Orientador: Wílson Mello Júnior / Resumo: O carcinoma de bexiga urinária é o tipo de câncer mais prevalente no sistema urinário em seres humanos e apresenta altos índices de morbidade e mortalidade. Seu desenvolvimento está relacionado a diversos fatores de risco, como o hábito de fumar, a exposição a agentes químicos industriais e metais pesados e o estresse crônico. As experiências adversas no início da vida estão relacionadas à modulação de eixos hormonais importantes, como o hipotalâmicohipofisário-adrenal (HHA) e hipotalâmico-hipofisário-gonadal (HHG). Alterações na resposta do eixo HHA, componente central da resposta fisiológica ao estresse, levam a desordens fisiológicas, como aumento da proliferação celular, angiogênese e prejuízo da resposta imune, que podem ser responsáveis pelo aumento da susceptibilidade do indivíduo a uma série de doenças na vida adulta, incluindo o câncer. Paralelamente, alterações na fisiologia de hormônios esteroides como andrógenos, estrógenos e glicocorticoides, bem como seus receptores, também têm sido relacionadas ao desenvolvimento de enfermidades como o câncer de bexiga urinária. Em ratos, o comportamento materno funciona como um fator importante na modulação desses eixos hormonais, modificando suas respostas na vida adulta, e estudos recentes têm relacionado os cuidados recebidos por filhotes à incidência de tumores. O objetivo deste estudo foi avaliar a influência do comportamento materno e sua consequente modulação nos eixos HHA e HHG, no desenvolvimento do câncer de bexiga u... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The urinary bladder carcinoma is the most prevalent type of cancer in the urinary tract in humans, with high morbidity and mortality. Its development is related to several risk factors such as smoking, exposure to industrial chemicals and heavy metals and chronic stress. Adverse experiences early in life are related to modulation of important hormonal axes, as the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG). Changes in the response of the HPA axis, a central component of the physiological response to stress, lead to physiological disorders, such as increased cell proliferation, angiogenesis, and impaired immune response, which may be responsible for the increased susceptibility of the individual to a number of diseases in adult life, including cancer. Similarly, changes in the physiology of steroid hormones such as androgens, oestrogens and glucocorticoids and their receptors have also been related to the development of diseases such as bladder cancer. In rats, maternal behavior serves as an important factor in the modulation of hormonal axes, modifying their responses in adulthood, and recent studies have linked the care received by pups and the incidence of tumors. The aim of this study was to evaluate the influence of maternal behavior and its consequent modulation in HPA and HPG axes in the development of bladder cancer. The results showed that rats reared by the negligent mothers had a greater susceptibility to the development of chemica... (Complete abstract click electronic access below) / Mestre

Arsenic is a human carcinogen associated with urinary bladder transitional cell carcinoma and other cancers. Arsenic is also a strong comutagen and cocarcinogen. One possible mode of action for arsenic carcinogenesis/cocarcinogenesis is inhibition of DNA damage repair. In laboratory animals, urinary bladder transitional cell carcinoma has only been observed in dimethylarsinic acid [DMA(V)]-exposed F344 rats. The goal of the present studies was to investigate inhibition of DNA repair as a mode of action for arsenic carcinogenesis/ cocarcinogenesis in the urinary bladder. Methods were first developed to harvest only transitional cells, the target cell type of arsenic carcinogenesis, suitable for RNA extraction or for DNA damage detection by Comet assay. Morphological studies established that DMA(V) in drinking water at 40 ppm was cytotoxic to the urothelium of Sprague-Dawley and F344 rats, and mitochondria were targeted by DAM(V). To investigate whether DMA(V) decreases the expression of DNA repair genes, mRNA levels of DNA repair genes in transitional cells were next measured in F344 rats exposed to up to 100 ppm DMA(V) in drinking water for 4 weeks. The mRNA levels of Ataxia Telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/Xeroderma Pigmentosum B (ERCC3/XPB), and DNA polymerase beta genes were not altered, as measured by real time RT PCR. These results suggested either that DMA(V) affects DNA repair without affecting the baseline expression of DNA repair genes or that DMA(V) does not affect DNA repair in the bladder. Arsenic effects on DNA repair were further investigated in F344 rats given 100 ppm DMA(V) or arsenate in drinking water for 1 week. DNA damage levels in transitional cells and micronuclei frequency (MN) in bone marrow were measured. Dimethylarsinic acid did not affect in vivo cyclophosphamide-induced DNA damage, and neither DMA(V) nor arsenate inhibited in vitro repair of hydrogen peroxide- or formaldehyde-induced DNA damage, as measured by Comet assay. Neither DMA(V) nor arsenate increased MN or elevated in vivo cyclophosphamide-increased MN. These results suggest inhibition of DNA repair by arsenic, in the transitional epithelium, may not be a major mechanism responsible for carcinogensis/cocarcinogenesis in the bladder. / Ph. D.

<p>This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy.</p><p>The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated <i>ex vivo</i>.</p><p>Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3⁺ to CD3⁺ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications.</p><p>Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. <i>In vitro</i> expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4⁺ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048).</p><p>In conclusion, human solid tumours are recognised in sentinel nodes and <i>in vitro</i> expanded sentinel node-acquired CD4⁺ T lymphocytes seem useful in the treatment of patients with disseminated cancer.</p>

This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy. The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated ex vivo. Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3+ to CD3+ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications. Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. In vitro expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4+ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048). In conclusion, human solid tumours are recognised in sentinel nodes and in vitro expanded sentinel node-acquired CD4+ T lymphocytes seem useful in the treatment of patients with disseminated cancer.

Background: At the time of diagnosis, approximately 75% of all bladder cancers are Non-Muscle Invasive Bladder Cancers (NMIBC) - the standard treatment for these cancers is a Transurethral Resection of the Bladder Tumour (TURBT). Although, the vast majority of these cancers are not life-threatening, they have a high risk of recurrence (and progression, particularly in higher risk NMIBC), despite the use of adjuvant intravesical chemotherapy. Consequently, patients are kept on long term cystoscopic surveillance with endoscopic removal if recurrences are detected - this impacts on patients' quality of life and contributes to the high cost for the healthcare provider. Aims: The fundamental aim of this series of clinical studies, spanning 12 years, was to identify and implement, means of improving the efficiency in both processing and operating on patients with NMIBC to not only reduce recurrence, but also to reduce the duration of follow up and repeat operations. It was an evolutionary process where the findings in the preceding studies formed the basis of the subsequent one - while the aim of the individual studies were different, there was a clear link to the essential principles, thus forming a coherent collection of studies. Methods and results: The project was carried out in 3 phases (with 2 or 3 main studies in each phase, augmented by 1 to 2 linked studies - making the entire submission for PhD by publications a series of 12 studies, to date): Phase 1 (5 studies in this phase): The aim was to demonstrate the natural history of non-invasive bladder cancer and identify sub-categories of patients who could be discharged from surveillance at 5 years. This was initially achieved by evaluating a prospectively maintained cohort of non-invasive bladder cancer patients diagnosed between 1978 and 1984 at the Western General Hospital, Edinburgh. This study identified the importance of the recurrence rate at the first follow up cystoscopy (RRFFC) as an essential prognostic marker. This finding was further validated using 2 separate cohorts from a different Centre (the Royal Infirmary, Edinburgh) managed in the 80s and the 90s, respectively. The data confirmed that over the decades, recurrence patterns do change, possibly as a result of differing techniques and improvements in optics and instruments; however, what remained the same was the prognostic value of the RRFFC. Phase 2 (3 studies in this phase): The early recurrence was deemed to be the result of missed and tumours left behind at the initial TURBT, i.e. a marker of quality. However, RRFFC was only known 3 months after the initial surgery. Since the RRFFC was such an important prognostic factor, the aim of this phase was to determine the surgical factors contributing to the quality of TURBT and subsequently implement changes to the principles in carrying out the surgery to improve this quality. This was achieved by prospective collection of information regarding all patients undergoing TURBT for new bladder cancers, recording the tumour features, surgeon experience, if the resection was deemed to have been complete or not, and the pathological results. We identified that the detrusor muscle in the resected specimen and the experience of the surgeon were independent determinants of TURBT quality. This finding was validated in a further study using cohorts from another time period and another Centre - this allowed me to develop the concept of Good Quality White Light TURBT (GQWLTURBT) as the benchmark for the white light TURBT. Phase 3 (4 studies in this phase): Photodynamic Diagnosis assisted TURBT (PDDTURBT) was demonstrated in randomised controlled trials as a technique that reduces the recurrences in NMIBC. In the absence of evidence with this technique in the 'real life' setting nor comparisons with standardised, benchmarked white light TURBT technique, we performed a prospective controlled study comparing PDD-TURBT and GQ-WLTURBT, evaluating early and delayed recurrence rates in 2 separate studies. I also performed a multicentre UK study on the outcomes with PDD-TURBT and collaborated with other experts in Europe in producing a review article around Photodynamic Diagnosis and the cost effectiveness of this technique. Summary: This coherent series of studies has contributed to knowledge in bladder cancer surgery by, among others: (a) mapping the individual patient natural history of non-invasive bladder cancer; (b) confirming the importance of early recurrence as a strong prognostic indicator; (c) identifying predictors of this early recurrence and the quality of TURBT; (d) introducing the concept of the benchmark Good Quality White Light TURBT and (e) demonstrating the benefits of photodynamic diagnosis within a 'real life' setting.

L'objectiu d'aquest tesi és avaluar els diferents passos en el camí que va des de l'exposició a contaminants atmosfèrics/PAHs fins a la malaltia, el càncer de bufeta urinària. Es van mesurar partícules ultrafines a Barcelona. S'ha avaluat l'exposició a contaminació atmosfèrica en un estudi cas-control, recollint informació sobre la història residencial incloent diversos indicadors de l'exposició a contaminació atmosfèrica i altres factors de risc potencials. Es va dur a terme una revisió sistemàtica de la literatura per avaluar si els nivells de metabòlits del pirè i els aductes d'ADN i de proteïnes es correlacionaven amb nivells baixos d'exposició a PAHs. Vam mesurar els nivells d'aductes d'ADN en un subgrup d'individus de l'estudi cas-control amb la tècnica del radioetiquetatge amb fòsfor-32, tractament de la nucleasa P1. Vam analitzar 22 SNPs en set gens de la via de reparació de l'ADN per excisió de nucleòtids. / The aim of this thesis is to evaluate the different steps in the pathway from exposure (air-contaminants/PAHs) to disease (bladder cancer). We measured ultrafine particles in Barcelona. We evaluated the exposure to air pollutants in a case-control study, collecting information on the residential history with proxies for exposure to air pollution and other potential risk factors. We did a systematic review of the literature to evaluate if pyrene metabolites and DNA and protein adducts are correlated with low level exposure to PAHs. We measured bulky DNA adducts in a subgroup of subjects of the case-control study using 32P-Postlabeling, nuclease P1 treatment. We analyzed 22 SNPs in 7 genes of the nucleotide excision repair pathway.

Paclitaxel is one of the most successful drugs for the treatment of cancer because of its ability to target tubulin, block cell cycle progression at mitosis, and induce apoptosis. Despite the success of Paclitaxel, the development of drug resistance hampers its clinical applicability. Paclitaxel is used in malignant tumors in the present research, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), head-neck scale epitheliomatous, urinary bladder cancer, tumor of the reproduction organ and so on. Clinical treatment of paclitaxel be injected 250mg/m2 to previously non-treated patients of small lung cancer its effect about 34% and previously non-treated patients of non-small lung cancer its effect about 21% to 24% . On the other hand we had established a taxol-resistant human lung carcinoma subline A549R by paclitaxel to compare the different proteins with A549 wild type and treat the different concention of paclitaxel with MTT assay so as to observe the tolerance dosage of subline growth.We obtained the patient¡¦s specimens of lung cancer to treat with paclitaxel some of resistant and some of non-resistant to compare differentially expressed proteins between normal and tumor. When we cultured taxol-resistant human lung carcinoma subline in which paclitaxel and calcium regulate growth, owing to the proteins of changes result to resistance. The extraction cell subline and specimens were analyzed by 2-D electrophoresis patterns and we found that interact paclitaxel with calcium it is the important factor of drug resistant. Verified from clinical treatment might have hypercalcemia in malignant tumors and calcium ion may increase paclitaxel drug resistance and hypercalcemia patient will be more insensitive to paclitaxel treatment.

This thesis addresses the three principal questions concerning the development of CT urography for investigating haematuria and each question is the subject of a separate chapter. The questions are: What is the reasoning behind using CT urography? What is the optimum diagnostic strategy using CT urography? What are the problems with using CT urography and how may solutions be provided? Haematuria can signify serious disease such as urinary tract stones, renal cell cancer, upper tract urothelial cancer (UTUC) and bladder cancer (BCa). CT urography is defined as contrast enhanced CT examination of kidneys, ureters and bladder. The technique used here includes unenhanced, nephrographic and excretory-phases for optimized diagnosis of stones, renal masses and urothelial cancer respectively. The reasoning behind using excretory-phase CT urography for investigating haematuria is based on results showing its high diagnostic accuracy for UTUC and BCa. Patients with haematuria are classified as low risk or high risk for UTUC and BCa, by a risk score, determined by the presence/absence of risk factors: age > 50 years, visible or nonvisible haematuria, history of smoking and occupational exposure. The optimum diagnostic strategy for patients at high risk for urothelial cancer, uses CT urography as a replacement test for ultrasonography and intravenous urography and as a triage test for flexible and rigid cystoscopy, resulting in earlier diagnosis and potentially improving prognosis. For patients at low risk, ultrasonography, unenhanced and nephrographic-phase CT urography are proposed as initial imaging tests. Problems with using CT urography include false positive results for UTUC, which are eliminated by retrograde ureteropyelography-guided biopsy, an innovative technique, for histopathological confirmation of diagnosis. Recommendations for the NHS and possible future developments are discussed. CT urography, including excretory-phase imaging, is recommended as the initial diagnostic imaging test before cystoscopy for patients with haematuria at high risk for urothelial cancer.