Titre de la thèse : Développement de thérapies adjuvantes associées à l'anti-cytoadhérence. Résumé : L'antigène de surface PfEMP-1 (P. falciparum Erythrocyte Membrane Protein-1) encodé par 60 gènes de la famille var du parasite, est un facteur de virulence du paludisme touchant l'Homme. Les différents variants de PfEMP-1 sont impliqués dans la cytoadhérence des érythrocytes infectés par P. falciparum (iRBCs) avec plusieurs récepteurs de l'endothélium vasculaire de l'hôte. Parmi eux, l'interaction avec ICAM-1 semble être liée à des manifestations sévères de la maladie telles que le paludisme cérébral. La majorité de la mortalité due à un paludisme sévère est observée dans les 24 heures après admission à l'hôpital, malgré l'utilisation d'antipaludiques efficaces, soulignant ainsi un besoin urgent en thérapies adjuvantes ciblant spécifiquement la cytoadhérence.Le site d'interaction d'ICAM-1 avec les érythrocytes infectés a été identifié au niveau du « BED side » de son domaine N-terminal, similaire à celui des immunoglobulines. Les variants antigéniques de P. falciparum capables de se lier à ICAM-1 présentent des différences subtiles au niveau des résidus responsables de cette interaction. La boucle DE semble être l'élément commun des sites de fixation à ICAM-1 pour trois variants de P. falciparum (ITO4-A4, ITO4-C24 and ItG-ICAM) et fut ainsi choisie par le groupe de Matthias Dormeyer comme cible pour un criblage in silico. Une librairie de structures de petites molécules fut criblée en utilisant une technique d'alignement moléculaire fournie par le programme 4Scan. Grâce à cette étude, la molécule (+)-epigalloyl-cathechin-gallate (EGCG) fut identifiée comme un inhibiteur d'interaction très spécifique et dose-dépendant pour deux des trois variants.Notre projet fut de développer des composés anti-adhésifs capables d'inhiber ou de supprimer l'interaction des hématies infectées avec les récepteurs endothéliaux. Tout d'abord, nous avons réalisé une série de substitues tetrahydroisoquinolines, analogues au composé naturel EGCG précédemment découvert. Ensuite, nous avons basé notre recherche sur la synthèse de composés peptidiques simulant des régions spécifiques d'ICAM-1 qui peuvent être impliquées dans l'interaction avec les variants de PfEMP1. Le design de ces peptides a été dirigé par le groupe du Prof. Tramontano sur la base d'analyses in silico de l'interaction moléculaire entre ICAM-1 (dont la structure cristallographique est connue) et un modèle de PfEMP1. Nous avons étudié les propriétés d'inhibition de la cytoadhérence de plusieurs de ces peptides afin de créer une base pour le design de molécules peptidomimétiques dotées de meilleures propriétés médicamenteuses.Finalement, afin d'élargir notre étude, EGCG a été criblé contre un panel de nouveaux isolats de P. falciparum provenant de patients et capables d'interagir avec ICAM-1. Ses propriétés anti-adhésives ont été étudiées afin de connaitre l'impact des variations de ces souches sur l'interaction et pour guider le design d'un inhibiteur de cytoadhérence à large spectre.INTITULÉ ET ADRESSE DE L'U.F.R. OU DU LABORATOIRE : - Prof. Giuseppe Campiani - Università degli Studi di Siena - Dipt. Farmaco-Chimico Tecnologico. Via Aldo Moro, 2, 53100, Siena (Italy) - Prof. Alister Craig – Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3, 5QA, UK. / Thesis title-: Development of anti-cytoadherence adjunct therapies.Thesis abstract-Parasite derived surface antigen PfEMP-1 (P. falciparum erythrocyte membrane protein-1) encoded by 60 var genes, is a virulence factor of the human malaria parasite. PfEMP-1 variants have been implicated in the cytoadherence of P. falciparum infected erythrocytes (iRBCs) to several binding receptors on host vascular endothelium. Among them, binding to ICAM-1 seems to be related to severe manifestations of the disease such as cerebral malaria. The majority of the mortality with severe malaria is seen within 24 hours of hospital admission despite the use of effective anti-parasite drugs, therefore the development of adjunctive therapies specifically targeting cytoadherence is urgently needed.The binding site for iRBC has been mapped to the BED side of the N-terminal immunoglobulin-like domain of ICAM-1, and shows subtle differences in the contact residues used by ICAM-1 binding P. falciparum antigenic variants. The DE loop appears to be a common feature of the ICAM-1 binding sites for three P. falciparum variants (ITO4-A4, ITO4-C24 and ItG-ICAM) analyzed and was selected by Matthias Dormeyer group for in silico screening of a small-molecule structures library using a molecular-alignment technique based on the program package 4Scan. From this study, (+)-epigalloyl-cathechin-gallate (EGCG) was found to inhibit binding of two variant ICAM-1 binding parasites in a highly specific, dose-dependent manner.Our approach to this need has been the development of anti-adhesive compounds to inhibit and reverse the binding of iRBCs to endothelial receptors. Firstly, we developed a series of substituted tetrahydroisoquinolines as analogues of the natural compound EGCG previously identified. Secondly, we based our research on the synthesis of peptidic compounds mimicking specific ICAM-1 regions hypothesized to be involved in the binding with disease-relevant PfEMP1 variants. The design of the peptides has been conducted by the group of Prof. Tramontano on the basis of an in silico analysis of the molecular interaction between ICAM-1 (whose crystal structure is known) and a homology model of PfEMP1. We have evaluated several peptides for their cytoadherence blocking properties and the results of this studies could form the basis for the design of peptidomimetics endowed with better drug-like properties. Finally, to extend previous studies, EGCG has been screened against a panel of new ICAM-1 binding patient isolates of P. falciparum for its anti-adhesive properties to investigate the impact of iRBC strain variation and to guide the design of a broad-spectrum cytoadherence inhibitor.TITLE AND ADDRESS OF LABORATOIRE : Prof. Giuseppe Campiani - Università degli Studi di Siena - Dipt. Farmaco-Chimico Tecnologico. Via Aldo Moro, 2, 53100, Siena, Italy. Prof. Alister Craig – Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3, 5QA, UK.

Cancer has become the major factor causing death in Taiwan. Although brain tumor take a few ratio in cancer, the current therapy shown not effect well. In previous studies, EGCG will promote cancer cells to go apoptosis after treatment with EGCG. But the mechanism in brain tumor is unknown. So, we use EGCG from green tea extract to investigate cell death of human brain tumor cell lines GBM8401, U87MG and U251. The metabolic rates of GBM8401, U87 and U251 are decrease with EGCG treatment, and it is significant in GBM8401. We also examine some genes and proteins related apoptosis by RT-PCR and Western blotting. The accumulation of p21, p27 and p53 are increasing with concentration and time course treatments. EGCG can inhibit the ability of brain tumor to form foci in anchorage-independent growth assay. Our data show that EGCG can inhibit brain tumor cell lines GBM8401, U87 and U251. It is validated that EGCG has the potential of cancer therapy and prevention.

Endometrial cancer is one of the most significant gynaecological malignancies that affect women from New Zealand and the rest of the world. One of the critical stages in the development of a tumour is the onset of hypoxia. The malignancy responds by having raised levels of Hypoxia Inducible Factor (HIF) that in turn induces increased production of Vascular Endothelial Growth Factor (VEGF). VEGF is a potent angiogenic factor that will mediate vascular supply of nutrients and oxygen to the developing tumour. The aim of this study was to investigate whether two compounds found in extracts of plant materials, Resveratrol (Resveratrol) and Epigallocatechin gallate (EGCG), altered the levels of VEGF in the supernatant of cultured endometrial cancer cells. Resveratrol is a phytoalexin that is found in many foods, such as grapes, nuts and berries, as well as in high concentrations in some red wines. 100 µM of resveratrol was added to cell cultures for 24 hours. VEGF levels in the supernatant were then analysed using ELISA. Resveratrol was found to have significant inhibitory effects in both primary endometrial cancer cell cultures and immortalised endometrial cancer cell cultures. Resveratrol was also shown to reverse the increase in VEGF caused by the hypoxia mimic cobalt chloride (CoCl₂). Epigallocatechin gallate (EGCG) is an antioxidant catechin extracted from green tea. The effect of EGCG was analysed using the same method as for resveratrol. 100 µM of EGCG was also shown to have a significant inhibitory effect on the level of VEGF in the supernatant of cultured endometrial cancer cells, as well as reducing the effect of CoCl₂. These results suggest that selected food compounds, resveratrol and EGCG, can reduce VEGF levels by inhibiting HIF. Further investigation This may have anti-tumour effects in women with endometrial cancer.

Ovarian cancer is the deadliest gynaecologic cancer in New Zealand. Its high mortality rate is due to the fact that it is usually diagnosed at an advanced stage. Advanced ovarian cancer is less responsive to current cytotoxic treatment. Thus, there is an urgent need for novel anti-cancer drugs that can improve patient longevity and quality of life. One of the clinical features of advanced ovarian cancer is the growth of secondary tumours due to the highly metastatic nature of the disease. Cancer cells disseminate from the ovary, some form cell clusters that travel through the abdominal cavity by physiological movement of body fluid and then deposit on the abdominal wall and internal organs to generate secondary tumours. The exact mechanisms of how these cells metastasize are unclear, but prognosis typically worsens if levels of vascular endothelial growth factor (VEGF) are elevated. This study investigated the anti-tumour activities of naturally occurring food compounds resveratrol, acetyl resveratrol and (-)-Epicatechin-3-gallate (EGCG), in cell spheroids/clusters of ovarian cancer. It also examined the protein expression of various proteins involved in the NF-κB signalling pathway. This pathway has been suggested to mediate the secretion of VEGF and is a possible target for the naturally occurring compounds. Results show that resveratrol and acetyl resveratrol reduce cell growth and cellular metabolism in a dose-, time- and cell line- dependent fashion. In addition, the reduction of VEGF is also dose-, time- and cell line- dependent. Paradoxically, another angiogenic protein interleukin-8 (IL-8) secretion is increased. Resveratrol and acetyl resveratrol attenuate the expression of NF-κB but this effect is cell line specific. EGCG has limited effect on cell growth, cellular metabolism and the secretion of VEGF and IL-8. These findings suggest that resveratrol and its derivative may have the ability to supress the angiogenic activity of ovarian cancer cells and warrant further in vivo study.

Endometrial cancer is one of the most significant gynaecological malignancies that affect women from New Zealand and the rest of the world. One of the critical stages in the development of a tumour is the onset of hypoxia. The malignancy responds by having raised levels of Hypoxia Inducible Factor (HIF) that in turn induces increased production of Vascular Endothelial Growth Factor (VEGF). VEGF is a potent angiogenic factor that will mediate vascular supply of nutrients and oxygen to the developing tumour. The aim of this study was to investigate whether two compounds found in extracts of plant materials, Resveratrol (Resveratrol) and Epigallocatechin gallate (EGCG), altered the levels of VEGF in the supernatant of cultured endometrial cancer cells. Resveratrol is a phytoalexin that is found in many foods, such as grapes, nuts and berries, as well as in high concentrations in some red wines. 100 µM of resveratrol was added to cell cultures for 24 hours. VEGF levels in the supernatant were then analysed using ELISA. Resveratrol was found to have significant inhibitory effects in both primary endometrial cancer cell cultures and immortalised endometrial cancer cell cultures. Resveratrol was also shown to reverse the increase in VEGF caused by the hypoxia mimic cobalt chloride (CoCl₂). Epigallocatechin gallate (EGCG) is an antioxidant catechin extracted from green tea. The effect of EGCG was analysed using the same method as for resveratrol. 100 µM of EGCG was also shown to have a significant inhibitory effect on the level of VEGF in the supernatant of cultured endometrial cancer cells, as well as reducing the effect of CoCl₂. These results suggest that selected food compounds, resveratrol and EGCG, can reduce VEGF levels by inhibiting HIF. Further investigation This may have anti-tumour effects in women with endometrial cancer.

Cisplatin is a commonly used chemotherapeutic agent for multiple solid tumors. However, cisplatin-induced neurotoxicity, nephrotoxicity and hearing loss hamper its use in clinical setting. Although, neurotoxicity and nephrotoxicity can be prevented, there is no cure for cisplatin-induced hearing loss. Cisplatin-induced hearing loss results from damage to outer hair cells (OHCs) in basal turn of the cochlea, to spiral ganglion neurons (SGN), stria vascularis (SV) and fibrocytes of spiral ligament (SL). At the cellular level, cisplatin produces profound increases in reactive oxygen species (ROS) that stimulate cell signaling pathways leading to cochlear inflammation, apoptosis and permanent hearing loss. Thus, potential otoprotective drugs should target oxidative stress and inflammatory mechanisms without interfering with cisplatin chemotherapeutic efficacy. In this study, I characterized the otoprotective actions of the green tea extract, epigallocatechin 3-gallate (EGCG), which possesses anti-oxidant, anti-inflammatory and anti-tumor properties. Oral administration of EGCG to male Wistar rats reduced cisplatin-induced hearing loss, assessed by auditory brainstem responses. These changes were associated with a reduction in cisplatin-induced loss of OHCs primarily in the basal region of the cochlea, along with reduced oxidative stress, inflammatory and apoptotic markers. In addition, EGCG protected against cisplatin-induced decrease in inner hair cell (IHCs) ribbon synapses, labeled with CtBP2. EGCG also protected against cisplatin-induced loss of Na+/K+ ATPase α1 immunoreactivity in the stria vascularis and spiral ligament. In vitro studies using University Bristol/Organ of Corti-1 (UB/OC-1) cells showed that EGCG reduced cisplatin-induced ROS generation and the activation of ERK and STAT1, while it preserved the activity of STAT3 and levels of Bcl-xL. Moreover, EGCG suppressed oxidative stress, inflammatory and apoptotic markers in cisplatin-treated UB/OC-1 cells. Co-administration of EGCG did not alter cisplatin-induced apoptosis of human-derived head and neck cancer cells, ovarian cancer cells or colon cancer cells. In studies using a xenograft model of head and neck cancer in severe combined immunodeficient (SCID) mice, I showed that EGCG did not interfere with cisplatin chemotherapeutic efficacy. These data suggest that EGCG is a potential otoprotective agent for treating cisplatin-induced hearing loss without compromising its chemotherapeutic efficacy.

Made available in DSpace on 2018-08-01T21:35:04Z (GMT). No. of bitstreams: 1 tese_12334_Dissertação_Gisele Pereira Diniz Schuenck.pdf: 3345641 bytes, checksum: 72a76070052bd5a46fdb845951b416b3 (MD5) Previous issue date: 2018-04-19 / As nanopartículas de ouro (AuNPs) tem se mostrado uma plataforma atraente para combinar uma variedade de propriedades biofisicoquímicas em um nanodispositivo unificado com grande potencial terapêutico. Neste estudo investigamos a capacidade do polifenol natural, epigalocatequina-3-galato (EGCG), de permitir a redução química de sais de ouro em nanopartículas de ouro e a estabilização em uma única etapa. As nanopartículas de ouro foram sintetizadas por oxirredução, com base em um Planejamento Fatorial Fracionado (triagem) e em seguida um Planejamento Composto Central (otimização) com o objetivo de determinar os pontos ótimos para a síntese. A caracterização do coloide sintetizado foi realizada com análises de UV-Vis, Infravermelho, Raman, Potencial Zeta, ICP-MS e Microscopia Eletrônica de Transmissão. Além disso, foi examinada a atividade citotóxica em modelo de macrófagos de camundongos. O pico de absorbância ocorreu em 533 nm e o diâmetro médio das nanopartículas foi de 14,54 nm. A nanoformulação apresentou boa estabilidade in vitro ao longo de mais de 6 meses e seu potencial zeta foi de -34,89 mV, sugerindo que o revestimento da EGCG previne a agregação. A análise de ICP-MS mostrou que a concentração de ouro reduzida com a síntese verde foi de 29,05 mg/L. Os dados obtidos pela espectroscopia do Infravermelho e Raman confirmaram a presença da EGCG nas nanopartículas de ouro. O coloide não apresentou efeito citotóxico para o modelo testado. A concentração inibitória referente a 50% de sobrevivência das células (IC50) para AuNPs-EGCG foi de 156,9 mg/L. Não foram utilizados produtos químicos, com exceção dos sais de ouro, neste processo verde de nanotecnologia biogênica, abrindo excelentes oportunidades para aplicações biomédicas.

L’hépatite C est un problème majeur de santé publique qui touche environ 160 millions de personnes dans le monde. L’agent étiologique responsable de cette maladie, le virus de l’hépatite C (HCV), est un petit virus enveloppé dont le génome est codé par un acide ribonucléique (ARN) simple brin de polarité positive. Actuellement, il n’existe aucun vaccin contre ce pathogène et les traitements utilisés sont insatisfaisants du fait de leur spécificité d’action limitée. Ainsi, afin d’établir une thérapie antivirale efficace évitant l’apparition et la sélection de mutants de résistance aux antiviraux, l’utilisation de plusieurs agents antiviraux ciblant directement la particule virale (direct acting antiviral agents ou DAAs) en combinaison est préconisée. C’est pourquoi la découverte de nouveaux DAAs à large spectre d’action ciblant diverses étapes du cycle viral infectieux est indispensable.Au cours de ma thèse, nous avons identifié un nouvel inhibiteur de l’entrée du HCV : l’épigallocatéchine-3-gallate (EGCG). Cette molécule, extraite du thé vert, inhibe l’infection des cellules par le HCV. Plus précisément, en utilisant des particules rétrovirales pseudotypées avec les glycoprotéines d’enveloppe E1 et E2 du HCV, nous avons démontré que cette catéchine naturelle, agit à une étape très précoce de l’entrée virale, indépendamment du génotype. De même, en nous servant du virus produit en culture cellulaire, nous avons montré que cette molécule agit directement sur la particule virale. Puis, par RT-PCR quantitative (quantitative real-time polymerase chain reaction), nous avons confirmé l’inhibition de la liaison du virus à la surface cellulaire, en présence d’EGCG. Par conséquent, nos travaux suggèrent que l’EGCG interagit avec la particule virale, probablement en se liant aux glycoprotéines d’enveloppe virales, bloquant ainsi une étape initiale d’attachement entre le virus et les facteurs cellulaires présents à la surface de l’hépatocyte. Puis, en inhibant la transmission libre du virus, à l’aide, soit d’agarose, soit d’anticorps neutralisants, nous avons démontré que l’EGCG inhibe la transmission du virus de cellule à cellule. Enfin, nous avons montré que l’EGCG élimine le virus présent dans le surnageant de culture cellulaire après quatre passages successifs sur des cellules naïves.La concentration d’EGCG nécessaire pour inhiber la moitié de l’infection virale (IC50) en culture cellulaire est 11 µM. Ainsi, afin d’identifier de nouvelles molécules présentant un mode d’action similaire à celui de l’EGCG et possédant une meilleure activité antivirale, nous avons sélectionnés différentes molécules naturelles et les avons testés pour leur potentiel effet anti-HCV. C’est ainsi que le chlorure de delphinidine, une anthocyanidine, a également été identifié en tant que nouvelle molécule inhibitrice de l’entrée du HCV. De même que l’EGCG, le chlorure de delphinidine cible directement la particule virale à une étape précoce de l’entrée, indépendamment du génotype, probablement en inhibant l’attachement du virus à la surface cellulaire et sans affecter ni l’étape de réplication, ni l’étape d’assemblage/maturation. De plus, le chlorure de delphinidine présente une activité anti-HCV améliorée avec une IC50 de 3 µM.Finalement, au cours de cette thèse, nous avons identifié deux nouvelles molécules naturelles inhibant l’étape d’entrée virale du HCV. Ces molécules pourraient être utilisées comme nouveau traitement en combinaison avec d’autres DAAs et pourraient également servir d’outil afin d’étudier les mécanismes d’entrée du HCV dans l’hépatocyte. / Hepatitis C is a major global health burden with 160 million infected individuals worldwide. This long-term disease, caused by a small positive-strand ribonucleic acid (RNA) enveloped virus, namely hepatitis C virus (HCV) evolves slowly. Nowadays, no vaccine is available and current treatments are unsatisfactory due to their restricted spectrum of action. For this reason, it is suggested that the combination of several drugs will prevent viral resistance and might conduct to an efficient antiviral therapy. Thus, the discovery of new direct acting antiviral agents (DAAs), with a broad spectrum of action, targeting different steps of the virus life cycle is still needed. Here, we identified (-)-epigallocatechin-3-gallate (EGCG) as a new inhibitor of HCV entry. Epigallocatechin-3-gallate, extracted from green tea, inhibits HCV infection. More precisely, this natural catechin molecule acts at a very early step of entry regardless of the genotype as illustrated with HCV pseudoparticles expressing HCV envelope glycoproteins E1 and E2 assays and cell-cultured HCV assays. Moreover, this molecule inhibits the docking of the virus to the cell surface as showed by the quantification of bound viruses during the attachment step using quantitative real-time polymerase chain reaction. Furthermore, EGCG inhibits viral cell-to-cell transmission as demonstrated by inhibiting cell-free transmission using agarose or neutralizing antibodies assays. Finally, EGCG clears HCV from cell culture supernatants after four passages.The half maximal inhibitory concentration (IC50) of EGCG in cell culture is approximately 11 µM. In order to identify new molecules exhibiting an enhanced anti-HCV activity and displaying similarities from EGCG scaffold, a series of natural compounds were selected and were tested for their anti-HCV activities. Thus, the anthocyanidin delphinidin chloride was identified as another inhibitor of HCV entry. Like EGCG, delphinidin chloride acts directly on the virus at a very early step of entry, regardless of the genotype, probably by inhibiting the docking of the virus to the cell surface without affecting replication or viral assembly/secretion. Finally, with an IC50 of 3 µM, delphinidin chloride displays a more potent anti-HCV activity.Together, these data indicate that EGCG and delphinidin chloride are new interesting anti-HCV molecules that inhibit entry and might be used as a new treatment in combination with other DAAs. Furthermore, these two inhibitors might be novel tools to further dissect the mechanisms of HCV entry into the hepatocyte.

No description available.

No description available.