Maintenance of a topological homeostasis by introduction and removal of the supercoils to relieve excessive strain on the DNA is a hallmark of topoisomerase function in the cell. The requirement of the topoisomerases during DNA transaction processes marks a ubiquitous presence of the enzymes in all the life forms. Different reactions carried out by the enzymes include relaxation of positive and negative supercoils required majorly during DNA replication and transcription, decatenation at the end of DNA replication to separate the daughter chromosomes and removal of lethal knots generated in the circular chromosome. In eubacteria, the enzymes introduce negative supercoils to facilitate easier strand separation for DNA transaction processes. However, in thermophiles, a different enzyme maintains the genome in a positively supercoiled form to protect from denaturation by excessive heat. These varied functions are carried out by different topoisomerases. Therefore, each organism maintains a minimum required set of the enzymes and the absence of a certain enzyme may be compensated for by topoisomerases with dual functions. For example, Mycobacterium tuberculosis and many other slow growing mycobacteria do not possess topoisomerase IV or its homologs. In these organisms, the DNA gyrase is suggested to carry out both negative supercoiling and decatenation reactions. Therefore, the mycobacterial DNA gyrase must be able to manage between both the functions in vivo. In contrast, Mycobacterium smegmatis and few other mycobacteria contain an additional type II topoisomerase which does not resemble any known type II enzyme but could catalyze relaxation and decatenation reactions. Importantly, the enzyme displays a unique ability to introduce limited positive supercoils and may have certain functions inside the cell which remains to be studied. Owing to the indispensability for bacterial survival topoisomerases present themselves as important drug targets. A large number of inhibitors have been found to inhibit the enzyme and thereby killing the bacterial. Among these, quinolones are successfully being used as broad spectrum antibacterial drugs. Although the commonly used quinolones inhibit many bacterial pathogens, a reduced susceptibility is exhibited by some of the pathogens e.g. Mycobacterium tuberculosis. To circumvent the lower efficacy of existing drugs, new and modified quinolones have been developed which are highly effective against mycobacteria. The difference in the susceptibility may be conferred by a difference in the chemical property of the drug and the interacting residues present in the enzyme. In the present thesis efforts have been made to understand the mechanism of the type II topoisomerases from mycobacteria and drug action on these enzymes. The thesis is divided into four chapters. In Chapter I of the thesis an introduction is provided on the topoisomerases, their classification and different reactions catalyzed by these enzymes. As the work in present thesis has been carried out with type II topoisomerases, introduction of type II enzymes, their structure and mechanisms is elaborated. DNA gyrase, its mechanism of reaction and in vitro and in vivo functions are explained in great detail. DNA gyrase and topoisomerase IV are targeted by a range of different inhibitors. These different classes of inhibitors and their mechanism of action are described. Finally, the mechanism of mycobacterial DNA gyrase with structural information and the current understanding of quinolone action on the enzyme are explained. The chapter ends with the objective of the study in the present thesis. In chapter II, the studies are aimed at understanding the molecular basis for decatenation carried out by mycobacterial DNA gyrase. Previous work from the laboratory showed that the enzyme can carry out decatenation more efficiently than its homolog from E. coli. It was shown that the mycobacterial enzyme binds two DNA molecules in trans in a length dependent manner. The ability to bind the second DNA is conferred upon the holoenzyme by ATPase subunit (GyrB) subunit which alone can bind DNA. Similar studies using topo IV from E. coli, the strongest known decatenase showed binding of two DNA molecules and the second DNA binding by ATPase (ParE) subunit. However, GyrB subunit from E. coli DNA gyrase, a weaker decatenase, does not bind second DNA molecule efficiently. The results provide a general mechanism for decatenation by type II enzymes in which efficient binding of second DNA is important. In Chapter III, studies have been carried out using topo NM, an atypical type II topoisomerase from Mycobacterium smegmatis. The enzyme has been characterized previously in the laboratory. In addition to efficient decatenation and relaxation, the enzyme exhibits a unique ability to introduce positive supercoils into the DNA. As demonstrated for the mycobacterial DNA gyrase and topo IV in the Chapter II, the ATPase subunit (Topo N) of topo NM, binds second DNA efficiently. The binding of both gate and transport segments increases with the length of the DNA. Binding of two DNA molecules by the holoenzyme appears to be a cumulative effect of DNA binding to individual subunits. In the absence of any inhibitor, the enzyme accumulates cleaved DNA products with shorter DNA but not with larger DNA. The cleavage of the shorter DNA is supported only in the presence of Mg2+ and Mn2+. Another important property of the enzyme is to introduce positive supercoils which appears to be due to its efficient utilization of ATP and a high rate of reaction. Chapter IV deals with the interaction of mycobacterial gyrase with fluoroquinolones (FQs). Although DNA gyrase is the sole target of the FQs in M. tuberculosis, the lower susceptibility to commonly used FQs have led to the studies to find out more effective quinolones. Previous studies from the laboratory showed a lower susceptibility of the mycobacterial gyrase to ciprofloxacin, but moxifloxacin could inhibit the enzyme efficiently. The better inhibition by moxifloxacin appears to be due to efficient trapping of the enzyme-DNA covalent complex. Both ciprofloxacin and moxifloxacin bind the DNA gyrase from mycobacteria, E. coli and E. coli topo IV, independent of DNA. The extent of binding also correlates with the inhibition potential of the drug against a given enzyme. A general model of quinolone enzyme interaction is provided wherein the quinolones are shown to interact with GyrA subunit or holoenzyme or the enzyme- DNA complex which would finally result in the trapping of the covalent complex.

No experimento I, objetivou-se avaliar os efeitos de aditivos microbianos e tempo de armazenamento na qualidade de silagens de milho. A cultura do milho foi ensilada sem aditivos (Controle) ou com inoculantes contendo bactérias láticas homofermentativas (Lactobacillus plantarum + Enterococcus faecium + Pediococcus acidilactici) + enzimas celulolíticas e hemiceluloliticas ou heterofermentativas (Lactobacillus buchneri) aplicados na dose 1 × 105 ufc/g. As silagens foram armazenadas em silos experimentais durante 3, 7, 15, 30, 60, 210, 390, 480 ou 570 dias. Foram avaliados: composição químico-bromatológica, produtos de fermentação, perfil microbiológico, perdas fermentativas, estabilidade aeróbia e degradabilidade ruminal in situ. Os inoculantes não afetaram a maior parte das variáveis estudadas. Entretanto, L. buchneri aumentou a concentração de ácido acético das silagens e diminuiu a deterioração aeróbia, confirmada pelo menor acúmulo térmico durante o ensaio de estabilidade aeróbia. O teor de carboidratos solúveis diminuiu ao longo do tempo de armazenamento, reflexo do metabolismo dos açúcares em produtos de fermentação. As concentrações da prolamina como esperado diminuíram e as concentrações de nitrogênio amoniacal e proteína solúvel aumentaram com os tempos de armazenagem prolongados, reflexo da ocorrência de proteólise na silagem de milho. O pH da silagem diminuiu rapidamente nos primeiros sete dias de armazenamentos mantendo-se estável para os demais tempos de estocagem, diferente do ocorrido para a concentração de ácido acético, que aumentou com o tempo de armazenamento. Verificou-se diminuição na contagem de bactérias láticas e leveduras ao longo do armazenamento. A produção de gás e a perda de matéria seca aumentaram com o tempo de armazenamento. O tempo de armazenamento aumentou todas as variáveis de EA. Os ganhos mais significativos para a EA se deram até aproximadamente 60 dias de armazenamento. A degradação ruminal de amido e, consequentemente de MS, foi incrementada ao longo do armazenamento. No experimento II, objetivou-se avaliar o desempenho de vacas leiteiras em função da estratégia de descarregamento da silagem de milho em silo do tipo trincheira: silagem de milho oriunda da metade superior do silo (topo) ou silagem de milho oriunda da metade inferior do silo (base). Foram utilizadas 24 vacas alocadas em 12 blocos casualisados, com arranjo de reversão simples com períodos de 21 dias. Os animais foram alojados em confinamento tie-stall. As dietas foram iso-protéicas (16,5%) e iso-amiláceas (17,0%), com 60% de silagem de milho (% MS). O consumo de matéria seca, produção e composição do leite foram determinados entre os dias 15 e 21 de cada período experimental. Apesar da silagem oriunda da base do silo levar à maior digestibilidade da dieta e menor concentração de nitrogênio ureico do leite (8,95 e 11,35 mg/dL) não houve efeito da dieta no consumo de matéria seca nem na produção de leite. Sob condições ótimas de manejo, a estratégia de descarregamento da silagem de milho não afeta o desempenho de vacas leiteiras. / In the experiment I, the aim was to evaluate the effects of microbial additives and length of storage on the quality of corn silage. Whole-corn plants were ensiled without or with inoculants containing homofermentative (Lactobacillus plantarum + Enterococcus faecium + Pediococcus acidilactici + cellulolytic and hemicellulolytic enzymes) or heterofermentative lactic acid bacteria (Lactobacillus buchneri) applied at 1 × 105 cfu/g. Treated forages were packed and stored in experimental silos for 3, 7, 15, 30, 60, 210, 390, 480, and 570 days. Samples were evaluated for chemical composition, fermentation end-products, microbial counts, fermentation losses, aerobic stability, and ruminal degradability in situ. Inoculants did not affect most of the variables studied. However, L. buchneri increased acetic acid concentration and decreased aerobic deterioration of silages, as indicated by the lower heat accumulation during the exposure to air. Soluble carbohydrates decreased across the storage period, reflecting the conversion of soluble sugars to fermentation end-products. Concentrations of prolamin decreased, whereas ammonia and soluble protein concentrations increased over the storage period, indicating the proteolysis. The silage pH declined rapidly in the first seven days of storage and remained stable for the remaining storage period, unlike for acetic acid concentration that increased with storage period. On the other hand, counts of lactic acid bacteria and yeasts decreased during the storage. Gas production and dry matter loss increased with the length of storage. The storage period increased all variables stability aerobic. Most of significant improvements in stability aerobic were observed during the first 60 days of storage. Ruminal degradability of starch and, consequently, dry matter increased along the storage. In the experiment II, the aim was to evaluate the influence of strategy of silage unload on the performance of dairy cows. Corn silage from a bunker silo was separated at unloading as silage from the upper half of the silo (top) or from the lower half of the silo (bottom) and used to compose total mixed rations fed to 24 lactating cows allocated in 12 randomized blocks, arranged in a cross-over design with 21 periods. Cows were housed in a tie-stall barn. All diets contained 60% of corn silage and were iso-nitrogen (16.5% CP) and iso-starch (17.0% of starch). Dry matter intake, milk yield and composition were determined from day 15 to 21 in each period. Although silage from bottom led to higher total tract DM digestibility and lower milk urea nitrogen concentration (8.95 vs. 11.35 mg/dL), most of evaluated variables were not affected by treatments. Under optimal silo management, the strategy used to unload corn silage does not affect the performance of dairy cows.

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Una dieta scorretta incrementa il rischio di malattie come l’insulino resistenza e l’obesità. Questa tesi ha l’obiettivo di valutare l’effetto di diete sbilanciate sulla fisiologia ed espressione genica in topi e suini allo svezzamento. Topi C57BL/6 sono stati sacrificati dopo 2 settimane, dopo essere stati alimentati con dieta iper-lipidica e dieta controllo. L’espressione genica è stata stimata usando la tecnologia microarray. Quattro dei sette geni identificati differenzialmente espressi tra il controllo e l’iper-lipidico sono coinvolti nella regolazione della via metabolica del sistema circadiano, che recentemente è stato mostrato avere effetti sul metabolismo lipidico e processo infiammatorio. Il secondo studio ha avuto lo scopo di capire gli effetti dello svezzamento con o senza l’aggiunta di acidificante nella dieta. I suinetti allo svezzamento (T0) sono stati comparati con i suinetti dopo una settimana (T1). Il gruppo post-svezzamento è stato alimentato con una dieta convenzionale, e metà di questi hanno ricevuto un supplemento di acido sorbico. L’aggiunta di acido sorbico nella dieta non ha causato nessuna differenza a livello fisiologico e di espressione genica. 205 geni sono stati identificati come differenzialmente espressi in T1 comparato con T0, evidenziando una forte risposta all’adattamento metabolico e agli stress subiti durante lo svezzamento. / An incorrect diet increases the risk of diseases as insulin resistance and obesity. This thesis aims at assessing the effects of unbalanced diets on gut physiology and gene expression in pig and mouse during weaning. The first research explored the impact of a high fat diet in C57BL/6 mice. High-fat-fed mice and control-fed mice were sacrificed after two weeks of treatment. Gene expression level was assessed by 90K Combimatrix microarray technology. Four of seven genes found differentially expressed between control and high fat diet mice are involved in the regulatory pathway of the circadian clock system, which was recently shown to affect lipid metabolism and inflammatory processes. Those genes were successfully validated by real time PCR. The second study aimed at understanding the weaning effect with or without acidifier addition in the diet. Piglets at weaning (T0) were compared to piglets after one week (T1). The post-weaning group was fed a conventional diet, half of which received in addition sorbic acid. The sorbic acid supplementation evidenced no effects in terms of physiology and gene expression. 205 genes were significantly differentially expressed in T1 when compared with T0, evidencing a response to the metabolic adaptation and the stress suffered during weaning.

Indiana University-Purdue University Indianapolis (IUPUI) / Colorectal cancer is the third most common type of cancer and the third most common cause of cancer-related mortality. There are three types of treatment available to patients, either individually or in combination. Treatments are radiation, chemotherapy, and surgery. In a Phase II clinical trial at IUSM, a multimodality approach was chosen. The patients with locally advanced rectal cancer received preoperative treatment with capecitabine and irinotecan (CPT-11) combination followed by chemoradiation with capecitabine and finally surgery to improve response and decrease local recurrence. Irinotecan and Capecitabine are both prodrugs activated in vivo to SN-38 and 5-FU, respectively. Identification of the molecular markers for 5-FU and Irinotecan efficacy and toxicity is important for the development of more efficient and less toxic treatment strategies for patients with colorectal cancer. The goal of this study was to determine the expression levels of the genes involved in activation and metabolism of capecitabine and irinotecan in pre and post treatment specimens from these patients. The genes quantitated by real-time PCR were carboxylesterase 1 and 2 (CES1 and CES2), thymidylate synthase (TS), β-glucoronidase (β-GUS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and topoisomerase I (Topo I). The UGT1A1*28 polymorphism in UDP glucuronosyltransferase 1 is associated with SN-38 toxicity. Therefore, the UGT1A1*28 polymorphism status in patients was determined by PCR-sequencing. Correlative analysis of gene expression and UGT1A1*28 mutation with clinical outcome in this Phase II study was completed.

[pt] Os deslizamentos ocorridos em grandes escalas ao redor do mundo decorrentes, principalmente, de eventos chuvosos extremos criaram um sinal de alerta para a necessidade de compreensão dos mecanismos de deflagração desses movimentos de massa. No entanto, a influência de variáveis fundamentais, como a espessura de solo e as condições iniciais de umidade, no estudo de estabilidade de bacias hidrográficas ainda não foi totalmente esclarecido. Nesta dissertação, simulações numéricas de fluxo e análise de estabilidade de encostas são realizadas nas bacias hidrográficas dos rios Quitite e Papagaio. Primeiramente, dois modelos analíticos de espessura de solo são utilizados em simulações envolvendo o fluxo tridimensional saturado-não saturado com o uso do método dos elementos finitos. A condição inicial de umidade também é analisada utilizando-se dados de precipitação por um período de spin-up. Posteriormente, o fator de segurança (FS) da região, via talude infinito, é analisado após a imposição de um evento chuvoso extremo. Dessa forma, é possível verificar que os intervalos de espessuras de solo de 0 a 3 m são os que mais se assemelham às cicatrizes mapeadas, sugerindo uma abordagem mais realística de espessura média para a bacia. Além disso, é verificado que, em eventos extremos, o uso do spin-up time para condição inicial de umidade variável pode não ter muita influência no fator de segurança, pois a intensidade e duração da chuva serão os agentes deflagradores. / [en] The large-scale landslides that have occurred around the world, mainly due to extreme rainfall events, serve as a warning signal for the need to understand the triggering mechanisms of these mass movements. However, the influence of fundamental variables, such as soil thickness and initial moisture conditions, in the study of watershed stability has not been fully clarified. In this dissertation, numerical flow simulations and slope stability analysis are carried out for the hydrographic basins of the Quitite and Papagaio rivers. In this study, two analytical soil thickness models are applied to saturated-unsaturated three-dimensional finite element flow simulations. The initial moisture condition is analyzed using precipitation data for a spin-up period. Subsequently, the safety factor (SF) is analysed, using the infinite slope method, after the imposition of an extreme rainfall event. From these results, it is possible to verify that the ranges of soil thicknesses from 0 to 3 m are the ones that most resemble the mapped scars, suggesting a more realistic approach for considering the average basin thickness. In addition, it is verified that, in extreme events, the use of spin-up time for initial variable humidity conditions may not have much influence on the safety factor, since the intensity and duration of the rain will be the triggering agents.

No description available.