Sources of arsenic exposure include air, water, and food from both natural and anthropogenic sources. Arsenic is categorized as a human carcinogen, and is associated with pleiotropic toxicities including cancers of the skin, lung, and bladder. Despite arsenic's long recognition as a human carcinogen, the exact mechanisms of arsenical-induced carcinogenesis are unknown. Arsenic exposure has been shown to cause DNA damage. However, because arsenic does not directly react with DNA, genotoxicity is generally considered to result from indirect mechanisms. The generation of arsenical-induced reactive oxygen species and the inhibition of critical DNA repair systems are believed to contribute to arsenical-induced carcinogenicity. The DNA damaging effects of arsenical exposure and alterations in DNA repair processes were examined within the human bladder urothelial cell line, UROtsa, following continuous exposure to the arsenic metabolite, monomethylarsonous acid [MMA(III)]. Chronic, low-level MMA(III) exposure results in the induction of DNA damage that remains elevated following the removal of MMA(III). Furthermore, data presented herein, defines the critical period in which continuous low-level MMA(III) exposure causes the malignant transformation of the UROtsa cell line. Results indicate that malignant transformation of UROtsa cells is irreversible following 12 wk of low-level MMA(III) exposure. Assessment of the MMA(III)-induced biological alterations leading to the malignant transformation of UROtsa cells following 12 wk of exposure suggest two potential interdependent mechanisms in which MMA(III) may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation. These mechanisms include MMA(III)-induced DNA damage via the production of reactive oxygen species and the MMA(III)-induced inhibition of poly(ADP-ribose) polymerase-1 as a result of the direct MMA(III)-mediated displacement of zinc.

No description available.

<p>Urinary tract infections (UTIs) are typically caused by bacteria that colonize different regions of the urinary tract, mainly the bladder and the kidney. Approximately 25% of women that suffer from UTIs experience a recurrent infection within 6 months of the initial bout, making UTIs a serious economic burden resulting in more than 10 million hospital visits and $3.5 billion in healthcare costs in the United States alone. Type-1 fimbriated Uropathogenic E. coli (UPEC) is the major causative agent of UTIs, accounting for almost 90 % of bacterial UTIs. The unique ability of UPEC to bind and invade the superficial bladder epithelium allows the bacteria to persist inside epithelial niches and survive antibiotic treatment. Persistent, intracellular UPEC are retained in the bladder epithelium for long periods, making them a source of recurrent UTIs. Hence, the ability of UPEC to persist in the bladder is a matter of major health and economic concern, making studies exploring the underlying mechanism of UPEC persistence highly relevant. </p><p>In my thesis, I will describe how intracellular Uropathogenic E.coli (UPEC) evade host defense mechanisms in the superficial bladder epithelium. I will also describe some of the unique traits of persistent UPEC and explore strategies to induce their clearance from the bladder. I have discovered that the UPEC virulence factor Alpha-hemolysin (HlyA) plays a key role in the survival and persistence of UPEC in the superficial bladder epithelium. In-vitro and in-vivo studies comparing intracellular survival of wild type (WT) and hemolysin deficient UPEC suggested that HlyA is vital for UPEC persistence in the superficial bladder epithelium. Further in-vitro studies revealed that hemolysin helped UPEC persist intracellularly by evading the bacterial expulsion actions of the bladder cells and remarkably, this virulence factor also helped bacteria avoid t degradation in lysosomes. </p><p>To elucidate the mechanistic basis for how hemolysin promotes UPEC persistence in the urothelium, we initially focused on how hemolysin facilitates the evasion of UPEC expulsion from bladder cells. We found that upon entry, UPEC were encased in “exocytic vesicles” but as a result of HlyA expression these bacteria escaped these vesicles and entered the cytosol. Consequently, these bacteria were able to avoid expulsion by the cellular export machinery. </p><p>Since bacteria found in the cytosol of host cells are typically recognized by the cellular autophagy pathway and transported to the lysosomes where they are degraded, we explored why this was not the case here. We observed that although cytosolic HlyA expressing UPEC were recognized and encased by the autophagy system and transported to lysosomes, the bacteria appeared to avoid degradation in these normally degradative compartments. A closer examination of the bacteria containing lysosomes revealed that they lacked V-ATPase. V-ATPase is a well-known proton pump essential for the acidification of mammalian intracellular degradative compartments, allowing for the proper functioning of degradative proteases. The absence of V-ATPase appeared to be due to hemolysin mediated alteration of the bladder cell F-actin network. From these studies, it is clear that UPEC hemolysin facilitates UPEC persistence in the superficial bladder epithelium by helping bacteria avoid expulsion by the exocytic machinery of the cell and at the same time enabling the bacteria avoid degradation when the bacteria are shuttled into the lysosomes. </p><p>Interestingly even though UPEC appear to avoid elimination from the bladder cell their ability to multiple in bladder cells seem limited.. Indeed, our in-vitro and in-vivo experiments reveal that UPEC survive in superficial bladder epithelium for extended periods of time without a significantly change in CFU numbers. Indeed, we observed these bacteria appeared quiescent in nature. This observation was supported by the observation that UPEC genetically unable to enter a quiescence phase exhibited limited ability to persist in bladder cells in vitro and in vivo, in the mouse bladder. </p><p>The studies elucidated in this thesis reveal how UPEC toxin, Alpha-hemolysin plays a significant role in promoting UPEC persistence via the modulation of the vesicular compartmentalization of UPEC at two different stages of the infection in the superficial bladder epithelium. These results highlight the importance of UPEC Alpha-hemolysin as an essential determinant of UPEC persistence in the urinary bladder.</p> / Dissertation

Bladder pain syndrome (BPS)/interstitial cystitis (IC) is a chronic pain disorder characterized by at least six weeks of lower urinary tract symptoms and unpleasant sensations (pain, pressure and discomfort) thought to be related to the urinary bladder and not meeting exclusion criteria. While the etiology is not known, BPS/IC may involve a "vicious circle" of uroepithelial dysfunction, inflammation and peripheral and central sensitization. We propose that the urinary bladder inflammatory insult partly mediates voiding dysfunction and visceral neurogenic pain characteristic of BPS/IC. Several studies from our laboratory have already demonstrated the role(s) of cytokines and their downstream targets in the functional alterations in micturition reflex pathways following chemically (cyclophosphamide, CYP)-induced cystitis. More recently, the pleiotropic protein, TGF-β, has been implicated in the pathogenesis of CYP-induced cystitis. TGF-β is activated locally at the initial site of injury by protease-dependent or protease-independent mechanisms to initiate a proinflammatory milieu. Depending on its contextual cues, TGF-β may then aid in resolving the primary immune response and support tissue repair. Though TGF-β is necessary to maintain normal immunological function, its aberrant expression and activation may have detrimental effects on responding tissues and cell types. A sustained increase in peripheral TGF-β reactivity, such as what may be observed in chronic inflammatory bladder conditions, may influence bladder afferent excitability to amplify nociceptive transmission and CNS input. The subsequent sensitization of peripheral afferent nociceptors at the level of the DRG or urothelium may promote spinal cord "wind-up" and cascade into visceral hyperalgesia and allodynia. In the first aim of this dissertation we investigated the functional profile of TGF-β isoforms and receptor (TβR) variants in the normal and inflamed (CYP-induced cystitis) urinary bladder with qRT-PCR, ELISA, IHC and in vivo cystometry. Our studies determined (i) the involvement of TGF-β in lower urinary tract neuroplasticity following urinary bladder inflammation, (ii) a functional role for TGF-β signaling in the afferent limb of the micturition reflex and (iii) urinary bladder TβR-1 as a viable target to reduce voiding frequency with cystitis. In the second aim of this dissertation we investigated the sensory components of the urinary bladder that may underlie the pathophysiology of aberrant TGF-β activation with bladder-pelvic nerve electrophysiology and luciferin-luciferase assays for ATP measurement. Our studies determined that TGF-β1 increased bladder afferent nerve excitability by stimulating ATP release from the urothelium via vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels. Furthermore, blocking aberrant TGF-β signaling in CYP-induced cystitis with TβR-1 inhibition decreased afferent nerve excitability with an equivalent decrease in ATP release. Taken together, these results establish a causal link between an inflammatory mediator, TGF-β, and intrinsic signaling mechanisms of the urothelium that may contribute to the altered sensory processing of bladder filling to facilitate increased voiding frequency. The distinct interactions of multiple mediators underscore the challenges for single target therapies and support the development of combinatory therapeutics for bladder dysfunction. Ultimately, these studies have increased our understanding of functional disorders and visceral pain and have the potential to improve the health of those suffering from inflammation-associated bladder syndromes.

Overactive bladder (OAB), involuntary contractions during bladder filling, is a common condition affecting 17% of the adult population worldwide, and in the U.S. ranks ahead of diabetes in a list of the 10 most common chronic disorders (Mullins 2009). Mechanical mechanisms contributing to OAB are not completely understood and because of the unique function and broad volume range of the bladder, there may be mechanical characteristics that distinguish detrusor smooth muscle (DSM) in bladder from other smooth muscles. Recent studies have shown that the length-passive tension curve in DSM exhibits adjustable passive stiffness (APS) characterized by a passive curve that can be shifted along the length axis as a function of strain history and activation history; however, the mechanical mechanisms responsible for APS remain to be determined. Also, whether DSM exhibits a dynamic length-active tension relationship, as has been identified in airway and vascular smooth muscles, has not been investigated. This dissertation focused on both the passive and active length-tension relationships in DSM and the mechanical mechanisms responsible for these relationships. The first objective was to study the impact of APS on the length-total tension relationship and identify the mechanical mechanisms responsible for generating APS. The second objective was to determine whether the length-active tension relationship is adaptive and identify specific mechanical mechanisms contributing to any adaptive behavior. The results showed that a shift in the length-passive tension curve due to APS corresponded with a shift in the length-total tension curve in DSM, and that APS was 27.0±8.4% of active tension at the optimum length for active tension generation. Most importantly, low-grade rhythmic contraction (RC), which can occur spontaneously in rabbit and human bladders, regenerated APS. Results also showed that the length-active tension curve shifted due to stretch to and then activation at long lengths, as well as either multiple KCl-induced maximal contractions or RC. Thus, DSM exhibits length adaptation, and RC may contribute to both APS and length adaptation. Because increased RC has been correlated with OAB, understanding RC, APS and length-adaptation in bladder may enable the identification of specific targets for new treatments for OAB.

Ce travail de thèse est centré sur l'établissement de modèles prédictifs de toxicité radio-induite et sur l’étude de leur intérêt en cas de radiothérapie par modulation d’intensité. Six modèles NTCP ont été implémentés et leur paramètres identifiés pour la prédiction des toxicités rectale et vésicale tardives dans le cancer de la prostate. Leur capacité prédictive a été démontrée pour les deux organes. Par ailleurs, le modèle LKB a été utilisé pour la prédiction de l’œsophagite aiguë en cas de radiothérapie du cancer bronchique non à petites cellules. Ensuite, le bénéfice tiré de l’incorporation du paramètre de dose équivalent uniforme (EUD) pour la planification inverse de la radiothérapie par modulation d’intensité (IMRT) a été évalué. L’évaluation de cette approche a montré une baisse significative de la dose dans les parois vésicale et rectale. L’incorporation de plusieurs modèles biologiques dans le processus d’optimisation de l’IMRT a aussi été réalisée. Des fonctions objectif ont été établies pour les différents facteurs biologiques comme le NTCP, l’EUD et le TCP. Les résultats dosimétriques obtenus montrent la supériorité de l’optimisation basée sur des facteurs biologiques sur celle reposant uniquement sur des facteurs physiques. Enfin, les modèles NTCP classiques ont été améliorés en intégrant un paramètre radiobiologique supplémentaire, le rapport α/β. Ce rapport α/β a été identifié pour différents types de toxicité. Avec ce nouveau paramètre, les modèles NTCP peuvent finalement être étendus à des patients traités suivant différents fractionnements, les traitements hypofractionnés étant de plus en plus utilisés. / This thesis is focused on the predictive models of irradiation induced toxicities in intensity modulated radiotherapy. Six different NTCP models were implemented and their parameters were identified at predicting late rectal and bladder toxicities in prostate cancer. Their predictive skills have been demonstrated on both organs. Second, LKB model was used to predict the irradiation induced acute esophagitis after nun-small-cell lung cancer. Then, the benefit of using EUD in prostate cancer IMRT inverse planning was evaluated. The evaluation of the proposed approach proved that the use of EUD significantly decreased both the dose in the bladder and rectum walls. Then, the incorporation of different biological models in IMRT optimization process has been realized. Objective functions were established for different biological factors like NTCP, EUD and TCP. Obtained results show the superiority of the optimization based on biological factors over the optimization relying only on physical factors. Finally, classical NTCP models were corrected to deal with another radiobiological parameter, the α/β ratio. With this additional factor, NTCP models can be extended to predict toxicity for patients with different dose fractionation, these kinds of treatments being more and more clinically used.

INTRODUÇÃO: A tuberculose urogenital é rara, tem diagnóstico tardio e é potencialmente destrutiva ao trato urogenital masculino. A bexiga contraída ocorre nas fases tardias de sua evolução e está associada à alta freqüência de exclusão renal unilateral, refluxo vésico-ureteral, estenose ureteral e insuficiência renal. A ampliação vesical é o tratamento padrão para estes casos. O presente trabalho avalia o seguimento tardio de 25 pacientes com bexiga contraída por tuberculose submetidos à ampliação vesical. CASUÍSTICA E MÉTODOS: Vinte homens e cinco mulheres, com idade mediana de 40 (12 a 60) anos foram estudados. Antes da ampliação, três pacientes estavam em insuficiência renal crônica em programação para transplante renal. Os demais pacientes possuíam exclusão renal funcional unilateral. Em oito casos, a ampliação foi feita com segmento ileocecal não destubulizado, em quatro com sigmóide não destubulizado e em 13 com sigmóide destubulizado. Os pacientes foram submetidos à avaliação clínica, radiológica e urodinâmica pós-operatória. Foi considerado bom resultado, após a ampliação, a presença de intervalo miccional diurno maior que duas horas e a satisfação do paciente avaliada pela pergunta sobre qualidade de vida do questionário \"ICSmaleSF\". RESULTADOS: O seguimento pós-operatório médio foi de 11,1 ± 9,1 (1 a 36) anos com 68% dos pacientes com seguimento maior que cinco anos e 52% maior que 10 anos. Um paciente morreu por um adenocarcinoma na bexiga ampliada após 25 anos de ampliação e seis anos de transplante renal. Após a ampliação, houve manutenção da mesma função renal em todos os pacientes, com exceção de dois casos de evolução para insuficiência renal crônica. Em sete (28%) casos, houve alto resíduo pós miccional com resolução após desobstrução cirúrgica em três casos e autocateterismo nos demais. Bom resultado foi encontrado em 80% dos pacientes operados. O mau resultado foi associado estatisticamente com a utilização do sigmóide não destubulizado (p <= 0,05) e tendeu a se associar com a presença de prostatite por tuberculose (p = 0,09). A comparação dos pacientes de mau com os de bom resultado mostrou que estes apresentaram, ao exame urodinâmico, bexiga ampliada com maior capacidade (p < 0,01), maior complacência (p < 0,01) e sensibilidade normal (p = 0,03). Entretanto, não houve diferença na presença de contrações involuntárias (p = 0,27) entre os dois grupos. Nos pacientes com bom resultado, as contrações iniciaram-se com maior volume de enchimento vesical (p = 0,02). CONCLUSÕES: No seguimento tardio da ampliação da bexiga contraída por tuberculose, 80% dos pacientes atingem intervalo miccional maior que duas horas e a ampliação vesical não contribui para a piora da função renal. O cólon sigmóide deve ser destubulizado, mas o segmento ileocecal pode ser utilizado na sua forma original sem destubulização para a ampliação vesical. O bom resultado com intervalo miccional maior que duas horas necessita de bexiga ampliada com capacidade maior que 250 ml, complacência maior que 20 ml/cm H2O e sensibilidade normal, sem influência da presença de contrações involuntárias. / INTRODUCTION: Urogenital tuberculosis is a rare disease with delayed diagnosis and is potentially destructive for the male urogenital tract. Chronic tuberculous cystitis is a late event in the tuberculosis evolution and is associated with high frequency of unilateral nonfunctioning kidney, ureteral reflux or stenosis and renal failure. Bladder augmentation is the standard treatment in these cases. The present study analyzes the late results of 25 patients with chronic tuberculous cystitis submitted to bladder augmentation. PATIENTS AND METHODS: Twenty men and five women, with median age of 40 (12 to 60) years were evaluated. Prior to augmentation, three patients had chronic renal failure and were in kidney transplantation program. The remaining patients had unilateral nonfunctioning kidney. In eight cases the augmentation was performed with tubularized ileocecal segment, in four with tubularized sigmoid and in 13 with detubularized sigmoid. All patients were submitted to postoperative clinical, radiological and urodynamic evaluation. It was considered a good result the miccional diurnal frequency of more than two hours and the patient?s satisfaction evaluated by the quality of life question from the ICSmaleSF questionnaire. RESULTS: The median follow-up time was 11,1 ± 9,1 (1 a 36) years. In 68% of the patients this time was higher than five years and in 52% higher than ten years. One patient died due to an adenocarcinoma in the augmented bladder 25 years after bladder augmentation and six years after kidney transplantation. After augmentation, all but two patients had the renal function preserved. In seven (28%) cases there was high post-void residue treated by surgery for bladder outlet obstruction in three cases and by intermittent self-catheterisation in the others. Good results were achieved in 80% of the patients. Bad results were statistically associated to augmentation performed with tubularized sigmoid (p <= 0,05) and in patients with prostatitis as a tendency (p = 0,09). The good result patients showed augmented bladder with higher capacity (p < 0,01), higher compliance (p < 0,01) and normal bladder sensation (p = 0,03) in comparison to the bad result patients. However, there was no difference in the frequency of involuntary contractions (p = 0,27) between these two groups. In good result patients the contractions started with higher bladder filling volume (p = 0,02). CONCLUSIONS: At late follow-up of bladder augmentation 80% of the patients with chronic tuberculous cystitis achieve miccional diurnal frequency of more than two hours and the augmented bladder does not contribute to the worsening of the renal function. The sigmoid has to be always detubularized but the ileocecal segment can be used in the tubularized form to augment the bladder. Augmented bladder with capacity of more than 250 ml, compliance of more than 20 ml/cm H2O and normal sensation is necessary to achieve miccional diurnal frequency of more than two hours and there is no interference of the presence of involuntary contractions.

Introdução: Ocupação foi identificada como o segundo fator de risco mais importante para o câncer de bexiga depois de fumar sendo responsável por até 20% de todos os cânceres de bexiga em países industrializados. Apesar dos esforços consideráveis para investigar ocupações em relação ao risco de câncer de bexiga, muitas não foram encontrados de forma consistente. Material e Métodos: Foram incluídos 200 pacientes com diagnóstico de câncer de bexiga entre os anos de 2009 e 2013. Foi aplicado um questionário para obter informações sobre a profissão, tempo de exposição e hábitos diários, sintomas, e também dados de doenças incluindo estágio, grau e número e tamanho de lesões. Os pacientes do Grupo 1 foram aqueles sem emprego previamente associados com o risco de câncer de bexiga. Grupo 2 representado pacientes em risco devido a profissões. Resultados: Os pacientes do Grupo 2 apresentaram uma proporção significativamente maior de pT2 CaB (P = 0,037), enquanto que os pacientes do grupo 1 apresentaram significativamente mais pTa (p = 0,002) da doença. Analisando preditores de pT2, a presença de ocupação aumento de alto risco por 2,80 vezes a chance de desenvolver uma doença invasiva. Ao analisar o grau do tumor descobriram que um tempo de exposição de 10 anos ou mais aumenta o risco de tumores de alto grau em 4,28 vezes (p = 0,001). Conclusão: Pacientes com história de exposição a agentes cancerígenos devido à sua atividade profissional podem estar em maior risco de desenvolvimento de tumores invasivos e aqueles que estão expostos a estes agentes para mais de 10 anos podem desenvolver doença de alto grau com mais freqüência. População em risco pode, portanto, beneficiar de rastreamento para o câncer de bexiga / Occupation was identified as the second most important risk factor for bladder cancer after smoking accounting for up to 20% of all bladder cancers in industrialized countries. Despite considerable efforts to investigate occupation against the risk of bladder cancer, many have not been found consistently. We included 200 patients diagnosed with bladder cancer between 2009 and 2013. A questionnaire was applied to obtain information about the profession, exposure time and daily habits, symptoms, and also diseases of data including stage, grade and number and lesion size. Patients in Group 1 were those without jobs previously associated with the risk of bladder cancer. Group 2 represented patients at risk because of professions. Group 2 patients had a significantly higher proportion of pT2 CaB (P = 0.037), whereas patients in group 1 had significantly more pTa (p = 0.002) of the disease. Analyzing predictors of pT2, the presence of high-risk occupation increases by 2.80 times the chance of developing invasive disease. In considering the degree of tumor found that a time of 10 or more years of exposure increases the risk of high-grade tumors in 4.28 times (p = 0.001). Patients with a history of exposure to carcinogens because of their duties may be at greater risk of developing invasive tumors and those who are exposed to these agents for more than 10 years can develop high-grade disease more often. Population at risk can therefore benefit from screening for bladder cancer

O carcinoma da bexiga urinária (CB) é a segunda doença maligna mais frequente do trato urinário. Devido a baixa eficácia dos tratamentos intravesicais atuais (imunoterapia com BCG e quimioterapia), seja pelo baixo tempo de residência do fármaco na bexiga ou pela baixa permeabilidade no urotélio, novas estratégias que aumentem esse tempo de residência do fármaco e sua penetração na bexiga têm sido investigadas. Dentre estas estratégias pode-se citar os sistemas de liberação sustentada nanoestruturados, que liberam o fármaco gradativamente, protegem o fármaco encapsulado, aumenta a biodisponibilidade, aumentando a eficácia da terapia e diminuindo os efeitos adversos. Nesta linha, o objetivo desse trabalho foi produzir e utilizar carreadores lipídicos nanoestruturados (CLN) como sistema de carreamento de doxorrubicina (DOXO) e a sua avaliação biológica em células de câncer de bexiga. Os CLN, compostos por manteiga de Illipê (lipídeo sólido), ácido oleico (óleo) e o estabilizante Pluronic F68, foram preparados pelo método de emulsão a quente e sonicação. A caracterização físico-química do CLN foi realizada determinando o diâmetro hidrodinâmico médio e potencial zeta (carga superficial) por espalhamento de luz dinâmico (DLS), cristalinidade por calorimetria exploratória diferencial (DSC), eficiência de encapsulamento por espectrofotometria UV-vis, ensaio de citotoxicidade em células RT4 e análise de permeação ex vivo e in vivo das formulações aplicadas em bexiga de porco por microscopia confocal. O diâmetro hidrodinâmico médio dos CLN sem o fármaco foi de 103 nm e seu PdI (índice de polidispersão) igual a 0,2. O encapsulamento da DOXO aumentou o diâmetro dos CLN para 112 nm e o valor de PdI foi de 0,2. O baixo valor de PdI indica formulações com baixa polidispersão. Os valores de potencial zeta dos CLN sem e com DOXO foram ambos negativos, variando de -5 mV a -25 mV. Nos termogramas das amostras de CLN-DOXO não foi observado o pico referente a fusão da DOXO em 197,93ºC, indicando que o fármaco provavelmente está molecularmente disperso na matriz lipídica. Nos ensaios de citotoxicidade, a formulação CLN-DOXO mostrou-se mais citotóxica do que a DOXO livre em baixas concentrações (31-250 ng/mL). O valor de IC50 reduziu 2,1 vezes quando a DOXO foi encapsulada. Essa maior atividade antitumoral in vitro pode estar relacionada ao aumento do uptake celular como confirmado pelos ensaios de citometria de fluxo. No estudo de permeação ex vivo em bexiga de porco foram observadas permeações muito semelhantes das formulações de DOXO livre e encapsulada em CLN. Porém no ensaio in vivo a DOXO encapsulada permeou mais que a DOXO livre. Os resultados obtidos apontam para um promissor sistema de liberação de doxorrubicina para a terapia do câncer de bexiga. / Urinary bladder carcinoma (BC) is the second most common malignant disease of the urinary tract. Due to the low efficacy of current intravesical treatments (BCG immunotherapy and chemotherapy), either because of the low residence time of the drug in the bladder or due to low permeability in the urothelium, new strategies that increase the residence time of the drug and its penetration into the bladder have been investigated. These strategies include nanostructured sustained release systems, which release the drug gradually, protect the encapsulated drug, increase the bioavailability; increasing the effectiveness of the therapy and reducing side effects. In this line, the aim of this work was to produce and apply nanostructured lipid carriers (NLC) as a doxorubicin delivery system (DOXO) and its biological evaluation in bladder cancer cells. The NLC, composed by Illipe butter (solid lipid), oleic acid (oil) and Pluronic F68 stabilizer, were prepared by the hot emulsion and sonication method. The physical-chemical characterization of these NLC was performed by measured the mean hydrodynamic diameter and zeta potential (surface charge) by dynamic light scattering (DLS), crystallinity by differential scanning calorimetry (DSC), encapsulation efficiency by UV-vis spectrophotometry, cytotoxicity assay in RT4 cells and ex vivo and in vivo permeation assay of formulations applied to pig bladder by confocal microscopy. The mean hydrodynamic diameter of NLC without the drug was 103 nm and its PdI (polydispersity index) was 0.2. The encapsulation of DOXO increased the NLC diameter to 112 nm and the PdI value was 0.2. The low value of PdI indicates formulations with low polydispersion. The zeta potential values of the NLC without and with DOXO were both negative (-5 mV to -25 mV). In the thermograms of the NLC-DOXO sample the fusion peak of DOXO at 197.93 was not observed, indicating that the drug is molecularly dispersed in the lipid matrix. In the cytotoxicity assays, the NLC-DOXO formulation was more cytotoxic than free DOXO at low concentrations (31-250 ng/mL). The IC50 value was reduced 2.1 fold when DOXO was encapsulated. This increased in vitro antitumor activity may be related to increased cell uptake as confirmed by flow cytometry analysis. In the ex vivo permeation study in the pig bladder, a very similar permeation was observed between free and encapsulated DOXO. However, in the in vivo assay the encapsulated DOXO permeated more than free DOXO. The results indicates a promising release system of doxorubicin to bladder cancer therapy.

Introdução: O câncer de bexiga é a segunda neoplasia maligna mais frequente do trato urinário, com 386.000 casos estimados e 150.000 mortes para 2011 no mundo. Noventa e cinco por cento são carcinomas uroteliais (CUB) papilíferos não músculo-invasivos de baixo grau, que apresentam altas taxas de recidiva, mas raramente progridem. Tumores invasivos de alto grau representam 10-20% dos diagnósticos, são altamente agressivos levando à mortalidade elevada. O conhecimento das vias moleculares envolvidas na carcinogênese dessa neoplasia é importante para a identificação de novos marcadores para diagnóstico, acompanhamento, prognóstico e desenvolvimento de novas terapias alvo. Micro RNA (miRNA) são pequenas sequências não codificantes de RNA que regulam a expressão dos genes inibindo a tradução da proteína ou promovendo a degradação do RNA mensageiro, estando atualmente envolvidos em vários processos celulares fisiológicos e patológicos, incluindo o câncer. Objetivos: Caracterizar o perfil de expressão de miRNA no CUB, relacionando-o com os parâmetros prognósticos clássicos para a doença: grau histológico e estadiamento. Além disso, relacionar esse padrão de comportamento dos miRNA com a recidiva tumoral e sobrevida câncer-específica em pacientes tratados cirurgicamente para CUB. Material e Métodos: Catorze miRNA (miR-100, miR-10a, miR-21, miR-205, miR-let7c, miR- 125b, miR-143, miR-145, miR-221, miR-223, miR-15a, miR-16-1, miR-199a e miR- 452) foram isolados de espécimes cirúrgicos de 60 pacientes divididos em 2 grupos: 30 pacientes com CUB não invasivo (pTa) de baixo grau submetidos à RTU de bexiga, 30 com CUB invasivo (pT2-3) de alto grau submetidos à cistectomia radical. O grupo controle é representado por cinco pacientes portadores de bexiga normal sem CUB que realizaram tratamento cirúrgico aberto para tratamento da hiperplasia prostática benigna (HPB). O processamento dos miRNA envolveu três fases: (1) extração do miRNA com kit específico, (2) geração do DNA complementar e (3) amplificação do miRNA por PCR quantitativo em tempo real (qRT-PCR). A expressão de cada miRNA foi obtida através do cálculo 2- CT e os RNU-43 e RNU-48 foram utilizados como controles endógenos. Testes estatísticos foram aplicados para estudar as variáveis envolvidas e curvas de Kaplan-Meyer foram usadas para avaliar a sobrevida livre de recidiva (SLR) e sobrevida câncer-específica (SCE). Resultados: Dos 14 miRNA estudados a maioria apresentou subexpressão nos dois grupos de tumor analisados, com exceção do miR-10a para o grupo pTa de baixo grau e do miR-100, 21 e 205 para os tumores pT2/pT3 de alto grau, onde demonstraram-se superexpressos. Essas diferenças de expressão de miRNA entre os dois grupos foram estatisticamente. Quando estudamos a relação entre expressão de miRNA e a evolução dos pacientes através de curvas de sobrevida, observamos que maiores níveis de expressão do miR-21 relacionou-se com menor SLR para tumores pTa. Ainda, maiores concentrações de miR-10a e miR-145 se associaram com menor SLR e maiores níveis de miR-10a com menor SCE para tumores pT2-3. Conclusões: Demonstramos um predomínio de subexpressão de miRNA em xv carcinomas de bexiga. Os miR-100, miR-10a, miR-21 e miR-205 demonstraram diferenças no perfil de expressão para grau e estadiamento dentro dos dois grupos de tumor, sendo capazes de diferenciá-los. Maiores níveis de miR-21 se relacionaram com menor SLR para tumores pTa de baixo grau, enquanto maiores concentrações de miR-10a estiveram associadas com menor SLR e SCE para tumores pT2/pT3 de alto grau / Introduction: Bladder cancer (BC) is the second most common malignancy of the urinary tract, with 386,000 cases estimated and 150,000 deaths in 2011. Urothelial carcinomas (UC) represent 95% of BC cases, and knowledge of the molecular pathways associated with BC carcinogenesis is crucial to identify new diagnostic and prognostic biomarkers, and development of new target molecular therapies. MicroRNAs (miRNAs) are short non-coding RNA molecules that play important roles in the regulation of gene expression by acting directly on mRNAs, leading to either mRNA degradation or inhibition of translation, involved in many physiological and pathological processes, including cancer. Objectives: To characterize miRNAs expression profiles in UC, associating with classic prognostic factors: grade and stage. Moreover, correlate miRNA expression with tumor recurrence and survival. Material and Methods: Fourteen miRNAs (miR-100, miR-10a, miR-21, miR-205, miR-let7c, miR-125b, miR-143, miR-145, miR-221, miR-223, miR-15a, miR-16-1, miR- 199a e miR-452) were isolated from surgical specimens from 60 patients classified in two groups: 30 patients with low-grade non-invasive pTa UC that underwent TURB, 30 with high-grade invasive pT2/pT3 UC underwent radical cystectomy. The control group consists in five normal bladder tissue taken from patients that underwent retropubic prostatectomy to treat benign prostatic hyperplasia (BPH). miRNA processing involved three phases: (1) miRNA extraction by specific kits, (2) cDNA generation (3) miRNA amplification through qRT-PCR. Expression profiles were obtained by relative quantification determined by 2-ct method. Endogenous control were RNU-43 and RNU-48. Statistic tests were used to study the prognostic variables and Kaplan-Meyer curves were constructed to analyze disease-free (DFS) and disease-specific (DSS) survivals. Results: All miRNAs were underexpressed in both groups, except miR-10a in pTa and miR-100, 21 and 205 in pT2/pT3 tumors, that where over-expressed. miR-100, miR-21, miR-10a and miR-205 differentialy expressed in both groups and this differences were statistically significant. The Kaplan-Meyer survival curves showed that higher levels of miR-21 were related to shorter DFS for pTa group. Also, higher levels of miR-10a and miR-145 were associated with shorter DFS and higher levels of miR-10a were also related to shorter DSS in pT2/pT3 group. Conclusions: The majority of miRNA were shown to be underexpressed in bladder UC. miR-100, miR-10a, miR-21 and miR-205 were differentially expressed considering tumor grade and stage. The miRNA profile was able to distinguish pTa low grade and pT2-3 high grade tumors. Higher levels of miR- 21 were related to shorter DFS in pTa, while higher levels of miR-10a were associated with shorter DFS and DSS in pT2-3, high grade UC