Nanoparticles were synthesized and modified for target drug delivery. The research involved the aqueous synthesis of near infrared (NIR) sensitive Au-Au₂S nanoparticles. An anti-cancer drug (<i>cis-platin</i>) was subsequently adsorbed onto the Au-Au₂S nanoparticle surface via the 11-mercaptoundecanoic acid layers. The results showed that the degree of adsorption of cis-platin onto Au-Au₂S nanoparticles was controlled by the pH value of solution, and the rate of drug release was sensitive to NIR irradiation. The results of the synthesis, drug-release properties and nanoparticle-cell interactions will be discussed. / Singapore-MIT Alliance (SMA)

No description available.

The statin family of hydroxymethylglutaryl coenzyme A reductase (HMGCR) inhibitors, used to control hypercholesterolemia, triggers apoptosis of various human tumour cells. HMGCR is the rate-limiting enzyme of the mevalonate (MVA) pathway, a fundamental metabolic pathway required for the generation of a number of biochemical end-products including cholesterol and isoprenoids, but the contribution of the MVA pathway to human cancer remains largely unexplored. Furthermore, as only a subset of tumour cells has been shown to be highly responsive to statins, the identification of appropriate subsets of patients will be required to successfully advance these agents as anticancer therapeutics. To this end, there were two major aims to this work: 1) Elucidate a molecular rationale for the observed therapeutic index of statin-induced apoptosis in normal and tumour cells; 2) Identify molecular determinants of sensitivity for statin-induced apoptosis in human tumour cells. To address the first aim we demonstrated that dysregulation of the MVA pathway, achieved by ectopic expression of either full length HMGCR (HMGCR-FL) or its novel splice variant lacking exon 13 (HMGCR-D13), increases transformation. Ectopic HMGCR promotes growth of transformed and non-transformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice. We also show that high mRNA levels of HMGCR and four out of five other MVA pathway genes correlate with poor prognosis in primary breast cancer, suggesting the MVA pathway may play a role in the etiology of human cancers. To address the second aim, we show that dysregulation of the MVA pathway is a key determinant of sensitivity to statin-induced apoptosis in multiple myeloma. In a panel of 17 distinct myeloma cell lines, half were sensitive to statin-induced apoptosis and the remainder were insensitive. Interestingly, in sensitive cells, the classic feedback response to statin exposure is lost, a feature we demonstrated could distinguish a subset of statin-sensitive primary myeloma cells. We further illustrated that statins are highly effective and well tolerated in an orthotopic model of myeloma using cells harboring a dysregulated MVA pathway. Taken together, this work provides a molecular rationale and determinants of sensitivity for statin-induced apoptosis of human tumour cells.

This study aimed to investigate the pharmacology of selected pyrrolobenzodiazepine (PBD) compounds shown to have cytotoxic activity with predicted DNA sequence selectivity. Research focused upon the PBD dimer, SJG-136, selected for clinical trials, and the novel PBD monomer compounds D709119, MMY-SJG and SJG-303. SJG-136, a novel sequence-selective DNA minor groove cross-linking agent, was shown to have potent tumour cell type selective cytotoxicity in in vitro assays. Pharmacokinetic studies in mice via both the i.p. and i.v. route (dosed at the maximum tolerated dose (MTD)) showed that SJG-136 reaches concentrations in plasma well in excess of the in vitro IC50 values for 1 h exposure, and was detected in tumour and brain samples also above the in vitro IC50 values. Furthermore, SJG-136 showed linear pharmacokinetics over a 3-fold drug dose range. Metabolism studies showed SJG-136 is readily metabolised in vitro by hepatic microsomes, predominantly to a monodemethylated metabolite; this metabolite could be detected in vivo. Analytical method development work was also conducted for the imminent Phase I clinical trial of SJG-136 resulting in a sensitive and selective bio-analytical detection protocol. Comet analysis showed that SJG-136 dosed at the MTD and ⅓MTD causes significant interstrand DNA cross-linking in lymphocytes in vivo. In vitro studies demonstrated that SJG-136 localises within the cell nucleus, and acts to disrupt cell division via a G2/M block in the cell cycle at realistic concentrations and exposure times that are achievable in vivo. In vivo pharmacokinetic studies of D709119 showed the compound is easily detectable in mouse plasma following i.p. dosing at the MTD, but could not be detected in either tumour or brain samples. In vitro cytotoxicity studies revealed D709119 to have potent activity across a selection of tumour cell lines. SJG-136, D709119, MMY-SJG, SJG-303 and DC-81 demonstrated a non-enzyme-catalysed reactivity with the biologically relevant thiol, reduced glutathione (GSH). Studies demonstrated that reactivity of the PBD compounds toward GSH was dependent on GSH concentrations. At levels of GSH found in plasma, the PBD compounds showed considerably lower reactivity with GSH than at intracellular GSH levels. SJG-136 and D709119 also showed favourable pharmacokinetic profiles in mice, and warrant further study for anti-tumour activity in vivo and progression to use in patients.

Le gène Gec1/Gabarapl1 a été identifié au sein de notre laboratoire comme un gène régulé par les estrogènes. Il appartient à la famille Gabarap incluant les gènes Gabarap, gabarap/2 et Gabarapl3 qui codent des protéines présentant de fortes homologies de séquences. L'étude fonctionnelle de Gabarapl 1 a montré que cette protéine est impliquée dans le transport des récepteurs et particulièrement les récepteurs Gabaₐ et des κ-opioïdes via son interaction avec la tubuline et la protéine NSF. Cependant, il a été décrit que certaines protéines de la famille Atg8 sont impliquées dans l' autophagie, un mécanisme de dégradation et de survie cellulaire, qui se caractérise par la formation de doubles membranes appelées autophagosomes. Les objectifs de mon travail étaient, d'une part, de caractériser le rôle de la protéine GABARAPL1 au cours de !'autophagie et, d'autre part, de caractériser son expression dans des lignées et tissus cancéreux et sa régulation en réponse à des composés anti-cancéreux. Tout d'abord, nous avons montré que Gabarapl1 est clivée par la protéase Atg4B au niveau de sa glycine 116 avant sa conjugaison à des phopholipides. Cette forme modifiée, lipidée, est localisée à la surface des autophagosomes et des lysosomes. Nous avons ensuite montré que Gabarapl1 est faiblement exprimée dans de nombreuses lignées cancéreuses, que son expression est altérée dans les méningiomes et qu'elle est régulée par des inhibiteurs du protéasome. Ces travaux ont montré, pour la première fois, que la protéine Gabarapl1 est associée à des vésicules autophagiques et permettront de poser les hypothèses de nos futurs travaux. / The Gec1 / Gabarapl1 gene was identified in our laboratory as an early estrogen regulated gene. Gabarapl1 belongs to the Gabarap family, also including Gabarap, Gabarapl2 and Gabarapl3 genes, that encode proteins which present high sequence homology with each other. A functional study of the Gabarapl 1 protein showed that this protein is involved in the transport of receptors such as the Gabaₐ and κ-opioid receptors via its interaction with tubulin and NSF. It has been reported that the Atg8 family proteins are involved in autophagy, a mechanism of degradation and cell survival that is charactenzed by the formation of double membranes called autophagosomes. The aims of my research were, firstly, to characterize the role of the Gabarapl1 protein during autophagy and, secondly, to study its expression in cancer cell lines and cancerous tissues and its regulation in response to anti-cancer drugs. First, we showed that Gabarapl1 is cleaved in the cells by the protease Atg4B at its 116 glycine residue prior to its conjugation to phospholipids. This modified form, lipidated, is located on the surface of autophagosomes and lysosomes. We then showed that Gabarapl1 expression is reduced in many cancer cell lines, and that its expression is also altered in meningiomas. Finally, we showed that Gabarapl1 expression is regulated by proteasom€: inhibitors. Thus, our results demonstrated for the first time that the Gabarapl1 protein is associatec with autophagie vesicles and allow us to propose hypothesis for future work

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-16T19:48:44Z No. of bitstreams: 2 Dissertacao_LuizHenriqueRauberRodrigues.pdf: 17209406 bytes, checksum: f1f5406222de4f0c348643f546a9a971 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-16T19:48:44Z (GMT). No. of bitstreams: 2 Dissertacao_LuizHenriqueRauberRodrigues.pdf: 17209406 bytes, checksum: f1f5406222de4f0c348643f546a9a971 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-03-30 / Dysplasia such as cancer can be identified by expression patterns involving mechanisms genome maintenance pathways (GMM). Activation pathways involved in the cell cycle (CC), in the DNA damage response (DDR) and apoptosis (APO), significantly contribute to tumor development. In previous studies, it was found that the precancerous activation process there is an anticancer barrier which is responsible for prevention of tumor progression. The identification of more expressed genes during activation of the anti-cancer barrier, with interactions associated GMM, is a complementary study of the way to evolution of cancer. In this work, the objective was investigate the anti-cancer barrier activation in pre-cancer and cancer, in tissues of adrenal gland, colon, pancreas and thyroid follicles, using networks of interaction between proteins. To describe this barrier was proposed modeling the interaction networks between proteins GMM routes with Cytoscape software. The results obtained with the most important genes in expression and quantity of interactions were compared with the results of previous publications and reconfirmed the relevance of genes CDKN1A, CHEK1, ATR, P53, MRE11A, BRCA1 and XRCC4. Through analysis allowed the identification of other genes, complementary to previous studies, as SKP2, CCNO, FADD, RAD50, NBN, BIRC3, CDK2 and XRCC6. These genes are associated with and complement activation studies of anti-cancer barrier. These considerations emphasize that it is important to observe all systemic biological context, soaking, as in nanoscience where the study makes sense to take into account the interactions. Analyses of interactions enable the development of future work, for example, treatment with drugs nanocapsules, activating or inhibitory acting proteins such as interlocking routes GMM, or nanosensors to monitor the development of cancer. / Displasias como o câncer podem ser identificadas por padrões de expressão envolvendo vias de mecanismos de manutenção do genoma (GMM). A ativação de vias GMM envolvidas em ciclo celular (CC), resposta ao dano no DNA (DDR) e apoptose (APO) contribuem significativamente para o desenvolvimento tumoral. Em estudos anteriores, verificou-se que em processos pré-cancerosos há ativação de uma barreira anti-câncer que é responsável pela prevenção da progressão tumoral. A identificação dos genes mais expressos durante a ativação da barreira anti-câncer, associadas as interações nas vias GMM, tornam-se uma complementariedade ao estudo da evolução do câncer. Neste trabalho, o objetivo foi investigar a ativação da barreira anti-câncer, em pré-câncer e em câncer, presentes em tecidos da glândula adrenal, cólon, pâncreas e folículos da tireoide, usando redes de interação entre proteínas. Para descrever esta barreira foi proposta a modelagem das redes de interação entre as proteínas das vias GMM usando o software Cytoscape. Os resultados obtidos com os genes mais destacados em expressão e quantidade de interações foram comparados com os resultados de publicações anteriores e reconfirmaram a relevância dos genes CDKN1A, CHEK1, ATR, TP53, MRE11A, XRCC4 e BRCA1. A análise por vias permitiu a identificação de outros genes complementares aos trabalhos anteriores como os genes SKP2, CCNO, FADD, RAD50, NBN, BIRC3, CDK2 e XRCC6. Estes genes estão associados e complementam os estudos sobre a ativação da barreira anti-câncer. Estas considerações realçam que é importante observar todo o contexto biológico sistêmico, imersivo, assim como ocorre na nanociência, onde o estudo tem sentido se levar em consideração as interações. As análises sobre as interações permitirão o desenvolvimento de trabalhos futuros, por exemplo, tratamentos com fármacos nanoencapsulados, atuando de forma ativadora ou inibidora de proteínas interconectadas nestas vias GMM, ou nanosensores, para o acompanhamento da evolução do câncer.

Natural products are bioactive secondary metabolites produced by living organisms and are prevalently utilized as pharmaceutical drugs. Marine adapted organisms are a promising source of new natural products possessing unique chemical structures and biological activities. By studying the biosynthetic pathways employed by living organisms to produce natural products, insights into new strategies to generate molecules to combat disease and overcome drug resistance may be gained. This thesis study aimed to uncover the biosynthetic pathway employed by a marine actinomycete, Nocardiopsis sp. CMB-M0232, to catalyze the assembly of the nocardioazines. These molecules are a group of 2,5-diketopiperazine natural products that feature structurally unique functional groups. Nocardioazine A, the hypothesized end product of the nocardioazine biosynthetic pathway, exhibits anticancer activity. Bioinformatics analyses revealed three biosynthetic gene clusters from Nocardiopsis encoding proteins with hypothesized roles in nocardioazine A biosynthesis. Two cyclodipeptide synthases (CDPSs), NozA and NcdA, were biochemically characterized in vivo and in vitro to reveal that both are substrate specific enzymes that utilize tryptophan-charged tRNA substrates to catalyze assembly of cyclo(L-Trp-L-Trp), a proposed precursor of nocardioazines. Fidelity is uncommon amongst characterized CDPSs, making NozA and NcdA important CDPS family additions. This study also aimed to characterize NozD and NozE, two cytochrome P450 homologs with predicted roles as diketopiperazine-tailoring enzymes. Heterologous expression of these enzymes in Streptomyces strains was not able to confirm the functions of NozD and NozE but set the stage for future studies to optimize conditions for probing their roles in nocardioazine A biosynthesis. The results gathered from this study, along with future work to better understand the engineering of unique functional groups from Nocardiopsis may provide opportunities to produce new bioactive molecules.

No description available.

No description available.

En dépit d’importants progrès dans le domaine de la thérapie anti-cancéreuse, les traitements actuels restent controversés, notamment en raison de la quantité importante d'effets secondaires induits. L'immunothérapie ciblée a récemment émergée en tant qu'alternative, afin d'améliorer les modalités de traitement des patients atteints du cancer. Malgré tout, seul un nombre limité d’approches sont aujourd’hui disponibles, et une grande partie des problèmes demeurent actuellement sans solution. C'est dans ce contexte que nous nous sommes intéressés à la conception de structures biomoléculaires innovantes et bifonctionnelles, capables de rediriger des anticorps endogènes, présents naturellement dans la circulation sanguine de l'homme, contre les tumeurs et, ce, sans immunisation préalable. Les anticorps naturels circulant étant polyspécifiques et ayant la capacité d’interagir avec des antigènes glycosylés, nous nous sommes plus particulièrement concentrés sur la conception de glycoconjugués multivalents, ligands d’anticorps endogènes. Une première partie de notre étude a consisté à synthétiser différents glycodendrimères multivalents, reposant sur des châssis peptidiques et obtenus par ligations chimiosélectives, tout en variant la nature du motif glycosylé et des plateformes, ainsi que la valence du conjugué. Puis, dans un second temps, des tests d’interaction par biopuce ont été mis en place avec une lectine modèle, la lectine Helix Pomatia Agglutinin (HPA). Des protocoles expérimentaux visant à calculer des constantes de dissociation de surface, ainsi que des IC50 ont été mis en place, permettant d’identifier de bons ligands de HPA avec des affinités de l’ordre du nanomolaire. Les tests par biopuce ont ensuite été confirmés avec d’autres méthodes d’analyses (BLI, ELLA). Finalement, afin d'identifier des architectures tri-dimensionnelles permettant une affinité optimale avec des anticorps, les tests d’interaction ont été adaptés au criblage de séra humains. Un large panel de glycoconjugués a alors été criblé par biopuce avec une vingtaine de séra, permettant la détermination de structures glycosylés prometteuses, qui pourront par la suite être utilisées dans le cadre de notre approche anti-cancéreuse. / Despite significant progress in anti-cancer therapy, current treatments are still controversial due to numerous side effects. Targeted immunotherapy recently emerged as an ideal alternative to improve treatment modalities for cancer patients. However, very limited approaches are available today and major issues remain to be addressed. In this context, we are interested in the design of biomolecular structures, innovative and bifunctional, able to hijack endogenous antibodies - which are naturally present in the human blood stream - toward cancer cells without pre-immunisation. Since natural circulating antibodies are polyspecific and have the ability to interact with multiple carbohydrate antigens, we focused on the design of multivalent glycodendrimers, as ligands for endogenous antibodies. The first part of our study consisted in synthesizing several multivalent glycoconjugates, based on peptide scaffolds and obtained by chemoselective ligations. To evaluate their influence on antibodies, the nature of both the carbohydrate and the scaffold, and the valency were varied. Then, in a second part of the study, microarray assays were developed with a model lectin, the Helix Pomatia Agglutinin (HPA). Experimental procedures were designed to determine surface dissociation constant and IC50 values, leading to the identification of high affinity ligands for HPA in the nanomolar range. Microarray assays were confirmed by other analytical methods (BLI, ELLA). Finally, the assays on slides were adapted to human sera screening, in order to identify tridimensional architectures highly affine to sera antibodies. A large panel of glycoconjugates were screened by microarray with around twenty sera, leading to the determination of promising glycosylated structures, as antibody ligands. The latter could be subsequently used for our anti-cancer approach.