Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T13:11:14Z (GMT). No. of bitstreams: 1 Antunes_MichelleBuenodeMouraPereira_D.pdf: 4387492 bytes, checksum: bf7a9e478bc9627b01a4e45548a6f170 (MD5) Previous issue date: 2012 / Resumo: Diversos tipos celulares respondem ao estresse mecânico ativando sinais que culminam com remodelamento e sobrevivência. O estresse mecânico pode atuar como agente modulador da homeostase celular e de numerosos processos patológicos. Evidências sugerem que a Quinase de Adesão Focal (FAK) medeia a resposta de miócitos cardíacos ao estresse mecânico. Contudo, os mecanismos moleculares que regulam a função da FAK ainda não são totalmente conhecidos. No presente trabalho foi demonstrado que a small heat shock protein ?B-Cristalina interage de forma direta e protege a FAK da degradação pela calpaína 2. Ensaios de pull down, cross-linking acoplado a espectrometria de massas, mutagênese sítio dirigida, docking e modelagem molecular demonstraram que as ?-hélices 1 e 4 do domínio FAT da FAK interage no sítio de ligação constituído pelas folhas ?4 e ?8 da ?B-Cristalina. Os dados funcionais e estruturais obtidos indicaram que ocorre um aumento da associação da ?B-Cristalina e o domínio FAT da FAK após mudanças conformacionais associadas com a fosforilação dependente de Src da tirosina 925. Experimentos de pull down demonstraram que a associação com a ?B-Cristalina protege a FAK da proteólise mediada pela calpaína 2. Miócitos cardíacos submetidos ao silenciamento gênico da ?B-Cristalina apresentaram uma menor quantidade de FAK detectada em 125 KDa, indicando que esta interação protege FAK da proteólise. A submissão dessas células ao estiramento cíclico revelou uma maior taxa de morte celular por apoptose, sendo que a superexpressão da FAK restaurou a viabilidade celular. Os achados deste trabalho indicam que o complexo formado entre FAK e ?B-Cristalina apresenta papel fundamental na proteção da FAK da proteólise durante o estresse mecânico, sendo importante na manutenção da sobrevivência celular / Abstract: Cell types of diverse function respond to mechanical stress by triggering downstream signals for remodelling and survival. As such, mechanical stress impacts organismal homeostasis and numerous pathologic processes. Evidence suggests that focal adhesion kinase (FAK) mediates the responses of myocytes to mechanical stress, yet the molecular mechanisms to regulate FAK function are unclear. We find that FAK is recognized and protected from calpain-induced degradation by the small heat shock protein alpha-B crystalline (CryAB). A model based in the pull down, crosslinking technology coupled with mass spectrometry, site-directed mutagenesis, molecular docking and molecular modeling indicated that a cleft formed by ?4 and ?8 sheets of ?B-Crystalline is critical to the interaction with ?-helix 1 and ?-helix 4 of FAK. Functional and structural data indicated that CryAB binds directly the FAT domain of FAK upon changes in conformation associated with Src-dependent phosphorylation of tyrosine 925 induced by cell stretch. Pulldown assay indicated that ?B-Crystalline interacts and protects from calpain-induced degradation FAK. Cardiomyocytes depleted of CryAB show reduced FAK quantity detected in 125KDa, indicating that this interaction protects degradation of FAK. The submission of such cells to stretch cyclic revealed a higher rate of cell death via apoptosis, whereas restoration of FAK expression restored cell viability. Our findings highlight a new role for CryAB in forming a complex with FAK that is essential for regulating cardiomyocyte survival in response to mechanical stress / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Ciências

Orientadores: Carlos Henrique Inácio Ramos, Celso Eduardo Benedetti / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T16:30:01Z (GMT). No. of bitstreams: 1 Mendonca_YurideAbreu_D.pdf: 5998927 bytes, checksum: dad9a44995521aa0e68ad9507bd61765 (MD5) Previous issue date: 2011 / Resumo: As bactérias patogênicas gram-negativas desenvolveram estratégias sofisticadas para infectar seus hospedeiros, utilizando sistemas de secreção especializados para translocar proteínas de virulência através da membrana das células eucarióticas para o citoplasma. Para que este processo seja eficiente, estas proteínas de virulência devem estar parcialmente enoveladas ou mesmo desenoveladas para que possam ser transportadas para o interior das células hospedeiras através desses sistemas de secreção. Uma vez dentro das células alvo, as proteínas de virulência são encaminhadas ao seu estado nativo e ativadas pela própria maquinaria de enovelamento da célula hospedeira. As proteínas responsáveis em auxiliar o enovelamento protéico nas células são as chaperonas, denominadas classicamente de proteínas de choque térmico (Hsps). Plantas, por serem organismos sésseis, são muito mais vulneráveis a fatores de estresse biótico e abiótico, tornando o papel das Hsps ainda mais relevante para a homeostase protéica e viabilidade celular. O estudo das proteínas da família Hsp90 é muito difundido devido ao seu papel fundamental desempenhado nas situações de infecção e em diferentes tipos de estresse. Neste trabalho, a proteína recombinante Hsp90 de laranja doce (Citrus sinesis) foi clonada e purificada com o objetivo de estudar o mecanismo geral de infecção do fitopatógeno bacteriano de espécies de citros Xanthomonas citri (Xac). Investigou-se a interação, combinando técnicas de western blot e ensaios de pull-down, entre a Hsp90 e todas as quatro variantes da PthA, principal fator de virulência de Xac, e as co-chaperonas da Hsp90 de laranja ciclofilina (Cyp) e uma proteína tiorredoxina-"like? (TDX). Estas proteínas já foram descritas por se apresentarem reguladas positivamente na laranja doce durante a infecção com Xac, apontando para um possível papel da Hsp90 na formação de um complexo de enovelamento capaz de ativar as proteínas de virulência de Xac no interior das células infectadas. Além disso, investigamos a estrutura e função da Hsp90 de laranja doce que apresentou-se enovelada e solúvel, como medido por dicroísmo circular (CD), espectroscopia de fluorescência intrínseca e espalhamento de luz dinâmico (DLS). A Hsp90 se apresentou como um dímero em solução com um raio de Stokes de 62 Å, e altamente resistente á desnaturação por temperatura, como medido por CD. A proteína mostrou-se funcional, como medido pela sua capacidade de proteger a agregação da citrato sintase in vitro em um ensaio de espalhamento de luz. O estudo do efeito de nucleotídeos na conformação e função da Hsp90 através de CD, fluorescência intrínseca e de espalhamento de raios-X a baixos ângulos (SAXS) mostram alterações na conformação da proteína na presença destes ligantes / Abstract: Gram-negative bacterial pathogens, which have developed sophisticated strategies to infect hosts, use specialized secretion systems to secrete and translocate virulence proteins across the eukaryotic cell membrane into the cytoplasm. The translocation process depends on unfolded or partially folded virulence proteins to be transportated through the secretion system into the target inner cell where they are folded by the host chaperone machinery. Auxiliary proteins are responsible to help folding in cells and are known as molecular chaperones, or heat shock proteins (HSPs). Plants, being sessile organisms, are much more vulnerable to biotic and abiotic stress factors, making the role of HSPs even more important for protein homeostasis and cell viability. The study of the Hsp90 family is widespread due to the key role played in situations of infection and under various types of stress. In this work, an Hsp90 sweet orange (Citrus sinesis) was cloned and purified in order to study the general mechanism of infection of the bacterial pathogen of citrus species, Xanthomonas citri (Xac). First, we investigated the interaction, by combining immunostaining and pull-down assays, between Hsp90 and all four variants of PthA, the major virulence factor of Xac, and the orange Hsp90 cochaperonas cyclophilin (Cyp) and a thioredoxin-like protein (TDX). These proteins have been described to be upregulated in sweet orange during infection with Xac, pointing to a possible role of Hsp90 in the formation of a folding complex able to activate the virulence proteins of Xac inside the infected cells. Furthermore, we took to investigate the structure and function of the Hsp90 from sweet orange, which was folded and soluble as measured by circular dichroism (CD), intrinsic fluorescence spectroscopy and dynamic light scattering (DLS). Hsp90 was a dimer in solution with a Stokes radius of about 62 Å, tolerating up to 90 °C without denaturation, as measured by CD. The protein was functional, as measured by its ability to protect the aggregation of citrate synthase in an light scattering assay. The study of the effect of nucleotides on the conformation and function of Hsp90 by CD, intrinsic fluorescence and small-angle X-ray scattering (SAXS) show evidence of conformation modulation by ATP and ADP / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.

Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein (Hsp70) and J protein chaperone complements in malaria parasites affecting humans, primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and structurally related proteins that play a crucial role in maintaining the structural integrity of proteins during normal and stress conditions. They are considered future therapeutic targets in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins for degradation. However, in P. falciparum, these classes of proteins have also been implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte, imparting properties that allow it to adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum encodes six Hsp70 homologues and a large number of J proteins that localize to the various intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding the Hsp70-J protein interactions and/or partnerships is an essential step for drug target validation and illumination of parasite biology. A review of these chaperone complements across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J protein complement compared to the rodent and primate infecting species. It further highlights how unique the P. falciparum chaperone complement is compared to the other Plasmodium species included in the analysis. In silico analysis showed that the genome of P. falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL containing J proteins are unique with no homologues in the human system and are considered as attractive drug targets. Very few of the predicted J proteins in P. falciparum have been experimentally characterized. To this end, cell biological and biochemical approaches were employed to characterize PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in literature regarding its localization formed the basis for the experimental work. This is the first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70 partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are described. It is also the first study that details the successful expression and purification of PfHsp70-3. Further, research findings have described for the first time the expression and localization of PFB0595w in the intraerythrocytic stages of P. falciparum development. Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone – cochaperone partnership was proposed that formed the basis for the in vitro experiments. PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1 pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need for further related experiments to support the findings. Gel filtration analysis showed that PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1 in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion. Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding of parasite biology.

La maladie d'Alzheimer est la démence la plus répandue dans le monde. Le nombre de cas augmente de manière exponentielle et il est donc important de comprendre les mécanismes moléculaires donnant lieu à cette terrible maladie. Une des hypothèses les plus supportées est celle suggérant que la production et dégradation déséquilibrées de l'amyloïde-beta (Aß), un peptide de 42 acides aminés trouvé dans tous les individus sains, est un événement clé dans le déroulement de la maladie d'Alzheimer. En effet, une production accrue ou une dégradation faible du peptide ont pour conséquence son agrégation et accumulation dans des plaques de fibres entre les neurones des régions spécifiques du cerveau. C'est pourquoi la modulation de l'agrégation du peptide Aß est une des approches envisageables pour modifier l'évolution de la maladie d'Alzheimer. Les protéines chaperons dont une des fonctions est d'assister d'autres protéines dans leur repliement, sont parmi les molécules les plus étudiées pour leur capacité modulatrice de l'agrégation des protéines (inclus le peptide Aß). Plusieurs chaperons ont montré la capacité d'inhiber la formation des fibres par l'Aß. Cependant, du fait que les chaperons sont des molécules conservées et peu spécifiques, leur surexpression ou administration directe peut avoir des conséquences négatives si les chaperons interagissent avec des protéines autres que la protéine cible. Dans ce travail, nous nous sommes intéressés à une protéine chaperon bactérienne possédant une forte activité " holdase " (i.e., elle empêche le repliement précoce des protéines) comme possible modulateur de l'agrégation du peptide Aß. Le chaperon sauvage a une très faible capacité d'inhibition de la formation de fibres par le peptide Aß. Cependant, nous avons démontré qu'en modifiant légèrement la surface de liaison du chaperon, la protéine devient un puissant inhibiteur de l'agrégation d'Aß. En parallèle, nous nous sommes intéressés à l'influence des ions métalliques sur l'agrégation du peptide Aß. [...] / Alzheimer's disease is the most frequent type of dementia. With an exponentially growing number of cases, understanding the underlying molecular events leading to this devastating condition is of crucial importance. Much evidence points to a disequilibrium in the production and degradation of amyloid beta (Aß), a normally physiological 42 amino acid peptide, as an early key event in Alzheimer's etiology. Whether Aß is overproduced or poorly degraded, the overall result is an abnormally large pool of peptide that gradually aggregates forming extracellular deposits of fibrils, called amyloid plaques, in specific brain regions. Hence, modulation of Aß aggregation process is one of the suggested approaches to control the evolution of Alzheimer's disease. Universally conserved molecular chaperones have been intensively studied for their capacity to prevent aggregation of disease-related proteins, and many of them have proven to efficiently modulate Alzheimer's Aß aggregation. In a scenario where chaperones are overexpressed or directly administered into the affected tissue, the universal conservation and the relatively poor client-specificity of generic chaperones can become a downside because of the risk of interaction with proteins other than the targeted one is not dismissible, and thus the consequences unpredictable. In the first part of this work, we looked upon a bacterial chaperone call SecB with an unusually robust holdase activity (i.e. it prevents early protein folding) as a promising modulator of Alzheimer's Aß peptide aggregation. [...]

No description available.

Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity. As such, mutations in this family of proteins result in a wide variety of diseases from cancers to cardiomyopathies. The sHSPs Beta-2 (HspB2) and alpha-beta crystalline (CryAB) are two of the ten human sHSPs and are both expressed in cardiac and skeletal muscle cells. A heart that cannot properly recover or defend against stressors such as extreme heat or cold, oxidative/reductive stress, and heavy metal-induced stress will constantly struggle to maintain efficient function. Accordingly, CryAB is required for myofibril recovery from ischemia/reperfusion (I/R) and HspB2 is required I/R recovery as well as efficient cardiac ATP production. Despite these critical roles, little is known about the molecular function of these chaperones. We have identified over two hundred HspB2-binding partners through both yeast two-hybrid and copurification approaches, including interactions with myofibril and mitochondrial proteins. There is remarkable overlap between the two approaches (80%) suggesting a high confidence level in our findings. The sHSP, CryAB, only binds a subset of the HspB2 interactome, showing that the HspB2 interactome is specific to HspB2 and supporting non-redundant roles for sHSPs. We have confirmed a subset of these binding partners as HspB2 clients via in vitro chaperone activity assays. In addition, comparing the binding patterns and activity of sHSP variants in comparison to wild type can help to elucidate how variants participate in causing disease. Accordingly, we have used Y2H and in vitro chaperone activity assays to compare the disease-associated human variants R120GCryAB and A177PHspB2 to wild type and have identified differences in binding and chaperone function. These results not only provide the first molecular evidence for non-redundancy of the sHSPs, but provides a useful resource for the study of sHSPs in mitochondrial and myofibril function.

La formation et le dépôt de fibres d'α-Synucléine dans le cerveau humain sont à l‟origine de la maladie de Parkinson. Cette thèse documente le rôle de deux chaperons moléculaires dans l‟assemblage en fibres de l'α-Syn : Hsc70 (protéine de choc thermique constitutivement exprimée chez l‟Homme) et Ssa1p (son équivalent chez la levure). Le but était d'élargir le catalogue d'effets connus des chaperons moléculaires sur α-Syn, pour éventuellement ouvrir la voie à des applications thérapeutiques. Nous avons montré que Hsc70 inhibe l'assemblage de l'α-Syn en fibres, en se liant avec une forte affinité à la forme soluble de l'α-Syn. Hsc70 se lie préférentiellement aux fibres de l'α-Syn, et cette liaison a un effet cytoprotecteur puisqu'elle rend les fibres moins toxiques pour les cellules de mammifères en culture. Pareillement à Hsc70, Ssa1p inhibe l'assemblage de l'α-Syn en fibres, et a une plus forte affinité pour les fibres que pour la forme soluble de l'α-Syn. En revanche, la liaison de Ssa1p aux fibres de l'α-Syn n'a pas d'effet cytoprotecteur, sûrement due aux différences entre les séquences du site de liaison aux peptides des deux chaperons moléculaires, qui fait que Ssa1p a une affinité plus faible que Hsc70 pour les fibres d'α-Syn. Nous avons fixé le complexe entre Ssa1p et α-Syn avec des agents pontants, pour ensuite établir une carte du site d'interaction entre les deux protéines en utilisant la spectrométrie de masse. Ceci est indispensable si un « mini » Ssa1p, constitué des éléments nécessaires et suffisants sera utilisé comme agent thérapeutique pour réduire la toxicité des fibres d'α-Syn. / The formation and deposition of α-Synuclein fibrils in the human brain is at the origin of Parkinson’s disease. The objective of my thesis was to document the role of two molecular chaperones on the assembly of α-Syn into fibrils: Hsc70, a constitutively expressed human heat shock protein, and Ssa1p, its yeast equivalent. The aim was to expand the catalogue of known effects of molecular chaperones on the PD implicated protein, which could have therapeutic significance. We showed that Hsc70 inhibits the assembly of α-Syn into fibrils, by binding with high affinity to the soluble form of α-Syn. We documented that Hsc70 binds preferentially to α-Syn fibrils and that this binding has a cytoprotective effect, as it renders the fibrils less toxic to cultured mammalian cells. Similarly to Hsc70, Ssa1p inhibits the assembly of α-Syn into fibrils, and has a higher affinity for fibrils than for the soluble form of α-Syn. On the other hand, binding of Ssa1p to α-Syn fibrils does not have a cytoprotective effect, almost certainly due to differences in the amino acid sequences of the peptide binding sites of the two molecular chaperones, which mean that Ssa1p has a lower affinity than Hsc70 for α-Syn fibrils. We stabilized the complex between Ssa1p and α-Syn using chemical cross-linkers, to then map the interaction site between the two proteins. This is indispensable if a “mini” Ssa1p, comprised of only what is necessary and sufficient of Ssa1p, is to be used as a therapeutic agent to decrease the toxicity of α-Syn fibrils. A therapeutic agent based on exogenous protein Ssa1p is less likely to trigger an autoimmune response than for example the endogenous protein Hsc70.

TCP-1是一种新型的定向于肿瘤血管的多肽，通过小鼠体内的噬菌体展示技术筛选得到。在之前的研究中，我们已证明TCP-1具有定向于肿瘤血管并有效靶向运输抗肿瘤药物和显像剂的特性。本研究的目的是进一步研究在原位结肠癌模型中定向运输抗肿瘤药物肿瘤坏死因子(TNFα)，以及在结肠癌临床样本中运输显像剂异硫氰酸荧光素(FITC)的能力。并对TCP-1与肿瘤坏死因子的融合蛋白TCP-1/TNFα的抗肿瘤机制进行阐述。 / 本研究中，我们首先尝试用TCP-1作为载体，将增强绿色荧光蛋白靶向运输至肿瘤血管。结果证明TCP-1可以成功将蛋白运输到在肿瘤血管而非其它正常的组织器官上。TCP-1还可以靶向运输肿瘤坏死因子并增强其抗肿瘤作用。和肿瘤坏死因子比较，融合蛋白TCP-1/TNFα处理组的凋亡细胞数量增多，肿瘤微血管数目减少，并且无明显毒副作用。与结肠癌的一线化疗药物5-氟尿嘧啶(5-FU)联合给药后，与TNFα与5-FU联合给药相比较，融合蛋白TCP-1/TNFα联合5-FU在以下方面具有更明显的作用：抑制肿瘤生长，增加肿瘤细胞凋亡和抑制肿瘤细胞增殖，促进肿瘤血管正常化，升高瘤内免疫细胞以及减轻骨髓和脾内的免疫抑制反应。经检测TCP-1的靶向运输增加了瘤内的TNFα以及5-FU的浓度。这些都表明TCP-1不但可以靶向运输TCP-1/TNFα至肿瘤血管，还可以增加CD8+细胞的浸润增加瘤内免疫反应以及增加血管对抗肿瘤药物的通透性。以上都对抗肿瘤起到重要作用。 / 在临床的结肠癌样本中，TCP-1对肿瘤血管的结合能力也得到了证实。48.98%的结肠癌样本对TCP-1的结合为阳性。统计学分析显示TCP-1的结合与结肠癌的分期和肿瘤位置有关，对于N2期，位于乙状结肠的肿瘤的结合尤为明显。本研究的主要目的是将分离鉴定出的TCP-1发展成为结肠癌的生物标记，并且作为运输抗肿瘤药物和显像剂的载体应用于结肠癌的诊断和治疗中。鉴于TCP-1的靶向运输特点，将会有机会研发更多的抗肿瘤药物，同时增强传统化疗药的抗肿瘤作用。这些都可以优化肿瘤治疗的方案。综上所述，TCP-1是一种在结肠癌治疗诊断中具有广阔前景的多肽。 / TCP-1 is a novel vasculature-targeting peptide. It was discovered through the in vivo phage library selection in mice. It was demonstrated that TCP-1 peptide exhibited a homing ability to the neovasculature of colon tumors and was capable of efficiently delivering imaging agents and chemotherapeutic drugs to this target site. The current study is to further investigate the targeting ability of TCP-1 to deliver a known immunomodulator, tumor necrosis factor α (TNFα) as an example of anti-cancer drug in an orthotopic colorectal cancer (CRC) model and fluorescein isothiocyanate (FITC) as imaging agent for testing the binding capacity for tumors in colorectal cancer patients. The mechanisms for the action of this novel biologic TCP-1/TNFα in the treatment of colon cancer in mice were also defined. / In this study, we observed that TCP-1 peptide delivered enhanced green fluorescent protein (EGFP) only to tumor blood vessel other than normal organs after TCP- 1/EGFP injection. This was not observed after EGFP injection. This finding showed that TCP-1 can deliver biologic protein to the tumor blood vessels. Furthermore, results from TNFα or TCP-1/TNFα targeted delivery experiments showed that TCP- 1/TNFα displayed stronger anti-cancer effects than TNFα alone on the induction of apoptosis and reduction in number of microvessels in the tumors, without significant effect in systemic toxicity. In the combined therapy with 5-fluorouracil (5-FU), a standard drug for colon cancer treatment, pretreatment with low dose (1 ng TNFα /mouse) of TNFα or TCP-1/TNFα potentiated the anti-cancer action of 5-FU. In this regard, TCP-1/TNFα could significantly reduce tumor size and weight, increase number of apoptotic cells, inhibit tumor cell proliferation, normalize tumor blood vessels, facilitate infiltration of immune cells to tumor mass and attenuate immunosuppression in bone marrow and spleen. Moreover, TCP-1 could significantly increase intratumoral levels of TNFα and 5-FU. It was also suggested that TCP-1 could selectively deliver TNFα to the tumor blood vessels and modulate the immune response by increasing CD8+ cells infiltration to tumors and increase vascular permeability to 5-FU. These observations may be the key actions to reduce tumor growth. / The binding ability of TCP-1 was also detected in clinical samples from colorectal cancer patients in which 24/49 (48.98%) tumor tissues were positive with TCP-1 binding signal. Statistical analysis showed that TCP-1 had a strong and significant binding with colorectal cancer at the N2 stage among the different colorectal cancer stages (P=0.028) and location in the colon at the sigmoid (P<0.001). / Our study also focused on the isolation and identification of the binding molecule of TCP-1 in order to develop it into a biomarker for CRC and using TCP-1 as a carrier in delivering anti-cancer drugs and imaging agents to colon tumors for cancer therapy and diagnosis. With the homing property of TCP-1 on colon tumor blood vessels, new types of anti-cancer drugs will be developed and their combinations with conventional chemotherapy drugs will optimize the therapeutic outcome and improve regimen of treatment for CRC. Taken together, TCP-1 peptide appears to be a promising agent in molecular imaging and drug delivery for CRC diagnosis and therapy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lu, Lan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 157-177). / Abstracts also in Chinese. / Lu, Lan.

La chaperonine CCT/TriC tient un rôle central dans le maintien de la protéostase cellulaire. Elle compte parmi ses clientes un grand nombre de protéines impliquées de près ou de loin dans la régulation du cycle comme l’actine, la tubuline et la cycline E. afin de mieux comprendre l’implication de la CCT/TriC dans la cancérogenèse, nous avons quantifié, dans 18 lignées cellulaires tumorales, une lignée issue d’un tissu sain et un tissu sain, ses taux d’expression et son activité. Nos résultats indiquent que l’expression de la CCT/TriC n’est pas toujours corrélée à son activité. Nous avons dans un second temps, documenté l’expression de ces partenaires/modulateurs d’activité, préfoldine, PhLP3, Hop/p60, Hsp/c70 et leur influence sur son activité. Nos résultats montrent que l’activité de la CCT/TriC pourrait être régulée à travers les variations des niveaux d’expression de la préfoldine et/ ou Hsp/c 70. Enfin, nous avons montré que dans les cellules qui se divisent le plus lentement, l’activité de la CCT/TRiC est la plus faible. Ces observations montrent que les variations de l’activité de la CCT/TriC pourraient constituer un point de contrôle dans la prolifération cellulaire maligne. / The molecular chaperone CCT/TRiC plays a central role in maintaining cellular proteostasis. It mediates the folding of lot of proteins involved in cell cycle regulation as the major cytoskeletal proteins tubulins and actins and the oncoprotein cyclin E. To assess the involvement of CCT/TRiC in tumor genesis, we quantified its expression levels and activity in 18 cancer, one non-cancer human cell lines and a non-cancer human liver. We show that the expression levels of CCT/TRiC in cancer cell lines are higher than that in normal cells. However, CCT/TRiC activity does not always correlate with its expression levels. We therefore documented the expression levels of CCT/TRiC modulators and partners PhLP3, Hop/P60, prefoldin and Hsp/Hsc70. Our analysis reveals a functional interplay between molecular chaperones that might account for a precise modulation of CCT/TRiC activity in cell proliferation through changes in the cellular levels of prefoldin and/or Hsc/p70 and CCT/TRiC client protein availability. Finally, our study reveals that cells with the longest doubling time host the smallest CCT/TRiC activity suggesting that CCT/TRiC-mediated client protein folding constitutes a bottle-neck in cancer cell proliferation. Our observation and approaches bring novel insights in the role of CCT/TRiC-mediated protein folding machinery in cancer cell development.