Cancer is a group of diseases in which normal cells are converted to cells capable of autonomous growth and invasion. In the chemotherapeutic control of cancer, drugs are usually given systemically so they reach toxic levels in healthy cells as well as cancer cells. This causes serious side effects. Another important problem with chemotherapy is resistance developed to cytotoxic drugs (multi drug resistance). Doxorubicin (Dox) occupies a central position in the treatment of breast cancer. However doxorubicin induced cardiac toxicity is associated with a high incidence of morbidity and mortality. Resistance of malignant tumors to Dox is another important cause of treatment failure in patients with cancer. One approach to overcome Dox-related toxicity is to use polymeric drug carriers, which direct the Dox away from heart tissue, and allow usage of lower dosages. In this present study two different anti-cancer drug delivery methods were evaluated. Dox was encapsulated in PLGA microparticles by single and double microemulsion solvent evaporation techniques. The highest entrapment of doxorubicin within PLGA microspheres obtained by optimization of process parameters. A sustained release of doxorubicin was obtained for 20 days. Several protein transduction domains are known to have the ability to pass through biological membranes. One such peptide is HIV-1 TAT. In this study TAT was evaluated for its ability to carry Dox into Dox resistant MCF-7 tumor cells. Dox peptide conjugate was more potent than free drug. The concentration of drug in resistant cancer cells was increased indicating a partial reversal of drug resistance.

Orientador: Marlus Chorilli / Resumo: O diabetes mellitus (DM) é uma síndrome metabólica caracterizada por deficiência na produção/secreção pancreática de insulina e/ou resistência à ação do hormônio nos tecidos alvo, resultando em hiperglicemia. Diversas pesquisas têm desencadeado o desenvolvimento de novos sistemas de administração de insulina que possibilitem a utilização de vias alternativas à parenteral, com destaque à administração de insulina por via nasal. Esta via tem-se mostrado promissora, pois pode promover uma rápida absorção do fármaco e aumentar a sua biodisponibilidade. Entretanto, existem mecanismos de depuração mucociliar que limitam a administração de fármacos, além da baixa permeabilidade do epitélio nasal, o qual dificulta a absorção de fármacos com alto massa molar. Uma estratégia para vencer tais barreiras é a utilização de sistemas nanoestruturados (lipossomas), pois são amplamente utilizados para o aperfeiçoamento da potencialização da ação terapêutica de fármacos. Além disso estes lipossomas foram funcionalizados com peptídeos de transdução de membrana (CPPs), tais como os peptídeos TAT e Penetratin (PNT), que atuam como promotores da penetração e absorção do fármaco, com posterior dispersão em hidrogel de hidroxietilcelulose. O objetivo deste trabalho foi desenvolver e caracterizar lipossomas contendo solução de insulina, funcionalizados com CPPs (TAT e PNT) e dispersos em hidrogel, avaliar o potencial pela via nasal, in vivo, para a melhora dos níveis séricos e efeito hipoglicemiante d... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Bacterial stationary phase is an interesting biological system to study, as the organism undergoes several metabolic changes during this period and new molecules are generated to support its survival. The stationary phase of mycobacteria has been extensively studied since the discovery of Mycobacterium tuberculosis, the causative agent of tuberculosis. The stationary phase of mycobacteria adds further complication as many antibacterial drugs become less effective. The M. tuberculosis infects the alveolar macrophages and dendritic cells or monocytes recruited from peripheral blood. Macrophages are supposed to provide an initial barrier against the bacterial infection, but fails. Mycobacteria have evolved several strategies to survive and set up an initial residence within these cells and grow actively inside the host. The host immune system tries to limit the bacterial growth and confines the organism to a latent state in which the organism can persist indefinitely, known as granuloma stage. During latency or granuloma stage mycobacteria can retain the ability to resume the growth in the future. Mycobacteria must adapt to a highly dynamic and challenging environment because the interior environment of granuloma is devoid of or in low level of oxygen, depleted nutrient, high carbon dioxide, and possess increased levels of aliphatic organic acids and hydrolytic enzymes. The survival of a bacterium in less nutrient supply or in depleted oxygen is important for its long-¬term persistence inside the host under harsh environmental conditions. Mycobacterium smegmatis is the closest non-¬pathogenic homologue of M. tuberculosis, and has been used widely as a model system to study gene regulation under such conditions. In these harsh environmental conditions bacteria need to sense the external environment to modulate their gene expression. More importantly, each individual cell should communicate with its neighbours, and the response takes place in a concerted manner, which is termed as quorum sensing. Thus, the quorum sensing is a cell-¬cell signaling process that allow the bacteria to monitor the presence of other bacteria in their surroundings by producing and responding to small signaling molecules, which are known as autoinducers. It is a density dependent phenomenon and regulates the expression of the genes in response to fluctuation in cell¬-population density. A minimum threshold level of autoinducers is necessary to detect the signal and respond to it. Quorum sensing enables bacteria to behave like multicellular organisms and controls group activities like biofilm formation, sporulation, bioluminescence, virulence, and pigment production, etc (Bassler, 1999; Camilli & Bassler, 2006; Fuqua et al., 1996; Miller & Bassler, 2001). In Gram-¬negative bacteria, small-¬molecules, which are known as autoinducers are produced. They are acyl homoserine lactones (AHLs), which are derived from S¬adenosyl methionine (SAM) and particular fatty acyl carrier protein by LuxI¬type AHL synthases (Fuqua et al., 1996). In Gram-¬positive bacteria small peptides autoinducers, 5¬12 amino acids long, play an active role in communication. These oligopeptides are post--translationally modified by the incorporation of lactone and thiolactone rings, lanthionines and isoprenyl groups. These oligopeptide autoinducers are detected by membrane-¬bound two-¬component signaling proteins, and signal transduction occurs by a phosphorylation cascade (Camilli & Bassler, 2006; More et al., 1996; Novick, 2003; Zhang et al., 2002). In bacteria, the cyclic adenosine monophosphate (cAMP), and guanosine pentaphosphate and/or tetraphosphate ((p)ppGpp) are well known second messengers, which play important role in relaying extracellular information, but recently cyclic diguanosine monophosphate (c-¬di¬-GMP) is being studied most comprehensively as a nucleotide-¬based second messenger. C-¬di¬-GMP was first discovered in Gluconacetobacter xylinus as a positive allosteric regulator of cellulose synthase (Ross et al., 1987; Tal et al., 1998; Weinhouse et al., 1997). The in vivo level of c-¬di-¬GMP in bacterial cell is maintained by the balance between diguanylate cyclase and phosphodiesterase activities. The GGDEF and EAL amino acids sequence are the signature motif for GGDEF and EAL domain protein within its active site, respectively. The GGDEF domain protein is involved in synthesis of c-¬di-¬GMP and the EAL domain protein is involved in the hydrolysis of c-¬di-¬GMP, and the majority of these proteins contain additional signal input domains (Paul et al., 2004; Ross et al., 1987; Ryjenkov et al., 2005; Tal et al., 1998). M. smegmatis has a single bi-¬functional protein having both the domains, GGDEF and EAL, for the diguanylate cyclase (DGC) and phosphodiesterase (PDE¬A) activities. In addition to GGDEF and EAL domain, one sensory domain, GAF, is also there at the N-terminal of MSMEG_2196 in M. smegmatis. In the present investigation, studies have been carried out to understand the regulation of c-¬di-¬GMP in M. smegmatis at protein and gene level. The entire study on mycobacterial MSMEG_2196 (msdgc¬1) can be broadly divided into five parts; the first part will cover the identification and biochemical characterization of MSDGC¬1 protein, responsible for the regulation of in vivo c-¬di-¬GMP concentration in M. smegmatis, and the presence of GGDEF¬EAL domain containing proteins in various mycobacterial species. The second part will cover the structure function relationship as a function of substrate, GTP and product, c-¬di-GMP, molecule using fluorescence spectroscopy as a tool, and the mutational and structural studies, which leads to the identification of a novel structural motif. The third part will cover the characterization of msdgc¬1 gene knockout and complementation studies in great detail. The fourth part will comprise in vivo and in vitro promoter characterization and regulation of the msdgc¬1 gene under nutritional starvation. The last chapter will cover the characterization of novel synthetic glycolipids, which are working as a growth and biofilm inhibitors in mycobacteria, and can be used as a new drug candidates. Chapter 1 outlines the signal transduction and quorum sensing mechanism, and small molecule signaling modules in brief. The importance of the study started with a brief introduction about the historical aspect of tuberculosis, the current scenario of the treatment of tuberculosis. The urgent need for new drug targets and drugs will be discussed. The important role of the novel second messenger, c-¬di¬-GMP has been explained in greater details in both Gram-¬positive and Gram-¬negative bacteria, and the information available on the different cellular targets has been documented. Chapter 2 describes the identification and biochemical characterization of M. smegmatis MSMEG_2196 protein. The domain architecture and individual domain role have been studied. The MSMEG_2196 proteins consist of three domains, GAF, GGDEF and EAL in tandem, and individual role of each domain has been studied. The diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL domains have been identified as the enzymes involved in the regulation of in vivo cellular concentration of c-¬di-¬GMP. GAF domain has been identified as a metal binding domain in other bacteria and may be playing a role in the regulation of synthesis and hydrolysis activities of c-¬di¬-GMP. The identification, cloning expression and purification of MSMEG_2196 and MSMEG_2774 have been discussed. We have reported that mycobacterial MSDGC¬1 protein has dual activity, which means that it can synthesize and hydrolyse c¬-di-¬GMP; and also full-¬length protein is necessary for its either of the activities. The synthesis and hydrolysis products, c-¬di-¬GMP and pGpG, of MSDGC¬1 protein have been identified and characterized using radiolabelled alpha [α¬32P]GTP and Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI). The effects of temperature and pH on the activities of MSDGC¬1 have been studied. The circular dichroism studies show that the MSDGC¬1 protein is predominantly α¬helical in nature, and secondary structure does not alter upon GTP binding. The kinetic parameters for MSDGC¬1 protein have been calculated as a function of substrate, GTP. The protein, MSDGC¬1, exist as a monomer and a dimer in solution. The MSDGC¬1 protein has four cysteines, and we have shown here using mass spectrometric analysis that none of the cysteines is involved in the disulphide linkage. Chapter 3 deals with the structure-¬function relationship as a function of GTP and c¬-di-GMP molecules using fluorescence spectroscopy as a tool. In order to do so we have generated several cysteine mutants using site directed mutagenesis, and protein was labelled with thiol-¬specific fluorophores. The labelled protein was checked for its DGC and PDE¬A activities and specificity of labelling was confirmed using MALDI and radiometric analysis. The Fluorescence Resonance Energy Transfer (FRET) has been carried out to observe domain-¬domain interaction as a function of GTP and c¬-di-¬GMP. The bioinformatics, structural, and mutational analysis suggest that cysteine at 579 position is important for DGC and PDE¬A activities, and may be involved in the formation of a novel structural motif, GCXXXQGF, which is necessary for synthesis and degradation of c-¬di-¬GMP. Chapter 4 describes the construction of a deletion mutation of MSMEG_2196 gene in M. smegmatis. The strategy for the construction of the knockout strain has been shown and confirmation of the knockout event has been carried out using PCR and Southern hybridization. The effect of deletion of msdgc¬1 has been studied in great detail, and it was noticed that biofilm formation is not affected, but long-¬term survival is significantly compromised. It is hypothesized here that c-¬di¬-GMP is involved in the regulation of cell population density in mycobacteria. We have successfully detected the c-¬di¬-GMP in the total nucleotide extract using HLPC coupled with MALDI, and we have shown here that level of c-¬di-¬GMP increases many fold in the stationary phase of growth under nutritional starvation. Chapter 5 deals with the identification and characterization of the promoter element of msdgc¬1 in M. smegmatis. The study was undertaken to understand the mechanism of regulation at promoter level. We have observed here that msdgc¬1 promoter is starvation induced, and expression of msdgc¬1 increases many fold in the stationary phase under nutritional starvation. We have also tried to establish the link between the ppGpp and c-di¬-GMP signalling, and possible role of c-¬di-¬GMP in the regulation of cell population density have been discussed. Further, the +1 transcription start site has been identified using primer extension method. The putative ¬10 hexamer region for the RNA polymerase binding has been identified and confirmed using site-¬directed mutagenesis. It was found to be TCGATA, which is 14 bp upstream from the +1 transcription start site. The msdgc-1 promoter is specific for mycobacteria and does not function in E. coli. Moreover, we have identified the sigma factors, which regulate the msdgc¬1 promoter in growth phase dependent manner. Chapter 6 begins with the screening of synthetic glycolipids as a novel drug candidate. The different glycolipids have been tested for their effect on growth, biofilm formation, and sliding motility of M. smegmatis, and we have screened few of them, which were found to be effective in inhibiting the microbial growth, biofilm formation, and sliding motility. Chapter 7 summarizes the work presented in this thesis. Appendix: The protein sequences of MSDGC¬1 and MSDGC¬2, and the multiple sequence alignments of MSDGC¬1 protein have been documented. The FORTRAN program, which was used to calculate spectral overlap integral J, and the diagrams of the plasmids used in this study have been provided.

No description available.

Mucopolysaccharidosis IIIB (MPS IIIB) is a rare, metabolic disorder that results from a deficiency in the lysosomal hydrolase, α-N-acetylglucosaminidase (Naglu). Naglu is a housekeeping enzyme involved in the degradation pathway of heparan sulfate. A deficiency in active Naglu leads to an accumulation of heparan sulfate within the lysosome, initiating a pathological cascade within the cell. Patients with MPS IIIB experience progressive central nervous system degeneration and die within the first few decades of life. Presently, enzyme replacement therapy, which is a standard of care for other lysosomal storage disorders, is an ineffective treatment for MPS IIIB. This is due to impermeability of the blood-brain barrier (BBB) to exogenous recombinant enzymes. A promising approach to this therapeutic obstacle is protein transduction domains. Protein transduction domains have been shown to facilitate the delivery of active enzyme across the BBB in mice. Previously, our laboratory used Spodoptera frugiperda (Sf9) insect cell system to express human recombinant Naglu fused to a synthetic protein transduction domain (PTD4). The purpose was to use PTD4 to the facilitate the delivery of Naglu across biological membranes, including the blood-brain barrier. However, a missing stop codon following PTD4 limited its transducibility. The stop codon was re-introduced and the improved fusion enzyme, Naglu-PTD4X, was stably expressed in Sf9 cells. The overarching goal of this project is to create a large-scale production of human recombinant Naglu that has the potential to be used to treat the neuropathology of patients with MPS IIIB. This project used a three-step purification system to purify Naglu-PTD4X. Uptake of Naglu-PTD4X was assessed in MPS IIIB fibroblasts using a fluorogenic activity assay, immunoblotting, and immunocytochemistry. Our purification system was successful at purifying Naglu-PTD4X to homogeneity with a 26% yield and specific activity of 84,000 units/mg. An increase in Naglu activity was detected in MPS IIIB fibroblasts following incubation with Naglu-PTD4X. Future directions will focus on optimizing immunodetection and conducting BBB penetration studies in murine models. / Graduate / 2020-06-21

No description available.

La compréhension des mécanismes moléculaires de différentes pathologies a permis la caractérisation de gènes et de protéines impliqués dans la pathogénèse et l'identification de cibles thérapeutiques intracellulaires. La nature hydrophobique de la membrane cellulaire empêche le passage des médicaments dans les cellules. Les Cell-Penetrating Peptides (CPP) ou domaines de transduction protéiques (PTD) sont des peptides qui permettent l'internalisation de macromolécules hydrophiles in cellulo et in vivo. Un nouveau peptide issu du facteur de transcription ZEBRA du virus Epstein-Barr, et qui possède des propriétés de transduction a été caractérisé récemment dans notre laboratoire. Des études par mutagénèse de délétion de la protéine ZEBRA ont permis d'identifier la région d'acides aminés (nommé ainsi MD) impliquée dans la pénétration cellulaire. Ce peptide traverse les membranes des cellules de mammifères par un mécanisme de translocation directe, même lorsqu'il est fusionné à des molécules telles que la protéine reportrice eGFP. Le mécanisme de pénétration directe représente un grand avantage pour les applications thérapeutiques: les molécules cargos peuvent être internalisées directement dans le cytoplasme cellulaire sans dégradation et sous une forme biologiquement active. L'objectif de cette thèse est d'étudier les propriétés de pénétration cellulaire du peptide MD et d'évaluer ses applications thérapeutiques comme système de vectorisation des protéines. Ce travail est structuré en trois parties. La première partie porte sur l'étude de l'optimisation de la séquence peptidique MD par réduction de taille et l'évaluation du rôle de sa composition en acides aminés dans le processus de translocation à travers la membrane cellulaire. Cette étude a conduit à l'identification d'une séquence plus courte MD (MD11) possédant une efficacité et un mécanisme de translocation inchangés. La deuxième partie décrit une approche thérapeutique basée sur MD11 visant à la complémentation protéique d'un dysfonctionnement identifiée dans la plupart des cancers. Les cellules tumorales présentent des altérations dans la machinerie de traduction résultant dans une prolifération cellulaire incontrôlée. Parmi les différents facteurs intervenant dans la régulation de ce processus, le facteur eucaryote d'initiation 3 (eIF3) contribue à l'oncogenèse et au maintien de l'état cancéreux. Ce complexe est composé de 13 sous-unités, désignées eIF3 a-m. L'expression de certaines sous-unités est altérée dans plusieurs cancers, et en particulier la sous-unité f (eIF3f) est significativement diminuée dans le mélanome, les cancers du pancréas, de la vulve, du sein, de l'intestin et de l'ovaire. L'expression ectopique par transfection transitoire du gène eIF3f inhibe la synthèse protéique et induit l'apoptose dans le mélanome et dans les cellules cancéreuses pancréatiques. A partir de ces observations, nous avons développé une approche thérapeutique innovante pour le traitement des cancers dans lesquels la protéine manquante eIF3f est produite sous forme recombinante fusionnée à la séquence de MD11, et ensuite internalisée dans les cellules cibles tumorales. Ces résultats démontrent que le système de transfert de eIF3f basé sur MD11 représente une stratégie efficace pour supprimer la prolifération des cellules tumorales. La dernière partie de cette thèse explore la propriété de pénétration de MD11 dans les cellules de levure, et en particulier dans le champignon pathogène Candida albicans. Les résultats obtenus démontrent la polyvalence de MD11, qui fonctionne comme vecteur de protéines à activité biologique aussi bien dans la levure que dans les cellules de mammifères. Le potentiel de MD11 comme système de transport et de relargage des protéines a donc été établis, toutefois certaines améliorations en ce qui concerne la formulation des protéines de fusion et des études in vivo doivent être réalisées afin de valider son efficacité thérapeutique. / In recent years, the understanding of disease molecular mechanisms has led to the identification of genes and proteins that are altered in disease state and many therapeutic targets have been found located within cells. The protective and hydrophobic nature of plasma membrane prevents therapeutic drugs from entering cells. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have emerged as a group of non-invasive delivery vectors for various hydrophilic macromolecules, and several in vitro and in vivo applications as pharmaceutical carriers have been reported. A novel cell-penetrating peptide deriving from the Epstein-Barr virus ZEBRA transcription factor has been recently characterized in our laboratory. A reductionist study of full-length ZEBRA protein has allowed to identify the amino acid region (named as Minimal Domain, MD) implicated in cellular uptake. This peptide is able to cross the mammalian cell membranes via a direct translocation mechanism even when fused to cargo molecules such as eGFP reporter protein. The direct penetration mechanism represents a great advantage for therapeutic applications as the cargo molecules can be directly delivered into cells cytoplasm in a biological active form. The aim of this thesis is to explore the cell-penetrating properties of the MD peptide and evaluate its applications as therapeutic protein delivery system. This work is structured in three parts.The first part describes the study on the optimization of MD peptide sequence by size-reduction and the evaluation of its amino acid composition role in the translocation process across the cell membrane. This study has led to the identification of a shorter MD sequence (MD11) with unvaried mechanism of translocation. The second section describes a MD11-based therapeutic approach aiming at repair a dysfunction of the protein synthesis identified in most cancers. The regulation of the protein synthesis has a crucial role in governing the eukaryotic cell growth and subtle defects in the translational machinery can alter the cellular physiology and lead to cell malignancy. Among the different factors intervening in the regulation of this process, the eukaryotic initiation factor 3 (eIF3) contributes to oncogenesis and maintenance of the cancer state. This complex is composed of 13 subunits (designated eIF3 a-m). The expression of eIF3 subunits is altered in several cancers, and in particular the f subunit (eIF3f) is significantly down-regulated in pancreas, vulva, breast, melanoma, ovary and small intestine tumors. The eIF3f ectopic expression by transient gene transfection inhibits cellular protein synthesis and induces apoptosis in melanoma and pancreatic cancer cells. Starting from these observations, we developed an innovative therapeutic approach for cancer treatment in which the missing eIF3f protein is produced in vitro in fusion to MD11, and delivered to cells. These results have demonstrated that the MD11- based eIF3f transfer system may represent a powerful strategy to suppress the tumor-cell proliferation. The last part of this thesis explores the cell-penetrating property of MD11 in yeast cells, and in particular in the pathogenic fungus Candida albicans. The presented results demonstrate the versatility of MD11, functioning as vectors in both yeast and mammalian cells and as carrier for proteins with biological activity.The MD11 potential as protein delivery system is evident; however some improvements regarding the fusion protein formulation and in vivo studies should be realized to validate the effectiveness of its therapeutic application.

Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB. / Graduate