Les protéoglycans (PGs) à héparane- (HS) et chondrol'tine-sulfate (CS) jouent un rôle essentiel dans la régulation de nombreux processus biologiques tels que la différenciation cellulaire, la signalisation, la prolifération et la morphogenèse. En effet, les PGs via leurs chaînes de glycosaminoglycanes (GAGs) agissent comme des récepteurs pour des facteurs de croissance, des enzymes et des protéines d'adhésion cellulaire, modulant ainsi leur biodisponibilité, la formation de gradient et leur activité. La synthèse des chaînes de CS et d'HS est initiée par le transfert d'un résidu xylose sur des sérines de la protéine "core" des PGs par les xylosyltransferases (XT), XT-I et XT-II. Ces enzymes catalysent une étape limitante régulant la biosynthèse des GAGs. En plus de la régulation par les XT, la synthèse des GAGs peut être régulée par la phosphorylation du xylose en position 2-0 par la kinase Fam20B. Il a été montré que la XT-I et la XT-II sont capables de restaurer la synthèse des PGs dans les cellules déficientes en activité xylosyltransférase, suggérant ainsi qu'elles sont fonctionnellement redondantes. Cependant, les rôles spécifiques de la XT­ I et de la XT-II et l'impact de leurs mutations génétiques sur la synthèse des CS et des HS ne sont pas connus. Au cours de cette thèse, nous avons montré que la XT-I initie la synthèse des PGs avec des chaînes de CS et d'HS de tailles plus longues que celle initiée par la XT-II et avons démontré que cela est lié à leurs localisations subcellulaires respectives. En outre, nous avons montré d'une part que les mutations génétiques de la XT-I réduisent fortement la capacité de l'enzyme à initier la synthèse des GAGs et d'autre part que deux mutations de la XT-II conduisent à la mislocalisation de l'enzyme et l'abrogation de sa capacité à initier la synthèse des chaînes de CS et d'HS. En outre, nous avons démontré en utilisant différentes lignées cellulaires et des mutants inactifs que la kinase Fam20B régule négativement le processus de synthèse des chaînes de GAGs et par conséquent que la phosphorylation du résidu xylose par Fam20B entraîne un blocage dans la polymérisation des chaînes de GAGs / Heparan- (HS) and chondroitin-sulfate (CS) proteoglycans (PGs) are essential regulators of many biological processes including cell differentiation, signalization, proliferation and morphogenesis. Indeed, PGs act through their glycosaminoglycan (GAG) chains as receptors for growth factors, enzymes and cell adhesion proteins, thereby modulating their bioavailability, gradient formation and biological activity. The assembly of HS and CS GAG chains is initiated by the transfer of xylose to serine residues of PG core protein by the xylosyltransferases (XT) enzymes, XT-I and XT-II. These enzymes catalyze a rate-limiting step in the biosynthesis pathway and therefore considered as a regulating factors in the GAG biosynthesis process. Beside the regulation by XT enzymes, GAG chain synthesis may also be regulated by phosphorylation of the xylose residue at 2-0 position by the kinase Fam20B. Ithas been shown that XT-I and XT-II are able to restore GAG-attached PG synthesis in xylosyltransferase-deficient cells, suggesting that they are functionally redundant. However, nothing is known of the specific roles of XT-I and XT-II ifany and of the impact of XT-I and XT-II mutations on the synthesis of CS- and HS-PG. Here, we showed that XT-I initiates PGs with large size CS- and HS-GAG chains compared to XT-II and demonstrated that this was linked to their subcellular localisation. In addition, we have addressed the question of whether genetic mutations of XT-I and XT-II associated with various diseases impact CS- and HS-PG synthesis and found that mutations in XT-I strongly reduced the capacity of the enzyme to initiate the synthesis of both CS and HS GAG chains. However, two mutations in XT-II abrogated the capacity of the enzyme to initiate CS and HS GAGs and led to the mislocalisation of the enzyme. Furthermore, we demonstrated using variouse cell lines and dead mutants that Fam20B negatively regulates GAG synthesis process and that phosphorylation of xylose residue by this kinase resulted in a blokage of the polymerisation procees of the GAG chain

INTRODUÇÃO: A matriz extracelular no endométrio fornece vasta gama de sinais envolvidos em diferentes processos celulares, tais como morte celular e proliferação. Neste sentido, os glicosaminoglicanos e os proteoglicanos, juntamente com os fatores de crescimento, modulam várias etapas da angiogênese, proliferação celular e remodelação do estroma, o que pode ser importante para o fluxo menstrual regular e a redução dos processos proliferativos. Além disso, são importantes para a adequada interação maternofetal. OBJETIVO: Avaliar a concentração de ácido hialurônico, das enzimas de biossíntese do ácido hialurônico - hialurônico sintases (HAS1, HAS2 e HAS3) e dos pequenos proteoglicanos ricos em leucina (decorim, biglicam, lumicam e fibromodulina) no endométrio de pacientes com síndrome dos ovários policísticos (SOP) e de mulheres eumenorreicas. MÉTODOS: Foram analisadas 20 amostras de endométrio, 10 provenientes de pacientes com SOP e 10 de mulheres com ciclos menstruais regulares na fase proliferativa do ciclo, com idade variando entre 20 e 35 anos, atendidas na Divisão de Clínica Ginecológica do Hospital das Clínicas da FMUSP (HC-FMUSP). A determinação do perfil e da concentração do ácido hialurônico foi efetuada por método bioquímico de ensaio fluorimétrico (ELISA-like). A sua localização no tecido endometrial, assim como as dosagens das enzimas sintases (HAS1, HAS2 e HAS3) e dos pequenos proteoglicanos ricos em leucina (decorim, biglicam, lumicam e fibromodulina e), foi feita por imunoistoquímica e \"western blotting\". Para a análise dos resultados foi utilizado o teste t de student (p<=0,05). Os cálculos foram realizados com auxílio do programa SPSS versão13 (SPSS, Chicago, IL). RESUTADOS: Houve maior concentração de ácido hialurônico no endométrio de mulheres eumenorreicas na fase proliferativa do ciclo menstrual do que no das com síndrome dos ovários policísticos. Com relação às sintases do ácido hialurônico, observou-se maior reatividade de HAS1 e HAS2 e menor reatividade de HAS3 no endométrio de mulheres com SOP em relação às mulheres com ciclos menstruais regulares na fase proliferativa. Quanto aos pequenos proteoglicanos ricos em leucina notou-se maior imunorreatividade de decorim e lumicam no endométrio de pacientes com SOP. CONCLUSÕES: As pacientes com SOP apresentam menor concentração de ácido hialurônico e maior reatividade ao decorim e lumicam em relação às mulheres com ciclos regulares na fase proliferativa. Esses dados sugerem que nas pacientes com SOP, o endométrio seria menos receptivo e teria mecanismos para evitar a proliferação excessiva / INTRODUCTION: Endometrium extracellular matrix provides wide range of signals at different cellular levels like cell death and proliferation. In this regard, glycosaminoglycans and proteoglycans, along with growth factors, modulate various stages of angiogenesis, cell proliferation and remodeling of the stroma, which can be important for regulating menses and reducing the proliferative processes. Additionally, it is important for proper fetal-maternal interactions. OBJECTIVE: Evaluate hyaluronic acid concentration, the enzymes of hyaluronic acid synthases (HAS1, HAS2 and HAS3) and small leucine-rich proteoglycans (decorin, lumican, fibromodulim and biglycan) in the endometrium of patients with polycystic ovary syndrome (PCOS) and eumenorrheic women. METHODS: A total of 20 endometrial samples from 10 patients with PCOS and 10 women with regular menstrual cycles in the proliferative phase, with ages ranging between 20 and 35 years, attended at Gynecology Division of Clinical Hospital of the FMUSP (HC-USP). Profile determination and the concentration of hyaluronic acid were performed by biochemical method of fluorimetric assay (ELISA-like). Its location in the endometrial tissue as well as the dosage of enzymes synthases (HAS1, HAS2 and HAS3) and small leucine-rich proteoglycans (decorin, lumican, fibromodulim and biglycan) was done by immunohistochemistry and western blotting. To analyze the results Student t test was used (p < 0.05). The calculations were performed with software SPSS version 13. RESULTS: A higher concentration of hyaluronic acid in eumenorrheic women endometrium in proliferative phase when compared with polycystic ovary syndrome. Regarding hyaluronic acid synthases, there was a higher HAS1 and HAS2 reactivity and lower HAS3 reactivity in the PCOS endometrium compared to women with regular menstrual cycles in the proliferative phase. Decorin and lumican showed higher immunoreactivity in PCOS endometrium. CONCLUSIONS: PCOS patients have a lower concentration of hyaluronic acid and greater reactivity to decorin and lumican than eumenorrheic women in proliferative phase. These data suggest that in patients with PCOS, the endometrium would be less receptive and have mechanisms to prevent excessive proliferation

INTRODUÇÃO: A matriz extracelular no endométrio fornece vasta gama de sinais envolvidos em diferentes processos celulares, tais como morte celular e proliferação. Neste sentido, os glicosaminoglicanos e os proteoglicanos, juntamente com os fatores de crescimento, modulam várias etapas da angiogênese, proliferação celular e remodelação do estroma, o que pode ser importante para o fluxo menstrual regular e a redução dos processos proliferativos. Além disso, são importantes para a adequada interação maternofetal. OBJETIVO: Avaliar a concentração de ácido hialurônico, das enzimas de biossíntese do ácido hialurônico - hialurônico sintases (HAS1, HAS2 e HAS3) e dos pequenos proteoglicanos ricos em leucina (decorim, biglicam, lumicam e fibromodulina) no endométrio de pacientes com síndrome dos ovários policísticos (SOP) e de mulheres eumenorreicas. MÉTODOS: Foram analisadas 20 amostras de endométrio, 10 provenientes de pacientes com SOP e 10 de mulheres com ciclos menstruais regulares na fase proliferativa do ciclo, com idade variando entre 20 e 35 anos, atendidas na Divisão de Clínica Ginecológica do Hospital das Clínicas da FMUSP (HC-FMUSP). A determinação do perfil e da concentração do ácido hialurônico foi efetuada por método bioquímico de ensaio fluorimétrico (ELISA-like). A sua localização no tecido endometrial, assim como as dosagens das enzimas sintases (HAS1, HAS2 e HAS3) e dos pequenos proteoglicanos ricos em leucina (decorim, biglicam, lumicam e fibromodulina e), foi feita por imunoistoquímica e \"western blotting\". Para a análise dos resultados foi utilizado o teste t de student (p<=0,05). Os cálculos foram realizados com auxílio do programa SPSS versão13 (SPSS, Chicago, IL). RESUTADOS: Houve maior concentração de ácido hialurônico no endométrio de mulheres eumenorreicas na fase proliferativa do ciclo menstrual do que no das com síndrome dos ovários policísticos. Com relação às sintases do ácido hialurônico, observou-se maior reatividade de HAS1 e HAS2 e menor reatividade de HAS3 no endométrio de mulheres com SOP em relação às mulheres com ciclos menstruais regulares na fase proliferativa. Quanto aos pequenos proteoglicanos ricos em leucina notou-se maior imunorreatividade de decorim e lumicam no endométrio de pacientes com SOP. CONCLUSÕES: As pacientes com SOP apresentam menor concentração de ácido hialurônico e maior reatividade ao decorim e lumicam em relação às mulheres com ciclos regulares na fase proliferativa. Esses dados sugerem que nas pacientes com SOP, o endométrio seria menos receptivo e teria mecanismos para evitar a proliferação excessiva / INTRODUCTION: Endometrium extracellular matrix provides wide range of signals at different cellular levels like cell death and proliferation. In this regard, glycosaminoglycans and proteoglycans, along with growth factors, modulate various stages of angiogenesis, cell proliferation and remodeling of the stroma, which can be important for regulating menses and reducing the proliferative processes. Additionally, it is important for proper fetal-maternal interactions. OBJECTIVE: Evaluate hyaluronic acid concentration, the enzymes of hyaluronic acid synthases (HAS1, HAS2 and HAS3) and small leucine-rich proteoglycans (decorin, lumican, fibromodulim and biglycan) in the endometrium of patients with polycystic ovary syndrome (PCOS) and eumenorrheic women. METHODS: A total of 20 endometrial samples from 10 patients with PCOS and 10 women with regular menstrual cycles in the proliferative phase, with ages ranging between 20 and 35 years, attended at Gynecology Division of Clinical Hospital of the FMUSP (HC-USP). Profile determination and the concentration of hyaluronic acid were performed by biochemical method of fluorimetric assay (ELISA-like). Its location in the endometrial tissue as well as the dosage of enzymes synthases (HAS1, HAS2 and HAS3) and small leucine-rich proteoglycans (decorin, lumican, fibromodulim and biglycan) was done by immunohistochemistry and western blotting. To analyze the results Student t test was used (p < 0.05). The calculations were performed with software SPSS version 13. RESULTS: A higher concentration of hyaluronic acid in eumenorrheic women endometrium in proliferative phase when compared with polycystic ovary syndrome. Regarding hyaluronic acid synthases, there was a higher HAS1 and HAS2 reactivity and lower HAS3 reactivity in the PCOS endometrium compared to women with regular menstrual cycles in the proliferative phase. Decorin and lumican showed higher immunoreactivity in PCOS endometrium. CONCLUSIONS: PCOS patients have a lower concentration of hyaluronic acid and greater reactivity to decorin and lumican than eumenorrheic women in proliferative phase. These data suggest that in patients with PCOS, the endometrium would be less receptive and have mechanisms to prevent excessive proliferation

A matriz extracelular (MEC) pode ser definida como um complexo de proteínas e glicoproteínas que envolve as células nos mais diversos tecidos e tem papel importante na diferenciação e atividade celular, bem como no processo de mineralização e nos processos neoplásicos. Os componentes não colágenos da MEC têm sido abundantemente estudados, visando conhecer os minuciosos detalhes da biologia dos tecidos e assim entender os mecanismos envolvidos em suas patologias. Neste contexto, o presente trabalho tem como objetivo estudar a expressão e distribuição dos seguintes componentes não colágenos da MEC dos tecidos dentais: biglican, decorin, fibromodulin, osteonectina (ONC), osteopontina (OPN), sialoproteína óssea (BSP) e osteocalcina (OCC) no ameloblastoma e no tumor odontogênico cístico calcificante (cisto de Gorlin). Para ta nto foi utilizada a técnica da imunoistoquímica, com o método da estreptavidina-biotina-peroxidase, e anticorpos contra as proteínas anteriormente citadas. Os resultados mostraram que o biglican, o decorin e a BSP foram expressos somente nas células epiteliais metaplásicas, nas células fantasmas e células fantasmas em processo de calcificação, no estroma dos ameloblastomas e no ectomesênquima neoplásico do tumor odontogênico cístico calcificante. Já o fibromodulin e a OC foram predominantemente negativos no componente epitelial e no mesenquimal, com exceção para as células fantasmas, células fantasmas em processo de calcificação e áreas de hialinização próximas ao epitélio. A ONC foi positiva na maioria das células epiteliais, com exceção das células estrelárias dos ameloblastomas folicular e acantomatoso, e também no componente mesenquimal de ambas neoplasias. Já a OPN apresentou positividade somente nos focos de calcificação presentes no tumor odontogênico cístico calcificante. As proteínas estudadas apresentaram distribuição semelhante em neoplasias caracterizadas por padrões de crescimento diferentes, levando a crer que, apesar de participarem ativamente do mecanismo de crescimento neoplásico intra-ósseo, isoladamente não exercem papel decisivo na determinação do tipo de padrão de crescimento. Outro fato digno de relevância é a baixa expressão dessas proteínas nas células epiteliais neoplásicas quando comparada com a expressão no estroma e ectomesênquima, levando-nos a crer que as células epiteliais atuem principalmente como estimuladores da expressão dessas proteínas, que, por sua vez, podem atuar de forma agonista ou antagonista ao crescimento neoplásico. / The extracellular matrix (ECM) can be defined as a complex of proteins and glycoproteins that involves the cells in all tissues. It has a key role in cell differentiation and activity, as well as in mineralization and neoplastic processes. The non-collagenous components of the ECM have been abundantly studied to know the details of the biology of tissues and thus to understand the mechanisms involved in its pathologies. The aim of the present work is to study the expression and distribution of the following noncollagenous components of the ECM of dental tissues: biglycan, decorin, fibromodulin, osteonectin (ONC), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OCC) in ameloblastoma and the calcifying cystic odontogenic tumour. The streptavidin-biotinperoxidase method of immunohistochemistry was used with antibodies against the antigens previously cited. The results show that biglican, decorin and BSP had been expressed only in metaplastic epithelial cells, in ghost cells and ghost cells in calcification process, stroma of ameloblastomas and neoplastic ectomesenchyma of the calcifying cystic odontogenic tumour. The fibromodulin and the OCC showed predominantly negative expression in the epithelial and mesenchymal components, with exception for the ghost cells, ghost cells in calcification process and hyalinization areas next to the epithelium. The ONC was positive in the majority of the epithelial cells, with exception of the central cells of follicular and acanthomatous ameloblastomas, and also in the mesenchymal component of both tumours. OPN presented positivity only in the calcification focus of the calcifying cystic odontogenic tumour. The proteins studied presented similar distribution in tumours characterized by different patterns of growth, leading to believe that although they participate actively of the mechanism of intraosseous growth, separately they do not exert a key role in the determination of the type of growth pattern. Another relevance fact is the low expression of these proteins in the neoplastic epithelial cells when compared to the expression in stroma and ectomesenchyma, which make us believe that the epithelial cells act mainly as stimulators of the expression of these proteins, which in turn can act as agonist or antagonist to the tumour growth.

Tse Pui Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves [191]-216). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABBREVIATIONS --- p.III / ABSTRACT --- p.VIII / 摘要 --- p.X / PUBLICATIONS --- p.XII / TABLE OF CONTENTS --- p.XIII / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Human Immune System and Cancer --- p.1 / Chapter 1.1.1 --- Brief Introduction of the Human Immune System --- p.1 / Chapter 1.1.2 --- Prevalence of Cancer in Hong Kong --- p.4 / Chapter 1.1.3 --- The Role of the Immune System in Tumorigenesis --- p.4 / Chapter 1.1.4 --- Cancer Treatment --- p.5 / Chapter 1.1.5 --- Cancer Prevention --- p.5 / Chapter 1.2 --- Mushroom Polysaccharides --- p.6 / Chapter 1.2.1 --- General Aspects of Mushroom Polysaccharides --- p.6 / Chapter 1.2.2 --- Structure of Mushroom Polysaccharides --- p.9 / Chapter 1.2.2.1 --- Beta (P)-D-glucans --- p.9 / Chapter 1.2.2.2 --- Heteroglucans and Protein-bound Polysaccharides --- p.10 / Chapter 1.2.2.3 --- Structure-Function Interactions of Polysaccharides --- p.12 / Chapter 1.2.3 --- Molecular Interactions of Polysaccharides --- p.14 / Chapter 1.2.4 --- Biological Activities of Polysaccharides --- p.15 / Chapter 1.2.4.1 --- Anti-tumor Activities of Polysaccharides --- p.15 / Chapter 1.2.4.2 --- Immunomodulatory Activities of Polysaccharides --- p.16 / Chapter 1.3 --- Yun Zhi (Coriolus versicolor) --- p.17 / Chapter 1.3.1 --- General Features of Yun Zhi --- p.17 / Chapter 1.3.2 --- Traditional Uses of Yun Zhi --- p.20 / Chapter 1.3.3 --- Active Ingredients of Yun Zhi --- p.20 / Chapter 1.3.3.1 --- "Origin, Properties and Composition of PSK" --- p.21 / Chapter 1.3.3.2 --- "Origin, Properties and Composition of PSP" --- p.22 / Chapter 1.3.4 --- Pharmacological Actions of PSP and PSK --- p.25 / Chapter 1.3.4.1 --- Immunomodulatory Activities --- p.25 / Chapter 1.3.4.2 --- Anti-tumor Activities --- p.32 / Chapter 1.3.4.2 --- Antiviral and Antimicrobial Activities --- p.35 / Chapter 1.3.4.3 --- Antioxidant Activities --- p.36 / Chapter 1.3.5 --- Human Clinical Studies on Yun Zhi --- p.36 / Chapter 1.3.6 --- Toxicology of Yun Zhi --- p.42 / Chapter 1.4 --- Danshen (Salvia miltiorrhiza) --- p.43 / Chapter 1.4.1 --- General Features of Danshen --- p.43 / Chapter 1.4.2 --- Traditional Uses of Danshen --- p.46 / Chapter 1.4.3 --- Active Ingredients of Danshen --- p.47 / Chapter 1.4.4 --- Pharmacological Actions of Danshen --- p.50 / Chapter 1.4.4.1 --- Cardiovascular Effects --- p.50 / Chapter 1.4.4.2 --- Scavenging Effects on Free Radicals --- p.52 / Chapter 1.4.4.3 --- Hepatoprotective Effects --- p.54 / Chapter 1.4.4.4 --- Anti-tumor Effects --- p.56 / Chapter 1.4.4.5 --- Renal Protective Effects --- p.56 / Chapter 1.4.5 --- Human Clinical Studies --- p.57 / Chapter 1.4.6 --- Toxicity of Danshen --- p.59 / Chapter 1.5 --- Aims and Scopes of This Investigation --- p.60 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Normal Subjects --- p.62 / Chapter 2.1.1 --- Inclusion and Exclusion Criteria of Recruitment --- p.62 / Chapter 2.1.2 --- Study Design and Procedure --- p.63 / Chapter 2.1.3 --- Treatment and Blinding --- p.65 / Chapter 2.1.4 --- Blood Sampling --- p.66 / Chapter 2.1.5 --- Blood Processing for Assessment of Immunological Functions --- p.67 / Chapter 2.2 --- Materials --- p.69 / Chapter 2.2.1 --- Endotoxin Assay --- p.69 / Chapter 2.2.2 --- Reagents for Whole Blood Assay --- p.69 / Chapter 2.2.2.1 --- Plain RPMI 1640 Medium --- p.69 / Chapter 2.2.2.2 --- Phosphate-Buffered Saline (PBS) --- p.69 / Chapter 2.2.2.3 --- Mitogens --- p.70 / Chapter 2.2.3 --- Reagents for Total RNA Extraction --- p.70 / Chapter 2.2.3.1 --- Ficoll-Paque Density Gradient Solution --- p.70 / Chapter 2.2.3.2 --- RNA Extraction Kit --- p.70 / Chapter 2.2.3.3 --- RNase-Free DNase Set --- p.71 / Chapter 2.2.3.4 --- β-Mercaptoethanol (β-ME) Solution --- p.71 / Chapter 2.2.4 --- Reagents for Flow Cytometric Analysis of T/B/NK Cell Ratios --- p.71 / Chapter 2.2.4.1 --- MultiTEST IMK Kit with TruCOUNT Tubes --- p.71 / Chapter 2.2.4.2 --- FACSFlo´wёØ Sheath Fluid --- p.74 / Chapter 2.2.4.3 --- CaliBRITE 3 and APC Beads --- p.74 / Chapter 2.2.5 --- Immunoassay Kits for Measuring Cytokines Level --- p.75 / Chapter 2.2.5.1 --- Enzyme-linked Immunosorbent Assay (ELISA) Kits of Cytokines --- p.75 / Chapter 2.2.5.2 --- Human Thl/Th2 Cytokine Cytometric Bead Array (CBA) Kit-II --- p.75 / Chapter 2.2.6 --- Reagents and Buffers for Gel Electrophoresis --- p.78 / Chapter 2.2.6.1 --- Ethidium Bromide (EtBr) --- p.78 / Chapter 2.2.6.2 --- Gel Loading Solution (5X) --- p.78 / Chapter 2.2.6.3 --- Tris-Acetate-EDTA (TAE) Buffer --- p.78 / Chapter 2.2.6.4 --- Agarose Gel --- p.78 / Chapter 2.2.6.5 --- 100 base pair DNA Ladder --- p.79 / Chapter 2.2.7 --- Kits and Reagents for Messenger RNA (mRNA) Expression Array --- p.79 / Chapter 2.2.7.1 --- Human Inflammatory Cytokine/Receptor GEArraýёØ Q Series Kit --- p.79 / Chapter 2.2.7.2 --- Deoxynucleoside Triphosphates (dNTPs) --- p.84 / Chapter 2.2.7.3 --- Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLVRT) --- p.84 / Chapter 2.2.7.4 --- Rnasin Ribonuclease Inhibitor --- p.84 / Chapter 2.2.7.5 --- Biotin-16-2'-deoxy-uridine-5'-triphosphate (Biotin-16-dUTP) --- p.85 / Chapter 2.2.7.6 --- Salmon Sperm DNA Solution --- p.85 / Chapter 2.2.7.7 --- 100 % Sodium Dodecyl Sulfate (SDS) Solution --- p.86 / Chapter 2.2.7.8 --- 20X SSC --- p.86 / Chapter 2.2.7.9 --- ECL Films (Hyperfilm 226}0ёØ ECL 226}0ёØ) --- p.86 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Endotoxin Assay --- p.87 / Chapter 2.3.2 --- Whole Blood Assay (WBA) --- p.88 / Chapter 2.3.3 --- Isolation and Preparation of Plasma and Peripheral Blood Mononuclear Cells (PBMC) from EDTA Blood --- p.88 / Chapter 2.3.4 --- Total RNA extraction --- p.89 / Chapter 2.3.5 --- Flow Cytometric Analysis of T/B/NK Cell Ratios --- p.90 / Chapter 2.3.6 --- Immunoassays of Plasma Samples or Culture Supernatant in WBA --- p.92 / Chapter 2.3.6.1 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.92 / Chapter 2.3.6.2 --- Human Thl/Th2 Cytokine Cytometric Bead Assay (CBA) --- p.93 / Chapter 2.3.7 --- mRNA Expression Study --- p.94 / Chapter 2.3.7.1 --- Agarose Gel Electrophoresis --- p.94 / Chapter 2.3.7.2 --- cDNA Expression Array Analysis --- p.95 / Chapter 2.3.8 --- Statistical Analysis --- p.96 / Chapter CHAPTER 3: --- ENDOTOXIN LEVEL OF YUN ZHI-DANSHEN CAPSULES & SAFETY MEASURES ON STUDY POPULATION IN THE CLINICAL TRIAL / Chapter 3.1 --- Introduction --- p.98 / Chapter 3.2 --- Results --- p.101 / Chapter 3.2.1 --- Endotoxin Level of the Yun Zhi and Danshen Active Capsule --- p.101 / Chapter 3.2.2 --- Study Population --- p.103 / Chapter 3.2.3 --- Dropout Cases --- p.103 / Chapter 3.2.4 --- Safety Parameters --- p.104 / Chapter 3.2.5 --- Compliance Rates --- p.104 / Chapter 3.3 --- Discussion --- p.109 / Chapter CHAPTER 4: --- FLOW CYTOMETRIC ANALYSIS OF T/B/NK CELL RATIOS OF HEALTHY SUBJECTS TAKING YUN ZHI-DANSHEN CAPSULES / Chapter 4.1 --- Introduction --- p.112 / Chapter 4.2 --- Results --- p.118 / Chapter 4.2.1 --- The Percentage and Absolute Count of T Lymphocytes (CD3+) --- p.118 / Chapter 4.2.2 --- The Percentage and Absolute Count of T Helper (TH) Lymphocytes (CD3+ CD4+) --- p.121 / Chapter 4.2.3 --- The Percentage and Absolute Count of Cytotoxic T (CTL) and T Suppressor (Ts) Lymphocytes (CD3+ CD8+) --- p.124 / Chapter 4.2.4 --- The Ratio of T Helper Lymphocytes (CD3+ CD4+) and Cytotoxic T (CTL) and T Suppressor (Ts) Lymphocyes (CD3+ CD8+) --- p.127 / Chapter 4.2.5 --- The Percentage and Absolute Count of B Lymphocytes (CD19+) --- p.129 / Chapter 4.2.6 --- The Percentage and Absolute Count of NK Lymphocytes (CD3- CD 16+ and/or CD56+) --- p.132 / Chapter 4.2.7 --- The Absolute Count of Lymphocytes (CD45+) --- p.135 / Chapter 4.3 --- Discussion --- p.138 / Chapter CHAPTER 5: --- PLASMA CONCENTRATION OF SOLUBLE CYTOKINE RECEPTOR AND EX VIVO CYTOKINE PRODUCTION OF HEALTHY SUBJECTS TAKING YUN ZHI-DANSHEN CAPSULES / Chapter 5.1 --- Introduction --- p.142 / Chapter 5.2 --- Results --- p.147 / Chapter 5.2.1 --- Plasma Concentration of Soluble IL-2 Receptor --- p.147 / Chapter 5.2.2 --- Ex vivo Cytokine Production --- p.147 / Chapter 5.2.3 --- Mitogen Induced IL-6 Production --- p.150 / Chapter 5.2.4 --- Mitogen Induced IFN- γ Production --- p.150 / Chapter 5.2.5 --- Mitogen Induced TNF- a Production --- p.153 / Chapter 5.2.6 --- Mitogen Induced IL-10 Production --- p.153 / Chapter 5.3 --- Discussion --- p.156 / Chapter CHAPTER 6: --- "GENE EXPRESSION OF CYTOKINES, CHEMOKINES AND RECEPTORS OF PBMC OF HEALTHY SUBJECTS TAKING YUN ZHI- DANSHEN CAPSULES" / Chapter 6.1 --- Introduction --- p.162 / Chapter 6.2 --- Results --- p.165 / Chapter 6.2.1 --- Gene Expression of IL-2 Receptor β chain --- p.165 / Chapter 6.2.2 --- Gene Expression of IL-2 Receptor γ chain --- p.165 / Chapter 6.2.3 --- Gene Expression of IL-6 Receptor --- p.166 / Chapter 6.2.4 --- "Gene Expression of Other Cytokines, Chemokines and Receptors" --- p.169 / Chapter 6.3 --- Discussion --- p.172 / Chapter CHAPTER 7: --- CONCLUDING REMARKS AND FUTURE / PERSPECTIVES --- p.176 / APPENDICES --- p.184 / REFERENCES --- p.192

A matriz extracelular (MEC) pode ser definida como um complexo de proteínas e glicoproteínas que envolve as células nos mais diversos tecidos e tem papel importante na diferenciação e atividade celular, bem como no processo de mineralização e nos processos neoplásicos. Os componentes não colágenos da MEC têm sido abundantemente estudados, visando conhecer os minuciosos detalhes da biologia dos tecidos e assim entender os mecanismos envolvidos em suas patologias. Neste contexto, o presente trabalho tem como objetivo estudar a expressão e distribuição dos seguintes componentes não colágenos da MEC dos tecidos dentais: biglican, decorin, fibromodulin, osteonectina (ONC), osteopontina (OPN), sialoproteína óssea (BSP) e osteocalcina (OCC) no ameloblastoma e no tumor odontogênico cístico calcificante (cisto de Gorlin). Para ta nto foi utilizada a técnica da imunoistoquímica, com o método da estreptavidina-biotina-peroxidase, e anticorpos contra as proteínas anteriormente citadas. Os resultados mostraram que o biglican, o decorin e a BSP foram expressos somente nas células epiteliais metaplásicas, nas células fantasmas e células fantasmas em processo de calcificação, no estroma dos ameloblastomas e no ectomesênquima neoplásico do tumor odontogênico cístico calcificante. Já o fibromodulin e a OC foram predominantemente negativos no componente epitelial e no mesenquimal, com exceção para as células fantasmas, células fantasmas em processo de calcificação e áreas de hialinização próximas ao epitélio. A ONC foi positiva na maioria das células epiteliais, com exceção das células estrelárias dos ameloblastomas folicular e acantomatoso, e também no componente mesenquimal de ambas neoplasias. Já a OPN apresentou positividade somente nos focos de calcificação presentes no tumor odontogênico cístico calcificante. As proteínas estudadas apresentaram distribuição semelhante em neoplasias caracterizadas por padrões de crescimento diferentes, levando a crer que, apesar de participarem ativamente do mecanismo de crescimento neoplásico intra-ósseo, isoladamente não exercem papel decisivo na determinação do tipo de padrão de crescimento. Outro fato digno de relevância é a baixa expressão dessas proteínas nas células epiteliais neoplásicas quando comparada com a expressão no estroma e ectomesênquima, levando-nos a crer que as células epiteliais atuem principalmente como estimuladores da expressão dessas proteínas, que, por sua vez, podem atuar de forma agonista ou antagonista ao crescimento neoplásico. / The extracellular matrix (ECM) can be defined as a complex of proteins and glycoproteins that involves the cells in all tissues. It has a key role in cell differentiation and activity, as well as in mineralization and neoplastic processes. The non-collagenous components of the ECM have been abundantly studied to know the details of the biology of tissues and thus to understand the mechanisms involved in its pathologies. The aim of the present work is to study the expression and distribution of the following noncollagenous components of the ECM of dental tissues: biglycan, decorin, fibromodulin, osteonectin (ONC), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OCC) in ameloblastoma and the calcifying cystic odontogenic tumour. The streptavidin-biotinperoxidase method of immunohistochemistry was used with antibodies against the antigens previously cited. The results show that biglican, decorin and BSP had been expressed only in metaplastic epithelial cells, in ghost cells and ghost cells in calcification process, stroma of ameloblastomas and neoplastic ectomesenchyma of the calcifying cystic odontogenic tumour. The fibromodulin and the OCC showed predominantly negative expression in the epithelial and mesenchymal components, with exception for the ghost cells, ghost cells in calcification process and hyalinization areas next to the epithelium. The ONC was positive in the majority of the epithelial cells, with exception of the central cells of follicular and acanthomatous ameloblastomas, and also in the mesenchymal component of both tumours. OPN presented positivity only in the calcification focus of the calcifying cystic odontogenic tumour. The proteins studied presented similar distribution in tumours characterized by different patterns of growth, leading to believe that although they participate actively of the mechanism of intraosseous growth, separately they do not exert a key role in the determination of the type of growth pattern. Another relevance fact is the low expression of these proteins in the neoplastic epithelial cells when compared to the expression in stroma and ectomesenchyma, which make us believe that the epithelial cells act mainly as stimulators of the expression of these proteins, which in turn can act as agonist or antagonist to the tumour growth.

Durante a gestação, em varias espécies de mamíferos, os fibroblastos endometriais são alvos de profundas modificações morfofuncionais que levam a aquisição de um fenótipo epitelial e à expressão de novas moléculas, formando uma nova estrutura no útero denominada decídua. Em camundongos, a reação decidual pode ser estimulada artificialmente (na ausência de embrião), resultando na formação do deciduoma, um modelo de grande relevância para a identificação de fatores oriundos ou não do embrião necessários para a promoção da decidualização. A decidualização também promove uma profunda remodelação da matriz extracelular (MEC) do endométrio, e ambos os processo são fundamentais para o sucesso da gestação. Existem evidencias, muitas das quais são oriundas dos estudos do Laboratório de Biologia da Reprodução e Matriz extracelular (LBR-MEC), mostrando que a remodelação da MEC do útero não grávido é modulada pelos hormônios ovarianos estrógeno (E2) e progesterona (P4). Faltam, entretanto, na literatura, estudos consistentes sobre a regulação da MEC endometrial na ausência de sinais parácrinos provenientes do embrião. Além disso, não se conhece detalhes sobre a ação dos hormônios ovarianos sobre a produção de componentes da MEC por células deciduais. Nesse contexto, o presente estudo teve dois objetivos centrais: (i) caracterizar por imuno-histoquímica a composição e organização da MEC durante o desenvolvimento do deciduoma, (ii) estudar por qPCR, Western blot, e imunolocalização o efeito dos hormônios E2 e Medroxiprogesterona (MPA) na dinâmica da expressão de RNAm, síntese e secreção de moléculas da MEC em culturas primárias de células obtidas de deciduoma. Observamos que, a distribuição do colágeno tipo I, III, IV, V e dos proteoglicanos decorim, biglicam e versicam no deciduoma, foi semelhante ao já observado na decídua. As análises in vitro, mostram que o hormônio E2 aumenta a expressão gênica, a síntese e a deposição de decorim enquanto o MPA tem como alvo o biglicam. Ambos hormônios modulam a expressão de desmina, um marcador de decidualização. O presente estudo também mostra que o padrão de remodelação das moléculas alvo do presente estudo, é similar ao observado durante a decidualização da gestação normal, Conclui-se, portanto, que a remodelação da MEC é um evento intrínseco do processo de decidualização quer na gestação quer na pseudogestação. Ou seja, não foram identificadas diferenças que indicassem a existência de controle pelo embrião. Mostramos ainda que, in vitro, os hormônios E2 e MPA regulam de modo específico a expressão gênica e a secreção do proteoglicanos decorim e biglicam. / During pregnancy in several species of mammals, including humans and mice, endometrial fibroblasts undergo extensive morphofunctional changes acquiring an epithelial phenotype. Those new cells form a new structure in the uterus called decidua. In mice, the decidual reaction can be artificially induced in pseudopregnant females resulting in the formation of a structure morphologically similar to the decidua called deciduoma, a relevant model to study the putative role of the embryo upon decidualization. Endometrial decidualization is an essential event for the success of pregnancy. A notable remodeling of the extracellular matrix (ECM) organization and molecular composition occurs during this process. There are evidences, many of them coming from studies of the Laboratory of Reproductive and Extracellular Matrix Biology (LBR-MEC), that estrogen (E2) and progesterone (P4) modulate the remodeling of the uterine ECM. Nevertheless, there is no consistent information about the role, if it exists, of the embryo on the regulation of the endometrial ECM. Furthermore, it was not yet clarified how the ovarian hormones act on the production of ECM components by decidual cells. Thus, the objective of present study was to identify the composition and organization of the ECM during the development of the mouse deciduoma; and to study by qPCR, Western blot and immunolocalization methods, the effect of hormones E2 and medroxiprogesterone (MPA) on synthesis and secretion of ECM molecules by primary cultures of mouse decidual cells obtained from deciduoma. We found that the distribution of collagen types I, III, IV, V and proteoglycans decorin, biglycan and versican in deciduoma, was similar to that previously observed in the decidua. The in vitro assays showed E2 increases the gene expression for the core protein of Decorin, while MPA increases the expression of the core protein of Biglycan. In addition, was observed that both hormones increase the expression of desmin a marker of decidualization. These results showed that in the endometrium of both pregnant and pseudopregnant animals ECM molecules such as collagens and proteoglycans are similarly modulated by ovarian hormones. At from the present study we may conclude that ECM remodeling is an intrinsic event that happens during decidualization modulated by E2 and MPA and this modualation independ of the presence of the embryo in the uterus. In adition we showed that in decidual cells in vitro the gene expression and the secretion of proteoglycans decorin and biglycan are differentially regulated by hormones E2 and MPA.

Includes bibliographical references (leaves 68-76) Aims to observe changes in the expression of syndecan-1 in both the developing epithelium of the rat oral mucosa, and in epithelial cell rests of Malassez in the developing periodontium of normal rat molars, from late crown development through to early eruption.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.