Background and Purpose: Primary nocturnal enuresis (PNE) is a heterogeneous disorder with various underlying pathophysiological mechanisms. Results of our recent studies focused on the relationship of bladder function, sleep and brain function demonstrated a simultaneous occurrence of bladder and brain dysfunction in children with severe refractory PNE. We therefore proposed to use an animal model with altered bladder function to evaluate if abnormalities in bladder function induce functional derangement in brainstem micturition centers and/or sleep-arousal centers. / Materials and methods: In general, the study was divided in to 6 parts. Male Wistar rats (~ 1.5 months) were used for the study. / Study I: Establishment of animal model —— Male Wistar rats (200-220 g) underwent either Sham surgery or surgical reduction of bladder volume (RBV). Animals were used for further Cystometry, EEG, MRS and Cognitive function studies 4-5 weeks postoperatively. / Study II: Conventional Fill Cystometry (CFC) to evaluate bladder functional changes in response to surgical bladder volume reduction —— Twenty-four rats (RBV=12, SHAM 12) were used for the study. CFC was performed under conscious condition for evaluating the functional changes in response to surgical bladder capacity reduction. / Study III: Radiotelemetered EEG study to assess the impact of bladder dysfunction on sleep architecture and cortical arousals in rats —— Twenty-four rats (RBV=12, SHAM 12) were used for the study. Radiotelemeters were implanted in both groups 4 weeks post-operatively. The EEG biopotential and bladder pressure were monitored for 48 hours. Sleep architecture and cortical arousals were then evaluated manually. / Study IV: Evaluation of cognitive function following surgical bladder volume reduction —— Ninety eight rats (RBV=50, SHAM =48) were used for the study. / Morris Water Maze task: A circular plastic translucent pool half-filled with 26 ± 2ºC water, was used in the Morris Animals were given 9 consecutive training (2/day) sessions of Morris Water Maze (MWM) at 4 weeks postoperatively. / 8-arm Radial Maze: Food pellets were randomly placed inside each arm of the maze and the rats were allowed to explore the maze freely for 5 minutes. The rat was allowed to explore the maze for 5 minutes. Total time spent in each arm, total distance traveled in the maze was recorded. / Study V: Magnetic Resonance Spectroscopy to detect functional changes in brain in response to bladder dysfunction elicited by surgical bladder volume reduction —— Proton magnetic resonance spectroscopy was employed to examine brain metabolic changes in 24 rats (RBV=12, SHAM=12). Single voxel 1 H MRS experiments were performed using a 7 T MRI scanner. MR spectra were then processed using the jMRUI software. / Phase VI: Enzyme -linked immunosorbent assay for the assessment of associated changes in neurotransmitters —— Animals were euthanized after MRS study and brain samples were collected. Serotonin and dopamine levels were assessed in 10 mg of tissue extracts from brainstem and cortex, with ELISA kits. / Results: Study I: Bladder reduction surgery did not affect the increase in body weight post -operatively. Average body weight of the RBV and the sham groups were 340.2 ± 47.2 g and 340.5 ± 67.9 g respectively at 4 weeks post operatively. / Study II: Compared to sham group, the maximum cystometric capacity in animals with RBV was remarkably reduced at week 4 (0.78 ± 0.12 ml vs. 1.46 ± 0.22 ml, RBV vs. Sham respectively; p<0.005). Moreover, maximum detrusor pressure during voiding was significantly increased in RBV group at week 4 post operatively (32.4± 2.14 vs.23.27±1.2 5 cm H2O, RBV vs. Sham respectively). / Study III: Light non-repaid eye movement sleep occurred significantly more in RBV rats compared to sham group (61.8% vs 35%). Deep sleep and rapid eye movement sleep occurred significantly less in RBV group compared to that of sham group (30.7% vs 53.4%). / Study IV: Results showed that the RBV group used a significantly longer latency to locate the platform compared to Sham group (24.4s vs 17.19s, RBV vs. Sham respectively, p<0.001).. Moreover, significantly more animals from the RBV group could not complete the visit of the 8 arms of radial maze than that of the sham group. / Study V: Seven metabolites were detected and quantified. The results demonstrated significant changes in the lactate (Lac) metabolism in some specific regions of rat brain. At 4 weeks post - operatively, level of lactate significantly decreased in the hippocampus (43%, P<0.001) cingulate and retrosplenial cortex (29%, p<0.05) of RBV rats compared to that of sham rats. / Study VI: Results demonstrated a significant increase in Serotonin level in the brainstem of RBV rats compared to that of SHAM rats (23.726 + 0.88 ng/ml vs. 1.88 + 0.302 ng/ml). Dopamine levels decreased significantly in brainstem samples of RBV group compared to sham group (2.85 + 0.10 ng/ml vs. 6.85 + 0.84 ng/ml). / Conclusion: Surgical bladder volume reduction of bladder capacity can induce functional changes in the central nervous system. An alteration of the sleep architecture occurred in response to surgical reduction of bladder volume in rats, suggesting that there exists a potential for central consequences of bladder dysfunction. Bladder disorder chronically altered brain energy metabolism. Furthermore, bladder disorder altered the central neurotransmission in the brainstem and cortex. The finding of bladder dysfunction induced significant impairments in cognitive function in RBV rats, suggesting that the alteration in brain energy metabolism may contribute to the behavioral and attention problems, impaired learning and cognitive performance. / 研究背景： 原發性夜間遺尿症（PNE）是一種異質性疾病，涉及多種潛在的病理生理機制。我們最近的研究主要集中在膀胱功能，睡眠和腦功能的關係，結果顯示膀胱和腦功能障礙同時出現在患有嚴重難治性的PNE的兒童。因此，我們建議採用一種已改變膀胱功能的動物模型來評估膀胱功能異常會否引起腦幹排尿中心和/或睡眠 - 覺醒中心的功能紊亂 / 研究工具和方法： 研究被分成6個部分。雄性Wistar大鼠（約1.5個月）被用於研究。 / 研究I： 動物模型的建立 —— 雄性Wistar大鼠（200-220克），會先接受假手術或手術降低膀胱容量（RBV）。手術後4至5週，動物會進行進一步的膀胱測壓，腦電圖，MRS和認知功能研究。 / 研究II： 以常規填充膀胱測壓（CFC）評估減少膀胱容量手術對膀胱功能的變化 —— 二十四隻大鼠（RBV=12，對照=12）被用於研究。 CFC是用以評估在有意識的條件下，膀胱因膀胱容量減少的手術而引起的功能變化。 / 研究III： Radiotelemetered腦電圖研究，以評估在大鼠膀胱功能失調對睡眠結構和皮質覺醒的影響 —— 二十四隻大鼠（RBV=12，對照=12）被用於研究。膀胱容量減少的手術4週後，Radiotelemeters被植入在兩個組別的大鼠，並監測其腦電生物電勢和膀胱內壓48小時，然後手動評估睡眠結構和皮層覺醒。。 / 研究IV： 評估在膀胱容量減少的手術後對認知功能的影響 —— 103個大鼠（RBV=56，對照= =47）被用於研究。 / Morris水迷宮任務： 一個圓形的塑料半透明池盛載半滿的水，溫度介乎26 - ±2℃，手術4週後，該池被用在莫里斯動物進行連續9次Morris水迷宮（MWM）培訓（每天2次）。 / 八臂迷宮： 食物顆粒被隨機放置在迷宮的每個臂內，大鼠可以自由地探索迷宮5分鐘。大鼠被允許探索迷宮5分鐘。在每個手臂所用的總時間，以及在迷宮行走的總距離都會被記錄。 / 研究V： 以磁共振波譜檢測膀胱容量減少的手術所引起的膀胱功能障礙對腦功能的改變 —— 以質子磁共振波譜研究24隻大鼠腦內的代謝變化(RBV=12，對照==12）。以7 T MRI掃描儀進行磁共振波譜實驗，然後使用jMRUI軟件處理MR譜。 / 第六期： 以酶聯免疫吸附測定法評估神經遞質的相關變化 —— 動物在進行MRS研究後實施安樂死，並收集其腦樣品。從腦幹和皮層提取10毫克組織提取物，使用ELISA試劑盒，以評估羥色胺和多巴胺水平。 / 結果： 研究I： 膀胱容量減少手術並沒有影響體重增加。手術4週後，利巴韋林和對照實驗組的平均體重分別為340.2±47.2克和340.5±67.9克。 / 研究II： 相比起對照實驗組的動物，RBV組的最大膀胱容量顯著降低（0. 0.78 ± 0.12毫升對1.46±0.22毫升），排尿頻率顯著增加（2.53±0.30 對.0.53±0.05/hr）。此外，排尿時最大逼尿肌壓力亦顯著升高（32.0.8±2.19 比.20.37±1.2 5厘米水分子） / 研究III： 相比起對照實驗組的動物，光非快速動眼期睡眠顯著地較多發生於RBV大鼠身上（61.8％對35.6％），深層睡眠和快速動眼期睡眠顯著地較少發生在RBV組（32.3％對52.8％） / 研究IV： 結果表明，RBV組使用了顯著較長的時間來定位平台（24.4s vs. vs.17.19s）。而且，在RBV組，顯著地較多動物無法完成行走8臂的放射狀迷宮。 / 研究V： 進行檢測和定量七種代謝物。結果顯示乳酸（LAC）代謝在大鼠大腦的某些特定區域出現顯著變化。在手術4週後，相比起對照實驗組的動物，RBV組大鼠在海馬體（43％，P <0.001），扣帶和夾肌皮質（29％，P <0.05）的乳酸水平均顯著減少。 / 研究VI： 結果顯示RBV大鼠腦幹的血清素水平較對照實驗組的顯著增加（23.726+0.88納克/毫升與1.88±0.302ng/ml）。RBV大鼠腦幹的多巴胺水平則較對照實驗組的顯著下降（2.850.10納克/毫升與6.85+0.84毫微克/毫升）。 / 結論： 外科膀胱容量減少可誘導中樞神經系統的功能變化。以外科手術減少膀胱容量的大鼠亦引起睡眠結構改變，這顯示膀胱功能障礙對中樞有潛在影響。膀胱疾病長期改變大腦的能量代謝。此外，膀胱疾病亦改變了在腦幹和大腦皮層的中樞神經遞質傳遞。研究發現膀胱功能障礙顯著地損害RBV大鼠的認知功能，顯示改變大腦的能量代謝亦可導致行為和專注力的問題，從而損害學習和認知能力。 / Yeung, Chung Kwong. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 199-230). / Abstracts also in Chinese. / Title from PDF title page (viewed on 14, September, 2016). / Yeung, Chung Kwong. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Orientador: Maria Luiza Cotrim Sartor de Oliveira / Banca: Carlos Márcio Nóbrega de Jesus / Banca: Heidge Fukumasu / Não disponível / Abstract: Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a herbicide that in a previous longterm study with Wistar rats fed at 2,500 ppm concentration showed high incidence of urothelial tumors in both genders. Accordingly, USEPA categorized Diuron as a "known/likely" human carcinogen. The accepted non-genotoxic mode of action (MOA) of Diuron encompasses urothelial necrosis induced by direct cytotoxicity, followed by regenerative cell proliferation and sustained urothelial hyperplasia that may favor neoplasia development. Scanning electron microscopy (SEM), light microscopy and labeling index are essentials tools for identification and classification of cytotoxic and proliferative changes in the bladder. The present study evaluated the dose-response of Diuron regarding urothelial lesions. Sixty male Wistar rats were fed Diuron for 20 weeks mixed in the diet at 0, 60, 125, 500, 1,250, or 2,500 ppm. Simple hyperplasia was significantly increased in the Diuron 1,250 and 2,500 ppm groups, and the cell proliferation at 2,500 ppm group. By SEM, the incidences and severity of lesions were significantly greater in the 500 and 1,250 ppm. Although numerically increased, the incidence of lesions in the 2,500 ppm group did not differ significantly from the control. The present study documented a doseresponse influence of Diuron on the rat urothelium, with a no observed effect level (NOEL) of 125 ppm. / Mestre

O diagnóstico de câncer e a necessidade de passar por vários tratamentos geram impactos e transformações na vida da pessoa. A reação individual para lidar com o itinerário da doença sofre influência do contexto sociocultural e repercute no modo de lidar com os sentimentos existenciais da experiência. Esses sentimentos podem ser expressos por termos diversos, entre eles, a depressão. O objetivo deste estudo foi interpretar os significados da depressão atribuídos pelos pacientes com câncer de bexiga em seguimento terapêutico, construídos com base em dados narrativos. Empregou-se a abordagem metodológica qualitativa, embasado pelo referencial teórico-metodológico da antropologia médica e do método da narrativa. Após aprovação ética para a pesquisa e consentimento das chefias da instituição coparticipante, foram convidados 12 participantes com câncer de bexiga, sem diagnóstico de depressão, em seguimento terapêutico no Ambulatório de Urologia Oncológica de um serviço de saúde de alta complexidade do interior do Estado de São Paulo, para participar do estudo. A coleta de dados ocorreu no período de janeiro 2014 a fevereiro de 2015, por meio de entrevistas semiestruturadas gravadas, observação direta e registros no diário de imersão, realizadas nos domicílios dos participantes e no serviço de saúde. Obteve-se uma amostra representativa do grupo, não intencional, homogênea quanto ao sexo, sendo a maioria acima de 60 anos, casados, com mais de sete anos de estudos e aposentados. Para tratamento, todos realizaram RTU com associação da BCG para a maioria, e houve três cistectomizados. A análise dos dados narrativos apoiou-se nos pressupostos da análise temática pelo processo indutivo. Identificaram-se as categorias com as quais elaboramos as unidades de sentidos do processo vivido com o câncer de bexiga e a depressão, compondo os modelos explanatórios dos pacientes. As unidades de sentidos serviram de guia para a construção de duas sínteses narrativas e seus significados: \"O paradoxo da vida com câncer de bexiga\" e \"A depressão como emoção no câncer de bexiga em seguimento terapêutico\". A primeira síntese aborda as dificuldades com o processo da doença e tratamento enquanto rupturas na vida, futuro incerto pela possibilidade de recidiva da doença, necessidade de continuidade do tratamento para o controle da doença e a lógica compensatória de controle emocional, relacionando-se com as ponderações contraditórias da vida atual. Assim, o significado desta síntese narrativa é de paradoxo. A segunda síntese aborda a incorporação do termo depressão ao senso comum, o qual revela a dimensão sociocultural da condição de ser sobrevivente oncológico em seguimento terapêutico. O seu significado revela a depressão no câncer como prática social e expressão de subjetividade, por meio da emoção de tristeza. Esta investigação permitiu-nos interpretar os significados da depressão atribuídos pelos adoecidos com câncer de bexiga segundo suas experiências. Como considerações finais, destacamos a importância dos enfermeiros em darem atenção à saúde mental do adoecido pelo câncer, pela escuta das suas subjetividades para promoveram intervenções adequadas, objetivando a integralidade do cuidado / The diagnosis of cancer and the need to undergo several treatments cause impact and change in the people\'s life. The personal reaction in coping with the itinerary of the disease suffers influence of the sociocultural context affecting the way of dealing with existential feelings from experience. These feelings can be related through different terms, among them, depression. The aim of study was to interpret the meanings of the depression attributed by patients with bladder cancer under therapeutic follow-up, based on their narratives. It was used the qualitative methodological approach, supported by theoretical-methodological referential of medical anthropology and narrative method. After ethical approval for the research and agreement of the heads of institutions, as coparticipants, 12 individuals were invited to participate of the study, they all with bladder cancer, without depression diagnosis, under therapeutic follow-up at the Oncological Urology Clinic that provide health service of high complexity in the countryside of São Paulo state . The data collection occurred from January 2014 to February 2015, through recorded semi-structured interviews, direct observations and it were registered in the immersion diary. These activities were held at the patients´ residences and in the health service. It was obtained a representative sample of the group, non-intentional, homogeneous regard to sex, being the most part of the patients over 60 years old, married, with more seven years of schooling and retired. For treatment, they all were submitted to TUR, with BCG association for the greater part and three of them by cystectomy. The analysis of narratives data was supported on the assumptions of thematic analysis by inductive process. The categories were identified and used to elaborate the units of senses of the experienced process by bladder cancer and depression as well as built the explanatory models of the patients. These units of senses served as guide for construction of two narratives syntheses and their meanings: \"The paradox of life with bladder cancer\" and \"The depression as emotion with bladder cancer under therapeutic follow-up\". The first synthesis addresses the difficulties with disease process and treatment as rupture of life, an uncertain future on the possibility of recurrence of the disease, the need of continuous treatment to control the disease and compensatory logic of the emotional control relating to contradictories considerations of the current life. Thus, the meaning of this narrative synthesis is paradox. The second synthesis approaches the incorporation of the depression term to common sense, which exposes the sociocultural dimension of the condition of being cancer survivor under therapeutic follow-up; its meaning reveals depression in cancer as social practice and subjectivity expression through emotion of sadness. This research allowed us to interpret the meanings of depression attributed by patients with bladder cancer according to their experiences. As final consideration, we emphasize the importance of the nurses to give attention to the mental health of the patients with cancer, by listening to their subjectivities to promote appropriate interventions, aiming care completeness

O diagnóstico de câncer e a necessidade de passar por vários tratamentos geram impactos e transformações na vida da pessoa. A reação individual para lidar com o itinerário da doença sofre influência do contexto sociocultural e repercute no modo de lidar com os sentimentos existenciais da experiência. Esses sentimentos podem ser expressos por termos diversos, entre eles, a depressão. O objetivo deste estudo foi interpretar os significados da depressão atribuídos pelos pacientes com câncer de bexiga em seguimento terapêutico, construídos com base em dados narrativos. Empregou-se a abordagem metodológica qualitativa, embasado pelo referencial teórico-metodológico da antropologia médica e do método da narrativa. Após aprovação ética para a pesquisa e consentimento das chefias da instituição coparticipante, foram convidados 12 participantes com câncer de bexiga, sem diagnóstico de depressão, em seguimento terapêutico no Ambulatório de Urologia Oncológica de um serviço de saúde de alta complexidade do interior do Estado de São Paulo, para participar do estudo. A coleta de dados ocorreu no período de janeiro 2014 a fevereiro de 2015, por meio de entrevistas semiestruturadas gravadas, observação direta e registros no diário de imersão, realizadas nos domicílios dos participantes e no serviço de saúde. Obteve-se uma amostra representativa do grupo, não intencional, homogênea quanto ao sexo, sendo a maioria acima de 60 anos, casados, com mais de sete anos de estudos e aposentados. Para tratamento, todos realizaram RTU com associação da BCG para a maioria, e houve três cistectomizados. A análise dos dados narrativos apoiou-se nos pressupostos da análise temática pelo processo indutivo. Identificaram-se as categorias com as quais elaboramos as unidades de sentidos do processo vivido com o câncer de bexiga e a depressão, compondo os modelos explanatórios dos pacientes. As unidades de sentidos serviram de guia para a construção de duas sínteses narrativas e seus significados: \"O paradoxo da vida com câncer de bexiga\" e \"A depressão como emoção no câncer de bexiga em seguimento terapêutico\". A primeira síntese aborda as dificuldades com o processo da doença e tratamento enquanto rupturas na vida, futuro incerto pela possibilidade de recidiva da doença, necessidade de continuidade do tratamento para o controle da doença e a lógica compensatória de controle emocional, relacionando-se com as ponderações contraditórias da vida atual. Assim, o significado desta síntese narrativa é de paradoxo. A segunda síntese aborda a incorporação do termo depressão ao senso comum, o qual revela a dimensão sociocultural da condição de ser sobrevivente oncológico em seguimento terapêutico. O seu significado revela a depressão no câncer como prática social e expressão de subjetividade, por meio da emoção de tristeza. Esta investigação permitiu-nos interpretar os significados da depressão atribuídos pelos adoecidos com câncer de bexiga segundo suas experiências. Como considerações finais, destacamos a importância dos enfermeiros em darem atenção à saúde mental do adoecido pelo câncer, pela escuta das suas subjetividades para promoveram intervenções adequadas, objetivando a integralidade do cuidado / The diagnosis of cancer and the need to undergo several treatments cause impact and change in the people\'s life. The personal reaction in coping with the itinerary of the disease suffers influence of the sociocultural context affecting the way of dealing with existential feelings from experience. These feelings can be related through different terms, among them, depression. The aim of study was to interpret the meanings of the depression attributed by patients with bladder cancer under therapeutic follow-up, based on their narratives. It was used the qualitative methodological approach, supported by theoretical-methodological referential of medical anthropology and narrative method. After ethical approval for the research and agreement of the heads of institutions, as coparticipants, 12 individuals were invited to participate of the study, they all with bladder cancer, without depression diagnosis, under therapeutic follow-up at the Oncological Urology Clinic that provide health service of high complexity in the countryside of São Paulo state . The data collection occurred from January 2014 to February 2015, through recorded semi-structured interviews, direct observations and it were registered in the immersion diary. These activities were held at the patients´ residences and in the health service. It was obtained a representative sample of the group, non-intentional, homogeneous regard to sex, being the most part of the patients over 60 years old, married, with more seven years of schooling and retired. For treatment, they all were submitted to TUR, with BCG association for the greater part and three of them by cystectomy. The analysis of narratives data was supported on the assumptions of thematic analysis by inductive process. The categories were identified and used to elaborate the units of senses of the experienced process by bladder cancer and depression as well as built the explanatory models of the patients. These units of senses served as guide for construction of two narratives syntheses and their meanings: \"The paradox of life with bladder cancer\" and \"The depression as emotion with bladder cancer under therapeutic follow-up\". The first synthesis addresses the difficulties with disease process and treatment as rupture of life, an uncertain future on the possibility of recurrence of the disease, the need of continuous treatment to control the disease and compensatory logic of the emotional control relating to contradictories considerations of the current life. Thus, the meaning of this narrative synthesis is paradox. The second synthesis approaches the incorporation of the depression term to common sense, which exposes the sociocultural dimension of the condition of being cancer survivor under therapeutic follow-up; its meaning reveals depression in cancer as social practice and subjectivity expression through emotion of sadness. This research allowed us to interpret the meanings of depression attributed by patients with bladder cancer according to their experiences. As final consideration, we emphasize the importance of the nurses to give attention to the mental health of the patients with cancer, by listening to their subjectivities to promote appropriate interventions, aiming care completeness

<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.

Η υπερδραστηριότητα του εξωστήρα μυ της κύστης, που αποτελεί το αντικείμενο της παρούσας μελέτης, είναι μια ουροδυναμική παρατήρηση. Κατά κανόνα, η εργαστηριακή αυτή παρατήρηση συνοδεύει ένα κλινικό σύνδρομο, που χαρακτηρίζεται από συνδυασμό συμπτωμάτων από το κατώτερο ουροποιητικό σύστημα, και ονομάζεται σύνδρομο της υπερδραστήριας κύστης ή σύνδρομο έπειξης ή σύνδρομο συχνουρίας-έπειξης. Αν και το κλινικό σύνδρομο είναι εξαιρετικά συχνό και αποτελεί αναπόσπαστο τμήμα του καθημερινού όγκου δουλειάς κάθε ουρολόγου ή και γενικού ιατρού, η χρήση της σχετικής ονοματολογίας για τα συμπτώματα, τα σημεία και εργαστηριακά ευρήματα είναι πολλές φορές καταχρηστική και, ως εκ τούτου, η προκαλούμενη σύγχυση αναπόφευκτη. Η αιτιολογία της υπερδραστηριότητας του εξωστήρα, με εξαίρεση τις περιπτώσεις όπου ανιχνεύσιμη νευρολογική βλάβη μπορεί να ενοχοποιηθεί αιτιολογικά (νευροπαθής υπερδραστηριότητα εξωστήρα) είναι άγνωστη (ιδιοπαθής) παρά τις σχετικές θεωρίες που έχουν διατυπωθεί για την ερμηνεία της. Όσον αφορά την αντιμετώπιση της σχετικής συμπτωματολογίας υπάρχει πληθώρα μεθόδων τόσο συντηρητικών όσο και επεμβατικών. Η ποικιλία των διαθέσιμων φαρμακευτικών σκευασμάτων και διαφόρων επεμβατικών τεχνικών, αν όχι ανεξάντλητη είναι σίγουρα μεγάλη και αποδεικνύει την έλλειψη ιδανικής θεραπευτικής προσέγγισης. Η εκτίμηση του αποτελέσματος της όποιας θεραπευτικής παρέμβασης γίνεται άλλοτε με αντικειμενικά και άλλοτε με υποκειμενικά κριτήρια. Αδιαμφισβήτητα η υποκειμενική εκτίμηση του αποτελέσματος είναι ιδιαίτερα μεγάλης σημασίας, καθώς, δικαιωματικά, ο ίδιος ο ασθενής αποτελεί τον τελικό αποδέκτη των ενεργειών και παρενεργειών της θεραπείας. Όμως, η επιστημονική μέθοδος, απαραίτητη για την βελτίωση της κατανόησης της αιτιολογίας , της παθοφυσιολογίας της φυσικής ιστορίας και της θεραπευτικής προσέγγισης οποιασδήποτε παθολογίας, απαιτεί αυστηρά αντικειμενικά κριτήρια . Γίνεται κιόλας φανερό ότι υπάρχουν αρκετά κενά όσον αφορά την ονοματολογία, την αιτιολογία και τη διάγνωση της υπερδραστηριότητας του εξωστήρα. Όμως και στον τομέα της θεραπείας ή έλλειψη τόσο προγνωστικών κριτηρίων για την ανταπόκριση στη θεραπεία όσο και γενικά αποδεκτών, κλινικά σημαντικών, παραμέτρων εκτίμησης του αποτελέσματος επιβάλλουν την περαιτέρω μελέτη για την ανεύρεσή τους. Σ’ αυτή την κατεύθυνση επικεντρώνεται και προσπάθεια της συγκεκριμένης μελέτης. / -

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Ο καρκίνος της ουροδόχου κύστης αποτελεί την 4η κατά συχνότητα μορφή καρκίνου στους άνδρες στο Δυτικό κόσμο, ακολουθώντας τον καρκίνο του προστάτη, του πνεύμονα και του κόλου. Η επιφανειακή μορφή του καρκίνου της κύστεως έχει το ιδιαίτερο χαρακτηριστικό των πολύ συχνών υποτροπών, οι οποίες ευθύνονται για τη μεγάλη νοσηρότητα της νόσου. Οι πολύ συχνές υποτροπές έχουν, όπως εύκολα γίνεται αντιληπτό, τεράστιο κοινωνικοοικονομικό κόστος, εφ’ όσον ένα σημαντικό τμήμα των πασχόντων αποτελεί μέρος του οικονομικά ενεργού πληθυσμού. Η καρκινογένεση δεν είναι μια απλή διαδικασία, αλλά μια αλληλουχία αλλαγών οι οποίες αφορούν τους κυτταρικούς μηχανισμούς αύξησης, διαφοροποίησης και απόπτωσης και οδηγούν στη μετατροπή ενός φυσιολογικού κυττάρου σε νεοπλασματικό. Ως «Χημειοπρόληψη» ορίζεται η χρήση ειδικών φυσικών ή συνθετικών χημικών ουσιών που μπορούν να παρέμβουν σε κάποια από τα μοριακά αυτά γεγονότα και να προλάβουν, καταστείλουν ή αναστρέψουν, την εξέλιξη προκαρκινικών βλαβών σε διηθητικό καρκίνο. Η διαφορά στην επίπτωση του νεοπλάσματος και τα διαφορετικά κλινικοπαθολογοανατομικά χαρακτηριστικά του καρκίνου της ουροδόχου κύστεως ανάμεσα στα δύο φύλα, υποδεικνύουν ένα σημαντικό ρόλο των ορμονών του φύλου στην παθογένεια του νεοπλάσματος. Υποθέσεις μόνο γίνονται για τη σημασία των οιστρογόνων στην ανάπτυξη καρκίνου της ουροδόχου κύστεως, αφού ακόμα ο ρόλος τους δεν έχει αποσαφηνιστεί. Τα οιστρογόνα ασκούν τη δράση τους μέσω των υποδοχέων τους (ERα, ERβ) οι οποίοι αποτελούν μέλη μιας υπεροικογένειας μεταγραφικών παραγόντων, των πυρηνικών υποδοχέων. Οι πυρηνικοί υποδοχείς ρυθμίζουν τη γονιδιακή έκφραση μέσω θετικής ή αρνητικής παρεμβάσεως στη δράση άλλων μεταγραφικών παραγόντων, όπως του NFκB, με τη βοήθεια ενός μηχανισμού που ονομάζεται cross-talk. Αν και η φλεγμονή με την καρκινογένεση έχουν αναγνωρισμένη σχέση από παλιά, συνδέθηκαν άμεσα με την παρατήρηση ότι υπερέκφραση του γονιδίου για το ένζυμο κυκλοοξυγενάση-2 (COX-2), αποτελεί πρώιμο γεγονός της καρκινογένεσης (Greten et al, 2004). Η COX-2, όπως επίσης και ο μεταγραφικός παράγοντας NFκB, που σχετίζεται με τη φλεγμονή, έχει διαπιστωθεί ότι συμμετέχουν στις διαδικασίες της καρκινογένεσης.. Η σύνδεση των οιστρογόνων στους οιστρογόνικους υποδοχείς επάγει τη δέσμευση συν-ρυθμιστικών παραγόντων, οι οποίοι διακρίνονται σε δύο μεγάλες κατηγορίες τους συν-ενεργοποιητές (coactivators), όπως p300 και τους συν-καταστολείς (corepressors), όπως NCoR. Κατά την παρούσα εργασία μελετήθηκε η μεμονωμένη όσο και η συνδυαστική έκφραση των πέντε παραπάνω μορίων στο φυσιολογικό επιθήλιο και στα καρκινώματα διαφόρων Grade, σε ιστικά δείγματα από 140 ασθενείς που υποβλήθηκαν σε διαγνωστική βιοψία διουρηθρική εκτομή νεοπλάσματος κύστεως ή ριζική κυστεκομή. Η μέθοδος που εφαρμόστηκε ήταν η ανοϊστοχημεία σε τομές παραφίνης, η οποία λόγω του μορφολογικού της χαρακτήρα επέτρεψε τη λήψη δεδομένων για τη σχετική εντόπιση των μορίων στους ενδοκυττάριους χώρους, τις ενδοεπιθηλιακές στιβάδες, τις φυσιολογικές ή παθολογικές ιστολογικές βαθμίδες και το επιθηλιακό ή μεσεγχυματικό διαμέρισμα. Ο παράγοντας NFκB (υπομονάδα p65) εμφάνισε μεικτή υποκυττάρια εντόπιση. Στην παρούσα ανοσοϊστοχημική μελέτη το επίπεδο της έκφρασης του NFκB στον πυρήνα των καρκινικών κυττάρων παρουσίαζε μια στατιστικά σημαντική συνολική αύξηση στα τρία επίπεδα διαφοροποίησης των καρκινωμάτων. Τα καρκινώματα χαμηλής διαφοροποίησης παρουσίαζαν ισχυρότερη ανοσοθετικότητα του NFκB από τα μετρίας και καλής διαφοροποίησης. Η αύξηση της πυρηνικής εντόπισης του NFκB συνδυάζεται με ταυτόχρονη ελάττωση της κυτταροπλασματικής, γεγονός που επιβεβαιώνει τη βιολογική δράση του. Ο πυρηνικός υποδοχέας ERβ, που εντοπίζεται στο πυρήνα των καλώς διαφοροποιημένων καρκινικών κυττάρων, είναι στατιστικά σημαντικά αυξημένος σε σχέση με λιγότερο διαφοροποιημένα νεοπλασματικά κύτταρα. Στην παρούσα ανοσοϊστοχημική μελέτη τα κύτταρα του φυσιολογικού επιθηλίου της ουροδόχου κύστης, εκφράζουν έντονα τον πυρηνικό υποδοχέα και κατά την πρόοδο της καρκινογένεσης η έκφραση του ελαττώνεται, παράλληλα με την απώλεια της διαφοροποίησης των καρκινικών κυττάρων. Στην εξέλιξη της καρκινογένεσης, η COX-2 επάγεται σταθερά, σύμφωνα με τα αποτελέσματα της παρούσας εργασίας, με διαδοχικές αυξήσεις που συνοδεύουν όλα τα στάδια της προοδευτικής αποδιαφοροποίησης των κυττάρων. Η πυρηνική έκφραση του p300 αυξάνεται σταδιακά καθώς τα καρκινώματα αποκτούν χαρακτήρες αποδιαφοροποίησης, συσχέτιση στατιστικώς σημαντική. Η πυρηνική έκφραση του NCoR ελαττώνεται σταδιακά καθώς τα καρκινώματα αποκτούν χαρακτήρες αποδιαφοροποίησης, συσχέτιση στατιστικώς σημαντική, σύμφωνα με τα ευρήματα της παρούσας μελέτης. Στις υπόλοιπες συσχετίσεις μελετήθηκε η συν-έκφραση πλέον των παραγόντων σε κάθε ασθενή, με σκοπό την εξαγωγή συμπερασμάτων για ενδεχόμενη αλληλεπίδραση τους. Αναλυτικότερα, παρατηρήθηκε στα καρκινώματα της ουροδόχου κύστεως ισχυρή θετική συσχέτιση του NFκB με την έκφραση της COX-2, υποδηλώνοντας τον υποστηρικτικό ρόλο των δύο αυτών παραγόντων στην πρόοδο της καρκινογένεσης. Από τη συσχέτιση NFκB και ERβ προέκυψε κατασταλτική επίδραση του πρώτου στην ογκοανασταλτική δράση του δεύτερου, υποδηλώνοντας σχέση ανταγωνισμού στη δέσμευση συνπαραγόντων και κατάληψης ίδιων περιοχών στους υποκινητές γονιδίων, ενώ δεν αναδείχθηκε συνομιλία ανάμεσα στον ERβ και COX-2. Τέλος αποκαλύφθηκε συνεργική δράση του NFκB με τον p300 στην καρκινογένεση, με τον ERβ και NCoR να χάνουν την ικανότητα πρόκλησης κυτταρικής διαφοροποίησης και άρα την προστατευτική επίδρασή τους. Η έκφραση του ERβ συσχετίστηκε με την ιστοπαθολογική βαρύτητα, ανά βαθμό έκφρασης των συνρυθμιστών p300 και NCoR, ώστε να διευκρινιστεί εάν η συγκέντρωση τους στα κύτταρα είναι καθοριστικός παράγοντας για την επίδραση του πυρηνικού υποδοχέα στον ιστολογικό φαινότυπο. Η επεξεργασία των δεδομένων φανερώνει αρνητική συσχέτιση της προόδου της καρκινογένεσης με την πυρηνική έκφραση του ERβ, αλλά μόνο όταν τα κύτταρα υπερεκφράζουν παράλληλα τον p300. Η απώλεια της έκφρασης του NCoR αναστέλλει την ενεργοποίηση του πυρηνικού υποδοχέα ERβ, γεγονός που διαπιστώθηκε και στην παρούσα μελέτη κατά τη διάρκεια της απώλεια διαφοροποίησης των καρκινικών κυττάρων της ουροδόχου κύστης. Επομένως, το τελικό αποτέλεσμα της δράσης του ERβ εξαρτάται από τον ανταγωνισμό των ενεργοποιητών και καταστολέων για τις ίδιες θέσεις σύνδεσης στο μόριο του ERβ. Συνολικά, η χρήση αγωνιστών των ERβ και NCoR με παράλληλη αναστολή των NFκB, COX2 και NCoR, θα είχε πιθανότατα ευνοϊκό αποτέλεσμα στην αναστροφή της καρκινογένεσης στην ουροδόχο κύστη. Ειδικές παράμετροι του χημειοπροληπτικού σχήματος, θα ήταν ωφέλιμο να τροποποιούνται ύστερα από εξατομικευμένη αξιολόγηση του δικτύου των πέντε παραγόντων. / Backround Bladder cancer is the forth most common malignancy among men in the Western World, following prostate, lung, and colon cancer. However, due to the highly recurrent nature of the disease, bladder cancer is the most prevalent and the most expensive per patient treated. Carcinogenesis is a complicated multistage process that gradually deprives normal cells of their natural phenotype, resulting in tissue disturbance, from which tumors finally emerge. During its lengthy course it is accompanied by an evenly prolonged inflammatory response. Chemoprevention pursues the arrest of both processes, by means of pharmacological targetting key molecules, involved in cell growth, differentiation and apoptosis, as well as in chronic inflammation. Nuclear Hormone Receptors are appropriate targets, as they are induced by ligand binding to mediate gene transcription. Epidemiological and molecular data support the possible role of ERβ and NFκB between the two collateral processes, providing evidence for target-specific chemopreventive strategies. ERβ promotes cellular differentiation and restriction of inflammation. Nuclear receptor coregulators provide a great level of sophistication in the dynamic process of transcriptional regulation. The transcriptional coactivator p300 is a ubiquitous nuclear protein and transcriptional cofactor with intrinsic acetyltransferase activity. NCoR is a protein that contain distinct functional domains responsible for interaction with NRs, and activation of HDAC proteins, ultimately resulting in targeted repression of transcription The inducible transcription factor NF-κB, immediately after being released from a cytoplasmic inhibitor, translocates into nucleus, where it enhances transcription of anti-apoptotic and pro-inflammatory genes. COX-2, an enzyme often induced in neoplastic conditions, perpetuates the chronic inflammatory state in the epithelium and its microenvironment, by means of prostaglandin synthesis. Elucidation of the molecular networks implicated in estrogen signaling is very important in view of the potential use of selective estrogen receptor modulators in chemoprevention and targeted anticancer therapy. Materials and Methods. In our retrospective study we included 111 consecutive patients (74 males and 37 females), aged 23-90 years (mean 70±10) diagnosed with TCC of the bladder by either biopsies, transurethral resection of bladder tumor, or radical cystectomies, between 2000 and 2002 from the Urological Department of Urology of University Hospital of Patras, Greece. None of the patients had received any preoperative intravesical therapy. Bladder tumors were graded and staged according to the World Health Organization (WHO) grading. Paraffin section immunohistochemistry was utilized and relative expression was estimated in intracellular compartments, intraepithelial layers, and histologic categories. NF-κB(p65 subunit) demonstrated mixed subcellular presence, COX2 cytoplasmic whereas ERβ, p300 and NCoR staining patterns were nuclear. NF-κB and COX-2, were constantly upregulated as tumorigenesis progressed. Results NF-κB, COX-2 and p300 expression correlated positively with progression of carcinogenesis, suggesting a potential involvement in bladder tumorigenesis. On the contrary, ERβ and NCoR were severely diminished in cancer, compared to normal epithelium, and they were affected by tumor Grade. The remaining correlations are based on coexpression analysis of the aforementioned factors, individually for each patient, to permit judgement of molecular interactions. In detail, an inverse staining between ERβ and nuclear p65 immunoreactivity was observed and we could suggest that there is a reciprocal transactivation between ERβ and activated NFκB. COX-2 was positively associated in bladder carcinomas with NFκB, a finding which may denote the nuclear factor contribution to the enzyme induction. A correlation has been established, when correlating the expression of ERβ with the coregulators, positive with NCoR and negative with p300, indicating a potential role of these key molecules in bladder carcinogenesis. Furthermore, p300 and NCoR, may not be strictly segregated and in bladder cancer cells interact directly, since, according to biochemical purification studies, p300 is capable of directed negative interaction with NCoR. Conclusions The inhibition of ERβ in combination with the antiapoptotic properties of NFκB may contribute to the pathogenesis of TCC. Selective ERβ and NCoR agonist and agents-inhibitors of NFκB, COX2 and p300 may represent a possible new treatment strategy, by virtue of their role in bladder carcinogenesis. Subtle variations in the chemopreventive regimen, based on personalized molecular profiling, would hopefully achieve a patient-tailored therapeutic approach.

A telenfermagem é a utilização dos recursos tecnológicos e dos sistemas de comunicação em prol do desenvolvimento da enfermagem. Vários países utilizam a telenfermagem na gestão de cuidados de saúde com resultados positivos. Na Enfermagem brasileira este é um campo a ser explorado, assim o objetivo desse trabalho foi de desenvolver estratégias de telenfermagem no atendimento a pacientes com bexiga neurogênica, usuários de cateterismo urinário intermitente limpo, atendidos em um centro de reabilitação de um hospital universitário. O estudo foi realizado em 3 etapas: etapa 1 - revisão integrativa da literatura para investigar na literatura a aplicação da telenfermagem; etapa 2 - elaboração e validação do protótipo de manual de telenfermagem para subsidiar o enfermeiro na implantação da intervenção de telenfermagem no atendimento ao paciente com bexiga neurogênica, usuário de cateterismo urinário intermitente limpo; etapa 3: estudo piloto sobre a implantação de intervenção de telenfermagem no atendimento ao paciente em uso de cateterismo urinário intermitente limpo. O manual de telenfermagem foi construído procurando contextualizar a telenfermagem no Brasil e no exterior, com seleção da teoria de Orem para direcionar o teleatendimento ao paciente, e um referencial sobre os cuidados para a realização de um teleatendimento. Descrição de recursos tecnológicos disponíveis que podem ser utilizados no teleatendimento, além de informações sobre os cuidados de saúde ao paciente com bexiga neurogênica e na realização do cateterismo urinário intermitente limpo. O manual foi validado em aparência e conteúdo por peritos com auxílio de um instrumento de avaliação. A intervenção de telenfermagem mostrou resultados importantes como forma de implementação do tratamento de saúde tradicional ao paciente, bem como evidenciou barreiras que precisam ser superadas para que esse tipo de atendimento seja realizado, como uma pesquisa efetiva com possibilidades de implementação posterior ao serviço de saúde. Os dados foram analisados através de estatística descritiva (frequência e porcentagem) com auxílio do programa SPSS (Statistical Package for Social Science), versão 15.0. O nível de concordância entre os peritos foi considerado de 70% para cada aspecto do instrumento utilizado / Telenursing is the use of technological resources and communication systems for the development of nursing. A great number of countries employ telenursing on healthcare management with positive results, however, it is a field yet to be explored in Brazilian nursing. The aim of this study was to develop telenursing strategies in the care to patients with neurogenic bladder, users of clean intermittent urinary catheterization, treated in a university hospital rehabilitation center. The study was carried out in 3 stages: stage 1 - integrative literature review to research the application of telenursing; stage 2 - development and validation of a telenursing booklet prototype to support nurses in implementing telenursing intervention on care to patients with neurogenic bladder, users of clean intermittent urinary catheterization; stage 3 - pilot study on the application of telenursing intervention in care to patients using clean intermittent urinary catheterization. The telenursing booklet was developed to contextualize telenursing in Brazil and abroad, selecting Orem\'s theory to guide the telecare to the patient and a framework about the care needed to carry out telecare. Description of available technological resources that can be used in telecare, as well as information about healthcare to patients with neurogenic bladder and in the procedure of clean intermittent urinary catheterization. The booklet underwent face and content validation by experts with aid from an assessment instrument. Telenursing intervention showed important results as a way to implement the traditional health treatment to the patient, also pointing out barriers that must be overcome in order to perform this kind of treatment, such as an effective research with possibilities of subsequent implementation at the health service. Data were analyzed through descriptive statistics (frequency and percentage) using SPSS (Statistical Package for Social Science) software version 15.0. The level of concordance considered among the experts was of 70% for each aspect of the used instrument