Understanding the cellular response thermal stress is important for improving thermoablative treatments of cancer. Cells generally respond to thermal stress by expressing heat shock proteins, or undergoing cell death by apoptosis or necrosis. Most of our detailed knowledge regarding these cellular phenomena has been gathered in vitro in two dimensional (2D) environments. Yet, little is known about how prostate cancer cells respond to thermal stress in a more physiologically relevant three dimensional (3D) environment. Several approaches were used to investigate this question, all of which focused on controlled heating of cells in both two dimensional (2D) and 3D culture. Tools and assays were developed to investigate cellular response to thermal stress in 2D and 3D environments. A computer-controlled heating apparatus was constructed to heat cell cultures to precise temperatures and durations. Three dimensional growth environments were produced using Matrigel, a commercially available extracellular matrix (ecm) mixture. Transcriptional expression of heat shock protein 70 (HSP70) was measured using a green fluorescent protein (GFP) reporter gene under the control of an HSP promoter. Apoptosis, necrosis and HSP70 transcription was measured using flow cytometry analysis. Quantitative polymerase chain reaction (qPCR) and microscopy revealed that transmembrane targets may be involved in the mechanism of the effect which 3D culture has on the cellular response to heat shock. The results herein demonstrate that the 3D growth environment, may be protective to the cell in that the percentage of cells that undergo apoptosis or necrosis when exposed to heat shock are reduced. Furthermore, HSP70 expression is enhanced in 3D culture at a specific thermal dose and integrins and heat shock proteins may be part of the mechanism by which the ecm exerts its protective effect against thermal stress. / text

Plasmodium falciparum is responsible for the most virulent form of human malaria. The biology of the intra-erythrocytic stage of P. falciparum is the most well studied as it is this stage that marks the clinical manifestation of malaria. To establish a successful infection, P. falciparum brings about extensive remodeling of erythrocytes, its host compartment. The infected erythrocytes harbor several parasite induced membranous structures. Most importantly, pathogenesis related structures termed knobs, which impart cytoadherence, appear on the cell surface of the infected erythrocytes. For bringing about such eccentric renovations in its host compartment, the parasite exports 8% of its genome (~400 proteins) to various destinations in the host cell. Studies from our lab have shown that proteins belonging to heat shock protein40 (Hsp40) and heat shock protein70 (Hsp70) group of chaperones are also exported to the host compartment. We and others have implicated these chaperones in important processes such as protein trafficking and chaperoning assembly of parasitic proteins into the cytoadherent knobs. As detailed above, malaria parasite invests a lot of energy in exporting a large number of proteins including chaperones in the red blood cell to meet its pathogenic demands. In order to do so, it heavily relies on its secretory pathway. However, it is known that the parasite experiences a significant amount of oxidative stress on account of heme detoxification, its own metabolism and the immune system of the host. The parasite also effluxes large quantities of reduced thiols such as glutathione and homocysteine into the extracellular milieu indicative of redox perturbation. Additionally, the parasite lacks Peroxiredoxin IV, which otherwise localizes in the ER and carries out detoxification of peroxide generated as a result of oxidative protein folding. Together, these factors indicate that maintaining redox homeostasis is a challenging task for the parasite. It also implies that the ER, where the redox balance is even more critical as it requires oxidising environment for protein folding, is predisposed to stress. In light of this fact and the importance of secretory pathway in malaria pathogenesis, we decided to address the ways and mechanisms used by the parasite to tackle perturbations in its secretory pathway. Examination of a canonical unfolded protein response pathway in P. falciparum ER-stress is a condition arising whenever the load of unfolded proteins increases the folding capacity of the ER. However, eukaryotes have evolved a fairly well conserved homeostatic response pathway known as unfolded protein response (UPR) to tackle ER-stress. This signal transduction pathway is composed of three arms involving three ER-transmembrane signal transducers namely; IRE1, ATF6 and PERK. IRE1 brings about splicing of a bZIP transcription factor, XBP1/Hac1 and ATF6 becomes activated upon getting proteolytically cleaved in the Golgi. These transcription factors then migrate to the nucleus where they bind onto the ER-stress elements thereby, leading to the transcriptional up-regulation of the UPR targets such as ER chaperones and components of ER associated degradation (ERAD) pathway which rescue the function of the ER. PERK on the other hand brings about translational attenuation by phosphorylating eIF2α, thereby providing parasite the benefit of time to recover. We started our examination on UPR in Plasmodium by carrying out in silico analysis of the major components of UPR in the parasite by using Homo sapiens protein sequences as the query. We found that the parasite lacks the homologues of all the transcriptional regulators of canonical UPR. Only PERK component of the UPR was found to be present in the parasite. To rule out the existence of the canonical UPR in P. falciparum, we examined the status of UPR targets by subjecting the parasites to treatment with DTT. DTT perturbs the disulfide oxidation in the ER and thereby inhibits protein folding leading to ER-stress. Owing to the missing components of a canonical UPR, we did not find up-regulation of known UPR targets such as ER-chaperones including PfBiP, PfGrp94, PfPDI and ERAD marker Derlin1 at transcript as well as protein level. Owing to the presence of a PERK homologue, phosphorylation of eIF2α followed by attenuation of protein synthesis was observed upon subjecting the parasites to DTT mediated ER-stress. In the absence of a canonical UPR, the parasites were found to be hypersensitive to ER-stress in comparison to the mammalian counterpart. In the presence of DTT, the parasites showed perturbation in the redox homeostasis as indicated by increase in the levels of ROS. Next, we sought to examine if the parasites resorted to any alternate means of increasing the availability of chaperones in the ER. For this, we analysed the involvement of another Hsp70 family member, Hsp70-x which is homologous to BiP and which is known to traverse the ER while getting exported to the erythrocyte compartment. Interestingly, we found that upon exposure to ER-stress, the export of this protein is partially blocked and around 30% of the protein is retained in the ER. On the other hand, there was no effect on the trafficking of another exported chaperone KAHsp40. This indicates that the parasite possibly recruits this pool of retained Hsp70-x for the chaperoning of unfolded proteins in the ER. Global response to ER-stress in P. falciparum To dig deeper into the parasite specific strategies employed for dealing with ER-stress at a global level, we carried out high throughput transcriptomic and proteomic analysis upon subjecting the parasites to DTT mediated ER-stress. Microarray based gene expression profiling was carried out upon subjecting the parasites to DTT mediated ER-stress. We found that the parasite mounts a transcriptional response as indicated by up-regulation of 155 transcripts. In congruence with our biochemical analysis, we did not find up-regulation of ER chaperones as well as ERAD proteins. Functional grouping of the up-regulated genes revealed large number of hypothetical proteins in our list of differentially expressed genes. The genes encoding exported proteins represent yet another abundant class. In the course of examining the involvement of Plasmodium specific transcriptional regulators mediating response to DTT induced ER-stress, we identified 4 genes belonging to the family of AP2 transcription factors. AP2 (Apetela-2) are specific transcription factors which are possessed by apicomplexa and bring about regulation of developmental processes and stress response in plants. On comparing our list of up-regulated genes with the previously known targets of AP2 factors, we found that an entire cascade of AP2 factors is up-regulated upon DTT-mediated ER stress. Thus, AP2 factors appear to be the major stress response mediators as they are together responsible for the up-regulation of 60% of genes identified in this study. In addition, another striking observation made, was the up-regulation of a few sexual stage specific transcripts. 2D Gel electrophoresis and 2D-DIGE based Proteomic analysis indicated an up-regulation of secretory proteins and some components of vesicular trafficking and secretory machinery possibly to overcome the block in the functions of the secretory pathway. ER-stress triggers stage transition in P. falciparum Intrigued by the up-regulation of a few sexual stage specific genes, we were curious to examine if there was a functional significance of this observation. To this end, we decided to investigate the effect of ER-stress on induction of gametocytes, the only sexual stage found in humans. Indeed, we found a two fold induction in the numbers of gametocytes formed upon challenging the parasite with DTT mediated ER-stress. The induction of gametocytogenesis was also observed by using a clinical isolate of P. falciparum for the assay. The DTT treated cultures progressed through the gametocytogenesis pathway normally forming all the five morphologically distinct stages. Then we sought to examine if this phenomenon could be simulated in the physiological scenario as well. For this, we made use of a rodent model of malaria, P. berghei. Two different treatment regimes involving 1) direct injection of increasing concentration of DTT into P. berghei infected mice and 2) injection of DTT pretreated P. berghei infected erythrocytes into healthy mice were followed. In both cases, a significant increase in the gametocyte induction was observed. Having seen that Plasmodium undergoes gametocytogenesis upon exposure to ER-stress not only in in vitro cultures but also in in vivo scenario, we wanted to identify the players involved in the commitment to sexual stage. Recently, a transcription factor belonging to AP2 class of transcription factors, referred to as AP2-G has been implicated in committing the asexual parasites for transition to gametocyte stage. To examine the role of this factor in the phenotype observed by us, we looked at the effect of DTT on AP2-G. Interestingly, we found around 6 folds up-regulation in the expression of AP2-G levels under ER-stress. The downstream targets of AP2-G, many of which are the markers of gametocyte were also found to be up-regulated upon being exposed to DTT mediated ER-stress indicating the launch of a transcriptional program which together works in the direction of transition to gametocytes. Having seen that P. falciparum undergoes ametocytogenesis in response to DTT treatment both under in vitro and in vivo conditions, we sought to look for probable physiological analogue of DTT. Since glutathione is the major cellular redox buffer, critical for redox homeostasis, we quantitated the levels of both oxidized and reduced forms of this non protein thiol using Mass Spectrometric approach. We found that the levels of reduced forms of glutathione significantly increased upon treating the parasites with DTT. This indicates that the levels of glutathione could be one of the physiological triggers of gametocytogenesis. Conclusion In conclusion, our study analyses the ways and mechanisms employed by malaria parasite to cope with perturbations to its secretory pathway. We have established the absence of a canonical UPR in this parasite and our results suggest that Plasmodium has developed a three stage response to cope with ER stress: 1) an early adaptation to increase the local concentration of chaperones in the ER by partially blocking the export of a Hsp70 family member, 2) activation of gene expression cascade involving AP2 transcription factors and 3) a consequent switch to the transmissible sexual stage. Hence, our study throws light on a novel physiological adaptation utilised by malaria parasite to tackle stress to its secretory pathway. Gametocytogenesis, which can be transmitted to the mosquito vector, could hence serve as an effective means to escape ER-stress altogether. Importantly, while it is widely known that stress brings about switch towards sexual stages in P. falciparum, the molecular triggers involved in this process remain obscure in the field of malaria biology. Therefore, our findings also address this long standing question by providing the evidence of ER-stress being one such trigger required for switching to the transmissible sexual stages.

Les plantes n'échappent pas à leur lieu de vie, elles doivent en permanence adapter leurs processus physiologiques pour répondre aux variations de leurs conditions environnementales. Durant ma thèse, j'ai étudié deux stress affectant le développement des plantes, les stress thermique (HS) et oxydant (OS), en ciblant des éléments clé de ces phénomènes (les protéines de choc thermique Hsp et Thiorédoxines TRX) afin d'apporter des éléments de réponse quant à l'interconnexion de ces stress et leur importance pour la plante.En utilisant le riz et le soja comme modèles, je montre que le HS suit une « signature Ca2+ » en provoquant une entrée de calcium de l'apoplaste vers le cytosol, assurant ainsi une rigidité à la paroi cellulaire et une cascade de signaux. J'identifie aussi une Pectine Methylesterase nécessaire au remodelage de la paroi cellulaire et à l'intégrité de la membrane. J'ai aussi recherché comment la plante perçoit les changements de température et transmet ce signal vers des effecteurs. Par des analyses d'expression de gènes, je montre qu'une CaM bien spécifique coordonne la réponse au HS, qui se traduit par l'expression spécifique de certaines petites Hsp nucléaires et cytosoliques.Je réalise enfin une étude moléculaire de TDX, une TRX suspectée d'agir dans la réponse au HS. Je montre que TDX interagit avec des Hsp70 de type cytosoliques/nucléaires de façon redox dépendante, que les stress HS et OS induisent une relocalisation nucléaire de TDX. Je montre enfin que TDX est essentielle pour la thermotolérance acquise et la transduction du signal oxydant. Ces résultats sont discutés et des modèles de transduction des signaux entre HS et OS sont proposés. / While being unable to escape their lands, plants are continuously submitted to the modifications of their environment, and need to adjust proper physiological processes in response to various stimuli. During this work, I devoted my studies on two major stresses affecting plant development, heat shock (HS) and oxidative stresses (OS), focusing on key elements in these pathways (HS chaperons and HS-related thioredoxins) in order to bring news elements of knowledge and interconnexion of these pathways.Using rice and soybean as mono- and dicotyledonous plant systems, I show how HS leads to calcium release from plant cell apoplast to the cytosol in a typical calcium signature, conferring cell wall rigidity and enhancing HS signaling pathway. I also identify Pectin Methylesterase as required in this pathway for cell wall remodeling and plasma membrane integrity. I further investigate how plant sense temperature increases and how they transmit the HS signal to downstream elements. Using systematic analyses of Calmodulin (CaM) and small heat shock protein (sHsp) gene expression, I identify one CaM as a coordinator of HS response, which I characterize as involving specific cytosolic/nuclear isoforms of the sHsp family.I latter perform the molecular analysis of TDX, a Thioredoxin suspected to be involved in heat shock response. I show that TDX interacts with cytosolic/nuclear members of the Hsp70 family in a redox dependent manner, both HS and OS inducing its nuclear relocation, and that TDX is required for both acquired thermotolerance and OS signaling.I finally discuss the data brought by this work and propose models with cross-talks between HS and oxidative stress signaling.

Sucrose fading was used to initiate self-administration of ethanol on an FR4 schedule in male Fischer 344 rats. Rats showed low response rates for ethanol alone. After administration of liquid diet containing ethanol, ethanol intake increased over levels prior to administration of the liquid diet. In situ hybridization compared mRNA for the inducible or constitutive 70 kD heat shock proteins in ethanol and nonethanol rats. Both inducible and constitutive mRNAs were found in nonethanol and ethanol tissues. In peripheral organs, radiolableling was higher in ethanol tissue. In brain regions, nonethanol tissues showed higher radiolabeling.

O GBM é o tipo mais agressivo de glioma, apresentando células indiferenciadas (CTGs), responsáveis pela proliferação, invasão e recidiva tumoral. Avaliamos o papel da proteína PrPC e seu ligante HOP na proliferação e autorrenovação de CTGs. Cultivamos linhagens de GBM humano em neuroesferas e geramos populações knockdown para PrPC e HOP. Observamos co-localização de PrPC e CD133 na superfície e sua internalização conjunta estimulada por cobre, sugerindo recrutamento de CD133 mediado por PrPC. O silenciamento de PrPC reduz a expressão de marcadores de células-tronco e autorrenovação, diminui a expressão de proteínas de adesão e afeta a migração celular. O silenciamento de HOP reduz a proliferação, recuperada com o tratamento com HOP em células PrPC+. A capacidade tumorigênica e proliferativa de neuroesferas knockdown para PrPC e/ou HOP in vivo é reduzida. Finalmente, um peptídeo de HOP que bloqueia a interação com PrPC inibe a proliferação e autorrenovação em células PrPC+, indicando potencial do complexo PrPC-HOP como alvo para terapias contra o GBM. / GBM is the most aggressive type of glioma, presenting undifferentiated cells (GSCs), responsible for proliferation, invasion and tumor recurrence. We evaluated the role of the PrPC and its ligand HOP in the proliferation and self-renewal of GSCs. We cultured human GBM lineages in neurospheres and generated knockdown populations for PrPC and HOP. We observed co-localization of PrPC and CD133 on the surface and their co-stimulated copper internalization, suggesting PrPC-mediated recruitment of CD133. PrPC silencing reduces the expression of stem cell markers and self-renewal, decreases adhesion proteins expression, and affects cell migration. HOP silencing reduces proliferation, recovered with HOP treatment in PrPC+ cells. The tumorigenic and proliferative capacity of neurospheres PrPC and/or HOP knockdown in vivo is decreased. Finally, a HOP peptide which blocks PrPC-HOP interaction inhibits proliferation and self-renewal in PrPC+ cells, indicating PrPC-HOP complex potential as a target for therapies against GBM.

The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response.

Malaria is caused by a protozoan parasite belonging to the genus Plasmodia. Plasmodium falciparum is responsible for the fatal form of human malaria. Spread of drug resistant parasites warrants for sound biological understanding of the parasite at both cellular and biochemical level. Heat shock proteins are highly conserved group of proteins required for correct folding, transport, and degradation of substrate proteins in vivo. Hsp60 is found in eubacteria, mitochondria, and chloroplasts, where in cooperation with Hsp10, it participates in protein folding. Keeping in mind the central importance of chaperones in biological processes, our lab has been interested in examining roles of heat shock proteins in malarial parasite during its asexual growth in human erythrocytes. During its life cycle, the parasite continually shuttles between a cold-blooded insect vector with the body temperature of 27°C and a warm-blooded human host with the body temperature of 37°C and parasite experiences episodes of heat shock periodically. Therefore malaria parasite serves as good model to study heat shock protein functions. Like all biological systems, the malaria parasite expresses several chaperones including proteins of the Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100 families. Towards this we have systematically characterized different families of stress proteins Hsp40, Hsp60, Hsp70, Hsp90 as well as Hsp100. In addition to cloning their genes we have studied their expression, localization, abundance, complexes and their biological roles. Earlier studies from our lab showed PfHsp90 is essential for parasite growth and survival in human erythrocytes. Our present study attempts to study heat shock protein 60 of the malarial parasite (PfHsp60). In this connection we have been successful to clone and express PfHsp60 gene from Plasmodium falciparum in E. coli and to raise antibodies specific to PfHsp60. We have examined its expression and import in the mitochondrion of malarial parasite during its asexual growth in human erythrocytes. Analysis of the total parasite lysates resolved by two-dimensional gel electrophoresis followed by western blotting using specific antibodies showed PfHsp60 exhibits an isoelectric point corresponding to its signal uncleaved precursor (pI - 6.2). Mass spectrometric analysis of the spot corresponding to precursor PfHsp60 confirmed the presence of signal peptide region. Co-immunoprecipitation analysis of total parasite lysates with antibodies specific to PfHsp60 showed precursor PfHsp60 to be associated with PfHsp70 and PfHsp90. Co-immunoprecipitation from the mitochondrial and cytoplasmic fraction confirmed the position of mature PfHsp60. Indirect immunofluorescence analysis also showed presence of a pool of PfHsp60 in the cytoplasm of the parasite, in addition to its expected localization in the mitochondrion. Treatment of parasite infected erythrocytes with an inhibitor of Hsp90 disrupted its association with cytoplasmic chaperones and targeted precursor Pfhsp60 for intracellular degradation. On the other hand treatment with the mitochondrial import inhibitor further inhibited the import of precursor PfHsp60 into the mitochondrion and stabilized its interaction with cytosolic chaperones. Previous reports have shown that there are four fold accumulations of PfHsp60 transcripts in heat shocked parasites. However, the expression of PfHsp60 was not induced upon heat shock in the blood stages of P.falciparum. Biochemical data indicate that the mitochondrion is not the source of ATP in the parasite. Furthermore the genome does not seem to encode the critical subunits of Fo-F1 ATP synthase. Yet, the active mitochondrial electron transport chain serves for regeneration of ubiquinone required for pyrimidine biosynthesis. The active electron transport chain is critical for parasite survival. Recent study with the lab-grown 3D7 strain of malaria parasite concluded that mitochondria are not required for energy conversion. Transcriptome analysis of the parasite derived directly from blood samples of infected patients showed that genes encoding the proteins of mitochondrial biogenesis, oxidative phosphorylation, respiration and highlighted the mean expression level for PfHsp60 is dramatically up regulated in parasites. Gene up regulation doesn’t always translate to increase in protein function or metabolic up regulation. When we analyzed the total parasite lysates of field isolates resolved by two-dimensional gel electrophoresis also showed presence of the precursor form of Pfhsp60 in the cytoplasm of the parasite. Overall, our observations indicated accumulation of precursor PfHsp60 in the parasite cytoplasm suggesting an inefficient mitochondrial protein import in the malarial parasite. The defect in mitochondrial protein import is possibly reflective of the compromised energy state of the parasite mitochondrion. This fits with the model that has been reported in mutant strains of yeast, Saccharomyces cerevisiae lacking functional F o-F1-ATPase. These strains were found to grow very poorly under anaerobic conditions and are known to accumulate Hsp60 protein in the cytoplasm mainly its precursor form. Under optimal growth conditions most eukaryotes maintain close co-ordination between gene expression, translation and translocation efficiently. As a result, mitochondrial precursor proteins are usually not found to accumulate in the cytoplasm. To our knowledge this the first report suggesting an inefficient co-ordination in the synthesis and translocation of precursor PfHsp60 and possibly other proteins during asexual growth of malarial parasite in human erythrocytes under optimal growth conditions. Finally, expression of the PfHsp60 gene in E.coli resulted in its association with bacterial GroEL subunits co-fractionating with a size of 920 kDa, corresponding to the tetra decameric form. The observation indicated possible existence of a hybrid chaperonin complex consisting of subunits from ectopically expressed PfHsp60 and endogenous GroEL.

The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response.

Thesis (Ph.D.). / Written for the Dept. of Plant Science. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Masters of Science / The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response. / South Africa