Introdução: Um dos principais obstáculos para compreensão dos eventos biológicos envolvidos no câncer é a falta de modelos adequados para o estudo in vitro em especial em relação ao câncer de próstata (CaP) e ao câncer de bexiga (CaB). Há um número limitado de linhagens celulares de CaP e de CaB sendo a maioria proveniente de tumores invasivos e metastáticos. Sabe-se ainda, que existem diferenças étnicas entre as populações quanto ao comportamento de neoplasias. Desta forma, a pesquisa baseada em linhagens de uma população homogênea seria fonte de resultados limitados, não contemplando a diversidade que sabidamente ocorre entre os diferentes grupos. Além desse aspecto, as linhagens celulares comerciais são na sua maioria adquiridas na Coleção Americana de Culturas de Tecido (ATCC, do inglês American Tissue Cell Culture) que apesar de serem bem padronizadas, requerem processos de importação com aumento do custo e demandas burocráticas que dificultam a pesquisa. Portanto, consideramos vital para a compreensão dos fenômenos relacionados à carcinogênese, assim como estudos de resistência a drogas, quimioprevenção e novas estratégias terapêuticas, o desenvolvimento de linhagens tumorais derivadas de tumores primários que acometem a nossa população, peculiarmente miscigenada. No presente trabalho, fragmentos de carcinoma urotelial da bexiga e de adenocarcinoma da próstata foram obtidos durante cirurgia para remoção de tumores primários de pacientes tratados e acompanhados na Divisão de Urologia da Faculdade de Medicina da Universidade de São Paulo (FMUSP) e no Hospital Sírio Libanês. As linhagens estabelecidas a partir destes fragmentos foram caracterizadas através da análise da cinética de crescimento, análises imunocitoquímicas e anormalidades cromossômicas incluindo cariótipo e hibridização in situ por fluorescência (FISH). Além disso, as linhagens obtidas foram submetidas a estudos de quimiossensibilidade com o uso dos compostos curcumin e Prima-1. Avaliamos ainda, a tumorigenicidade de nossas linhagens em camundongos atímicos. Os resultados deste trabalho demonstram o desenvolvimento de três linhagens de CaB e três linhagens de CaP sendo as mesmas não tumorigênicas em camundongos atímicos. Além disso, demonstramos que o curcumin na concentração de 50 M induziu morte celular em todas as linhagens estudadas, sendo seu efeito mais evidente nas linhagens de CaP. Por fim, Prima-1 reduziu a viabilidade celular independente do status de p53 nas linhagens de CaB / Introduction: One of the main obstacles for understanding biological events involved in cancer is the lack of appropriated models for in vitro studies especially for prostate cancer (PC) and bladder cancer (BC). There are a limited number of PC and BC cell lines being the majority originated from metastatic and invasive tumors. Also it is well known that there are ethnic differences between populations concerning the behavior of tumors. In such a way, the research based on cell lines derived from a homogenous population should be source of limited results, not contemplating the diversity known to occur among different groups. In addition the commercial cell lines are generally acquired at American Tissue Cell Culture (ATCC) that although wellestablished requires importation processes with cost increase and bureaucratic demands that difficult the research. Therefore we consider vital to the comprehension of the carcinogenesis phenomena, as well as drug resistance studies, chemoprevention and new therapeutic strategies, the development of tumor lineages derived from primary tumors that assail our miscigenated population. At the present work, fragments of bladder urothelial carcinoma and prostate adenocarcinoma were obtained by surgical resection of primary tumors from patients treated and followed in the Division of Urology of the Clinical Hospital of the São Paulo University (FMUSP) and Syrian Lebanese Hospital. The cell lines established from these fragments were characterized through growth kinetic, immunocytochemistry and chromosome abnormalities including karyotyping and Fluorescence in situ hybridization (FISH). Moreover, the cell lines were submitted to chemosensitivity studies using curcumin and Prima-1 and analyzed regarding their tumorigenicity in athymic mice. The results of this work show the development of three BC and three PC cell lines that were not tumorigenic in athymic mice. Curcumin at 50 M concentration induced cell death in all studied lineages, being more effective in PC cell lines. Finally, PRIMA-1 reduced the cellular viability independent of the p53 status in BC cell lines

by Poon Yam Kau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 174-183). / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Discovery of melatonin --- p.4 / Chapter 1.2 --- Biosynthesis of melatonin --- p.4 / Chapter 1.3 --- Physiology of melatonin and other pineal indoles --- p.5 / Chapter 1.4 --- Relationship between pineal indoles and cancers --- p.6 / Chapter 1.5 --- Macrophages --- p.9 / Chapter 1.6 --- Lymphocytes --- p.11 / Chapter Chapter 2 --- Effects of different light/dark cycles on serum melatonin level in mice and effect of melatonin-feeding on serum glutamate-oxaloacetate transaminase (GOT) activity in mice / Chapter 2.1 --- Introduction --- p.14 / Chapter 2.2 --- Materials and methods --- p.15 / Chapter 2.3 --- Results --- p.22 / Chapter 2.4 --- Discussion --- p.23 / Chapter Chapter 3 --- Actions of endogenous and exogenous melatonin on murine peritoneal macrophages / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.2 --- Materials and methods --- p.28 / Chapter 3.3 --- Results --- p.33 / Chapter 3.4 --- Discussion --- p.36 / Chapter Chapter 4 --- Actions of endogenous and exogenous melatonin on murine splenic lymphocytes / Chapter 4.1 --- Introduction --- p.55 / Chapter 4.2 --- Materials and methods --- p.56 / Chapter 4.3 --- Results --- p.62 / Chapter 4.4 --- Discussion --- p.69 / Chapter Chapter 5 --- In vitro effects of melatonin on murine peritoneal macrophages and splenic lymphocytes / Chapter 5.1 --- Introduction --- p.105 / Chapter 5.2 --- Materials and methods --- p.106 / Chapter 5.3 --- Results --- p.109 / Chapter 5.4 --- Discussion --- p.113 / Chapter Chapter 6 --- Effects of methoxytryptamine on murine peritoneal macrophages and splenic lymphocytes / Chapter 6.1 --- Introduction --- p.125 / Chapter 6.2 --- Materials and methods --- p.126 / Chapter 6.3 --- Results --- p.129 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7 --- In vitro effects of pineal indoles on cultured tumor cell lines / Chapter 7.1 --- Introduction --- p.145 / Chapter 7.2 --- Materials and methods --- p.146 / Chapter 7.3 --- Results --- p.148 / Chapter 7.4 --- Discussion --- p.152 / Chapter Chapter 8 --- General Discussion --- p.170 / References --- p.174

Finally, the pharmacodynamic effects of propranolol powder formulation with different buffering were carried out in two healthy male subjects. The maximal reduction in heart rate was found at the saliva pH of 7.6, which corresponded to the pHmax of propranolol. A buffered propranolol sublingual tablet was then prepared to achieve the saliva pH around 7.6. The preliminary investigation confirmed that the sublingually administrated buffered propranolol tablet produced a faster and more pronounced heart rate reduction than the non-buffered commercial propranolol tablet. / Firstly, the use of the HO-1-u-1 cell culture for screening sublingual drug delivery was validated. The cells were seeded on cell culture inserts. The integrity of cell layers, inter-passage variation and directionality were assessed by measuring the resistance and the permeability of standard markers, beta-blockers and calcium channel blockers. The effect of pH, osmolarity and a permeation enhancer (GDC) were also studied. The results showed that HO-1-u-1 cells grown on inserts formed stratified and epithelial-like structure that preserved the typical histological feathers of the normal human sublingual epithelium. The maximal integrity was reached in 23 days. The Papp of beta-blockers and calcium channel blockers ranged from 2.89+/-0.17 x 10 -6 cm/s to 6.37+/-0.37 x 10-6 cm/s. The permeability of selected beta-blockers under different pH, osmolarity and GDC revealed that enhancing effects were significant for hydrophilic compounds but less for lipophilic compounds. / Secondly, fresh porcine sublingual mucosa was prepared and compared to the cell line model. Good correlations were obtained for both the Papp of beta-blockers and the enhancement ratios of pH and GDC between the two models. / The aims of the present study are (1) to develop and validate a human sublingual epithelial cell line model and (2) to demonstrate the application in sublingual development of cardiovascular drugs. / Thirdly, the steady-state flux (Jss) at various pH levels were measured. Results show that saturated propranolol solution at pH 7.0--7.6 resulted in a much higher Jss than the solution at other pHs. These data led to the development of theoretical equations for predicting the optimum pH (pHmax) for ionizable compounds. The calculation fitted well with the experimental data. / Wang Yanfeng. / Advisers: Moses S. S. Chow; Zhong Joan Zuo. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 184-). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

<p>The symptoms of Parkinson's disease occur due to degeneration of dopamine neurons in substantia nigra. It has been demonstrated that glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor when it comes to protect and enhance survival of dopamine neurons in animal models of Parkinson's disease. The aim of this study was to evaluate short- and long-term effects of GDNF on survival and nerve fiber outgrowth of dopamine cells and astrocytic migration in mouse fetal ventral mesencephalic (VM) tissue. Primary tissue cultures were made of mouse fetal VM tissue and evaluated at 7 and 21 days in vitro (DIV) in terms of dopaminergic nerve fiber outgrowth and astrocytic migration when developed with GDNF present, partially, or completely absent. The results revealed that VM tissue cultured in the absence of GDNF did not exhibit any significant differences in migration of astrocytes or dopaminergic nerve fiber outgrowth neither after 7 DIV nor after 21 DIV, when compared with tissue cultured with GDNF present. Migration of astrocytes and dopaminergic nerve fiber outgrowth reached longer distances when tissue was left to develop for 21 DIV in comparison with 7 DIV. In order to study the long-term effects of GDNF, mouse fetal dopaminergic tissue was transplanted into the ventricles of adult mice and evaluated after 6 months. No surviving dopamine neurons were present in the absence of GDNF. In contrast dopamine neurons developed with GDNF did survive, indicating that GDNF is an essential neurotrophic factor when it comes to long-term dopamine cell survival. More cases have to be assessed in the future in order to strengthen the findings. Thus, transplanted dopamine neurons will be assessed after 3 and 12 months in order to map out when dopamine neurons deprived of GDNF undergo degeneration.</p>

<p>The relationship between Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) is not entirely elucidated and a clonal relation may be present more often than previously believed. Mechanisms of tumour progression and resistance to therapy are poorly understood.</p><p>Between 1974 and 1994 all individuals in Sweden with both HL and NHL were identified. Thirty-two cases were studied using clinical, histopathological and immunohistochemical methods. The second lymphoma often appeared in an aggressive clinical form and a significant correlation between the expression of p53 and LMP-1 in the first and second lymphoma was demonstrated.</p><p>The treatment outcome for 307 patients with advanced stages of HL, in an unselected population was in accordance with the treatment results of large centres world-wide. Some patients were successfully selected for a shorter chemotherapy-regimen without inferior treatment results.</p><p>In 124 patients with relapse, the survival of those primarily treated with radiotherapy according to the National guidelines was in accordance with the survival of patients of initially advanced stages. A worse outcome was found for those who received both chemotherapy and radiotherapy initially, probably because of a higher frequency of bulky disease in this group. </p><p>Immunohistochemical analysis of the tumour suppressor protein p53 and retinoblastoma protein (Rb) of paired samples at diagnosis and at relapse in 81 patients did not reveal any specific staining pattern affecting survival.</p><p>A novel B-cell line (U-2932) was established from a patient with a diffuse large B-cell lymphoma previously treated for advanced stage and subsequent relapses of HL. An identical rearranged IgH gene was demonstrated in tumour cells from the patient and in U-2932. A p53 point mutation was detected and over-expression of the p53 protein was found. A complex karyotype with high-level amplifications of the chromosomal regions 18q21 and 3q27, i.e. the loci for <i>bcl-2</i> and <i>bcl-6</i> were demonstrated. </p>

<p>Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. </p><p>In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate.</p><p>In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36.</p><p>In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice.</p><p>In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.</p>

<p>Primary hyperparathyroidism (pHPT) due to parathyroid tumours with hypersecretion of parathyroid hormone and hypercalcaemia is a common disease with incompletely understood etiology affecting more than 1 % of the population, primarily postmenopausal women. In secondary hyperparathyroidism (sHPT), parathyroid tumours develop in response to calcium and vitamin D deficiency generally in patients with uraemia. HPT is usually treated by surgical removal of enlarged parathyroid glands.</p><p>The aim of this thesis was to examine the Wnt/β-catenin signalling pathway in parathyroid tumours.</p><p>Aberrantly accumulated β-catenin was found in all analysed pHPT and sHPT tumours, with a stabilising homozygous mutation (Ser37Ala) in 7.3% of the pHPT tumours. Truncation of the APC protein was not found. MYC, a β-catenin target gene was overexpressed in a substantial fraction of pHPT and sHPT parathyroid tumours. </p><p>A parathyroid tumour cell line (sHPT-1) was established from a hyperplastic gland removed at operation of a patient with sHPT. The cells produced parathyroid hormone and grew with a doubling time of approximately 72 hours. Stabilised nonphosphorylated transcriptionally active β-catenin was expressed. Efficient transfection of siRNA against β-catenin decreased expression of cyclin D1 and MYC, and inhibited cell growth with ensuring cell death. </p><p>The Wnt coreceptor LRP5 was found expressed with an internal deletion of 142 amino acids (LRP5Δ) in 86% and 100% of pHPT and sHPT tumours, respectively. Stabilising mutation of β-catenin and expression of LRP5Δ was mutually exclusive. Expression of LRP5Δ was required to maintain the nonphosphorylated transcriptionally active ß-catenin level, MYC expression, parathyroid cell growth in vitro, and tumour growth in transplanted SCID mice. Wnt3 ligand and LRP5Δ strongly activated transcription, and LRP5Δ was insensitive to inhibition by DKK1.</p><p>Aberrant accumulation of β-catenin by stabilising mutation or expression of LRP5Δ appears as a common pathogenic pathway for hyperparathyroid disease. LRP5Δ in particular presents a potential target for therapeutic intervention.</p>

The relationship between Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) is not entirely elucidated and a clonal relation may be present more often than previously believed. Mechanisms of tumour progression and resistance to therapy are poorly understood. Between 1974 and 1994 all individuals in Sweden with both HL and NHL were identified. Thirty-two cases were studied using clinical, histopathological and immunohistochemical methods. The second lymphoma often appeared in an aggressive clinical form and a significant correlation between the expression of p53 and LMP-1 in the first and second lymphoma was demonstrated. The treatment outcome for 307 patients with advanced stages of HL, in an unselected population was in accordance with the treatment results of large centres world-wide. Some patients were successfully selected for a shorter chemotherapy-regimen without inferior treatment results. In 124 patients with relapse, the survival of those primarily treated with radiotherapy according to the National guidelines was in accordance with the survival of patients of initially advanced stages. A worse outcome was found for those who received both chemotherapy and radiotherapy initially, probably because of a higher frequency of bulky disease in this group. Immunohistochemical analysis of the tumour suppressor protein p53 and retinoblastoma protein (Rb) of paired samples at diagnosis and at relapse in 81 patients did not reveal any specific staining pattern affecting survival. A novel B-cell line (U-2932) was established from a patient with a diffuse large B-cell lymphoma previously treated for advanced stage and subsequent relapses of HL. An identical rearranged IgH gene was demonstrated in tumour cells from the patient and in U-2932. A p53 point mutation was detected and over-expression of the p53 protein was found. A complex karyotype with high-level amplifications of the chromosomal regions 18q21 and 3q27, i.e. the loci for bcl-2 and bcl-6 were demonstrated.

Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate. In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36. In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice. In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.

Primary hyperparathyroidism (pHPT) due to parathyroid tumours with hypersecretion of parathyroid hormone and hypercalcaemia is a common disease with incompletely understood etiology affecting more than 1 % of the population, primarily postmenopausal women. In secondary hyperparathyroidism (sHPT), parathyroid tumours develop in response to calcium and vitamin D deficiency generally in patients with uraemia. HPT is usually treated by surgical removal of enlarged parathyroid glands. The aim of this thesis was to examine the Wnt/β-catenin signalling pathway in parathyroid tumours. Aberrantly accumulated β-catenin was found in all analysed pHPT and sHPT tumours, with a stabilising homozygous mutation (Ser37Ala) in 7.3% of the pHPT tumours. Truncation of the APC protein was not found. MYC, a β-catenin target gene was overexpressed in a substantial fraction of pHPT and sHPT parathyroid tumours. A parathyroid tumour cell line (sHPT-1) was established from a hyperplastic gland removed at operation of a patient with sHPT. The cells produced parathyroid hormone and grew with a doubling time of approximately 72 hours. Stabilised nonphosphorylated transcriptionally active β-catenin was expressed. Efficient transfection of siRNA against β-catenin decreased expression of cyclin D1 and MYC, and inhibited cell growth with ensuring cell death. The Wnt coreceptor LRP5 was found expressed with an internal deletion of 142 amino acids (LRP5Δ) in 86% and 100% of pHPT and sHPT tumours, respectively. Stabilising mutation of β-catenin and expression of LRP5Δ was mutually exclusive. Expression of LRP5Δ was required to maintain the nonphosphorylated transcriptionally active ß-catenin level, MYC expression, parathyroid cell growth in vitro, and tumour growth in transplanted SCID mice. Wnt3 ligand and LRP5Δ strongly activated transcription, and LRP5Δ was insensitive to inhibition by DKK1. Aberrant accumulation of β-catenin by stabilising mutation or expression of LRP5Δ appears as a common pathogenic pathway for hyperparathyroid disease. LRP5Δ in particular presents a potential target for therapeutic intervention.