Rozprawy doktorskie na temat „Immunosuppressive agents Mechanism of action”

Bibliography: leaves 234-257. Three separate clinical studies in organ transplant recipients are presented. The aims are to examine fundamental questions regarding the clinically and economically important pharmokinetic interaction between diltiazem and cyclosporin, an interaction widely utilised in organ transplantation. The data contained should assist the development of soundly based policies that will ensure a benefit exists before a sparing agent is coprescribed, and that the lowest effective dose of sparing agent is used.

Thesis (Ph. D.)--Ohio State University, 2005.<br>Title from first page of PDF file. Document formatted into pages; contains xiii, 127 p.; also includes graphics (some col.) Includes bibliographical references (p. 113-127). Available online via OhioLINK's ETD Center

The Best M.Phil Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize, 2001-2003.<br>published_or_final_version<br>abstract<br>toc<br>Medicine<br>Master<br>Master of Philosophy

Both acquired and intrinsic drug resistance are established clinical problems in many areas of medicine. This is particularly evident with the growing resistance to platinum chemotherapy agents in cancer treatment programmes. New, alternative treatments for platinum-resistance patients are needed, with comparable potency, no platinum cross- resistance, better safety profiles and which target non-repairable areas of the cell, reducing acquired resistance. This thesis focuses on osmium- and iridium-based organometallic anticancer agents to fill this clinical need. Previous work has validated their potency, safety and activity in platinum-resistant cancers, however, their mechanism of action (MOA) was yet to be identified. Knowing the MOA of new compounds is essential for personalising and stratifying cancer treatment, allowing for better patient selection and prediction of treatment outcomes. Often identifying the biological target of a new therapeutic is not essential. Instead, quantifying the cellular response to that treatment, and identifying cell types which hold beneficial biological properties to optimise compound effects, is more effective. This thesis has applied the principles of systems biology to study the whole cell effect of osmium and iridium compounds in epithelial ovarian cancer. Cells were studied at the transcriptional, translational and structural level to investigate compound response, integrating a selection of these findings using novel statistical modelling. Results propose that these compounds induce oxidative stress in cancer cells, and subsequently damage DNA to exert antiproliferative effects at submicromolar concentrations. This is the first example of studying organometallic compounds using this combination of techniques, and is a promising work flow for future efforts in this area.

Over the past 200 years, much attention has been paid to natural products for their great contribution in the industry of drug development as many of them have been shown effective against various diseased conditions in humans by virtue of their structural diversity and biological potency. Therefore, they are undeniably a rich resource for the discovery of novel bioactive compounds. To date, many of the mainstay anticancer agents often lead to undesirable side effects and/or develop rapid emergence of drug resistance. Therefore, new therapeutic remedies are desperately needed. In fact, many active compounds are derived from macrocyclic natural products. The identification of their molecular targets may assist researchers to synthesize biological agents for combating particular diseased conditions. Cycloheptapeptides that modulate specific molecular pathways in suppressing the proliferation of cancer cells are potential candidates for anticancer therapeutics and/or chemopreventive agents. In the current research project, we have demonstrated that MV-A, a novel cycloheptapeptide with the unique amino acid DMCPA isolated from Maytenus variabilis (Loes.) C. Y. Cheng (Celastraceae), showed potent cytotoxic activities against a panel of human cancer cell lines, and is worthy for further investigation. Objectives--The objectives of this study were to i) evaluate the anticancer effect, ii) elucidate the mechanism of action, and iii) identify the binding target(s) of the natural cycloheptapeptide MV-A. Methods--We first carried out various kinds of cellular and animal studies for validating the in vitro and in vivo anticancer efficacy of MV-A. Next, we performed a number of bioassays to ascertain the inhibitory effect of MV-A on several major cancer-associated pathways, including apoptosis, cell cycle arrest, senescence and metastasis. The biochemical assays included sulforhodamine B colorimetric assay, flow cytometric analyses of apoptosis and cell cycle arrest, Western blotting, real-time polymerase chain reactions (qPCR) arrays, senescence-associated β-galactosidase staining, phospho-specific protein arrays, as well as migration and invasion staining experiments. Lastly, we also identified the potential protein targets of MV-A by biochemical means, particularly the drug affinity responsive target stability (DARTS) approach. Results--MV-A is a potent anti-proliferative agent against a variety of cancer cells. It inhibited the proliferation of the human colorectal carcinoma (CRC) HCT116 cells with an IC50 value of 2.28 nM. However, the application of MV-A at 2.68 nM did not induce significant apoptosis; rather it caused a notable cell-cycle arrest at the G1 phase. Moreover, the treatment with this compound (0.68 to 2.68 nM) led to a remarkable senescence in cancer cells as well as a mitigated cellular migration. Meanwhile, the expression levels of some components of the p16 cascade and PI3K-AKT pathway, so as several epithelial-to-mesenchymal transition (EMT) molecules were suppressed by MV-A. Furthermore, HSP90, calnexin, EF2, 14-3-3 and annexin A1 were identified as the direct binding targets of MV-A in our DARTS analysis.Conclusions--In the present study, our results indicated that the novel cycloheptapeptide MV-A inhibited proliferation and migration of CRC HCT116 cells via the induction of cellular senescence and modulation of multiple pathways, including the p16/Rb, PI3K-AKT and EMT signaling pathways. These results revealed a potential role of MV-A in cancer therapy. The direct binding targets of MV-A further uncovered its molecular actions against different diseased conditions. Our findings strongly support the development of MV-A as a therapeutic agent for combating cancerous pathologies, explicitly CRC.

Application of chemotherapeutic agents in current cancer treatment has been limited by adverse effects as poor selectivity results in systemic toxicity; most chemotherapy approaches also experience inherited or acquired drug resistance which lead to reduced treatment outcome. Research efforts have focused on the discovery of novel chemotherapies that overcome the limitations mentioned above. Ru(II) polypyridyl complexes with anti-cancer properties have been extensively studied as traditional cytotoxic agents and photodynamic therapy agents due to their photophysical and photochemical characteristics. Most research has focused on the design of Ru(II) polypyridyl complexes that have affinities to nucleic acids as inspired by the classic small molecule metal complex cisplatin. Though modifying the structures of ligands on the ruthenium metal center, the hydrophilicity, charge state and photochemical properties can be tuned, resulting to Ru(II) polypyridyl complexes that act through cellular targets other than DNA. Understanding the mechanism of action and identifying functional targets remain the challenging and complex research topic in the design and study of novel medication or candidates. With the development of semi-high throughput cytological profiling in a bacterial system, rapid investigation of the mechanism of action can be achieved to distinguish anti-cancer agents which possess different mechanisms of actions. Ru(II) polypyridyl complexes with different scaffolds have been studied and suggested to have anti-cancer properties through DNA damage response, and/or translational inhibition.

The search for more effective treatment strategies in melanoma led to many new innovative approaches aiming at different molecular targets. Chemotherapy still remains the most effective treatment and many efforts are put in order to improve targeting and delivery of the chemotherapeutic agents. Among these, peptide conjugates of anticancer drugs were designed to increase stability, cell penetration, specificity and accumulation in cancer cells. We as well as others evaluated such a conjugate, termed PSF (L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine-ethylester) in terms of its cytotoxicity in vitro and in vivo using a human melanoma tumor as a model, its stability, transport, and metabolisation. <p>By comparing the cytotoxicity of PSF and melphalan towards different cancer primary melanoma cell cultures, we noticed some interesting observations: PSF displayed the same toxicity pattern both in short (2h) and long term (24h) cell exposures whereas melphalan and m-sarcolysin needed long term exposure to reach the same toxicity. This could indicate that PSF very quickly penetrates the cells in accordance with what has been shown with red blood cells (RBCs). PSF has shown a much better and quicker penetration into the cells in vitro as compared to melphalan. <p>In this present work, the cytotoxic effect of PSF was further evaluated in vivo using a standardized nude mice tumor model bearing a human melanoma. First, the acute toxicity in rats and mice and the maximum tolerated dose were determined. After a dose-escalation study one dose was singled out and tested as a single dose and as a fractionated dose. PSF was able to reach the tumor site and a dose-response relationship was observed. The IP administration of fractionated doses of PSF had significantly better effect on tumor growth inhibition, regression and regrowth than single dose administration and this without any evidence for general toxicity monitored by animal weight loss. We also compared the efficacy of PSF to its parent drug m-sarcolysin, melphalan and cyclophosphamide and observed that PSF was much more active than both melphalan and m-sarcolysin at the same molar doses.<p>Body distribution of the 14C-labelled PSF revealed ratios of 2.4 and 1.5 compared to muscle tissue for the two melanoma tumors evaluated with no significant and stable accumulation in any vital organ. The amount of tracer was still high in the blood after 24 hours explaining the high radioactivity in the kidney and partly in the liver. Interestingly, the spleen had an unusual high radioactivity uptake reflecting the exceptional binding of the tracer to blood cells (BC), while the pancreas very high load was an indicator of protease-mediated specific delivery and strongly support our hypothesis elaborated on the basis of in vitro results. <p><p>Our in vitro data point to a particular mechanism of action of PSF based on the transport of PSF through the body by the rapid binding to blood cells and the delivery at the tumor site by the subsequent release of its active metabolites due to cleavage by tumor-associated proteases.<p>Concerning the binding of PSF to membranes and its transport the following observations were made: while PSF was stable in human plasma, it disappeared very quickly in whole blood along with the generation of a main metabolite: m-sarcolysin. The presence of BC membranes was required for both binding and generating the metabolites. Binding to natural or artificial membranes was achieved and only competition with melanoma cells or proteolytic enzymes such as dispase, led to the generation of active metabolites. The different metabolites were isolated using preparative LC and were then identified using Electrospray Ionisation Mass Spectrometry (ESI). Three metabolites, of which m-sarcolysin was the main one, were identified all bearing the chloroethyl alkylating group. <p>Enzymatic catalysis was further supported by a set of experiments where the enzymatic activity was non-specifically and specifically inhibited. In order to look at the effect of extracellular matrix proteases on PSF, three representatives of ECM proteases were incubated with PSF: collagenase A had no effect, but both dispase and trypsine were able to process PSF. <p>The following data indicate the higher processing of PSF in the presence of cells with a higher proteolytic activity and thus the delivery of the blood cell-bound PSF. When comparing BC with melanoma cells (MC), the latter showed a higher ability to bind and process PSF both by membrane-associated and most interestingly soluble proteases. A lot of families of enzymes are reported to be overexpressed by melanoma cells including: metalloproteases, cysteine cathepsins, serine proteases and aminopeptidases. All the melanoma cells and cell lines evaluated were able to generate PSF active metabolites. <p>To identify the families of enzymes expressed on the membrane of melanoma cells that might be involved in the mechanism of action of PSF, we performed 2D-gel electrophoresis on their membrane extracts. The 2D-gels experiments revealed the presence of proteins compatible with enzymes known to be important in melanoma and further work is needed to identify the individual enzymes involved by using mass spectrometry and Western blotting. <p><p>Both our in vitro and in vivo findings strongly suggest that not only melanoma tumor cells and tumor sites but other types of tumors as well may be targets for the toxic activity of PSF owing to their much higher load in proteolytic enzymes that are closely related to their invasive potential. The transport of PSF by the blood cells and the release of its metabolites at the tumor site result in a low amount of drug in its free soluble form within the blood and this may explain the relatively lower side-effects observed. PSF is thus expected to have a much better therapeutic index than conventional alkylating agents. This original mechanism of drug delivery may well be extended to other cancer and non-cancer drugs than alkylating agents.<p><br>Doctorat en Sciences biomédicales et pharmaceutiques<br>info:eu-repo/semantics/nonPublished

Adverse drug reactions (ADR) are a major concern for the pharmaceutical industry and health practitioners as they can cause morbidity and in severe cases mortality. ADRs are one of the major reasons why drugs fail during clinical trials so research directed at predicting ADRs to minimise failure is essential. The CDDO (2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oate) and the synthetic endoperoxide series are two promising classes that have potential for the treatment of cancers and malaria and may revolutionise treatment, within their fields, if approved for clinical use. The two main aims that are presented in this thesis are to; (i) design and synthesise novel analogues and chemical probes to identify potential molecular targets for both the CDDO and endoperoxide series (ii) develop appropriate in vitro test systems to help define the molecular mechanism of each class of drug. CDDO-Me (methyl 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oate) is one of the most potent inducers of Nrf2, a transcription factor that regulates the expression of numerous cell defence genes in mammalian cells. Nrf2 is sequestered in the cytosol by Keap1, which ‘senses’ chemical and oxidative stress via its 27 cysteine residues. Although CDDO-Me is one of the most potent inducers of Nrf2, the molecular target and chemical mechanism is still not defined. Current literature suggests that a reversible 1,4 conjugate addition to specific cysteine residue(s) located on the Keap1 protein results in an increase in Nrf2 levels. In order for SAR work to be performed a synthetic route to CDDO and analogues was developed which involved nine steps using oleanolic acid as starting material. Highlights of the chemistry included addition of the ketone using mCPBA and incorporation of the cyano group in steps 3 and 7 of the synthesis. In addition to preparing the target molecule CDDO a number of additional molecules were prepared to define the importance of functional groups in the A and C rings of CDDO. Genetically modified H4IIE rat hepatoma cells transiently transfected with the an Nrf2-sensitive luciferase reporter gene were used to screen the CDDO-Me analogues, including DDO-Me which lacks a cyano group on the A ring, for their ability to induce Nrf2. NMR studies with model thiols were performed to determine the ability of these compounds to form reversible or non-reversible adducts. Mass spectrometry (MS) was used to confirm the NMR data and interpretations. In total, four probes were identified that reacted in a non-reversible fashion: DDO-Me, DDO-Al and DDO-Az (click probe versions of the parent DDO-Me that can be used to facilitate proteomic studies) and CDDO-Epox (a probe with similar overall structure to CDDO-Me but can react at the β-carbon in a non-reversible fashion; this feature should aid proteomic approaches to reactive cysteine residue identification). To further investigate if these compounds were reactive to cysteine residues within a model protein, recombinant human GSTP1 was used as a model protein for chemically reactive molecules. Cys-47 located on GSTP1 has been shown to react with other electrophones and during our studies LCMS has confirmed that all four of the synthesised active probes were capable of attaching covalently to Cys-47 of GSTP1. The emergence of malaria parasite resistance to most available drugs, including the semi-synthetic artemisinin derivatives artemether and artesunate, has led to efforts to create new synthetic peroxides as potential antimalarial agents. Leading examples of synthetic endoperoxides include OZ277 (arterolane), a molecule in phase III clinical trials in combination with piperaquine, and OZ439, a second generation derivative with improved pharmacokinetics and enhanced in vivo antimalarial activity. 1,2,4,5-Tetraoxanes are another class of endoperoxide with proven excellent antimalarial profiles against both chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum and oral activity in murine models of the disease. It is currently widely accepted that endoperoxides have a similar antimalarial mechanism to artemisinin, whereby Fe2+ medicated generation of cytotoxic carbon-centred radicals, results in death of the parasite. It is presumed that C-radicals can react with important key proteins; however, the specific molecular target(s) that leads to eventual parasite death are still unknown. A chemical synthesis of tetraoxane probes that contain a UV chromophore was performed and analogues were subsequently screened for antimalarial activity. The most active tetraoxane identified was exposed to a range of Fe2+ salts and conditions developed to mimic the biological environment. Primary, secondary and novel carbon-centred radical derived products (surrogate markers of bioactivation) were purified using UV-HPLC, characterised and submitted as chemical probes and standards for biological studies. In order for proteomic studies to be initiated, an allyl or azide group was incorporated into a semi-synthetic artemisinin skeleton. The azide (and alkyne) functional group within these probes provides a handle for protein pull down via click chemistry. Azide and acetylenes were chosen over direct linkage to the biotin group to reduce steric hindrance in the semi-synthetic probe. The synthesised click probes were tested for antimalarial activity and were submitted for protein pull down and identification of potential molecular targets. Similarly DDO allyl and azide were synthesised and were tested for Nrf2 induction and further confirmed as viable probes via NMR experiments with simple thiols and GSTP1. In summary, novel CDDO non reversible probes were synthesised and have shown potential as chemical tools to identify the molecular targets/mechanisms by which these compounds activate Nrf2. Tetraoxanes also have been prepared along with artemisinin click probes and the latter have been submitted for click chemistry pull down experiments, within Plasmodium falciparum parasites, to identify potential molecular targets.

Le paludisme reste une des maladies infectieuses les plus importantes au monde. Récemment, le laboratoire de Dr E. Davioud-Charvet a conçu des 3-Benzyl-Ménadiones substituées (benzylMD) comme agents antipaludiques prometteurs. Les études sur le mode d'action ont mis en évidenceque ces molecules déstabilisent l'équilibre redox des érythrocytes infectés en agissant comme agent catalytique redox (redox-Cycler), une stratégie prometteur pour le développement de nouveaux agents antipaludiques. Le travail de thèse présenté a caractérisé l'activité in vitro et le mécanisme d'action de tête de série, la 3-[4-(trifluorométhyl)-Benzyl]-Ménadione 1c, ce qui représente une partie principale du développement des benzylMDs. Une deuxième partie explorait les relations structure-Activité de benzylMD dérivés. Dans l'ensemble, les résultats démontrent l'activité in vitro très prometteuse de la benzylMD 1 cet soutiennent l'amélioration de benzylMDs comme nouveaux candidats-Médicaments antipaludiques<br>Malaria is still one of the most important infectious diseases worldwide. Previously, the laboratory of Dr. E. Davioud-Charvet presented the chemical design of very promising antimalarial agents, 3-[substituted-Benzyl]-Menadiones (benzylMD). Studies on the mode of action evidenced that these agents disturb the redox balance of the parasitized erythrocyte by acting as redox-Cyclers - a promising strategy for the development of new antimalarial agents. The presented PhD work characterized the in vitro potency and the mechanism of action of the lead agent, the 3-[4-(trifluoromethyl)benzyl]-Menadione 1 c, which represents an essential part of the lead optimization stage of the benzylMD drug development process. A second part of this work focused on the structure-Activity relationships benzylMD derivatives. Overall, the presented findings demonstrate the promising in vitro potency of lead benzylMD 1c and highly support the further development of benzylMDs as antimalarial drug candidates

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.<br>Medicine, Faculty of<br>Pathology and Laboratory Medicine, Department of<br>Graduate

Bcr-abl expression being associated with anti-apoptotic signaling and expression of DNA repair enzymes, we surmised that single molecules capable of blocking abl tyrosine kinase (TK) function and damaging DNA should lead to compounds with potency superior to that of GleevecRTM. To this end, we designed novel agents termed "combi-molecules" programmed to not only behave as bcr-abl inhibitors on their own, but also to further degrade to another inhibitor and a DNA damaging species. The released inhibitor was designed to sustain bcr-abl inhibition following degradation of the combi-molecule and the DNA damaging species to activate pathways leading to apoptosis. To model this strategy termed "combi-targeting", we synthesized ZRCM5 (a monoalkyltriazene) that showed antiproliferative activity superior to that of the classical DNA damaging agent TemodalRTM, but not to that of Gleevec RTM. This result was imputed to the rather weak bcr-abl inhibitory activity of ZRCM5 and its strong DNA damaging property. Another prototype designed to contain an aniline mustard moiety (AK04) was a strong bcr-abl inhibitor but a poor DNA alkylating agent. Its cytotoxic activity was again stronger than that of the clinical alkylating agent chlorambucil but inferior to that of GleevecRTM. Further chemical studies directed at structural modification of the benzamide moiety led to the synthesis of ZRF1 with strong potency against bcr-abl TK and strong DNA damaging property. This novel optimized combi-molecule showed a 1.6-3-fold greater potency than GleevecRTM against bcr-abl expressing cells. Further investigation with ZRF1, showed that its cytotoxic potency was dependent on the p53 wild-type status of the cells. In cells expressing wild-type p53, p21 transactivation was associated with cell cycle arrest and that of Bax with apoptosis. In addition to, the pro-apoptotic effect of bcr-abl inhibition, these multiple mechanisms of action may synergistically enhance the cytotoxic potency of ZRF1 in p53 wild-type cells. The study conclusively demonstrated that p53 is a major determinant for the cytotoxic advantage of the novel combi-molecular approach in chronic myelogenous leukemia (CML), a disease in which 70-85% of all cases express wild-type p53.

Depression and stimulant drug addiction each result in massive losses of health, productivity and human lives every year. Despite decades of research, current treatment regimes for depression are ineffective in approximately half of all patients. Therapy available to stimulant drug addicts is largely ineffective and moreover, dedicated treatments for drug dependence (including abuse of cocaine) are non-existent. Thus, there is a pressing need to further understanding of the molecular mechanisms underlying these disorders in order to develop novel, targeted therapeutic strategies. Chromatin remodeling, including changes in histone acetylation, has been proposed to play a role in both the etiology and treatment of depression and stimulant abuse. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate numerous cellular processes, including transcription, cell cycle progression and differentiation. Moreover, histone acetylation has been shown to regulate hippocampal neurogenesis, a cellular response associated with the pathogenesis and treatment of depression and stimulant abuse (Hsieh et al., 2004, Yamaguchi et al., 2004, Fischer et al., 2007). Ultimately, such basic cellular processes impact higher order function, namely cognition and emotion. Indeed, recent studies suggest that HDAC activity in selected forebrain regions, including ventral striatum and hippocampus, modulate stimulant- and antidepressantinduced behavior (Kumar et al., 2005, Tsankova et al., 2006a, Fischer et al., 2007). These reports highlight an association between chromatin remodeling and diverse behavioral changes, including changes induced by the pleiotropic HDAC inhibitor, sodium butyrate (SB), (Kumar et al., 2005, Tsankova et al., 2006a, Fischer et al., 2007). However, behavioral, brain-metabolic and molecular effects of SB treatment in the context of rodent models of depression, dopaminergic sensitization and repeated cocaine administration remained unclear. The work described in this thesis illustrates the potential for chromatin modifying drugs in mechanisms underlying the experimental pharmacology of depression and stimulant addiction. Specifically, the data presented here support the view that treatment with the short chain fatty acid, sodium butyrate enhances: (1) antidepressant-like behavioral effects of the selective serotonin reuptake inhibitor (SSRI), fluoxetine (2) locomotor sensitization induced by repeated administration of the dopamine D1/D5 receptor agonist SKF82958; and(3) brain metabolic activation upon repeated cocaine administration as evidenced by fMRI in awake rats. Furthermore, this report provides evidence that these treatment paradigms will result in chromatin modification changes associated with active transcription, in addition to increased mRNA levels of plasticity-associated genes, including brain-derived neurotrophic factor (BDNF) at key brain regions implicated in the pathogenesis of depression and stimulant addiction. To date, little is known regarding the underlying mechanisms of action mediating the enhancing effects of sodium butyrate on the various antidepressant- and stimulantrelated paradigms. Our findings underscore the potential of chromatin-modifying drugs to profoundly affect the behavioral response of an animal to antidepressant and stimulant drugs and warrants consideration in the context of developing novel therapeutic strategies.

Les bactéries Gram-négatif possèdent une structure d'enveloppe, ainsi que des pompes d'efflux (i.e., systèmes de transport actif permettant de détoxifier la cellule bactérienne) qui les rendent naturellement résistantes aux antibiotiques. Ces deux caractéristiques constituent de véritables barrières qui s'opposent à l'accumulation d'une grande variété d'antibiotiques près de leur cible, à l'intérieur de la bactérie. La perturbation des mécanismes s’opposant à l’accumulation d’antibiotiques par des agents chimiosensibilisants représente une stratégie prometteuse.L’objectif de cette thèse était de mieux comprendre les mécanismes d'inhibition de la résistance qui s’oppose à l’accumulation d’antibiotiques chez les bactéries Gram- négatif.Dans un premier temps l'activité de divers agents chimiosensibilisants synthétiques a été caractérisée. Trois dérivés ont été identifiés pour augmenter significativement l'activité synergique avec les antibiotiques, préalablement observée avec le géraniol. Ces dérivés ont montré une activité inhibitrice des pompes d'efflux ou perméabilisatrice de la membrane externe, pouvant être à l'origine des synergies observées.Dans un second temps, une méthode de criblage a été mise au point, en permettant la détection spécifique des agents chimiosensibilisants tout en décrivant leur mécanisme d'action.Ces travaux de thèse ont participé à proposer une solution thérapeutique brevetée au stade pré-clinique. Ils ont en outre permis de mettre en place des outils originaux pour identifier de nouveaux chimiosensibilisants, mais aussi pour mieux comprendre comment perturber les barrières s'opposant à l'accumulation d'antibiotiques<br>Gram-negative bacteria are naturally resistant to many classes of antibiotics thanks to their ability to control the accumulation of drugs. Decreasing membrane barrier permeability and producing efflux pumps that expel drugs outside bacteria, represent the prevalent mechanisms of this resistance. One of the most promising solutions consists in restoring antibiotic activity by targeting such barriers to accumulation, with chemosensitizers.The purpose of my PhD was to better understand the inhibition of resistance that opposes the accumulation of antibiotics in Gram-negative bacteria.In the first stage of the study, the activity of various synthetic chemosensitizers has been characterized. Three compounds were identified to significantly increase the synergistic activity with antibiotics, that was previously observed with geraniol. These derivatives showed an efflux pump inhibition or an outer membrane permeabilization effect, that could be related to the observed synergy.In the second stage of the study, a screening method has been developed for the specific detection of chemosensitizers, while describing their mechanism of action.This work participated in proposing a patented therapeutic solution in the preclinical stage. This study has led to new tools to identify novel chemosensitizers, but also to better understand how to impair the barriers opposing the accumulation of antibiotics

Un récent rapport de l’OMS met en garde les autorités de santé sur l’émergence de résistances chez les bactéries et le développement de souches bactériennes multi-résistantes aux traitements antibiotiques actuels. La progression de ces phénomènes est dû à différents facteurs. L’environnement hospitalier concentre un usage important de traitements antibiotiques et représente ainsi un terreau favorable au développement de résistances chez les bactéries. Le staphylocoque doré, ainsi que sa souche résistante (SARM), s’avèrent problématiques et entraînent un grand nombre de maladies nosocomiales. Dans ce contexte, il est primordial de mettre au point de nouveaux agents antibactériens permettant de lutter contre ces bactéries. Les liquides ioniques (sels à point de fusion bas) démontrent d’importantes propriétés antibactériennes. Néanmoins les mécanismes de cet effet bactéricide n’ont pas encore été établis. Ainsi nous nous proposons, dans un premier temps, de synthétiser des liquides ioniques di-cationiques afin d’étudier les différents facteurs structuraux qui régissent leur activité antibactérienne. Dans un second temps, nous concevrons des liquides ioniques à base de phosphoniums fonctionnalisés avec une sonde fluorescente. En exploitant les propriétés spectroscopiques, nous tenterons d’observer leurs interactions avec les cellules bactériennes. Enfin, nous nous proposons d'utiliser les phosphoniums comme agents de fonctionnalisation de surface dans le but de mettre au point des surfaces aux qualités bactéricides intrinsèques. Pour ce faire nous utiliserons différentes méthodes comme la conception de monocouches auto-assemblées ou l’électropolymérisation<br>A recent WHO report warns the health authorities about the emergence of new bacterial resistances and the development of multi-resistant strains against current antibiotics treatments. The growth of those resistances is due to several factors. The hospital environment concentrates a significant use of antibiotics and disinfectant representing a favorable ground for bacterial resistance development. Among them the Staphylococcus aureus and its methicillin resistant strain (MRSA) represent a crucial issue in care environments and is a major cause of hospital acquired infections. In this context, it is essential to develop new antibacterial agents to fight against these bacteria. Ionic liquid are low melting point salts, they show significant antibacterial properties. However, the fact that the mechanisms of action of their bactericidal effect have not been established yet constitutes a major obstacle to their development as bactericidal agents. Thus, we propose to synthetize ammonium- and phosphonium-based di-cationic ionic liquids in order to study the different structural factors that govern their antibacterial activity. Then we will develop phosphonium based ionic liquids functionalized with a fluorescent probe. By taking advantage of their spectroscopic properties we will try to observe their interactions with bacterial cells. Finally, we propose to use the phosphonium salts as surface functionalization agents in order to design surfaces with intrinsic antibacterial properties. To do so, we will use innovative methods such as conception of self-assembled monolayers or electropolymerization technics

Le règne végétal est une ressource renouvelable d'une large gamme de métabolites secondaires biologiquement actifs. Ces travaux de thèse proposent une stratégie multidisciplinaire d'évaluation du potentiel de composés phénoliques antimicrobiens d'origine végétale pour la conservation des aliments. Un criblage de l'activité antimicrobienne in vitro vis-à-vis de 8 souches de la flore pathogène et d'altération des aliments d'une centaine de molécules pures et une soixantaine d'extraits végétaux a d'abord permis de sélectionner les plus actifs. Différents mécanismes d'action vis-à-vis de S. aureus ont pu être mis en évidence par cytométrie de flux couplée à l'utilisation de marqueurs de l'état physiologique des bactéries pour quelques uns des composés actifs sélectionnés. En vue d'une application à de la viande bovine, l'activité antibactérienne des composés phénoliques ou extraits végétaux les plus actifs a été réévaluée dans des milieux de culture plus complexes mimant leur teneur en protéines et en matières grasses. Les résultats de ce criblage et un suivi microbiologique de viande hachée de bœuf avec 1% (m/m) d'extrait ajouté ont permis d'observer que les pertes d'activité antibactérienne observées étaient notamment corrélées aux interactions des composés phénoliques avec les protéines ou les matières grasses. L'incorporation des composés phénoliques ou extraits végétaux dans des matériaux d'emballage en contact alimentaire a constitué la seconde voie de mise en œuvre envisagée. Des films plastiques conservant une activité antibactérienne ont ainsi pu être élaborés par voie fondue<br>The plant kingdom is a renewable resource of a wide range of biologically active secondary metabolites. This thesis proposes a multidisciplinary strategy for evaluating the potential of plant-derived antimicrobial phenolic compounds for food preservation. A screening of the antimicrobial activity in vitro against 8 strains of foodborne pathogenic and spoilage microorganisms of a hundred pure molecules and about sixty plant extracts allowed to select the most active. Different mechanisms of action with respect to S. aureus could be demonstrated by flow cytometry coupled with the use of probes of the physiological state of the bacteria for some of the selected active compounds. For application to beef, the antibacterial activity of the most active phenolic compounds or plant extracts has been re-evaluated in more complex culture media mimicking their protein and fat content. The results of this screening and a microbiological monitoring of minced beef with 1% (m / m) of added extract made it possible to observe that the observed losses of antibacterial activity were in particular correlated with the interactions of the phenolic compounds with the proteins or fat. Incorporation of phenolic compounds or plant extracts into packaging materials in contact with food constituted was the second proposed route of implementation. Plastic films that retain antibacterial activity have thus been able to be prepared by melting

by Wong Yick Fu.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 1990.<br>Bibliography: leaves 201-224.<br>Acknowledgments --- p.V<br>Summary --- p.vi<br>Publications --- p.ix<br>Statement of Originality --- p.X<br>List of Abbreviations --- p.xi<br>Chapter Chapter 1 --- Introduction --- p.1<br>Chapter 1.1 --- Preamble --- p.2<br>Chapter 1.2 --- History of Trichosanthin --- p.6<br>Chapter 1.3 --- Preparation of Trichosanthin --- p.8<br>Chapter 1.4 --- Chemistry of Trichosanthin --- p.10<br>Chapter 1.4.1 --- Primary structure --- p.10<br>Chapter 1.4.2 --- Three dimensional structure --- p.12<br>Chapter 1.5 --- Pharmacology of Trichosanthin --- p.14<br>Chapter 1.5.1 --- Pharmacologic action --- p.14<br>Chapter 1.5.2 --- Pharmacokinetics --- p.18<br>Chapter 1.5.3 --- Toxicity --- p.21<br>Chapter 1.6 --- Clinical Use of Trichosanthin --- p.24<br>Chapter 1.6.1 --- Clinical application --- p.24<br>Chapter 1.6.2 --- Mechanism of action --- p.29<br>Chapter 1.6.3 --- Adverse reactions --- p.31<br>Chapter 1.6.4 --- Contraindications --- p.33<br>Chapter 1.7 --- Objectives of Project and Organization of Thesis --- p.35<br>Chapter Chapter 2 --- Anti-tumour Activity of Trichosanthin In Vitro and In Vivo --- p.37<br>Chapter 2.1 --- Cytotoxic Effects of Trichosanthin on Cultured Tumour Cells --- p.38<br>Chapter 2.1.1 --- Introduction --- p.38<br>Chapter 2.1.2 --- Materials and methods --- p.40<br>Chapter 2.1.3 --- Results --- p.49<br>Chapter 2.1.4 --- Discussion --- p.59<br>Chapter 2.2 --- Effects of Trichosanthin on Co-cultured Cell Lines In Vitro --- p.64<br>Chapter 2.2.1 --- Introduction --- p.64<br>Chapter 2.2.2 --- Materials and methods --- p.65<br>Chapter 2.2.3 --- Results --- p.66<br>Chapter 2.2.4 --- Discussion --- p.77<br>Chapter 2.3 --- Combination Effects of Trichosanthin with Adriamycin and Cisplatin on Cultured Tumour Cells --- p.80<br>Chapter 2.3.1 --- Introduction --- p.80<br>Chapter 2.3.2 --- Materials and methods --- p.81<br>Chapter 2.3.3 --- Results --- p.84<br>Chapter 2.3.4 --- Discussion --- p.93<br>Chapter 2.4 --- Effects of Trichosanthin on Choriocarcinoma Cells In Vivo --- p.96<br>Chapter 2.4.1 --- Introduction --- p.96<br>Chapter 2.4.2 --- Materials and methods --- p.97<br>Chapter 2.4.3 --- Results --- p.101<br>Chapter 2.4.4 --- Discussion --- p.107<br>Chapter 2.5 --- Effects of Trichosanthin Protein and Polysaccharide on Choriocarcinoma Cells In Vitro --- p.110<br>Chapter 2.5.1 --- Introduction --- p.110<br>Chapter 2.5.2 --- Materials and methods --- p.112<br>Chapter 2.5.3 --- Results --- p.114<br>Chapter 2.5.4 --- Discussion --- p.117<br>Chapter Chapter 3 --- Mechanism of Action of Trichosanthin on Tumour Cells --- p.119<br>Chapter 3.1 --- Morphological Study of Effects of Trichosanthin on Cultured Choriocarcinoma Cells --- p.120<br>Chapter 3.1.1 --- Introduction --- p.120<br>Chapter 3.1.2 --- Materials and methods --- p.121<br>Chapter 3.1.3 --- Results --- p.124<br>Chapter 3.1.4 --- Discussion --- p.133<br>Chapter 3.2 --- Binding of Radiolabelled Trichosanthin with Tumour Cells In Vitro --- p.137<br>Chapter 3.2.1 --- Introduction --- p.137<br>Chapter 3.2.2 --- Materials and methods --- p.138<br>Chapter 3.2.3 --- Results --- p.146<br>Chapter 3.2.4 --- Discussion --- p.154<br>Chapter 3.3 --- Effects of Trichosanthin on Macromolecule Synthesis of Choriocarcinoma Cells In vitro --- p.159<br>Chapter 3.3.1 --- Introduction --- p.159<br>Chapter 3.3.2 --- Materials and methods --- p.160<br>Chapter 3.3.3 --- Results --- p.163<br>Chapter 3.3.4 --- Discussion --- p.167<br>Chapter Chapter 4 --- General Discussion --- p.169<br>Chapter 4.1 --- Anti-tumour Activity of Trichosanthin --- p.170<br>Chapter 4.2 --- Mechanism of Action of Trichosanthin --- p.180<br>Chapter 4.3 --- Prospects of Research on Trichosanthin --- p.195<br>References --- p.201<br>Appendix 1 --- p.225<br>Appendix 2 --- p.242

Tese de mestrado em Química, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017<br>O cancro é um termo genérico para um vasto grupo de doenças que podem afetar qualquer parte do nosso corpo. Esta doença é definida pela proliferação anormal de células. Estas células anómalas podem invadir outros tecidos e órgãos formando assim metástases. O cancro, considerado uma doença mundial e que afeta diversas faixas etárias, continua a ser uma preocupação para a população e, nomeadamente, para os cientistas. A investigação nesta área já é longa e felizmente conta já com importantes avanços. No entanto, apesar de todos os progressos, continuam a existir obstáculos para o tratamento cem por cento eficaz. Um desses obstáculos é a resistência das células cancerígenas aos fármacos, o que limita consideravelmente a eficácia dos mesmos. Esta resistência deve-se a vários fatores sendo, um deles, a existência de um tipo de proteínas transportadoras, denominadas transportadores ABC, que se encontram sobre expressas nas células cancerígenas e que atuam sobre os fármacos levando ao seu rápido efluxo para fora da célula limitando, assim, a sua capacidade de ação sobre as células cancerígenas. A resistência a fármacos refere-se à capacidade das células cancerígenas para resistirem a uma variedade estrutural de fármacos anticancerígenos, levando a um dos maiores problemas da quimioterapia. Na realidade, este tipo de resistência é responsável pelo fracasso de mais de 90 % dos tratamentos em cancro. A família ABC (ATP binding cassette) é constituída por várias proteínas, sendo que atualmente as mais conhecidas, e aqui estudadas são: P-gp ou ABCB1, MRP1 ou ABCC1, MRP2 ou ABCC2 e ABCG2 ou BCRP. Apesar de existirem várias teorias que procuram explicar os seus mecanismos de ação, a certeza é que estas proteínas transportadoras permitem a expulsão dos fármacos, aumentando, em consequência, a resistências das células cancerígenas a estes fármacos. Os estudos de elucidação dos mecanismos bioquímicos que permitem combater esta resistência aos fármacos têm-se centrado principalmente na identificação de inibidores seletivos destas proteínas que bloqueiem a passagem dos fármacos para o exterior da célula cancerígena. A maior limitação até agora tem sido encontrar inibidores específicos para cada transportador, que ao mesmo tempo apresentem baixa citotoxicidade para células saudáveis e de alta eficiência. Por isso, a investigação nesta área continua a ser uma prioridade. Foi neste âmbito que o Laboratorio de Química Organometálica da Faculdade de Ciências da Universidade de Lisboa, Portugal, juntamente com o “Drug Resistance and Membrane Proteins team” em Lyon, França, avaliou, durante a realização desta tese de Mestrado, o papel que diversos transportadores ABC têm no mecanismo de ação de uma família de complexos organometálicos de ruténio ciclopentadienilo, “RuCp” (Cp = η5-C5H5). Foram estudados sete compostos, todos contendo o fragmento ‘Ru(η5-CpR)(PPh3)(bipiridina-R)’, com potencial atividade anticancerígena e anteriormente desenvolvidos pelo Laboratório de Química Organometálica. Entre eles, encontram-se os compostos de ruténio-polímero PMC78 e PMC85 que foram escolhidos devido ao seu elevado peso molecular que permite uma maior facilidade de acumulação destes compostos no interior das células pelo efeito de EPR (“enhanced permeation and retention effect”). Para além disso, estes compostos revelaram melhores citotoxicidades que a cisplatina para as linhas celulares do ovário A2780 e mama MCF7 e MDA-MB-231, e parecem ser capazes de ultrapassar os mecanismos de resistência de células cancerígenas (resultados obtidos por comparação entre a linha celular A2780 sensível e A2780CisR, resistente à cisplatina) . Para além destes compostos, foi também escolhido o composto PMC79, composto parental dos anteriores, com a mesma estrutura, mas sem as cadeias de polímero na sua estrutura. O composto PMC79 apresenta uma boa citotoxicidade relativamente à cisplatina para as mesmas linhas celulares. No entanto, para este composto o nível de acumulação nas células A2780 sensíveis foi muito superior que nas resistentes. Devido a estes resultados, o PMC79 foi também escolhido para este trabalho para se tentar perceber em maior detalhe qual o(s) transportadores ABC responsáveis por este efeito. O composto LCR134, [Ru(η5-Cp)(PPh3)(bipiridina-biotina)][CF3SO3], foi também escolhido uma vez que é baseado no PMC79, mas onde foram adicionadas duas moléculas de biotina (vitamina H ou B7) à bipiridina. A inclusão desta biomolécula poderá ser vantajosa devido à capacidade de se ligar a recetores da membrana celular das células cancerígenas. A biotina é essencial para o nosso organismo e tem sido frequentemente utilizada em diversos estudos reportando a sua facilidade de transporte para dentro das células cancerígenas. Os três compostos restantes, pertencem à subfamília de ruténio η5-metilciclopentadienilo e foram escolhidos com o objetivo de se conseguir obter uma correlação entre a sua atividade biológica e os substituintes na bipiridina. Desta forma, para se estudar o papel dos transportadores ABC no mecanismo de ação destes compostos, utilizaram-se diversas técnicas, tais como o teste de viabilidade celular para avaliar a citotoxicidade de cada composto através do cálculo do IC50, citometria de fluxo para verificar a percentagem de inibição de cada composto para os transportadores ABC, citometria de massa para quantificar a percentagem de acumulação do ruténio nas células, e docking molecular para a caracterizar a ligação de compostos ao sitio ativo da proteína P-gp. Todos os compostos obtiveram bons resultados ao nível da citotoxicidade para a linha celular cancerígena 2008C (1.1 - 4.5 μM), assim como bons níveis de internalização celular de ruténio. Os resultados obtidos permitiram concluir que compostos mesmo estruturalmente muito similares, possuem atividades biológicas distintas. Verificou-se que os compostos de ruténio-polímero, PMC78 e PMC85, são mais citotóxicos para células sobre expressas com transportadores (P-gp e MRP1, respetivamente) do que sem transportadores. O PMC78 demonstrou também que seria um bom inibidor para a P-gp. Todos estes fatores levaram a indicar que o uso do polilactídeo poderá potenciar a ação anticancerígena de compostos não poliméricos. Observou-se também que o uso do fragmento da bipiridina funcionalizada com duas moléculas de biotina poderá potenciar a capacidade anticancerígena dos compostos, visto que o complexo LCR134 revelou ser muito bom inibidor da P-gp. Cálculos de docking molecular mostram que é possível que haja competição entre o LCR134 e o conhecido substrato Rodamina 123 pelo centro ativo da P-gp . Os compostos LCR136 e RT11, pertencentes à família η5-MeCp, foram os compostos que revelaram os melhores resultados ao nível das suas atividades inibidoras e a melhor internalização para as linhas com os transportadores ABC estudados, sugerindo uma correlação entre as suas atividades e a sua internalização celular. Para além disso, revelaram melhor citotoxicidade para células sobre expressas. Os compostos PMC79 e RT12, são os compostos estruturalmente mais parecidos, onde a única diferença é a existência do grupo metil no ciclopentadienilo para o RT12. Os resultados mostraram que estes dois compostos têm atividades biológicas muito parecidas. Ambos são mais citotóxicos para as células sem sobre expressão de transportadores do que para as células sobre expressas e parecem não terem qualquer efeito inibitório para este tipo de células resistentes, contrariamente aos outros compostos estudados. Concluindo, pode-se afirmar que o grupo -CH2OH, comum aos dois compostos e que os distingue dos restantes, terá um papel importante no efluxo dos mesmos, tornando-os substratos dos transportadores ABC. Decorrente da avaliação dos estudos biológicos realizados, foi sintetizado com sucesso um novo complexo de ruténio, [Ru(η5-(Me-C5H4)(PPh3)(bipiridina-biotina)][CF3SO3] (Ru2). Este composto foi analisado por técnicas espetroscópicas como o RMN (1H, 31P, 13C e técnicas bidimensionais), UV-Vis e FT-IR, e a sua pureza foi determinada por análises elementares. O complexo revelou também adequada estabilidade em meio celular (variação menor que 5 % às 24 h) e caráter lipofílico (logPo/w= 1,6), o que nos assegurou continuação para os estudos biológicos neste novo composto. Foi então avaliado, para Ru2, a viabilidade celular nas linhas celulares utilizadas anteriormente. Contrariamente aos resultados previamente obtidos, este novo complexo de ruténio é muito menos citotóxico para NIH3T3 WT, NIH3T3-P-gp e 2008C, sendo que não é citotóxico para as outras linhas celulares estudadas. Percebe-se também que este composto é um substrato para a P-gp e não tem qualquer efeito inibitório para esta ou outra proteína transportadora. Concluindo, pode-se afirmar que a coordenação da biotina e do grupo η5-MeCp na mesma estrutura parece modificar a capacidade inibitória para P-gp e MRP2 como tinham os compostos LCR134, RT11 e LCR136. Este resultado revelou ser muito interessante, e como tal deve ser explorado em trabalhos futuros. Deste modo este trabalho apresenta pela primeira vez o estudo de novos compostos de ruténio com fragmento ‘Ru(η5-CpR)(PPh3)(bipiridina-R)’ em células sobre expressas por transportadores ABC. A descoberta de que estes complexos de ruténio são inibidores para proteínas transportadoras abre novas possibilidades relativamente aos seus mecanismos de ação. Para além disso, tal como observado para outros compostos da literatura, verificou-se que pequenas alterações estruturais desencadeiam respostas biológicas muito diferentes mostrando a importância deste tipo de estudos que relaciona a estrutura com a atividade. O objetivo deste trabalho foi então concluído com sucesso revelando que compostos de ‘Ru(η5-CpR)(PPh3)(bipiridina-R)’ poderão constituir uma ferramenta importante para o combate ao cancro, especialmente em cancros resistentes.<br>Cancer is a global disease that affects most of the age ranges and is still one of the biggest concerns for the scientists worldwide. The research in this area is exhaustive and, fortunately, important developments are done year after year. However, there are some obstacles for the successful treatment such as multidrug resistance (MDR) that limits the drug efficacy. The main reason for this resistance lies in one type of proteins called ABC transporters. These proteins are overexpressed in cancer cell lines and allow the efflux of the drug out from the cell. P-gp or ABCB1, MRP1 or ABCC1, MRP2 or ABCC2 and ABCG2 or BCRP are the most studied proteins belonging to the ABC family. Although the transport mechanism of each pump is still missing, one thing that the scientists are sure is that these proteins are responsible for the efflux of molecules out of the cells. To try to avoid this efflux, the identification of selective inhibitors that block the drugs efflux is being explored. The main challenge of this research is to find compounds that can act as high effective inhibitors while presenting low toxicity for healthy cells. Within this frame, the Organometallic Chemistry Laboratory from Faculdade de Ciências da Universidade de Lisboa, Portugal, and the Drug Resistance and Membrane Proteins in Lyon, France, studied the role of several ABC transporters on the mechanism of action of new ruthenium cyclopentadienyl compounds “Ru(η5-Cp)”. All the complexes were cytotoxic for the cell lines overexpressed and not overexpressed with ABC transporters and also for one cancer cell line, 2008C. Four compounds (PMC78, LCR134, RT11, LCR136) exhibited specific inhibitory activity for some of the ABC transporters studied. The amount of ruthenium internalization on the cell lines was also quantified by mass cytometry (CyTOF), indicating that, in all cases, the compounds are internalized. A molecular docking study was also carried out for one of the structures (LCR134) in P-gp protein revealing that a competition between LCR134 and the P-gp substrate might happen. With the aim of optimizing the inhibitory activity of this family of compounds, a new ruthenium complex was synthesized, [Ru(η5-MeCp)(PPh3)(bipy-biot)][CF3SO3] Ru2, bearing the structural features inducing the best inhibition effects: a biotin molecule and a η5-MeCp ligand. This compound was characterized by the usual techniques (NMR, UV-Vis and IR spectroscopies) and its purity was assessed by elemental analyses. Ru2 was found to be very stable in cell medium (less than 5% variation over 24 h) and it has an hydrophobic character (logPo/w= 1.6), allowing us to carry on with the biological evaluation. The new compound was evaluated in the same cell lines as the previous compounds. Interestingly, this compound is much less cytotoxic for NIH3T3 WT, NIH3T3-P-gp and 2008C cell lines than the previously compounds studied, and is non-cytotoxic for all the other cell lines. Moreover, it seems that this compound is a substrate for P-gp pumps and does not have any inhibitory effect. To conclude, we can say that the biotin and η5-MeCp motifs in the same complex do not improve the inhibitory potential, resulting, in contrast, in the loss of the inhibitory capacity. Altogether, the proposed aims for this work were successfully achieved and allowed us to unravel an unprecedented mechanism of action for ruthenium cyclopentadienyl complexes that can be used as tool to fight the multidrug resistance in cancer.

Tese de doutoramento, Ciências Biomédicas (Bioquímica Médica), Universidade de Lisboa, Faculdade de Medicina, 2012<br>Antimicrobial peptides (AMPs) are currently looked as new candidates to overcome the bacterial resistance against therapeutic antibiotics. However, their mechanism of action remains unclear, despite several theories having been proposed. The understanding of the processes that govern AMPs activity is the best way to provide rational design of new antibiotics for further clinical use against bacterial infections. Human AMPs are part of the innate immune system and synthetic versions of these AMPs are good candidates, due to their low toxicity and high antimicrobial activity. It is believed that the mode of action of these AMPs involves their action at the membrane level. This thesis is focused on the study of the interaction of a fragment based on the N-terminal region of a human antimicrobial protein, the bactericidal/permeability increasing protein (BPI), with biomembrane model systems and bacterial cells. The fragment, named rBPI21, has antimicrobial properties and neutralizes the effect of the lipopolysaccharide (LPS) during bacterial infections. The thesis describes the use of biophysical strategies in order to unravel the fundamental steps involved in the bactericidal activity of rBPI21. Membrane selectivity was quantified using a range of biophysical techniques and reported in Chapters III and V. Chapter III reveals that rBPI21 prefers negatively charged liposome systems containing phosphatidylglycerol, which mimic bacterial membranes. The preference for the anionic phosphatidylglycerol membranes is followed by membrane aggregation/fusion and, at higher rBPI21 concentrations, there is a leakage of liposome content, as described in Chapter IV. Liposomes fusion leads to multilamellar membrane structures, as studied in Chapter V by small angle X-ray scattering. Previous studies revealed that rBPI21 was able to induce membrane perturbations on negatively charged membrane model systems mimicking the membranes of bacteria. On the other hand, membrane model systems that mimic eukaryotic membranes remain unaffected by the presence of rBPI21. In Chapter VI, the interaction of rBPI21 with bacteria was studied using atomic force microscopy, and the results were correlated with those obtained with membrane model systems in order to unravel the possible mechanism of action of the peptide. rBPI21 was shown to induce membrane perturbations, culminating in bacterial cells content leakage, both on the Gram-negative bacteria Escherichia coli and on the Gram-positive Staphylococcus aureus. The interaction of rBPI21 with bacteria was decreased in the presence of free lipopolysaccharide aggregates, demonstrating the affinity of rBPI21 for free LPS, as studied by force spectroscopy. The overall observed results potentiate the use of the rBPI21 in clinics against bacterial infections. Also, the development of new synthetic peptides based on rBPI21 structure is a valuable route to develop new therapeutic agents with antibacterial properties.<br>O aumento considerável das resistências bacterianas aos antibióticos em uso na prática clínica têm vindo a deixar as comunidades científica e clínica, alarmadas. O desenvolvimento de novas classes de antibióticos é urgente, para que se possa combater a resistência bacteriana adquirida. Os péptidos antibacterianos são vistos como novos candidatos para uso terapêutico nesta área. No entanto, ainda existem dúvidas e necessidades de esclarecimento quanto ao seu mecanismo de acção. A aquisição deste conhecimento, ainda em falta, irá permitir o desenvolvimento de novos péptidos para uso clínico contra infeções bacterianas e a optimização das suas aplicações e modo de administração. Os péptidos antibacterianos podem-se encontrar em quase todos os organismos vivos expostos à ação dos micróbios, desde os humanos até plantas e insetos. Estes agentes fazem parte da resposta rápida e eficaz contra patogénios. No caso dos humanos, o sistema imune inato é rico em péptidos antibacterianos, o que faz destes agentes bons candidatos para terapêutica, devido à baixa toxicidade e elevada actividade antibacteriana. A maioria dos péptidos antibacterianos são caracterizados principalmente por, em condições fisiológicas, possuírem carga global positiva devido à presença na sua constituição de grande quantidade de aminoácidos catiónicos, como é o caso de argininas e lisinas. É também conhecida a tendência para estas moléculas adquirirem uma estrutura anfipática, ou seja, são moléculas que apresentam uma região hidrófila e uma outra hidrófoba. Em concordância com estas características, estudos demonstram que a actividade antibacteriana destes péptidos ocorre maioritariamente ao nível da membrana. A selectividade destes agentes para as bactérias em detrimento das células humanas, por exemplo, deve-se sobretudo à presença de algumas moléculas específicas na superfície das bactérias, como é o caso dos lipopolissacáridos, e também de uma elevada percentagem de fosfolípidos aniónicos na membrana. É a carga negativa da membrana que potencia a atracão electrostática entre os péptidos antibacterianos e as membranas das bactérias. ação antibacteriana destes agentes ao nível das membranas das bactérias faz com que seja difícil para as bactérias adquirirem resistência. Isto implicaria alterações significativas na constituição membranar, o que levaria a uma alteração no potencial de membrana com custos energéticos e funcionais para a bactéria. De forma a desenvolver os péptidos antibacterianos como fármacos é necessário o estudo pormenorizado da ação dos mesmos em membranas. No entanto, as membranas biológicas são sistemas muito complexos para poderem ser usados na análise da actividade dos péptidos antibacterianos. Uma alternativa comummente usada e bastante viável prende-se com o uso de sistema modelo de membranas, nomeadamente o uso de vesículas lipídicas. É possível usar diferentes misturas de lípidos na realização destas vesículas permitindo aproximar da composição biológica das membranas das bactérias e humanas. Estes sistemas modelo de membranas podem ser usados com diferentes tamanhos, tendo sido nesta tese estudadas as interacções com vesículas lipídicas unilamelares grandes, de aproximadamente 100 nm, e também vesículas multilamelares com tamanhos compreendidos entre 1 e 100 micrometros. A ação sistémica dos antibióticos aquando da morte bacteriana é acompanhada pela libertação de certos componentes bacterianos em circulação sanguínea. Uns destes componentes são os lipopolissacáridos, que são libertados das membranas das bactérias Gram-negativas. Estas moléculas em circulação tornam-se agentes infecciosos que em baixas concentrações mantém o organismo em alerta contra patogénios, mas em concentrações elevadas originam uma produção excessiva de citocinas que podem culminar no choque séptico e daí resultando a morte. No caso de péptidos com actividade antibacteriana contra bactérias Gramnegativas é muito importante que, além da acção antibacteriana, os péptidos sejam capazes de neutralizar o efeito endotóxico do lipopolissacárido. A bactericidal/permeability increasing protein (BPI) é uma proteína homóloga da lipopolysaccharide binding protein (LBP) e que se encontra nos grânulos azurófilos dos neutrófilos. Além da sua actividade antibacteriana descrita é também conhecido o seu efeito neutralizador da ação do lipopolissacárido livre em circulação. Estas actividades estavam associadas à região amina da proteína. Desta forma foi desenvolvido um fragmento baseado nesta região, denominado rBPI21. O rBPI21 possui uma massa molecular de 21 kDa tendo actividade antibacteriana e também é capaz de se ligar ao lipopolissacárido, neutralizando a toxicidade provocada pelo mesmo. Esta tese de doutoramento está focada na interacção do rBPI21 com sistemas modelo de membranas e bactérias. Os estudos realizados ao longo deste trabalho foram essencialmente biofísicos, utilizando técnicas, como exemplo, espectroscopias dispersão de luz, de fluorescência e de força atómica. Nos Capítulos III e V desta tese de doutoramento é determinada, através da espectroscopia de fluorescência e da calorimetria de titulação isotérmica, a selectividade do rBPI21 para sistemas modelo de membranas com carga negativa conferida pelo fosfolípido fosfatidilglicerol. Estas membranas carregadas negativamente são um modelo aproximado das membranas das bactérias Gramnegativas. A preferência do rBPI21 para membranas contendo fosfatidiglicerol é seguida da formação de agregados membranares, estudada por espectroscopia de dispersão de luz, e também da fusão membranar. No entanto, a elevadas concentrações do rBPI21 ocorre a lise do sistema modelo de membrana. Estes estudos estão descritos no Capítulo IV da tese. No Capítulo V da tese mostra-se através do uso da dispersão de raios-X de pequeno ângulo que a interacção do rBPI21 com sistemas modelo de membranas é seguida da formação de sistemas multilamelares aquando da fusão membranar. Após os estudos efectuados com sistemas modelo de membranas, no Capítulo VI foi descrita a interacção do rBPI21 com bactérias, de forma a corroborar o mecanismo de acção observado nos sistemas modelo. Os estudos foram feitos com a bactéria Gram-negativa Escherichia coli e com a Gram-positiva Staphylococcus aureus. Estudos por microscopia de força atómica e espectroscopia de força mostram que o rBPI21 é capaz de causar a lise de ambas as bactérias e que a interacção do péptido com as mesmas é reduzida na presença de agregados livres de lipopolissacárido, evidenciando a afinidade do péptido para o lipopolissacárido na forma livre. Os resultados revelam para os determinantes da selectividade do rBPI21 para as membranas das bactérias, em detrimento das membranas de células humanas. Desta forma, o rBPI21 é um bom candidato para uso clínico, assim como um bom modelo para, através da sua estrutura, serem desenhados novos péptidos com maior actividade antibacteriana e reduzida toxicidade.<br>Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/41750/2007)