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.

There is a need for informative biomarkers in localised urological cancers. At present, no method can accurately distinguish between indolent and aggressive prostate cancers, and men often require repeated biopsies. Patients with muscle invasive bladder cancer undergo neo-adjuvant chemotherapy (NAC) to improve survival. However many do not respond to NAC, delaying definitive treatment. Cell-free mutant DNA (mutDNA) analysis represents an opportunity for non-invasive monitoring of cancer through tumour genome analysis. MutDNA derived from plasma can monitor tumour burden. There is emerging evidence that mutDNA can identify mutations from multiple clones and is abundant in adjacent body fluids. This work explores the utility of plasma and urinary mutDNA in localised prostate and bladder cancers. This thesis describes the optimisation of urinary mutDNA analysis by assessing urinary DNA processing and extraction methods using healthy volunteer and bladder cancer patient urine samples. Primer panels were designed and validated to target frequently mutated regions in prostate and bladder cancers, as well as for analysis of patient-specific mutations. Sequencing-based methods and dPCR were employed to analyse clinical samples including plasma and urine, to detect and quantify mutDNA. Molecular and clinical data were integrated to explore potential areas of application of mutDNA analysis. For bladder cancer, mutDNA was analysed from liquid-biopsy samples including plasma, cell pellets from urine and urine supernatant from multiple time-points of 17 MIBC patients undergoing NAC. I showed that mutDNA was more frequently detected and was present at higher AFs in urine compared to plasma samples. Of potential clinical relevance, I showed that the presence of mutDNA after starting NAC was associated with disease recurrence. This original contribution to knowledge could offer patients an opportunity to expedite surgical resection in a timely manner, if corroborated in large-scale trials. For prostate cancer, a TP53 specific panel was applied to men with metastatic disease, to demonstrate that clones containing TP53 mutations, which are dominant in at the metastatic stage were present in historical prostatectomy samples taken when then patient was believed to have localised disease only. Furthermore, I showed that these TP53 mutations could be detected at the localised stage of disease. To investigate the ability of mutDNA detection private clonal mutations I developed a method for higher sensitivity analysis (MRD-Seq). This was applied to a clinical cohort of 2 men with multi-focal localised prostate cancer to demonstrate the though the overall levels of mutDNA is low, private clonal mutations may be detectable. Taken together, these original contributions to knowledge could allow for less invasive surveillance of men with low risk prostate cancer and warrants further investigation. In this thesis, I used a range of molecular methods were applied to small cohorts of clinical samples from patients with urological malignancies, in an exploratory analysis. The molecular data was analysed in conjunction with clinical information to draw hypotheses on the biology and natural history of these cancer, and to suggest possible utility of mutDNA analysis in their clinical management. Some of the findings suggest areas of potential utility, which merit further validation or investigation in larger cohorts or clinical studies.

Guang Fu Chen. / "May 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 194-221). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Os efeitos da obstrução uretral parcial na musculatura da bexiga urinária de coelhos foram investigadas usando as ferramentas estereológicas. Foram utilizadas 12 fêmeas de coelhos da raça Norfolk, com três meses de idade e peso corporal variando de 2,5-3,0 kg. O procedimento cirúrgico consistiu de celiotomia mediana retro-umbilical para exposição da bexiga urinária. A parede dorsal da uretra foi divulsionada de sua íntima associação com o útero e vagina, o suficiente para a passagem de fio nylon 2-0. Um pino de Steinmann (3 mm de diâmetro) foi interposto temporariamente entre a uretra e o fio para determinar indiretamente o grau de obstrução uretral. Após três, sete e doze semanas os animais foram ortotanasiados e comparados com o grupo de animais controle (não obstruídos). Os fragmentos da bexiga foram preparados para microscopia de luz. Cortes seriados foram realizados para o estudo morfométrico e estereológico. Os três eixos: crânio-caudal (CC), dorso-ventral (DV) e latero-lateral (LL) aumentaram em todos os grupos analisados: controle, 3, 7 e 12 semanas. Os valores para CC foram estatisticamente similares para 3, 7 e 12 semanas. O mesmo foi observado no eixo DV. Os valores para o eixo LL foram similares para os grupos de 7 e 12 semanas. O estudo morfométrico baseou-se em determinar o tamanho da fibra (área seccional) e comprimento da fibra muscular. Nos animais do grupo de 3, 7 e 12 semanas foi observado um aumento de 4,63x, 4,32x e 7,10x no tamanho celular e um decréscimo de 2,55x, 1,94x e 4,04x no comprimento da fibra muscular quando comparados ao grupo controle. O estudo estereológico baseou-se em estimar o volume referência (Vref), a densidade numérica (Nv), o número total de fibras musculares (N), a densidade de volume (Vv) e o volume da fibra muscular (Vn). O Vref apresentou um aumento de 11,07x, 7,98x e 31,7x quando comparado com o grupo controle. A densidade numérica (Nv) aumentou 0,06x e 0,05x para os grupos de 3 e 7 semanas, respectivamente, em relação ao grupo controle. O grupo de 12 semanas, no entanto, apresentou um decréscimo de 0,01x em comparação com o grupo controle. Os grupos de 3, 7 e 12 semanas apresentaram, respectivamente, um aumento de 0,81x, 12,56x e 38,43x em número total de células. A densidade de volume (Vv) para os grupos de 3, 7 e 12 semanas apresentou um aumento de 0,97x, 0,56x e 0,86x em relação ao grupo controle. E finalmente, o volume médio da fibra muscular apresentou um aumento de 0,62x, 0,81x e 0,82x, respectivamente para os animais de 3, 7 e 12 semanas. Os dois mecanismos: hipertrofia e hiperplasia ocorrem na bexiga urinária de coelhos, porém não sabemos a seqüência exata em que aparecem. / The effects of partial urethral obstruction on rabbit´s urinary bladder musculature were investigated using stereological designed methods. A total of 12 female Norfolk rabbits weighing from 2.5 to 3 kg were used. A retro-umbilical celiotomy was made to expose the urinary bladder. The urethra´s dorsal wall was isolated from its association with the uterus. A 3mm-Steinmann-pin was positioned on the urethra to produce a standard degree of obstruction and a ligature was tied up around it, using a 2-0 nylon silk. Three, seven and twelve weeks after the surgery procedures the rabbits were euthanised. Bladder fragments were prepared for light microscopy. Serial sections were performed to morphometric and stereological study. In relation to the bladder axis: cranio-caudal (CC), dorso-ventral (DV) and latero-lateral (LL) increased in all groups analysed: control, 3, 7 and 12 week-obstructed animals. Values for CC were statistically similar for 3, 7 and 12-week-obstructed groups. The same was observed for DV axis. The LL axis showed values statistically similar for 7 and 12-week-obstructed groups. The morphometric study was based on the muscle fibre size (sectional area) and the muscle fibre length. In 3, 7 and 12-week-obstructed animals, it was observed a 4.63, 4.32 and 7.10-fold cell size increase and a 2.55, 1.94 and 4.04-fold decrease in length, respectively, when compared to control group. As for the stereological study. Vref presented a 11.07, 7.98 and 31.7-fold increase when compared to control subjects. Numerical density (Nv) increased by 0.06 and 0.05 in 3 and 7-week-obstructed groups, respectively, in relation to control group. Twelve week-obstructed group. Presented however a 0.01x-decrease compared to control animals. Three, seven and twelve-week-obstructed groups presented, respectively, 0.81, 12.56 and 38.43-fold increase in total number of cells (N). Volume density presented a 0.97, 0.56 and 0.86-fold increase in 3, 7 and 12-week-obstructed groups, respectively. And finally, mean muscle cell volume (Vn) presented a 0.62, 0.81 and 0.82-fold in 3, 7 and 12-week obstructed groups, respectively. Both mechanisms: hypertrophy and hyperplasia happened to occur on rabbit´s urinary bladder, thought we do not know the exact sequence in which they appear altogether.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-08-25T16:00:45Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:58:38Z (GMT) No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:58:46Z (GMT) No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-28T20:58:56Z (GMT). No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-03-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O Mycobacterium bovis BCG é utilizado mundialmente como vacina contra tuberculose há mais de meio século, com altos níveis de segurança. O BCG possui características que o tornam um promissor vetor para a produção de vacinas recombinantes multivalentes. Modificações genéticas podem aprimorar as propriedades imunogênicas e adjuvantes do BCG. Uma dessas modificações permite que o BCG escape do endossoma de células apresentadoras de antígenos. Desta maneira, os imunógenos de BCG acessam vias de apresentação cruzada de antígenos, ampliando a sua imunogenicidade. Este trabalho teve como objetivo desenvolver uma cepa de BCG ΔleuD expressando listeriolisina (Hly) de Listeria monocytogenes e avaliar seu potencial citotóxico na linhagem celular 5637 de câncer de bexiga. Para isso, o BCG ΔleuD foi transformado com o plasmídeo recombinante pUP410 contendo o gene hly. A cepa obtida demonstrou um nível de citotoxicidade similar à cepa BCG Pasteur parental, quando avaliada na linhagem celular de câncer de bexiga. Esta cepa necessita ser avaliada em outras linhagens de câncer para comprovar seu possível efeito citotóxico potencializado. / Mycobacterium bovis BCG has been used worldwide as a vaccine against tuberculosis for over fifty years with an outstanding safety record. BCG has features that make it a promising vector for production of recombinant multivalent vaccines. Genetic modification can enhance the adjuvant and immunogenic properties of BCG. One of these modifications would allow BCG to escape the endosome of antigen presenting cells. Therefore, theimmunogens of BCG can access antigens cross priming routes, increasing their immunogenicity. The aim of this work was to develop a strain of BCG ΔleuD expressing listeriolysin (Hly) of Listeria monocytogenes and evaluate its cytotoxic potential on cell line 5637 of bladder cancer. Thus, BCG ΔleuD was transformed with recombinant plasmid pUP410 containing the hly coding sequence. The recombinant BCG strain expressing listeriolysin showed a similar level of cytotoxicity when compared to the parental BCG Pasteur. This strain needs to be evaluated in other cancer lines in order to confirm its possible potentiated cytotoxic effect.

Lesões na inervação do trato urinário inferior ocasionado por traumatismo raquimedular afetam geralmente o músculo detrusor e o esfíncteres uretrais. Estas alterações acarretam problemas basicamente de incontinência urinária e aumento da pressão intravesical, decorrente deste traumatismo, trazendo consequências para o funcionamento do sistema urinário superior. Quantificar os elementos fibrosos da matriz extracelular e fibras musculares das bexigas neurogênicas hiper-reflexas comparando-as com bexigas normais. Foram utilizadas 6 amostras de bexigas neurogênicas de indivíduos que foram submetidos a cirurgia de reparação por cistoenteroplastia realizados pelo serviço de urologia do Hospital Municipal Souza Aguiar, estas amostras foram fixadas imediatamente em solução tamponada de formalina a 10%. O controle com amostras iguais as do estudo extraída de cadáveres cuja causa morte não relacionava-se ao sistema urogenital macroscópicamente. O material foi submetido as seguintes técnicas histoquímicas: H&E, van Gieson e Resorcina Fucsina resorcina de Weigert com prévia oxidação pela oxona. Imunohistoquímica: anti-elastina. A observação dos cortes corados pelo van Gieson demonstrou uma diminuição significativa do músculo liso de 13% e aumento do colágeno em 72% e as fibras do sistema elástico um aumento de 101%. Conclusão. Nas bexigas neurogênicas hiper-reflexas o músculo detrusor e os elementos fibrosos da matriz foram profundamente modificados. As fibras do sistema elástico foram as mais afetadas. / Lesions on lower urinary tract innervations caused by spinal cord injuries usually affect the detrusor muscle and urethral sphincter. Beside the smooth muscle fibers, the collagen fibers and elastic system fibers, fibrous components of the extracellular matrix of the bladder wall, are strongly related to vesicle bladder compliance. For this reason the aim of this work is to quantify the fibrous elements of the extracellular matrix and muscle fibers of the neurogenic bladder hyperreflexia. Samples of neurogenic bladder were obtained from six men who had previously undergone surgical repair. The control group samples (n=6) were similarly obtained from patients whose deaths were not related to the urogenital system. The samples were stained using the following histochemical techniques: H&E, Van Gieson, Weigert and Sirius Red. Sections stained with Sirius Red were observed under polarization light microscopy to characterize possible different kinds of collagen. Immunohistochemical technique was used to characterize and quantify the elastic system fibers. Quantification analysis was performed by stereological methods. An increase of 72% of the collagen was observed. Nevertheless, the most significant difference observed was the raising of 101% of the elastic system fibers. Contrary the smooth muscle fibers showed a decrease of 13%. In the neurogenic bladder with detrusor hyperreflexia the fibrous elements of the extracellular matrix and smooth muscle fibers were greatly modified. The elastic system fibers seem to be the most affected in this disease.

A complacência da bexiga depende de músculos lisos, fibras colágenas, fibras elásiticas e suas relações. O objetivo deste trabalho é determinar a composição da matriz extracelular em amostras de bexigas normais através de análise bioquímica de colágeno e glicosaminoglicanos em amostras obtidas de mulheres em diferentes grupos de idade, analisando separadamente as camadas urotelial e muscular. Avaliamos 17 amostras de bexiga divididas em três grupos: infância (N=5), menacme (N=6) e pós-menopausa (N=6). As bexigas foram analisadas para concentração de GAG total e colágeno e para análise qualitativa de GAG por eletroforese em gel de agarose. Na camada muscular, não houve diferença entre os grupos tanto para GAG quanto para colágeno. Na camada urotelial, a análise da concentração de colágeno não mostrou diferença entre os grupos, mas a concentração de GAG no grupo da pós-menopausa (0.21 0.12 μg de ácido hexurônico/mg de tecido seco) apresentou diferença em relação aos grupos do menacme (1.78 1.62 μg de ácido hexurônico/mg de tecido seco) e da infância ( 2.29 1.32 μg de ácido hexurônico/mg de tecido seco).Nosso trabalho concluiu que a concentração de GAG está substancialmente diminuída na camada urotelial da bexiga de mulheres na pós-menopausa. / Bladder compliance is dependent on smooth muscle, collagen fibers, elastic fiber and their ratios. The luminal surface of the urothelium is covered by an adhering glycosaminoglycan (GAG) layer. The aim of this study was to determine the composition of the extracellular matrix (ECM) in normal samples of women bladders through biochemistry analysis of collagen and GAG on samples obtained from individuals from different age groups, analyzing separately the urothelial and muscular layers. We studied samples taken from bladders of 17 patients divided in three different groups: childhood (N=5), menacme (N=6) and menopause (N=6). Bladders were analyzed for total GAG and collagen concentration per mg dry tissue and for the contents of GAG species, as determined by agarose electrophoresis and reported as the percent of total sulfated GAG. In muscular layer, collagen and GAG concentration showed no difference between groups. In urothelial layer, collagen concentration showed no difference between groups but GAG concentration in menopause ( 0.21 0.12 μg hexuronic acid /mg dry tissue) was different from menacme (1.78 1.62 μg hexuronic acid /mg dry tissue) and childhood ( 2.29 1.32 μg hexuronic acid /mg dry tissue). There was no difference between sulfated GAG in three groups.In conclusion, GAG concentration in urothelial layer was substantially lower in menopause women.

A radioterapia é frequentemente utilizada no tratamento de tumores da próstata, porém durante esse procedimento a bexiga sadia usualmente sofre efeitos colaterais. Através do uso de um modelo animal para irradiação pélvica, avaliamos se a suplementação nutricional com L-glutamina poderia prevenir possíveis danos na parede da bexiga, especialmente em suas camadas mais superficiais. Ratos Wistar adultos machos com idade entre 3 e 4 meses foram separados em grupos de 8 animais: grupo controle que não recebeu a irradiação; grupos somente irradiados que foram mortos 7 (R7) e 15 dias (R15) após a irradiação (dose única de 10 Gy na região pélvico-abdominal); grupos irradiados e suplementados com L-glutamina (0,65g/kg de peso por dia), que foram mortos 7 (RG7) ou 15 após a irradiação. Células e vasos sanguíneos da lâmina própria, bem como o urotélio, foram avaliados com métodos histológicos. No urotélio foram feitas análises da altura e densidade nuclear e na lâmina própria densidade celular, densidade vascular e o número de mastócitos. Os resultados mostraram que em R7, a altura e densidade nuclear do urotélio e a densidade celular da lâmina própria não foram alterados significativamente. Entretanto a densidade dos vasos sanguíneos foi reduzida em 48% (p<0,05) e essa alteração foi evitada pela glutamina (p <0,02). No grupo R15, a densidade celular do epitélio aumentou em 35% (p<0,02). A densidade celular da lâmina própria não apresentou diferença estatística entre os grupos. Os mastócitos na lâmina própria foram reduzidos em R7 e R15. Apesar de ainda reduzidos em RG7 em RG15 houve aumento no número desse tipo celular o que sugere uma ação positiva da glutamina. Células &#945;-actina positivas na lâmina própria formam uma camada suburotelial e foram identificadas como miofibroblastos. A espessura dessa camada aumentou em R7, mas foi semelhante ao controle em RG7, enquanto alterações em R15 e RG15 foram menos evidentes. Esses resultados mostraram que a utilização da L-glutamina antes e após a radioterapia deve ser considerada para uso humano na proteção da bexiga contra os efeitos da radiação. / Radiotherapy is often used to treat prostate tumors, but the normal bladder is usually adversely affected. Using an animal model of pelvic radiation, we investigated whether glutamine nutritional supplementation can prevent radiation-induced damage to the bladder, especially in its more superficial layers. Male rats aged 3 to 4 months were divided into groups of 8 animals each: controls, which consisted intact animals; radiated-only rats, which were sacrificed 7 (R7) or 15 (R15) days after a radiation session (10 Gy aimed at the pelvico-abdominal region); and radiated rats receiving L-glutamine supplementation (0.65 g/kg body weight/day), which were sacrificed 7 (RG7) or 15 (RG15) days after the radiation session. Morphological and morphometric analysis of the urothelium were made. Nuclear density, lamina propria cell density and mast cells numbers per area were counted. The results showed that, in R7, epithelial thickness, epithelial cell density, and cell density in the lamina propria were not significantly affected. However, density of blood vessels in R7 was reduced by 48% (p < 0.05) and this alteration was mostly prevented by glutamine (p < 0.02). In R15, density of blood vessels in the lamina propria was not significantly modified. However, epithelial thickness was reduced by 25% (p < 0.05) in R15, and this effect was prevented by glutamine (p < 0.01). In R15, epithelial cell density was increased by 35% (p < 0.02), but glutamine did not protect against this radiation-induced increase. Cell density in the lamina propria was likewise unaffected in R15. Density of mast cells in the lamina propria was markedly reduced in R7 and R15. The density was still reduced in RG7, but a higher density in RG15 suggested a glutamine-mediated recovery. Alpha-actin positive cells in the lamina propria formed a suburothelial layer and were identified as myofibroblasts. Thickness of this layer was increased in R7, but was similar to controls in RG7, while changes in R15 and RG15 were less evident. In conclusion, pelvic radiation leads to significant acute and post-acute alterations in the composition and structural features of the vesical lamina propria and epithelium. Most of these changes, however, can be prevented by glutamine nutritional supplementation. These results emphasize, therefore, the potential use of this aminoacid as a radioprotective drug.

Orientador: Márcia Rita Fernandes Machado / Banca: Antonio Carlos Alessi / Banca: Sheila Canevese Rahal / Resumo: Neste estudo, implantou-se peritônio bovino ou biomembrana de látex natural em defeito (1,5 x 1,5 cm) induzido na parede ventral da bexiga de coelhos, com o intuito de se avaliar o processo de reparação tecidual no que se refere à biocompatibilidade, capacidade de reparação e possíveis complicações. Utilizaram-se 36 coelhos, albinos, raça Nova Zelândia Branco, adultos, machos inteiros, divididos em grupos experimentais: grupo Biomembrana (GB), grupo peritônio bovino (GP) e grupo controle (GC), cada um com 12 animais. Aos sete, 14, 30 e 60 dias de pós-operatório, os animais foram eutanasiados mediante o emprego de uma dose elevada de tiopental sódico para avaliações macroscópica e histopatológica da interface do implante com o tecido nativo. Macroscopicamente, em ambos os grupos (GB, GP e GC), observaramse, em todos os períodos, aderências de estruturas adjacentes ao local do implante, presença de cálculos e ausência de sinais de rejeição. No grupo biomembrana, aos 30 e 60 dias de pós-operatório, constatou-se a presença do biocompósito solto na luz vesical. Sob microscopia de luz, aos sete dias verificaram-se intensa reação inflamatória, neovascularização e presença de fibroblastos proliferados. Aos 14 dias não havia epitélio, porém observaram-se escassos feixes musculares, intensa neovascularização, fibroblastos e infiltrado inflamatório. No grupo GP, aos 30 dias de observação, o implante estava presente, havia fibroblastos, feixes de fibras musculares, moderada população inflamatórias e ausência de epitélio e de lâmina própria. No grupo GB, neste mesmo período, todas as camadas vesicais estavam reconstituídas. Em ambos os grupos, aos 60 dias de pós-operatório, todas as camadas vesicais encontravam-se reparadas e a membrana peritônio bovino não foi observada. No grupo GC, em todos os tempos de avaliação verificou-se integridade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In this study, bovine peritoneum or biomembrane of natural latex were utilized to replace a section of ventral face of the bladder of rabbits, with purpose of evaluation of the tissue repair process in regard of its biocompatility, scar formation ability and possible complications. Thirty-six New Zealand rabbits, adult and entire males were divided in three experimental groups: biomembrane group (GB), bovine peritoneum group (GP) and control group (GC), each one with 12 animals. The animals were euthanized at seven, 14, 30 and 60 days post operative, by use of an elevated dosage of sodium tiopental, followed by macroscopic and histopathological analysis of the implant interface with the native tissue. Macroscopically, in both groups (GB, GP and GC), it was observed, in all time periods, structure adherence to the implant site, presence of calculi and absence of rejection signs. In the GB group, since 30 until 60 days of post operative, it was evidenced the presence of the biomembrane inside the bladder. Under light microscopy, at seven days, there were intense inflammatory reaction, new vessels and proliferated fibroblasts. In 14 days, there was no urotelium, however it saw muscle fibers, intense inflammatory cells, new vessels and fibroblasts. In the GP group, at 30 days the implant was still present, there were fibroblasts, muscle fibers, moderate population of inflammatory cells and absence of urotelium and sheath. In the GB group, in the same time period, all the vesical layers were reconstituted. In both groups, at 60 days of post operative, bladder wall was repaired and the bovine peritoneum membrane was not observed. In the GC group, all the evaluation times, integrity of the vesical wall was verified... (Complete abstract click electronic access below) / Mestre

Submitted by PPG Engenharia e Tecnologia de Materiais (engenharia.pg.materiais@pucrs.br) on 2018-08-08T14:30:34Z No. of bitstreams: 1 Disserta??o Luisa Alban.pdf: 1572475 bytes, checksum: 51e47c0f80b15d05eda24af2e3b8fde5 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-08-09T11:13:00Z (GMT) No. of bitstreams: 1 Disserta??o Luisa Alban.pdf: 1572475 bytes, checksum: 51e47c0f80b15d05eda24af2e3b8fde5 (MD5) / Made available in DSpace on 2018-08-09T11:23:16Z (GMT). No. of bitstreams: 1 Disserta??o Luisa Alban.pdf: 1572475 bytes, checksum: 51e47c0f80b15d05eda24af2e3b8fde5 (MD5) Previous issue date: 2018-08-01 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The interest in nanostructures such as titanate nanotubes (TNTs) has grown notably in recent years due to their biocompatibility and economic viability, which makes them promising for application in the biomedical field. Quercetin (Qc) has been reported to have great potential as a chemopreventive agent widely used in the study of the treatment of diseases such as bladder cancer. Therefore, this work aimed to study the incorporation of quercetin in sodium TNTs (NaTNT) and zinc (ZnTNT), as well as characterize the nanostructures formed. In addition, it was intended to conduct Qc release tests and biological and antitumor activities in T24 lineage cells. The nanostructures of TNTs were synthesized and characterized by FTIR, MEVFEG, EDS, MET DRX and TGA techniques. The results showed that the nanostructures have a tubular structure and the exchange of Na+ ions by Zn2+, as well as the incorporation of quercetin in the structure do not alter this morphology. In addition, the interaction established between Zn and Qc increases the thermal stability of nanostructures. The release test showed that the maximum delivery of Qc occurs after 24h and the presence of Zn controls the release of the flavonoid. Biological assays have shown that the NaTNTQc and ZnTNTQc nanostructures decrease the cellular viability of T24 after 48h in high concentrations. Furthermore, NaTNT, NaTNTQc and ZnTNT reduce the number of T24 lineage cells when combined with irradiation after 48h showing that the combination of nanostructures and ionizing energy is an attractive object of study in the treatment of bladder cancer. / O interesse por nanoestruturas como nanotubos de titanatos (TNTs) tem crescido notavelmente nos ?ltimos anos devido a sua biocompatibilidade e viabilidade econ?mica, o que os tornam promissores para a aplica??o na ?rea biom?dica. A quercetina (Qc) tem sido relatada como tendo grande potencial como agente quimiopreventivo amplamente utilizada no estudo do tratamento de doen?as como c?ncer de bexiga. Neste sentido, este trabalho pretendeu estudar a incorpora??o de quercetina em TNTs de s?dio (NaTNT) e de zinco (ZnTNT), bem como, realizar a caracteriza??o das nanoestruturas formadas. Al?m disso, pretendeu-se conduzir testes de libera??o de Qc e atividades biol?gicas e antitumorais em c?lulas da linhagem T24. As nanoestruturas de TNTs foram sintetizadas e caracterizadas por t?cnicas de FTIR, MEV-FEG, EDS, MET DRX e TGA. Os resultados mostraram que as nanoestruturas apresentam estrutura tubular, e a troca de ?ons Na+ por Zn2+, bem como a incorpora??o de quercetina na estrutura n?o alteram esta morfologia. Al?m disso, a intera??o estabelecida entre o Zn e Qc aumenta a estabilidade t?rmica das nanoestruturas. O ensaio de libera??o mostrou que a entrega m?xima de Qc ocorre ap?s 24h e apresen?a de Zn controla a libera??o do flavonoide para o meio. Os ensaios biol?gicos mostraram que as nanoestruturas NaTNTQc e ZnTNTQc diminuem a viabilidade celular de T24 ap?s 48h em altas concentra??es. Ainda, NaTNT, NaTNTQc e ZnTNT reduzem o n?mero de c?lulas da linhagem T24 quando combinadas com irradia??o ap?s 48h mostrando que a combina??o entre as nanoestruturas e energia ionizante se apresenta como um objeto de estudo atrativo no tratamento de c?ncer de bexiga.