Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-20Bitstream added on 2014-06-13T20:21:26Z : No. of bitstreams: 1 leme_dm_dr_rcla_parcial.pdf: 198180 bytes, checksum: 4c280b4c68a010c361d6ef4e0da11146 (MD5) Bitstreams deleted on 2015-06-25T13:00:52Z: leme_dm_dr_rcla_parcial.pdf,. Added 1 bitstream(s) on 2015-06-25T13:03:16Z : No. of bitstreams: 1 000638858_20151201.pdf: 243435 bytes, checksum: d1e6915fdf00c6f43d70add5c4248acb (MD5) Bitstreams deleted on 2015-12-01T09:08:34Z: 000638858_20151201.pdf,. Added 1 bitstream(s) on 2015-12-01T09:09:26Z : No. of bitstreams: 1 000638858.pdf: 2789008 bytes, checksum: 13a14092763ec9a00dd9bfbff49af27a (MD5) / A questão energética é hoje um tema de preocupação mundial. A limitação das fontes de energia fóssil e seus efeitos indesejáveis ao meio ambiente têm estimulado a busca de novas fontes alternativas, desde que renováveis. Em diversos países, como no Brasil, têm sido destinados grandes incentivos ao desenvolvimento do setor de biocombustíveis, no qual se inclui o biodiesel. O futuro promissor do biodiesel está relacionado não apenas ao fato de ser um combustível renovável, mas também às suas contribuições na redução da emissão de poluentes atmosféricos e à sua maior degradabilidade, em relação aos combustíveis fósseis. No entanto, poucos são os estudos realizados com o biodiesel para avaliar os seus possíveis impactos, quando usados puro ou em misturas ao óleo diesel, sobre o ambiente (principalmente corpos d’águas e solos) e sobre os organismos vivos, em caso de uma possível contaminação ambiental. O presente estudo teve como objetivo avaliar a citotoxicidade, genotoxicidade e mutagenicidade do biodiesel e de suas misturas com diesel (B5, B20 e B50), por meio de simulações de vazamento em água e solo. As simulações foram realizadas no verão, para caracterizar um vazamento em condições tropicais, sendo aplicados diferentes ensaios biológicos [teste de Allium cepa, ensaio Salmonella/microssoma, ensaios de mutagenicidade com células CHO-K1 e HepG2 in vitro, ensaio para detecção de indução de morte celular (alterações do ΔΨm, externalização da fosfatidilserina), avaliação da citotoxicidade em tempo real (sistema xCELLigence™)] nas amostras de água e solo obtidas nos experimentos. Foi observada no presente estudo uma significativa citotoxicidade, que foi relacionada com constituintes do óleo diesel, mais especificamente, com os hidrocarbonetos policíclicos aromáticos (PAHs). Já os efeitos genotóxicos/mutagênicos, observados... / The energy-related issue is currently a subject that causes world concern. The limitations of fossil energy sources and their undesirable effects upon the environment have encouraged the search for new alternative sources, as long as they are renewable ones. In several countries, like Brazil, great incentives have been allocated to the development of the biofuel sector, and that includes biodiesel. The promising future of biodiesel is related not only the fact of being a renewable biofuel, but also to its contribution to reducing release of air pollutants and its higher degradability when compared to fossil fuels. Nevertheless, not enough studies have been conducted with biodiesel to evaluate their possible impacts – either when they are used crude or in biodiesel blends – upon both the environment (especially water bodies and soils) and living organisms, should there be environmental contamination. The aim of this study was to assess toxicity, genotoxicity and mutagenicity of biodiesel and its diesel blends (B5, B20 and B50), by simulating spills into water and soil. The simulations were carried out in the summer in order to characterize a spill under tropical conditions, and different assays [Allium cepa test, Salmonella mutagenicity assay, in vitro mutagenicity assays with CHO-K1 and HepG2 cells line, detection of cell death inductions (changes in ΔΨm, phosphatidylserine externalization), cytotoxicity assessment by xCELLigence™ system] were conducted using soil and water samples obtained in the experiment. In this study, significant citotoxicity was observed, which was found to be related to biodiesel contaminants, even more specifically, to its polycyclic aromatic hydrocarbons (PAHs). On the other hand, the genotoxic/mutagenic effects observed in the other assays done with diesel and biodiesel proved to result from the action of pollutants present in the raw material used... (Complete abstract click electronic access below)

The thesis “Ancillary Ligand Effects on the Anticancer Activity of Ruthenium (II) Piano Stool Complexes” is an effort to design better antitumor metallodrugs based on ruthenium(II) complexes with various H-bond donor/acceptor ligands and to understand their mechanism of action. Chapter 1 presents a brief review of metallodrugs and their mechanism of action. Different classes of metallodrugs are discussed. A short discussion on ruthenium based anticancer drugs and their established mechanism of action is also included in this chapter. Chapter 2 deals with the synthesis, characterization and anticancer activity of Ru(II) complexes with P(III) and P(V) ligands. The effect of a strong hydrogen bond acceptor on the cytotoxicity of the complexes has been investigated which allows comparison of complexes with ligands possessing a strong hydrogen bond donor or hydrogen bond acceptor. Partial oxidation of the tertiary phosphine ligands leads to a decrease in cytotoxicity of the ligand, while coordination to ruthenium resulted in a significant increase in the cytotoxicity. A molecular mechanism of action for these complexes was suggested on the basis of various biophysical studies. These complexes bind DNA through non-intercalative interactions which lead to the destabilization of the double helix of the DNA and also unwinding of the negatively supercoiled DNA. Results show that the presence of a hydrogen bond acceptor on the ligand is not capable of enhancing interactions with DNA in comparison with hydrogen bond donor groups. Cellular studies of these complexes showed that inhibition of DNA synthesis and apoptosis occur on treatment with these complexes. Interestingly, these complexes are found to be not only cytotoxic but also antimetastatic. Chapter 3 deals with the synthesis, characterization and anticancer activity of Ru(II) complexes with biologically active S containing heterocyclic ligands and their mechanistic study. Complexation of ruthenium with mercaptobenzothiazole (MBT) gave the most cytotoxic complex (H3) in the series. Heterocyclic Ru(II) complexes behave differently as evidenced by cellular and biophysical studies. Unlike phosphine complexes, H3 shows biphasic melting of DNA at higher concentrations which suggests two different types of interaction with DNA. Chapter 4 deals with synthesis and characterization of water soluble multiruthenated hydrophilic ruthenium(II) complexes with urotropine. An increase in cytotoxicity and binding affinity has been observed with increase in the number of ruthenium atoms per molecule. The complex with three ruthenium atoms showed the best activity. However cytotoxicity of the complexes decreases with decrease in the lipophilicity of the complexes. Chapter 5 describes studies on the interaction of Ru complexes with water, ss-DNA, AMP, GMP and GSH by various spectroscopic techniques. Hydrolysis of Ru-Cl bond in the complexes correlates with the cytotoxicity. Chapter 6 reports the summary of the observations of the thesis and the future prospects of metallodrugs.

Durant une infection pulmonaire, les porcs sont souvent infectés par plus d’un microorganisme. Actinobacillus pleuropneumoniae et le virus du syndrome reproducteur et respiratoire porcin (VSRRP) sont des pathogènes qui peuvent infecter de manière simultanée les porcs. L’objectif du présent projet est d’étudier l’interaction entre ces pathogènes. Les deux lignées cellulaires permissives au VSRRP utilisées sont les cellules « St-Jude porcine lung » (SJPL) et MARC-145. Les cellules ont été pré-infectées avec le VSRRP, puis infectées avec A. pleuropneumoniae. Un dosage de la lactate déshydrogénase a montré qu’une co-infection VSRRP-A. pleuropneumoniae comparée à une infection simple augmente significativement la cytotoxicité. Dans les mêmes conditions expérimentales, une pré-infection virale ne semble pas affecter l’adhérence d’A. pleuropneumoniae aux cellules. À l’aide de tests ELISA, il a été possible de démontrer la production d’IL-8 et d’INF-γ lorsqu’il y a infection des cellules. Pour ce qui est du TNF-α, d’IL-6 et d’IL-10, ces cytokines ne sont pas détectées en présence des pathogènes étudiés. Des expériences de pré-infection bactérienne suivie d’infection virale ont également été réalisées. Il a été démontré que la pré-infection avec A. pleuropneumoniae diminuait la réplication du VSRRP chez la lignée cellulaire SJPL, mais cela n’est pas observé avec la lignée cellulaire MARC-145. Les résultats préliminaires ont démontré que cette diminution de la réplication serait causée par une molécule de faible poids moléculaire sécrétée dans le surnageant bactérien et celle-ci serait résistante à la chaleur. Les lignées cellulaires SJPL et MARC-145 représentent de bons modèles pour l’étude des infections mixtes des voies respiratoires du porc. / The respiratory tract of pigs is often colonized by more than one pathogen during an infection. Actinobacillus pleuropneumoniae and the porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that can be associated with co-infection. The objective of this project was to study interactions between A. pleuropneumoniae and PRRSV during mixed infection. The PRRSV-permissive cell lines, St-Jude porcine lung (SJPL) and MARC-145 were used. In the first part, cells were pre-infected with PPRSV followed by an infection with A. pleuropneumoniae. Results obtained with a lactate dehydrogenase test showed that a co-infection resulted in a greater cytotoxicity then the single infections. The adherence of A. pleuropneumoniae to non-infected or PRRSV-infected cells was similar. Based on ELISAs tests, it was found that the cells produced IL-8 and IFN-γ when they were infected, but TNF-α, IL-6 and IL-10 were not detected. In the second part, cells were pre-infected with A. pleuropneumoniae followed by viral infection. The results showed that a pre-infection with A. pleuropneumoniae decreased PRRSV replication in SJPL cells, whereas A. pleuropneumoniae did not impair PRRSV replication in MARC-145 cells. Preliminary results indicate that a molecule secreted by A. pleuropneumoniae is the factor impairing PRRSV replication in SJPL cells. The factor is probably a small molecular weight molecule that is heat-resistant. In conclusion, both cell lines allowed the study of A. pleuropneumoniae and PRSSV interactions during a mixed-infection and these models could be adapted to study interactions of other swine pathogens.

The pathogenesis of oral submucous fibrosis (OSMF) is due to a complex interplay between the production and degradation of extracellular matrix (ECM) protein components. In tissue fibrosis, there is a net accumulation of collagen as a result of an imbalance between enhanced production, deposition and impaired degradation of ECM components. OSMF is a chronic inflammatory condition of the oral cavity and regulation of a number of pro-inflammatory and fibrogenic cytokines such as interleukine-1, -6 and -8 isoforms, TGF-β, PDGF, bFGF, IFN-γ and TNF-α has been reported in OSMF tissues. The expression of these growth factors has a bearing on the epithelial changes as well as proliferation and differentiation of oral fibroblasts into ECM protein producing myofibroblast cells. One key modulator of fibrosis in several organs has been TGF-β. Overproduction of TGF-β mRNA and protein has been reported in several fibrotic disorders including that of skin, lungs, liver, kidney and heart. This is mainly due to stimulation of ECM genes by TGF-β. Although there have been few reports suggesting the over production of TGF-β in OSMF tissues, the specific isoforms involved or the mechanisms are poorly understood. Areca nut components, especially arecoline have been implicated in the pathophysiology of OSMF. Few reports indicate the involvement of arecoline in the regulation of collagen production and activity of collagenases and their inhibitors in oral fibroblast cells. Moreover, the alkaloid is involved in initiating epithelial inflammation by inducing COX-2, prostaglandin E2, IL-1α, IL-6 and IL-8 in KB oral carcinoma cells and oral fibroblast cells. These and other reports strongly suggest that changes in gene expression mediated by Arecoline may be central to the progression of OSMF. Not much is known about arecoline-mediated cellular signaling events except for few recent reports that suggest the activation of MAPK pathways. In neuronal and colonic smooth muscle cells of mouse, rat and rabbit, the actions of Arecoline have been reported to be through the activation of muscarinic acetylcholine receptors. Direct binding of arecoline to human M1, 2 and 3 muscarinic receptor isoforms have been shown in brain tissues. Stimulation of these receptors alters the intracellular levels of Ca+2 and cAMP, which are important second messengers. The cholinergic potential of arecoline may be important for their roles in arecoline-mediated signaling events. The expression of muscarinic acetylcholine receptors has been reported in several cell types besides neuronal and excitatory cells. Although several gene expression changes have been reported following Arecoline treatment of a variety of cells, the mechanism of such regulations is not established. Hence in order to understand the role of arecoline in OSMF disease process, we undertook studies that provide insights into arecoline action in epithelial and fibroblast cells and possible molecular mechanisms. The objectives are to study: 1. The role of arecoline in cellular proliferation, cell-cycle regulation and apoptosis in human normal keratinocytes. 2. Mechanism of regulation of gene expression by arecoline in normal keratinocytes. 3. Mechanism of regulation of gene expression by arecoline in human normal oral fibroblasts. In order to achieve the above objectives, a human keratinocyte cell line, HaCaT and an oral periodontal fibroblast cell line (PDC) were utilized. The cells were treated with arecoline and a variety of assays including RT-PCR analysis of mRNA of several genes, phosphorylation status of MAPK pathway intermediates, cell cycle analysis and other cellular and molecular methods have been employed. Following arecoline treatment, there is induction of oxidative stress, growth arrest and epithelial cell death. Since actions of TGF-β are central to most fibrotic disorders and arecoline has been implicated in OSMF, it is hypothesized that arecoline may influence fibrosis via TGF-β pathway. Towards this, several TGF-β target genes that may have a possible role in fibrosis have been studied in arecoline treated epithelial and fibroblast cells. Since arecoline mediated oxidative stress has been reported, the regulation of genes that are involved in stress response pathway have been studied for induction by arecoline in epithelial cells. The results presented in this thesis suggest the up regulation of oxidative stress-responsive genes in HaCaT cells including HOX-1, FTL, G6PD, GCLC and GRD in HaCaT cells. Oxidative stress is a major inducer of inflammatory response in the epithelial tissues. The expression of IL-1α, an important inflammatory cytokine is induced by arecoline in HaCaT cells in response to oxidative stress via the activation of p38 MAPK pathway. Interestingly, activation of MAPK pathways by arecoline is involved in the regulation of common target genes of arecoline and TGF-β and also in the induction of TGF-β−responsive promoter reporter construct, p3TP-lux activity in HaCaT cells. Due to the involvement of TGF-β in fibrosis, regulation of TGF-β pathway genes by arecoline has been studied both in HaCaT and PDC cells. In HaCaT cells, arecoline induces the expression of TGF-β2 mRNA while TβRII expression is down regulated. The expression of the rest of TGF-β/SMAD pathway genes including TGF-β1, β3, TβRI, SMAD1, 2, 3, 4 and 7 are not affected by arecoline in HaCaT cells. Over expression of TGF-β2 is also observed in most of the OSMF tissues compared to normal oral tissues. However, in normal oral fibroblast cells, we observed that the TGF-β/SMAD pathway genes are not regulated by arecoline. These results suggest the possible involvement of arecoline-mediated induction of TGF-β2 in epithelial cells in OSMF disease development. We investigated the signaling pathways involved in the regulation of TGF-β2 and found that stimulation of M3 muscarinic receptor by arecoline leads to the induction of TGF-β2 expression in HaCaT cells via PKC pathway. TGM-2 is an important TGF-β target gene involved in the cross linking of ECM proteins. Arecoline-mediated induction of TGM2 mRNA and transglutaminase activity are observed in oral fibroblast cells, PDC. The induction of TGM-2 was found to be independent of oxidative stress and TGF-β, but dependent on muscarinic acid receptor activation by arecoline and involves cytosolic cAMP. When tested in OSMF tissues, there was an increased expression of TGF-β2, TSP1 and TGM2 as compared to normal tissues suggesting a possible role of these genes in arecoline-mediated progression of OSMF. Interleukin-8 (IL-8), which is involved in inflammation has been reported to be regulated by TGF-β in a cell type specific manner. In several cell types including human endometrial stromal cells, LnCaP (prostate cancer cells), human retinal pigment epithelial cells and rat lung alveolar epithelial (LM5) cells etc., TGF-β up regulates the expression of IL-8 mRNA. Arecoline was found to down regulate IL-8 expression in PDC cells as measured by RT-PCR. Interestingly, the presence of serum along with arecoline induces the expression of IL-8 in PDC cells suggesting the modulation of arecoline-mediated gene regulation by a serum activated signaling pathway. Intriguingly, arecoline treatment led to down regulation of collagens in PDC cells. However, collagen genes are induced in PDC cells in the presence of HaCaT spent medium by arecoline suggesting a role for factor(s) secreted by epithelial cells in the regulation of collagen genes by arecoline. This factor could be an isoform of TGF-β as shown by blocking the induction of collagens by the TGF-β inhibitor, βLAP. Taken together, all these results indicate the ability of arecoline to cause fibrosis in a tissue environment where both epithelial and fibroblasts respond to arecoline and mutually contribute to the disease manifestation. Major conclusions from this study includes, 1] cell death in epithelial cells due to oxidative stress following arecoline treatment, 2] regulation of gene expression by arecoline involves MAPK, PKC pathways, 3] muscarinic acid receptor and oxidative stress are also important for regulation gene expression by arecoline. The most important inference from this study is the possible paracrine influence of TGF-β isoforms secreted by epithelial cells on the oral fibroblasts in determining the progression of OSMF. In summary, in this thesis, an attempt has been made to study the molecular mechanisms and role of arecoline, an alkaloid in conferring gene expression changes that may lead to the initiation and progression of oral sub mucous fibrosis.

Durant une infection pulmonaire, les porcs sont souvent infectés par plus d’un microorganisme. Actinobacillus pleuropneumoniae et le virus du syndrome reproducteur et respiratoire porcin (VSRRP) sont des pathogènes qui peuvent infecter de manière simultanée les porcs. L’objectif du présent projet est d’étudier l’interaction entre ces pathogènes. Les deux lignées cellulaires permissives au VSRRP utilisées sont les cellules « St-Jude porcine lung » (SJPL) et MARC-145. Les cellules ont été pré-infectées avec le VSRRP, puis infectées avec A. pleuropneumoniae. Un dosage de la lactate déshydrogénase a montré qu’une co-infection VSRRP-A. pleuropneumoniae comparée à une infection simple augmente significativement la cytotoxicité. Dans les mêmes conditions expérimentales, une pré-infection virale ne semble pas affecter l’adhérence d’A. pleuropneumoniae aux cellules. À l’aide de tests ELISA, il a été possible de démontrer la production d’IL-8 et d’INF-γ lorsqu’il y a infection des cellules. Pour ce qui est du TNF-α, d’IL-6 et d’IL-10, ces cytokines ne sont pas détectées en présence des pathogènes étudiés. Des expériences de pré-infection bactérienne suivie d’infection virale ont également été réalisées. Il a été démontré que la pré-infection avec A. pleuropneumoniae diminuait la réplication du VSRRP chez la lignée cellulaire SJPL, mais cela n’est pas observé avec la lignée cellulaire MARC-145. Les résultats préliminaires ont démontré que cette diminution de la réplication serait causée par une molécule de faible poids moléculaire sécrétée dans le surnageant bactérien et celle-ci serait résistante à la chaleur. Les lignées cellulaires SJPL et MARC-145 représentent de bons modèles pour l’étude des infections mixtes des voies respiratoires du porc. / The respiratory tract of pigs is often colonized by more than one pathogen during an infection. Actinobacillus pleuropneumoniae and the porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that can be associated with co-infection. The objective of this project was to study interactions between A. pleuropneumoniae and PRRSV during mixed infection. The PRRSV-permissive cell lines, St-Jude porcine lung (SJPL) and MARC-145 were used. In the first part, cells were pre-infected with PPRSV followed by an infection with A. pleuropneumoniae. Results obtained with a lactate dehydrogenase test showed that a co-infection resulted in a greater cytotoxicity then the single infections. The adherence of A. pleuropneumoniae to non-infected or PRRSV-infected cells was similar. Based on ELISAs tests, it was found that the cells produced IL-8 and IFN-γ when they were infected, but TNF-α, IL-6 and IL-10 were not detected. In the second part, cells were pre-infected with A. pleuropneumoniae followed by viral infection. The results showed that a pre-infection with A. pleuropneumoniae decreased PRRSV replication in SJPL cells, whereas A. pleuropneumoniae did not impair PRRSV replication in MARC-145 cells. Preliminary results indicate that a molecule secreted by A. pleuropneumoniae is the factor impairing PRRSV replication in SJPL cells. The factor is probably a small molecular weight molecule that is heat-resistant. In conclusion, both cell lines allowed the study of A. pleuropneumoniae and PRSSV interactions during a mixed-infection and these models could be adapted to study interactions of other swine pathogens.