Le cancer de la vessie est un cancer fréquent et extrêmement onéreux par patient puisque plusieurs patients subissent des récidives de cancer et ont recours parfois à des chirurgies complexes. Il est donc important de diagnostiquer efficacement ces cancers lors de la prise en charge initiale du patient. En effet, la procédure standard d’imagerie pour la détection du cancer est la cystoscopie de la vessie guidée par lumière blanche, toutefois cette méthode ne permet pas de bien distinguer les cellules qui sont propices à l’invasion musculaire des cellules de cancer de la vessie non infiltrant. Ce mémoire propose d’utiliser un nouvel immunoconjugué fluorescent ciblant la sous-unité alpha du récepteur de l’interleukine 5, un nouveau biomarqueur spécifique aux cellules du cancer de la vessie infiltrant, afin d’effectuer la cystoscopie de la vessie guidée par fluorescence. Pour ce faire, un protocole de conjugaison du fluorochrome cyanine-5 (Cy5) à un anticorps monoclonal a été développé. De plus, un protocole de conjugaison d’un peptide Cell Accumulator (ACCUM) sur cet anticorps fluorescent (A14-Cy5-ACCUM) a été optimisé. Ensuite, la capacité de cet immunoconjugué à marquer les cellules humaines de cancer de la vessie infiltrantes du muscle (MIBC), HT1376, a été testée. Par la suite, un nouveau modèle orthotpique murin de MIBC humain permettant la validation préclinique prochaine de l’A14-Cy5-ACCUM a été développé. Une banque de plasma et sérum sanguin, et d’urine de patients sains et atteints de cancer de la vessie a été compilé. Cette biobanque contient 111 échantillons de plasma sanguin et d’urine qui pourront être utilisé afin de tester l’hypothèse selon laquelle le niveau d’interleukine-5 sanguin pourrait être un facteur pronostique pour la progression du cancer de la vessie. Ce projet jette les bases pour l’évaluation potentielle de la cystoscopie guidée par fluorescence lors de la prise en charge initiale des patients atteints de cancer de la vessie afin d’améliorer la survie sans progression et la survie à long terme des patients atteints de MIBC. / Abstract: Bladder cancer is a frequent and extremely costly cancer when evaluated on a per-patient basis because of its high recurrence rate and patients undergoing complex medical procedures. It is of utmost importance to better identify the aggressiveness of this cancer at initial diagnosis. The standard procedure for bladder cancer detection is still white-light guided cystoscopy, which relies mostly on physicians experience in regard to identifying invasive malignancies. This memoir proposes the use of a new fluorescent immunoconjugate, targeting the alpha subunit of interleukin-5 receptor (IL-5R[apha]), a new biomarker specific to muscle-invasive bladder cancer (MIBC) cells for fluorescence-guided cystoscopy. To do so, a conjugation protocol to fluorescently label a monoclonal antibody with cyanine-5 fluorophores has been developped. Then, a conjugation protocol to attach Cell Accumulator (ACCUM) peptides to this fluorescent immunoconjugate (A14-Cy5-ACCUM) has been optimized. Moreover, the ability of A14-Cy5-ACCUM to stain MIBC cell line HT1376 has been tested. Most importantly, a novel orthotpic rat model of human MIBC for the future preclinical validation of fluorescence-guided cystoscopy in rat bladder has been developped. Finally, a new bladder cancer tissue repository at the CHUS has been established. This repository contains a total of 111 plasma and urine patient samples that will be helpful to evaluate if interleukin-5 blood levels could be used as a prognosis marker for bladder cancer progression. This project laid the basis for the potential evaluation of fluorescence-guided cystoscopy during initial diagnosis of bladder cancer patients to improve their disease-free and long-term survival.

Objectif: Définir l’effet des lipides et du traitement de la dyslipidémie sur les cancers de la prostate et de la vessie en utilisant différents devis d’étude et en tenant compte de la présence de plusieurs biais, particulièrement le biais du temps immortel. Devis: Le premier volet utilise un devis rétrospectif de type cas témoins. Un questionnaire semi-quantitatif de fréquence de consommation alimentaire validé a été utilisé. Le génotype COX2 de neuf polymorphisme nucléotidique unique (SNP) a été mesuré avec une plateforme Taqman. Des modèles de régression logistique non conditionnelle ont été utilisés pour comparer le risque de diagnostic d’un cancer de la prostate et l’interaction. Le deuxième volet utilise un devis rétrospectif de type cohorte basée sur les données administratives de la Régie de l’assurance-maladie du Québec (RAMQ). Des modèles de régression de Cox ont été employés pour mesurer l’association entre les statines et l’évolution du cancer de la vessie. Le troisième volet, porte un regard méthodologique sur le biais du temps immortel en examinant sa présence dans la littérature oncologique. Son importance est illustrée avec les données de la cohorte du deuxième volet, et les méthodes de correction possibles son appliquées. Résultats: L’étude du premier volet démontre qu’une diète riche en acides gras oméga-3 d’origine marine était fortement associée à un risque diminué de cancer de la prostate agressif (p<0.0001 pour la tendance). Le ratio de cote pour le cancer de la prostate du quartile supérieur d’oméga-3 était de 0.37 (IC 95% = 0.25 à 0.54). L’effet diététique était modifié par le génotype COX-2 SNP rs4648310 (p=0.002 pour l’interaction). En particulier, les hommes avec faible apport en oméga-3 et la variante rs4648310 avait un risque accru de cancer de la prostate (ratio de cote = 5.49, IC 95%=1.80 à 16.7), effet renversé par un apport en oméga-3 plus grand. L’étude du deuxième volet a observé que l’utilisation de statines est associée à une diminution du risque de progression du cancer de la vessie (risque relatif = 0.44, IC 95% = 0.20 à 0.96, p=0.039). Cette association était encore plus forte pour le décès de toute cause (HR = 0.57, 95% CI = 0.43 to 0.76, p=0.0001). L’effet des statines semble être dose-dépendant. L’étude du troisième volet démontre que le biais du temps immortel est fréquent et important dans les études épidémiologiques oncologiques. Il comporte plusieurs aspects dont certains sont mieux prévenus au stade du choix du devis d’étude et différentes méthodes statistiques permettent un contrôle de ce biais. Conclusion: 1) Une diète riche en oméga-3 aurait un effet protecteur pour le cancer de la prostate. 2) L’utilisation de statines aurait un effet protecteur sur la progression du cancer non invasif de la vessie. Les lipides semblent avoir un effet sur les cancers urologiques. / Purpose: To define the effects of dietary lipids and of treatment of dyslipidemia with statins on prostate and bladder cancers, using different epidemiologic study designs and accounting for biases, particularly immortal time bias. Study Design: The first part used a retrospective a case-control study design. Diet was assessed with a semi-quantitative food frequency questionnaire, and nine COX-2 tag single nucleotide polymorphisms (SNPs) were genotyped. We used logistic regression models to estimate odds ratios (ORs), 95% confidence intervals (CIs), and p-values for association and interaction. The second part used a retrospective cohort study design based on administrative databases of Québec, Canada. Cox regression models were used to measure association between statin use and bladder cancer evolution. The third part focuses on the immortal time bias by describing its presence in the oncologic literature. The importance of this bias is illustrated with data from the cohort used in the second part and statistical correction methods are applied. Results: The first part showed that an increasing intake of omega-3 fatty acids of marine origin was strongly associated with a decreased risk of aggressive prostate cancer (trend p<=0.0001). The OR (95% CI) for prostate cancer comparing the highest to the lowest quartile of omega-3 intake was of 0.37 (0.25 – 0.54). The dietary effect was modified by the rs4648310 COX-2 SNP (interaction p=0.02). This reflected the observation that men with low marine omega-3 intake and the variant rs4648310 SNP had an increased risk of disease (OR = 5.49; 95% CI: 1.80-16.7), which was reversed by increasing intake of marine omega-3. The second part showed that statin use was associated with a decreased risk of bladder cancer progression (HR = 0.44, 95% CI = 0.20 to 0.96, p=0.0388). The inverse association was even stronger for risk of mortality from all causes (HR = 0.57, 95% CI = 0.43 to 0.76, p=0.0001). The statin use effect appears dose-dependent. The third part showed that the immortal time bias is frequent and important in many epidemiological studies in oncology. It has many aspects and some of these are better prevented at time of study design selection. Various statistical methods also allowed control of this bias. Conclusion. 1) Dietary omega-3 appears to decrease prostate cancer risk. 2) Statin use appears to decrease risk of bladder cancer progression. Lipids seem to have an effect on urological cancers.

But: Cette étude a pour but de comparer : a)la morphologie du plancher pelvien (PP), du col vésical et du sphincter urogénital strié (SUS) par IRM et b) la fonction du PP par palpation digitale (PERFECT scheme) chez les femmes âgées continentes ou avec incontinence urinaire à l’effort (IUE) et mixte (IUM). Méthode: Les femmes ont appris à contracter correctement leur PP et la fonction de leur PP a été évaluée. Une séance d’IRM dynamique 3T a suivi. Résultats: 66 femmes ont participé à l’étude. Les groupes étaient similaires en âge, IMC, nombre d’accouchements vaginaux et d’hystérectomie. La validité et la fidélité des différentes mesures anatomiques utilisées ont été confirmées au début de cette étude. Afin de contrôler l’effet potentiel de la taille du bassin sur les autres paramètres, les femmes ont été appariées par la longueur de leur inlet pelvien. Les femmes avec IUM ont un PP plus bas et un support des organes pelviens plus faible, selon leurs ligne M, angle LPC/Ligne H et hauteur de la jonction urétro-vésicale (UV). Les femmes avec IUE ont un PP similaire à celui des continentes, mais présentent plus d’ouverture du col vésical et un angle UV postérieur plus large au repos que les autres groupes. Il n’y a aucune différence de morphologie du SUS entre les groupes. De plus, selon les résultats du PERFECT scheme, les femmes avec IU ont une force du PP plus faible que les continentes. Les femmes avec IUM montrent aussi une faible élévation des muscles du PP à la contraction. Les femmes avec IUE ont, quant à elle, un problème de coordination à la toux. Conclusion: Les déficits causant l’IUE et l’IUM sont différents, mais supportent tous le rationnel des exercices du PP pour le traitement de l’IUE et l’IUM. Ces résultats supportent le besoin de traitements de rééducation spécifiques aux déficits de chacun des types d’IU. / Aims: The study’s aim was to compare: a) pelvic floor muscle (PFM), bladder neck and urethral sphincter morphologies using MRI and b) PFM function using digital palpation (PERFECT scheme) in continent women, women with stress (SUI) and mixed (MUI) urinary incontinence. Method: Women were taught how to perform PFM contractions correctly and their PFM function was assessed, then each woman completed a dynamic 3T MRI session. Results: 66 women participated in the study. Groups were similar for age, BMI, vaginal deliveries and hysterectomies. Validity and reliability of the different anatomical measures used has been confirmed at the beginning of the study. To control for the potential effect of pelvic size on study parameters, women were matched based on pelvic inlet length. MUI women seemed to have lower PFM resting position and pelvic organ support at rest, based on their M-Line, PCL/H-Line angle and urethrovesical (UV) junction height. However, SUI women seemed to have a PFM morphology similar to that of continent women, but presented a greater occurrence of bladder neck funnelling and a larger posterior UV angle. There were no differences in urethral sphincter morphology between the 3 groups. Functionally, as shown by the PERFECT scheme results, both UI groups had poorer PFM strength on MVC then continent women. The MUI group also showed poor PFM elevation on contraction. Conversely, women with SUI had a timing problem on cough. Conclusion: The deficits in women with SUI and MUI appear to be very different. Notwithstanding, they all support the rationale for PFM exercise treatment in older women with SUI and MUI. However, the findings suggest the need for rehabilitation treatments specificity for each UI type.

Introdução: O seguimento de pacientes com câncer de bexiga superficial apresenta embargos financeiros e psicológicos ao paciente, devido à realização frequente de exames invasivos. Com fim de substituir ou diminuir os exames invasivos, busca-se biomarcadores urinários acurados, que permitam diagnosticar a recidiva tumoral e estratificar pacientes com maior risco de recidiva futura. O objetivo deste estudo é avaliar se expressão na urina de PAI-1, DJ-1, ApoA-1, MMP-9 e IL-8 permite identificar e antecipar a recidiva de câncer de bexiga. Método: A expressão da PAI-1, DJ-1, ApoA-1, MMP-9 e IL-8 foi mensurada por ELISA na urina de 152 pacientes tratados previamente de carcinoma urotelial superficial de bexiga e em seguimento. Os níveis das proteínas foram comparados entre pacientes com e sem recidiva de câncer de bexiga (1) no momento da coleta de urina e (2) durante o seguimento. A ocorrência de recidiva tumoral foi confirmada por análise histopatológica da biópsia de lesões suspeitas, investigadas quando havia alterações na cistoscopia, ultrassom ou citologia oncótica. Pacientes com recidiva diagnosticada no momento da coleta de urina foram excluídos da análise para avaliar o papel antecipatório das cinco proteínas. Foi avaliado se o uso prévio de BCG intra-vesical exercia influência no nível das cinco proteínas estudadas. Resultados: Entre os pacientes avaliados, 16 (10,5%) apresentaram recidiva de carcinoma urotelial no momento da coleta de urina e 21 (15,4%) apresentaram recidiva de carcinoma urotelial durante o seguimento. O seguimento mediano foi de 47 meses (interquartis de 39 e 50 meses). Um painel para o diagnóstico de recidiva tumoral incluindo três biomarcadores (ApoA-1, MMP-9 e IL-8) apresentou razão de risco de 12,9 (IC 95% =3,5-47,4) e um painel para prever pacientes que desenvolverão recidiva durante o seguimento incluindo dois biomarcadores (PAI-1 e IL-8) apresentou razão de risco de 4,1 (IC 95% =1,4-11,4). Os resultados dos painéis não foram influenciados pelo uso prévio de BCG intra-vesical. Conclusão: Os painéis apresentados permitem identificar pacientes com recidiva de carcinoma urotelial de bexiga e prever quais pacientes terão maior risco de desenvolver recidiva no futuro. O uso prévio de BCG intra-vesical não alterou a expressão dos biomarcadores / Purpose: To evaluate if the urinary levels of PAI-1, DJ-1, ApoA-1, MMP-9 and IL-8 can identify and predict tumor recurrence in patients on follow-up of superficial bladder cancer. Methods: We prospectively analyzed the urine of 152 patients previously treated of superficial bladder cancer on follow-up regimen. Five biomarkers (PAI-1, DJ-1, ApoA-1, MMP-9 and IL-8) were assessed by ELISA and compared among patients with and without bladder cancer recurrence (1) in the moment of urine collection and (2) during follow-up. Tumor recurrence was evaluated with cystoscopy, ultrasound and urine oncotic cytology and confirmed by pathological analysis. Patients with recurrence at urine collection were excluded from prediction analysis. A correlation between the level of the biomarkers and previous use of intravesical BCG was investigated. Results: Median follow-up was 47 months (IQR =39-50 months). Among patients included, 16 (10,5%; N =152) and 21 (15,4%; N =136) had bladder cancer recurrence diagnosed in the moment of urine collection and during follow-up, respectively. The panel to diagnose recurrence including 3 biomarkers (ApoA-1, MMP-9 and IL-8) presented OR =12,9 (CI =3,5-47,4), while the panel to predict patients who will have a recurrence during follow-up including 2 biomarkers (PAI-1 and IL-8) presented OR =4,1 (CI =1,4- 11,4). Previous use of intra-vesical BCG didn\'t influence urine biomarkers expression. Conclusions: The 3-biomarker panel can be used to identify patients with bladder cancer recurrence. The 2-biomarker panel can be used to predict patients at greater risk of bladder cancer recurrence during follow-up. Both are reliable in patients with previous use of intravesical BCG

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.

But: Cette étude a pour but de comparer : a)la morphologie du plancher pelvien (PP), du col vésical et du sphincter urogénital strié (SUS) par IRM et b) la fonction du PP par palpation digitale (PERFECT scheme) chez les femmes âgées continentes ou avec incontinence urinaire à l’effort (IUE) et mixte (IUM). Méthode: Les femmes ont appris à contracter correctement leur PP et la fonction de leur PP a été évaluée. Une séance d’IRM dynamique 3T a suivi. Résultats: 66 femmes ont participé à l’étude. Les groupes étaient similaires en âge, IMC, nombre d’accouchements vaginaux et d’hystérectomie. La validité et la fidélité des différentes mesures anatomiques utilisées ont été confirmées au début de cette étude. Afin de contrôler l’effet potentiel de la taille du bassin sur les autres paramètres, les femmes ont été appariées par la longueur de leur inlet pelvien. Les femmes avec IUM ont un PP plus bas et un support des organes pelviens plus faible, selon leurs ligne M, angle LPC/Ligne H et hauteur de la jonction urétro-vésicale (UV). Les femmes avec IUE ont un PP similaire à celui des continentes, mais présentent plus d’ouverture du col vésical et un angle UV postérieur plus large au repos que les autres groupes. Il n’y a aucune différence de morphologie du SUS entre les groupes. De plus, selon les résultats du PERFECT scheme, les femmes avec IU ont une force du PP plus faible que les continentes. Les femmes avec IUM montrent aussi une faible élévation des muscles du PP à la contraction. Les femmes avec IUE ont, quant à elle, un problème de coordination à la toux. Conclusion: Les déficits causant l’IUE et l’IUM sont différents, mais supportent tous le rationnel des exercices du PP pour le traitement de l’IUE et l’IUM. Ces résultats supportent le besoin de traitements de rééducation spécifiques aux déficits de chacun des types d’IU. / Aims: The study’s aim was to compare: a) pelvic floor muscle (PFM), bladder neck and urethral sphincter morphologies using MRI and b) PFM function using digital palpation (PERFECT scheme) in continent women, women with stress (SUI) and mixed (MUI) urinary incontinence. Method: Women were taught how to perform PFM contractions correctly and their PFM function was assessed, then each woman completed a dynamic 3T MRI session. Results: 66 women participated in the study. Groups were similar for age, BMI, vaginal deliveries and hysterectomies. Validity and reliability of the different anatomical measures used has been confirmed at the beginning of the study. To control for the potential effect of pelvic size on study parameters, women were matched based on pelvic inlet length. MUI women seemed to have lower PFM resting position and pelvic organ support at rest, based on their M-Line, PCL/H-Line angle and urethrovesical (UV) junction height. However, SUI women seemed to have a PFM morphology similar to that of continent women, but presented a greater occurrence of bladder neck funnelling and a larger posterior UV angle. There were no differences in urethral sphincter morphology between the 3 groups. Functionally, as shown by the PERFECT scheme results, both UI groups had poorer PFM strength on MVC then continent women. The MUI group also showed poor PFM elevation on contraction. Conversely, women with SUI had a timing problem on cough. Conclusion: The deficits in women with SUI and MUI appear to be very different. Notwithstanding, they all support the rationale for PFM exercise treatment in older women with SUI and MUI. However, the findings suggest the need for rehabilitation treatments specificity for each UI type.

Objectif: Définir l’effet des lipides et du traitement de la dyslipidémie sur les cancers de la prostate et de la vessie en utilisant différents devis d’étude et en tenant compte de la présence de plusieurs biais, particulièrement le biais du temps immortel. Devis: Le premier volet utilise un devis rétrospectif de type cas témoins. Un questionnaire semi-quantitatif de fréquence de consommation alimentaire validé a été utilisé. Le génotype COX2 de neuf polymorphisme nucléotidique unique (SNP) a été mesuré avec une plateforme Taqman. Des modèles de régression logistique non conditionnelle ont été utilisés pour comparer le risque de diagnostic d’un cancer de la prostate et l’interaction. Le deuxième volet utilise un devis rétrospectif de type cohorte basée sur les données administratives de la Régie de l’assurance-maladie du Québec (RAMQ). Des modèles de régression de Cox ont été employés pour mesurer l’association entre les statines et l’évolution du cancer de la vessie. Le troisième volet, porte un regard méthodologique sur le biais du temps immortel en examinant sa présence dans la littérature oncologique. Son importance est illustrée avec les données de la cohorte du deuxième volet, et les méthodes de correction possibles son appliquées. Résultats: L’étude du premier volet démontre qu’une diète riche en acides gras oméga-3 d’origine marine était fortement associée à un risque diminué de cancer de la prostate agressif (p<0.0001 pour la tendance). Le ratio de cote pour le cancer de la prostate du quartile supérieur d’oméga-3 était de 0.37 (IC 95% = 0.25 à 0.54). L’effet diététique était modifié par le génotype COX-2 SNP rs4648310 (p=0.002 pour l’interaction). En particulier, les hommes avec faible apport en oméga-3 et la variante rs4648310 avait un risque accru de cancer de la prostate (ratio de cote = 5.49, IC 95%=1.80 à 16.7), effet renversé par un apport en oméga-3 plus grand. L’étude du deuxième volet a observé que l’utilisation de statines est associée à une diminution du risque de progression du cancer de la vessie (risque relatif = 0.44, IC 95% = 0.20 à 0.96, p=0.039). Cette association était encore plus forte pour le décès de toute cause (HR = 0.57, 95% CI = 0.43 to 0.76, p=0.0001). L’effet des statines semble être dose-dépendant. L’étude du troisième volet démontre que le biais du temps immortel est fréquent et important dans les études épidémiologiques oncologiques. Il comporte plusieurs aspects dont certains sont mieux prévenus au stade du choix du devis d’étude et différentes méthodes statistiques permettent un contrôle de ce biais. Conclusion: 1) Une diète riche en oméga-3 aurait un effet protecteur pour le cancer de la prostate. 2) L’utilisation de statines aurait un effet protecteur sur la progression du cancer non invasif de la vessie. Les lipides semblent avoir un effet sur les cancers urologiques. / Purpose: To define the effects of dietary lipids and of treatment of dyslipidemia with statins on prostate and bladder cancers, using different epidemiologic study designs and accounting for biases, particularly immortal time bias. Study Design: The first part used a retrospective a case-control study design. Diet was assessed with a semi-quantitative food frequency questionnaire, and nine COX-2 tag single nucleotide polymorphisms (SNPs) were genotyped. We used logistic regression models to estimate odds ratios (ORs), 95% confidence intervals (CIs), and p-values for association and interaction. The second part used a retrospective cohort study design based on administrative databases of Québec, Canada. Cox regression models were used to measure association between statin use and bladder cancer evolution. The third part focuses on the immortal time bias by describing its presence in the oncologic literature. The importance of this bias is illustrated with data from the cohort used in the second part and statistical correction methods are applied. Results: The first part showed that an increasing intake of omega-3 fatty acids of marine origin was strongly associated with a decreased risk of aggressive prostate cancer (trend p<=0.0001). The OR (95% CI) for prostate cancer comparing the highest to the lowest quartile of omega-3 intake was of 0.37 (0.25 – 0.54). The dietary effect was modified by the rs4648310 COX-2 SNP (interaction p=0.02). This reflected the observation that men with low marine omega-3 intake and the variant rs4648310 SNP had an increased risk of disease (OR = 5.49; 95% CI: 1.80-16.7), which was reversed by increasing intake of marine omega-3. The second part showed that statin use was associated with a decreased risk of bladder cancer progression (HR = 0.44, 95% CI = 0.20 to 0.96, p=0.0388). The inverse association was even stronger for risk of mortality from all causes (HR = 0.57, 95% CI = 0.43 to 0.76, p=0.0001). The statin use effect appears dose-dependent. The third part showed that the immortal time bias is frequent and important in many epidemiological studies in oncology. It has many aspects and some of these are better prevented at time of study design selection. Various statistical methods also allowed control of this bias. Conclusion. 1) Dietary omega-3 appears to decrease prostate cancer risk. 2) Statin use appears to decrease risk of bladder cancer progression. Lipids seem to have an effect on urological cancers.

Karzinome der Gallenwege sind mit einer schlechten Prognose assoziiert. Eine potentiell kurative chirurgische Resektion ist bei der Mehrzahl der Patienten aufgrund des späten Zeitpunkts der Diagnosestellung nicht möglich, so dass derzeit vorrangig palliative Therapieansätze Anwendung finden. Das nur geringe Ansprechen auf konventionelle Radio- oder Zytostatikatherapie begründet die Notwendigkeit neuer Therapieansätze. Einen möglichen Angriffspunkt stellt hierbei die Polo-like-Kinase 1 (Plk1) dar, da ihre zentrale Rolle in der Regulation des Zellzyklus zunehmend erkannt und eine vermehrte Expression in malignem Tumorgewebe verglichen mit gesundem Gewebe nachgewiesen wurde. Das Dihydropteridinon BI 2536 ist ein poten-ter, niedermolekularer und selektiver Hemmstoff der Plk1 und sollte daher auf seine Wirksamkeit an Gallenwegskarzinomen untersucht werden. In der vorliegenden Arbeit konnte gezeigt werden, dass BI 2536 die untersuchten 14 Zelllinien von Gallenblasen- und Gallengangskarzinomen wirkungsvoll hemmt. Das Ansprechen unterschied sich zwischen den Zelllinien und ordnet sich vergleichbar zu Veröffentlichungen an anderen malignen Tumoren ein. Die Expression von Plk1 und dessen assoziierten Transkriptionsfaktor FoxM1 konnte bei Westernblot-Versuchen bei allen Zelllinien nachgewiesen werden, was eine Bedeutung in der Onkogenese vermuten lässt. Die Behandlung mit BI 2536 beeinflusste jedoch die Proteinmenge beider nicht. An für die folgenden Versuche ausgewählten drei Zelllinien zeigten sich in der reversen Transkription mit anschließender Echtzeit-Polymerase-Kettenreaktion (qRT PCR) ähnliche Ergebnisse in Bezug auf die exprimierte mRNA von Plk1. Westernblot-Analysen ermittelten keine signifikanten Veränderungen der an wichtigen intrazellulären Kaskaden beteiligten Proteine p42/44 und Akt sowie deren phosphorylierten Formen. Obwohl die Proteinmenge des Mitosemarkers Phospho-Histon H3 ebenso unverändert blieb, führte die Behandlung mit BI 2536 – dies zeigen Ergebnisse der Durchflusszytometrie – zu einer signifikanten, dosisabhängigen Zunahme der G2/M Fraktion des Zellzyklus und Zunahme der Apoptose-rate. Der maximale Hemmeffekt in der Behandlung von BI 2536 lag bei einer Inkubations-dauer von vier Tagen. Die Empfehlungen aus den klinischen Studien der Phase II von BI 2536 sowie dem Ziel der Vermeidung von Resistenzen ergibt sich die Notwendigkeit von Kombinationsversuchen mit Zytostatika, die in einer anderen Phase des Zellzyklus angreifen. Die in der Behandlung von Gallenwegskarzinomen etablierten Antimetaboliten 5-Fluorouracil und Gemcitabin wurden hierzu ausgewählt und es zeigten sich für 5 Fluorouracil synergistische, für Gemcitabin hingegen additive Kombinationseffekte. Zusätzlich wurde die Wechselwirkung mit dem IGF 1-Rezeptor-Inhibitor NVP-AEW541 untersucht, der ebenfalls einen neuen Behand-lungsansatz in der Krebstherapie darstellt und bei Gallenwegskarzinomen in vitro wirksam ist. Auch hier zeigen sich synergistische Effekte, die jedoch erst in höheren Behandlungs-dosen auftraten. Die Ergebnisse dieser Arbeit zeigen, dass die Hemmung der Plk1 bei Gallenblasen- und Gallengangskarzinomen einen wirksamen Behandlungsansatz darstellt. Auf der Grundlage der in dieser Arbeit beschriebenen Ergebnisse wird eine weitere präklinische und klinische Testung von selektiven Plk1-Hemmstoffen wie BI 2536 an Gallenwegskarzinomen empfohlen.

Made available in DSpace on 2014-07-29T15:07:47Z (GMT). No. of bitstreams: 1 Dissertacao_Gabriela_Uchoa.pdf: 897498 bytes, checksum: 9761f9b1b5b6c7f028d1d5674bbaa4d8 (MD5) Previous issue date: 2009-02-06 / Urinary disorders are an important discovery in the ileocystoplasties, especially uroliths and bladder crystals, and are partially related to the kind of material used the in suture itself. The purpose of this study is to verify if there are differences regarding the formation of uroliths or bladder crystals after dogs ileocystoplasties practices involving sutures using unabsorbed titanium staples and sutures with polyglatine 910, observing the impact of the procedure in the renal function, modifications in the urinary constituents and differences in the surgery time duration between the groups. For that, two experimental groups of animals, each one with six, have been considered. In one group it has been used a polyglactin 910 suture thread (group A) and in the other, a titanium stapler (group B). In each animal of both groups there was selected a terminal ileac segment with approximately 5 cm for bladder augmentation. In group A, a suture of the detubularized ileum segment in bladder was executed using a polyglactin 910. In group B, the bladder augmentation with the selected ileac segment was done by linear cutter stapler using titanium staples. It was observed the presence of struvite crystals in both groups, in 11 animals of the experiment. In group A the operation duration was longer if compared to group B . It was also observed the formation of urinary mucus in great quantity in all animals in the post-operation first days. It was confirmed the formation of calculi in two animals, one in each experimental group, but in the group A animal the calculi was free from lumen and in the group B animal the stone was adhered to the stapling zone, attached to a staple that got exposed to direct contact with the urine. In the parameters verified in the urinalysis, urea and seric creatinine there was no sign of renal alteration and in the verification of blood count, no alterations were noticed or considered significant. It was possible to conclude that no significant differences were observed between the groups as for the formation of urinary stones and crystals in ileocystoplasties after 100 days. If compared to group B , Group A presented a longer operation and there were not evidences of alteration in the renal function in any phase of the experiment in both groups / Alterações urinárias são um importante achado nas ileocistoplastias, sobretudo os urólitos e a cristalúria, e em parte estão relacionados ao tipo de material de sutura envolvido neste procedimento. O objetivo deste estudo foi verificar se existe diferença na formação de urólitos ou cristalóides urinários após ileocistoplastias em cães, realizadas por meio de sutura com grampos inabsorvíveis de titânio e sutura com poliglactina 910, observando o impacto do procedimento na função renal, alterações nos constituintes urinários e a diferença de tempo cirúrgico entre os grupos. Utilizou-se dois grupos experimentais com seis animais cada, um com fio de poliglactina 910 para a sutura da ileocistoplastia (grupo A), e outro usando grampos de titânio (grupo B). Em cada animal foi selecionado um segmento de aproximadamente 5 cm de íleo terminal para ampliação vesical. No grupo A realizou-se a sutura do segmento ileal detubulizado na bexiga com fio de poliglactina 910. No grupo B foi realizada a ampliação vesical com o segmento ileal selecionado com auxílio de grampeador linear cortante para detubulização e sutura grampos de titânio. Observou-se a presença de cristais de estruvita em 11 animais de ambos os grupos do experimento. No grupo A o tempo operatório foi maior se comparado ao grupo B . Foi possível observar a formação de grande quantidade de muco na urina de todos os animais já nos primeiros dias de pós-operatório. Verificou-se a formação de cálculos em dois animais, um de cada grupo experimental, sendo que no grupo A o cálculo estava livre no lúmen e no grupo B o cálculo estava aderido à zona de grampeamento, ligado a um grampo em contato direto com a urina. Dentro dos parâmetros avaliados na urinálise, uréia e creatina séricas, não houve qualquer sinal de alteração renal e nas avaliações de hemograma não foram percebidas alterações significativas. Foi possível concluir que não houve diferenças significativas entre os grupos quanto à formação de cristais urinários e urólitos após 100 dias das ileocistoplastias, o grupo A apresentou maior tempo cirúrgico se comparado ao grupo B e não houve evidências de alteração na função renal em nenhuma fase do experimento em ambos os grupos.

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.:1. Introduction............................................................................ 1 1.1. Anatomy and histology of the human urinary bladder..................... 1 1.1.1. Anatomy of the human urinary bladder..................................... 1 1.1.2. Structure of the human urinary bladder wall............................... 2 1.2. Normal bladder function and bladder dysfunction.......................... 3 1.2.1 Normal bladder function......................................................... 3 1.2.2 Sensory aspect.................................................................... 4 1.2.3 Overactivity or hypersensitivity of bladder.................................. 5 1.3 The role of functional cell types and interaction in urinary bladder... 6 1.3.1 The role of urothelium.......................................................... 7 1.3.2Theroleofsuburotheliamyofibroblast...................................... 7 1.3.3Theroleofdetrusorsmoothmusclecells.................................. 9 1.3.4 Possible interactions in urinary bladder cell types........................ 10 1.4 ATP function and Purinergic signalling in bladder........................... 11 1.5 Spontaneous activity of bladder................................................... 13 2. Objective.................................................................................. 15 3. Material and methods............................................................... 16 3.1. Ethics Statement........................................................................ 16 3.2. Cell preparation.......................................................................... 16 3.3. Solutions and chemicals............................................................. 19 3.4. Intracellular calcium measurements............................................. 20 2.4.1. Preparing cells for Calcium Imaging.......................................... 20 2.4.2. Preparing workspace of calcium imaging................................... 20 2.4.3. Calcium imaging recording...................................................... 22 3.5 Data analysis with automated Fluorescence analysis..................... 22 3.6 Confocal Immunofluorescence.................................................... 25 3.7 Statistics................................................................................. 26 4. Results.................................................................................. 27 4.1 Spontaneous calcium activity of sMF........................................... 27 4.2 ATP effects on calcium response in sMF...................................... 27 4.3 Analysis of purinergic receptors involved.................................... 30 3.3.1 Agonist stimulation.............................................................. 30 3.3.2 Signal inhibition by specific antagonists................................... 31 4.4 Confocal immunofluorescence of purinergic receptors.................. 32 5. Discussion............................................................................. 34 5.1 Myofibroblast identification....................................................... 34 5.2 Spontaneous activity in the bladder............................................ 36 5.3 ATP modulated calcium activity in sMF....................................... 37 5.4 purinergic signalling in sMF........................................................ 39 6. Summary................................................................................ 42 7. References.............................................................................. 45 Declaration............................................................................. 50 Acknowledgements................................................................. 51