced HSP27 and HSP70, secretion of VEGF (protein), and suppression of MMP-9 (gene) more than heating or tension alone. HSP70 silencing followed by heating (44°C, 8 minutes) enhanced expression of HSP27. Mitogenic activity was inhibited by heating with more significant decrease occurring by heating and HSP70 silencing. At 10 hours PH, TGF-β1, MMP-9, and ALP mRNA decreased in response to heating and HSP70 silencing. At 48 hours PH, heating following HSP70-silencing induced VEGF secretion significantly. In conclusion, effective application of individual or combinatorial conditioning utilizing heating, tension, and GFs could be beneficial as a bone healing-strategy by rapidly inducing stress proteins (HSPs), angiogenic factor (e.g. VEGF), anti-osteoclastogenic cytokines (e.g. OPG), and bone matrix proteins (e.g. OPN and OCN) with anti-resorptive activity by inhibiting MMP-9. / Ph. D.

β-Glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) catalyzes the hydrolysis of aryl and alkyl β-D-glucosides as well as glucosides with a carbohydrate moiety such as cellobiose and other beta-linked oligosaccharides. In maize (Zea mays L.), β-glucosidase exists as 120 kD homodimers, but also forms high-molecular-weight (HMW) aggregates in certain maize inbreds (nulls). In this study we show that the null β-glucosidase phenotype is caused by the formation of HMW enzyme aggregates (>1.5 X 10⁶ Daltons), caused by a β-glucosidase aggregating factor (BGAF). BGAF is a 32 kD protein that binds specifically to β-glucosidase and renders it insoluble during extraction. The data unequivocally demonstrate that BGAF is solely responsible for β-glucosidase aggregation and insolubility, and thus, the apparent null phenotype. Additionally, I have isolated the cDNA encoding BGAF and have identified BGAF as a member of the small heat-shock protein (sHsp) family. Interestingly, BGAF binds to both maize β-glucosidase isozymes (Glu1 and Glu2), but does not bind to their sorghum homolog Dhurrinase-1 (Dhr1; Sorghum beta-glucosidase), that shares 70% sequence identity with Glu1 and Glu2. Therefore, these proteins provide an excellent system to study functional differences at nonconserved residues and elucidate the mechanism of enzyme aggregation and insolubility. By examining the behavior of β-glucosidase chimeras in binding assays, I demonstrate that BGAF binding is conformation dependent, highly specific, and reminiscent of antigen-antibody interactions. Additionally, I have identified two disparate polypeptide segments in the primary structure of the maize beta-glucosidase isozyme Glu1 that form a BGAF binding site in the tertiary structure of the enzyme. / Master of Science

Due to the fact that bone cells are highly responsive to mechanical stimuli, shear stress has been extensively studied for its ability to enhance osteogenic differentiation through mechanotransduction. In addition, thermal stress has also been explored as a conditioning method to stimulate cellular proliferation, differentiation, and cytoprotection through heat shock protein induction. Despite the beneficial effects observed with individual stress on cells, there has been little focus on the potential of a combination of stresses to improve cellular response. Therefore, the aim of this study was to investigate the effect of combined shear and thermal stress on preosteoblasts to stimulate an enhanced osteogenic response. To achieve this, MC3T3-E1 cells were exposed to one of the following protocols for an hour: no stress (control), shear stress at 1 dyne/cm2 using a parallel plate flow chamber, thermal stress in a 42°C incubator, or combined shear and thermal stress (1 dyne/cm2 at 42°C). Stress treatments were applied on Day 2, Day 6, and Day 10. To assess the early response of cells to stress treatments, we measured metabolic activity, expression of signaling factors, and HSPs following stress on Day 2. Despite an initial decrease in metabolism, combined stress stimulated a strong response in VEGF (12.49 RFI) COX-2 (12.32 RFI), HSPs (2-4 RFI) and increased PGE accumulation. The long-term cellular response to stress treatments was measured on Day 15 by evaluating the ability of combined stress to stimulate late stage markers of differentiation. Combined stress increased OPN gene and protein expression, yet OCN was minimally affected by stress treatments. However, mineralization was significantly decreased with combined stress. Overall, combined stress was able to stimulate an enhanced effect across a majority of the bone-related markers measured, whereas individual shear stress or thermal stress were limited in their response. This suggests that combined stress can provide the appropriate cues to modify osteoblast differentiation and generate an enhanced osteogenic response. / Master of Science

Laser therapies can provide a minimally invasive treatment alternative to surgical resection of tumors. However, therapy effectiveness is limited due to nonspecific heating of target tissue, leading to healthy tissue injury and extended treatment durations. These therapies can be further compromised due to heat shock protein (HSP) induction in tumor regions where non-lethal temperature elevation occurs, thereby imparting enhanced tumor cell viability and resistance to subsequent therapy treatments. Introducing nanoparticles (NPs), such as multi-walled nanotubes (MWNTs) or carbon nanohorns (CNHs), into target tissue prior to laser irradiation increases heating selectivity permitting more precise thermal energy delivery to the tumor region and enhances thermal deposition thereby increasing tumor injury and reducing HSP expression induction. This research investigates the impact of MWNTs and CNHs in untreated and laser-irradiated monolayer cell culture, tissue phantoms, and/or tumor tissue from both thermal and biological standpoints. Cell viability remained high for all unheated NP-containing samples, demonstrating the non-toxic nature of both the nanoparticle and the alginate phantom. Up-regulation of HSP27, 70 and 90 was witnessed in samples that achieved sub-lethal temperature elevations. Tuning of laser parameters permitted dramatic temperature elevations, decreased cell viability, and limited HSP induction in NP-containing samples compared to those lacking NPs. Preliminary work showed MWNT internalization by cells, which presents imaging and multi-modal therapy options for NT use. The lethal combination of NPs and laser light and NP internalization reveals these particles as being viable options for enhancing the thermal deposition and specificity of hyperthermia treatments to eliminate cancer. / Master of Science

Le cancer de la prostate (CaP) est devenu un véritable problème de santé publique dans les pays industrialisés. L'hormonothérapie reste le traitement de première ligne le plus efficace dans les cancers avancés mais il n'empêche pas la progression vers un stade androgéno-indépendant (AI), pour lequel la chimiothérapie s'avère peu efficace. Une des stratégies pour améliorer les thérapies actuelles consiste à cibler des gènes de survie surexprimés dans les CaPs AI afin de restaurer la sensibilité aux traitements du CaPs. Hsp27, protéine surexprimée dans ces cancers, à un effet cytoprotecteur qui engendre une résistance aux traitements. Elle est maintenant reconnue comme une cible thérapeutique importante. Rocchi et al. ont développé un oligonucléotide antisense (ASO) de deuxième génération (OGX-427) qui cible l'ARNm d'Hsp27. OGX-427 est actuellement en essai clinique phase II chez des patients atteints de CaPs au Canada et aux Etats-Unis. Mon projet de thèse a porté sur l'étude des mécanismes d'action d'Hsp27 impliqués dans l'évolution AI du CaP. Cette étude a pour but d'améliorer la sureté pharmacologique d'OGX-427, mais aussi d'identifier de nouvelles cibles thérapeutiques visant spécifiquement les cellules tumorales. Mes travaux de thèse ont montré que lors d'un stress cellulaire induit par hormonothérapie et/ou chimiothérapie, Hsp27 interagit avec le facteur eucaryotique d'initiation de la traduction eIF4E et le protège de sa dégradation par la voie ubiquitine/protéasome. Ceci maintient la synthèse protéique et engendre une survie cellulaire impliquée en partie dans l'effet cytoprotecteur médié par Hsp27. / Prostate cancer (PC) has become a real public health issue in industrialized countries, mainly due to patients' relapse by castration-resistant (CR) disease after androgen ablation. One strategy to improve current therapies in advanced PC involves targeting genes that are activated by androgen withdrawal, either to delay or prevent the emergence of the CR phenotype. Hsp27 is over-expressed in this cancer and has been shown to play a cytoprotective role leading to treatments resistance. This protein is now considered as promising therapeutic target. Rocchi, P. et al. developed and patented a second generation antisens oligonucleotides (ASO) targeting Hsp27 that has been licensed (OGX-427) and phase II clinical trials are currently in process in PC in Canada and USA. My PhD project focused on the study of Hsp27 action mechanisms involved in CRPC progression. The present study aims to improve pharmacological safety of OGX-427 and to identify new therapeutic targets specific of CRPC cells. The results of my PhD have shown that during cell stress induced by hormone- and/or chemotherapy, Hsp27 interacts with eukaryotic translation initiation factor eIF4E and protects it from degradation by the ubiquitin/proteasome pathway. This maintains protein synthesis and leads to cell survival, partly involved in the cytoprotection mediated by Hsp27. Our work therefore concerned the characterization of the interaction site between Hsp27 and eIF4E in order to identify potential inhibitors of this interaction that could delay CRPC progression.

Le cancer de la prostate (CaP) représente la deuxième cause de mortalité par cancer chez l'homme. La suppression androgénique (castration-thérapie) demeure la seule thérapie efficace du CaP avancé du fait de son caractère castration-sensible. Cependant, elle n'empêche pas la progression castration-résistante (CR) de la maladie dans les 1 à 3 ans après le début du traitement hormonal. Récemment, l'implication d'Hsp27 (Heat shock protein 27) dans l'échappement thérapeutique des CaPs a été montrée et un oligonucléotide antisens inhibiteur d'Hsp27, OGX-427, est en cours d'évaluation clinique II/III pour le traitement des CaPs CR. Afin de comprendre le rôle d'Hsp27 dans le mécanisme de résistance à la castration, nous avons réalisé le criblage de l'ensemble des protéines partenaires d'Hsp27 par double hybride. Mes travaux de thèse ont permis d'identifier une nouvelle protéine cliente d'Hsp27, TCTP (translationally controlled tumor protein) dont l'expression est indétectable dans les cellules normales. J'ai également montré que la progression CR des CaPs corrélait avec une surexpression de TCTP, une perte de P53 et que l'inhibition de TCTP par un oligonucléotide antisens restaurait l'expression de P53. Cette étude suggère, pour la première fois, un lien direct entre P53 et la sensibilité à la castration des CaPs. De plus, l'étude de l'interactome d'Hsp27 a mis en évidence son implication dans de nouvelles fonctions telles que la réparation de l'ADN ou l'épissage alternatif des ARNm. L'ensemble de ces travaux ont permis de mieux comprendre les mécanismes d'action d'Hsp27 dans la progression CR des CaPs et de développer de nouvelles approches thérapeutiques. / Prostate cancer (PC) is the second most common cause of cancer-related mortality in men in the Western world. Androgen ablation (castration-therapy) is usually the initial therapy in patients with advanced or metastatic disease. Unfortunately, the disease gradually progresses to a metastatic castration-resistant (CR) state, which remains incurable. Recently, the involvement of Hsp27 (Heat Shock Protein 27) in CR progression has been identified and an oligonucleotide antisense (OGX-427), inhibitor of Hsp27 is currently in phase II/III clinical trials to treat CRPC. In order to understand Hsp27 mechanisms of action in CR progression, we started to screen for Hsp27 partner proteins by using two-hybrid system. My PhD work has reported that Translationally Controlled Tumor Protein (TCTP) was a new Hsp27 protein partner that mediated Hsp27 cytoprotection in CRPC and that TCTP expression was absent in normal prostate tissues. We have further found that CR progression correlated with TCTP overexpression, the loss of P53 and that TCTP silencing using an antisense was able to restore P53 expression and function. This study suggests for the first time that castration-sensitivity is directly linked to P53 expression. In addition, we revealed exciting new aspects of the Hsp27 involvement in essential metabolic and cellular processes such as DNA repair and mRNA splicing. In summary, my PhD results have provided an enriched understanding of Hsp27 mechanisms of cytoprotection contributing to CRPC progression and opened a new promising field of research for multi-target therapeutic approaches.

Molecular chaperones have emerged in recent years as major players in many aspects of cell biology. Molecular chaperones are also known as heat shock proteins (HSPs) since many were originally discovered due to their increased synthesis in response to heat shock. They were initially identified when Drosophila salivary gland cells were exposed to a heat shock at 37°C for 30 min and then returned to their normal temperature of 25°C for recovery. A “puffing” of genes was found to have occurred in the chromosome of recovering cells, which was later shown to be accompanied by an increase in the synthesis of proteins with molecular masses of 70 and 26 kDa. These proteins were hence named “heat shock proteins”. The first identification of a function for HSPs was the discovery in Escherichia coli that five proteins synthesized in response to heat shock were involved in λ phage growth. The products of the groEL and groES genes were found to be essential for phage head assembly while the dnaK, dnaJ and grpE gene products were essential for λ phage replication. It was later shown that GroEL and GroES are part of a chaperonin system for protein folding in the prokaryotic cytosol while DnaK is a member of the Hsp70 family that works in conjunction with the DnaJ (Hsp40) co-chaperone and the nucleotide exchange factor GrpE to promote phage replication by dissociating the DnaB helicase from the phage-encoded P protein. Since then, a large number of other proteins collectively referred to as HSPs have been discovered. However, heat shock is not the only signal that induces synthesis of heat shock proteins. Stress of any kind, such as nutrient deprivation, chemical treatment and oxidative stress among others causes increased production of HSPs and therefore, they are also known as stress proteins. The term “molecular chaperone” was originally used to describe the function of nucleoplasmin, a Xenopus oocyte protein that promotes nucleosome assembly by binding tightly to histones and donating the bound histone to chromatin. However, since then, chaperones have been defined as “a family of unrelated classes of proteins that mediate the correct assembly of other proteins, but are not themselves components of the final functional structure”. This view of molecular chaperones, though undoubtedly correct, doesn’t capture the multifaceted roles they have since been discovered to play in cellular processes. In recent years, molecular chaperones have been shown to perform other functions in addition to the maintenance of protein homeostasis: translocation of proteins across organelle membranes, quality control in the endoplasmic reticulum, turnover of misfolded proteins as well as signal transduction. As a result, many chaperones are also essential under non-stress conditions and play crucial roles in cell growth and development, cell-cell communication and regulation of gene expression. Heat shock protein 90 (Hsp90) is one of the most abundant and highly conserved molecular chaperones in organisms ranging from bacteria to all branches of eukarya. It has been shown to be essential for cell viability in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster. Although the bacterial homolog HtpG is dispensable under normal conditions, it is important for cell survival during heat shock. In addition to its role as general chaperone in protein folding following stress, Hsp90 has a more specialized role as a chaperone for several protein kinases and transcription factors. Many Hsp90 client proteins are signaling proteins involved in regulation of cell growth and survival. These proteins are critically dependent on Hsp90 for their maturation and conformational maintenance resulting in a key role for Hsp90 in these processes. Recent reports have also highlighted a role for Hsp90 in linking the expression of genetic and epigenetic variation in response to environmental stress with morphological development in Drosophila melanogaster and Arabidopsis thaliana. In Candida albicans, Hsp90 augments the development of drug resistance, implicating a role for Hsp90 in the evolution of infectious diseases. The malarial parasite, Plasmodium falciparum, is the causative agent of the most lethal form of human malaria. The parasite life cycle involves two hosts: an invertebrate mosquito vector and a vertebrate human host. As the parasite moves from the mosquito to the human body, it experiences an increase in temperature resulting in a severe heat shock. The mechanisms by which the parasite adapts to changes in temperature have not been deciphered. Our laboratory has been interested in investigating the role of heat shock proteins during acclimatization of the parasite to such temperature fluctuations. Heat shock proteins of the Hsp40, Hsp60, Hsp70 and Hsp90 families have been characterized in the parasite and are being examined in our laboratory. This thesis pertains to understanding the functional role of Plasmodium falciparum Hsp90 (PfHsp90) during adaptation of the parasite to fluctuations in environmental temperature. The parasite expresses a single gene for cytosolic Hsp90 on chromosome 7 (PlasmoDB accession no.: PF07_0029) coding for a protein of 745 amino acids with a pI of 4.94 and Mw of 86 kDa. Eukaryotic Hsp90 regulates several protein kinases and transcription factors involved in cell growth and differentiation pathways resulting in a crucial role for Hsp90 in developmental processes. A role for PfHsp90 in parasite development, therefore, seems likely. Indeed, PfHsp90 has previously been implicated in parasite development from the ring stage to the trophozoite stage during the intra-erythrocytic cycle. Pharmacological inhibition of PfHsp90 function using geldanamycin (GA), a specific inhibitor of Hsp90 activity, abrogates stage progression. These experiments suggest that PfHsp90 may play a critical role in parasite development. This is further substantiated by the fact that several pathogenic protozoan parasites such as Leishmania donovani, Trypanosoma cruzi, Toxoplasma gondii and Eimeria tenella depend on Hsp90 function during different stages of their life cycles. It appears, therefore, that a principal role of Hsp90 in protozoan parasites may be the regulation of their developmental cycles. However, the precise functions of PfHsp90 during the intra-erythrocytic cycle of the malarial parasite are not clear. In this study we have carried out a functional analysis of PfHsp90 in the malarial parasite. We have examined the role of PfHsp90 in parasite development during repeated exposure to febrile temperatures. We have investigated its involvement in parasite development during a commonly used synchronization protocol involving cyclical changes in temperature. We have examined the interaction of GA with the Hsp90 multi-chaperone complex from P. falciparum as well as the human host. Finally, we have carried out a systems level analysis of chaperone networks in the malarial parasite as well as its human host using an in silico approach. We have analyzed the protein-protein interactions of PfHsp90 in the chaperone network and predicted putative cellular processes likely to be regulated by parasite chaperones, particularly PfHsp90.