Introdução: A evolução dos marcadores sorológicos da hepatite B em pacientes com hepatite B crônica coinfectados com o vírus da imunodeficiência humana (HIV) tem sido pouco documentada. Objetivos: O objetivo deste estudo foi descrever a evolução dos marcadores sorológicos AgHBe e AgHBs, com ênfase na avaliação da frequência de perda definitiva ou transitória desses marcadores, neste grupo de pacientes. Buscamos, também, comparar as variáveis clínicas e demográficas desses pacientes segundo a evolução desses marcadores sorológicos. Pacientes e métodos: A população de estudo foi composta por pacientes atendidos em um ambulatório de referência para atendimento a pacientes infectados pelo HIV em São Paulo, Brasil. Todos os pacientes selecionados eram portadores de HIV e de hepatite B crônica. Foram incluídos nesse estudo pacientes AgHBs positivos, com confirmação da presença desse marcador em, no mínimo, duas sorologias consecutivas, com intervalo mínimo de seis meses entre elas. Variáveis clínicas foram coletadas: idade, sexo, fator de exposição ao HIV/VHB, contagem de células T CD4+, carga viral do HIV, níveis de alaninoaminotransferase (ALT), uso de terapia antirretroviral, incluindo lamivudina, tenofovir ou outras drogas com ação anti-VHB. Resultados: Entre 2.242 pacientes HIV positivos encontrados, foram identificados 105 (4,68%) pacientes com hepatite B crônica. O tempo de seguimento variou de 06 meses a 20,5 anos e o número de coletas variou de 2 a 18 por paciente no período. A maioria dos pacientes era do sexo masculino (97%) e 43,9% (46/105) tinha história de uma ou mais infecções oportunistas. Todos os pacientes tiveram terapia antirretroviral iniciada durante o seguimento. Entre os pacientes com hepatite B crônica, 58% (61/105) eram AgHBe positivos na primeira avaliação. Entre eles, 15% (16/105) apresentaram clareamento de AgHBs e 50% (8/16) dos que clarearam AgHBs apresentaram posterior reativação desse marcador durante a evolução clínica. Dentre os pacientes AgHBe positivos na primeira sorologia, 57% (35/61) apresentaram clareamento desse marcador, sendo que 28,5% (10/35) dos que clarearam AgHBe voltaram a apresentar este marcador durante a evolução clínica. Conclusão: Observamos uma significativa taxa de clareamento e posterior reativação dos marcadores AgHBs e AgHBe no grupo de pacientes avaliados. Estes resultados sugerem que o monitoramento frequente desses marcadores sorológicos deveria ser recomendado / Introduction: Evolution of hepatitis B serological markers among HIV co-infected patients has rarely been documented. Objectives: The aim of this study was to describe the evolution of HBsAg and HBeAg serological markers, with emphasis on the frequency of transient or permanent loss of these markers, among this group of patients. It was also our objective to compare patients\' demographic and clinical variables according to the evolution of these serological markers. Patients and methods: The enrolled patients were selected from those registered at a HIV-Outpatient Clinic in Sao Paulo, Brazil. All included patients were diagnosed with HIV infection and chronic hepatitis B. HBsAg patients who underwent at least two repeated HBV serological testing during clinical follow up , with tests taken at least six months apart, were included in the analysis. Clinical information was collected: age, sex, patient history regarding HIV/HBV transmission, CD4 T+ cell count, HIV viral load, alanine amino transferase (ALT) level, and use of antiretroviral drugs including lamivudine, tenofovir or other anti-HBV drugs. Results: Among 2,242 HIV-positive patients 105 (4.68%) patients were identified with chronic hepatitis B. Follow-up time for these patients varied from 06 months to 20.5 years and the number of serological testing for each patient varied from 2 to 18 along this period. Most patients were male (97%) and 43.9% (46/105) had a history of one or more opportunistic infections. All patients had initiated antiretroviral medication during follow-up. Among patients with chronic hepatitis B, 58% (61/105) were HBeAg reagent at the first assessment. Also, fifteen percent of them (16/105) underwent HBsAg clearance and 50% (8/16) of those who initially lost HBsAg underwent HBsAg reactivation during clinical follow up. Among HBeAg positive patients in the first serology, 57% (35/61) lost this marker during clinical follow up, whereas 28.5% (10/35) of those who initially cleared this serological marker underwent HBeAg reactivation. Conclusion: A significant rate of changes of HBsAg and HBeAg was observed, during clinical follow up among this group of patients. These results suggest that periodic monitoring of HBV serological markers should be recommended

Chi Keung Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 219-251). / Chapter CHAPTER I --- LITERATURE REVIEW / Chapter 1 --- The structure of bone --- p.2 / Chapter 1.1. --- The cortical bone --- p.3 / Chapter 1.2. --- The cancellous bone --- p.3 / Chapter 2 --- The composition of bone --- p.3 / Chapter 2.1. --- Bone minerals --- p.4 / Chapter 2.2. --- The organic matrix --- p.4 / Chapter 2.3. --- The bone cells --- p.9 / Chapter 2.3.1. --- The osteoblast and the osteocyte --- p.9 / Chapter 2.3.2. --- The osteoclast --- p.11 / Chapter 3 --- Bone turnover - modelling and remodelling of bone --- p.13 / Chapter 3.1. --- Postulated sequence of bone remodelling --- p.14 / Chapter 4 --- Regulation of bone resorption --- p.16 / Chapter 4.1. --- Role of osteoblast and the lining cell on bone resorption --- p.17 / Chapter 5 --- Regulation of bone formation --- p.19 / Chapter 6 --- Effects of systemic hormones and local factors on bone metabolism --- p.20 / Chapter 6.1. --- Parathyroid hormone --- p.20 / Chapter 6.2. --- "1,25-dihydroxyvitamin D3" --- p.22 / Chapter 6.3. --- Calcitonin --- p.23 / Chapter 6.4. --- Prostaglandins --- p.23 / Chapter 6.5. --- Sex hormones --- p.24 / Chapter 6.6. --- Glucocorticoid --- p.26 / Chapter 6.7. --- Growth hormone --- p.27 / Chapter 6.8. --- Insulin --- p.28 / Chapter 6.9. --- Thyroid hormones --- p.29 / Chapter 6.10. --- Other systemic and local factors --- p.30 / Chapter 7 --- Indices of bone turnover --- p.34 / Chapter 8 --- Non-biochemical indices of bone metabolism --- p.34 / Chapter 8.1. --- Radionuclide bone scan --- p.34 / Chapter 8.2. --- Radiokinetic assessment --- p.35 / Chapter 8.3. --- Bone biopsy --- p.35 / Chapter 8.4. --- Bone densitometry --- p.36 / Chapter 9 --- Biochemical indices of bone metabolism --- p.37 / Chapter 10 --- Biochemical markers of bone formation --- p.38 / Chapter 10.1. --- Alkaline phosphatase --- p.38 / Chapter 10.1.1. --- Role and origin of bone alkaline phosphatase isoenzyme --- p.39 / Chapter 10.1.2. --- Measurement of bone alkaline phosphatase --- p.41 / Chapter 10.1.2.1. --- Heat inactivation --- p.42 / Chapter 10.1.2.2. --- Chemical inactivation --- p.43 / Chapter 10.1.2.3. --- Immunological methods --- p.44 / Chapter 10.1.2.4. --- High performance liquid chromatography --- p.45 / Chapter 10.1.2.5. --- Gel electrophoresis --- p.45 / Chapter 10.1.2.6. --- Isoelectric focusing --- p.47 / Chapter 10.2. --- Osteocalcin --- p.48 / Chapter 10.3. --- Osteonectin --- p.51 / Chapter 10.4. --- Matrix Gla-protein --- p.51 / Chapter 10.5. --- Other non-collagenous proteins --- p.52 / Chapter 10.6. --- Urinary Gla concentration --- p.52 / Chapter 10.7. --- Collagen peptides and extension peptides --- p.54 / Chapter 11 --- Biochemical markers of bone resorption --- p.55 / Chapter 11.1. --- Urine hydroxyproline --- p.55 / Chapter 11.2. --- Pyridinium cross-links --- p.58 / Chapter 11.3. --- Acid phosphatase --- p.60 / Chapter 11.3.1. --- Acid phosphatase isoenzymes --- p.60 / Chapter 11.3.2. --- The band 5 acid phosphatase isoenzyme genetics and characteristics --- p.62 / Chapter 11.3.3. --- Band 5 acid phosphatase as marker of osteoclastic function --- p.64 / Chapter 11.3.4. --- Measurement of osteoclastic acid phosphatase --- p.67 / Chapter 11.3.4.1. --- Specific chemical inhibitor --- p.67 / Chapter 11.3.4.2. --- Electrophoresis --- p.67 / Chapter 11.3.4.3. --- Immunological methods --- p.68 / Chapter 12 --- Problems with current biochemical markers of bone metabolism --- p.68 / Chapter 13 --- Aims of this study --- p.70 / Chapter CHAPTER II --- PURIFICATION OF TARTRATE-RESISTANT ACID PHOSPHATASE AND THE DEVELOPMENT OF AN IMMUNOASSAY FOR IT'S MEASUREMENT / Chapter 1 --- Introduction --- p.72 / Chapter 2 --- Materials and methods --- p.75 / Chapter 2.1. --- Chemicals and reagents --- p.75 / Chapter 2.1.1. --- Apparatus --- p.76 / Chapter 2.2. --- Methods --- p.77 / Chapter 2.2.1. --- Cord serum --- p.77 / Chapter 2.2.2. --- Measurement of tartrate-resistant acid phosphatase activity --- p.77 / Chapter 2.2.3. --- Measurement of protein concentration --- p.80 / Chapter 2.2.4. --- Purification of TRACP from cord plasma --- p.82 / Chapter 2.2.4.1. --- Cation-exchange column chromatography --- p.83 / Chapter 2.2.4.2. --- Gel filtration column chromatography --- p.84 / Chapter 2.2.4.3. --- Concanavalin A-affinity column chromatography --- p.85 / Chapter 2.2.4.4. --- Preparative isoelectric focusing (IEF) --- p.86 / Chapter 2.3. --- Characterisation of purified TRACP --- p.90 / Chapter 2.3.1. --- Polyacrylamide gel electrophoresis (PAGE) --- p.91 / Chapter 2.3.2. --- "Optimum pH, substrate specificity and the effects of potential activators and inhibitors on TRACP activity" --- p.99 / Chapter 2.3.3. --- Amino acid composition of purified TRACP --- p.101 / Chapter 2.4. --- Methods for raising anti-human TRACP antibody and characterisation of the antiserum --- p.102 / Chapter 2.4.1. --- Production of rabbit anti-human TRACP antibody --- p.102 / Chapter 2.4.2. --- Determination of the titre of rabbit anti-human TRACP antibody --- p.103 / Chapter 2.4.3. --- Immunoblotting analyses for cross reactivity study --- p.103 / Chapter 2.4.4. --- Immunohistochemical study for antibody specificity --- p.105 / Chapter 2.4.5. --- Cross reactivity study of the rabbit anti-human TRACP antibody to some tissue preparations --- p.107 / Chapter 2.5. --- Enzyme linked immunosorbent assay for TRACP --- p.109 / Chapter 2.5.1. --- Optimisation and evaluation of the new ELISA method for TRACP --- p.111 / Chapter 3 --- RESULTS --- p.113 / Chapter 3.1. --- "Precision of methods for the determination of protein, TRACP and phosphate." --- p.113 / Chapter 3.2. --- Isolation and purification of TRACP --- p.113 / Chapter 3.2.1. --- Concanavalin A affinity chromatography --- p.120 / Chapter 3.2.2. --- Isoelectric focusing (IEF) --- p.120 / Chapter 3.3. --- Characterisation and homogeneity of purified TRACP --- p.128 / Chapter 3.3.1. --- Characterisation of purified TRACP --- p.128 / Chapter 3.3.2. --- Homogeneity of purified TRACP --- p.132 / Chapter 3.3.3. --- Amino acid composition --- p.136 / Chapter 3.4. --- Characterisation of the rabbit anti-human TRACP antibody --- p.136 / Chapter 3.4.1. --- Antibody specificity - immunoblotting study --- p.139 / Chapter 3.4.2. --- Antibody specificity - cross reactivity with partially purified non-cord plasma TRACP --- p.142 / Chapter 3.4.3. --- Antibody specificity - immunohistochemical study --- p.145 / Chapter 3.5. --- Enzyme linked immunosorbent assay for TRACP --- p.145 / Chapter 3.5.1. --- Optimal concentration of antigen for coating of microtitre plate --- p.145 / Chapter 3.5.2. --- Kinetics of reaction with the primary rabbit anti-human TRACP antibody --- p.149 / Chapter 3.5.3. --- "Precision, recovery and assay range" --- p.149 / Chapter 4 --- DISCUSSION --- p.155 / Chapter 4.1. --- Purification of cord plasma TRACP --- p.155 / Chapter 4.2. --- Characterisation of cord plasma TRACP --- p.158 / Chapter 4.3. --- Characterisation of rabbit anti-human TRACP antibody --- p.163 / Chapter 4.4. --- Enzyme immunoassay for TRACP --- p.165 / Chapter CHAPTER III --- STUDY OF SERUM TRACP IN HEALTHY SUBJECTS AND IN PATIENTS WITH BONE RELATED DISEASES / Chapter 1 --- Introduction --- p.168 / Chapter 2 --- Materials and methods --- p.171 / Chapter 2.1. --- Subjects --- p.171 / Chapter 2.1.1. --- Healthy subjects --- p.171 / Chapter 2.1.2. --- Patients --- p.172 / Chapter 2.1.2.1. --- Post-menopausal women on hormone replacement therapy --- p.172 / Chapter 2.1.2.2. --- Hip fracture patients --- p.173 / Chapter 2.1.2.3. --- Other patients --- p.174 / Chapter 2.3. --- Measurement of other biochemical parameters --- p.175 / Chapter 2.3.1. --- Bone alkaline phosphatase --- p.175 / Chapter 2.3.2. --- "Measurement of urine hydroxyproline, creatinine, calcium, osteocalcin, thyroid hormones and parathyroid hormone" --- p.176 / Chapter 2.4. --- Statistics --- p.178 / Chapter 3 --- RESULTS --- p.179 / Chapter 3.1. --- Healthy subjects --- p.179 / Chapter 3.2. --- Serum TRACP concentration in post-menopausal women before and after hormone replacement therapy --- p.185 / Chapter 3.3. --- TRACP concentration in elderly subjects with hip fractures --- p.189 / Chapter 3.4. --- Serum TRACP concentrations in patients with other bone related diseases --- p.190 / Chapter 3.4.1. --- Hyperthyroidism --- p.194 / Chapter 3.4.2. --- Hyperparathyroidism --- p.198 / Chapter 3.4.3. --- Haemodialysis --- p.201 / Chapter 4 --- DISCUSSION --- p.204 / GENERAL DISCUSSION --- p.216 / REFERENCES --- p.219

by Chak Chi Wai. / Thesis (M.Phil)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 142-155). / ACKNOWLEDGMENTS --- p.i / TABLE OF CONTENT --- p.ii / LIST OF ABBREVIATION --- p.viii / ABSTRACT --- p.x / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- INTRODUCTION TO ALKALINE PHOSPHATASE --- p.2 / Chapter 1.1.1 --- The Alkaline Phosphatase Isoenzymes --- p.2 / Chapter 1.1.2 --- The Properties of Alkaline Phosphatases --- p.5 / Chapter 1.1.3 --- Serum Alkaline Phosphatases --- p.7 / Chapter 1.1.3.1 --- Intestinal Alkaline Phosphatase --- p.8 / Chapter 1.1.3.2 --- Placental Alkaline Phosphatase --- p.8 / Chapter 1.1.3.3 --- Renal Alkaline Phosphatase --- p.9 / Chapter 1.1.3.4 --- Skeletal Alkaline Phosphatase --- p.9 / Chapter 1.1.3.5 --- Hepatic Alkaline Phosphatase --- p.10 / Chapter 1.1.3.6 --- Miscellaneous Alkaline Phosphatases --- p.10 / Chapter 1.1.4 --- Problems in Discriminating the Skeletal and Hepatic Alkaline Phosphatase in Serum --- p.12 / Chapter 1.1.5 --- Wheat Germ Lectin Precipitation of the Bone- Specific Alkaline Phosphatase --- p.13 / Chapter 1.2 --- STRUCTURE OF BONE AND MECHANISMS OF CALCIFICATION --- p.15 / Chapter 1.2.1 --- Gross Structure of Bone --- p.15 / Chapter 1.2.2 --- The Elements of Bone --- p.17 / Chapter 1.2.2.1 --- Bone Cells --- p.17 / Chapter 1.2.2.2 --- Organic Substances of Bone --- p.19 / Chapter 1.2.2.3 --- Inorganic Substances of Bone --- p.21 / Chapter 1.2.3 --- Mechanisms of Calcification --- p.22 / Chapter 1.3 --- BONE FRACTURE HEALING --- p.24 / Chapter 1.3.1 --- Types of Fracture --- p.24 / Chapter 1.3.2 --- The Process of Bone Fracture Healing --- p.26 / Chapter 1.3.2.1 --- Stage of Hematoma --- p.26 / Chapter 1.3.2.2 --- Stage of Subperiosteal and Endosteal Cellular Proliferation --- p.28 / Chapter 1.3.2.3 --- Stage of Fibrocartilaginous Callus --- p.28 / Chapter 1.3.2.4 --- Stage of Bony Callus --- p.30 / Chapter 1.3.2.5 --- Stage of Remodeling --- p.31 / Chapter 1.4 --- THE OSTEOBLASTIC CHARACTERS OF UMR-106 OSTEOSARCOMA CELL LINE --- p.32 / Chapter 1.4.1 --- Classification of Osteosarcoma --- p.32 / Chapter 1.4.2 --- Derivation of UMR-106 Osteosarcoma Cell Line --- p.33 / Chapter 1.4.3 --- Osteoblastic Characters of UMR-106 --- p.34 / Chapter 1.4.3.1 --- ALP Expression --- p.34 / Chapter 1.4.3.2 --- Hormone Responsive Adenylate Cyclase System --- p.35 / Chapter 1.4.3.3 --- "Cytosolic Receptors for 1,25-Dihydroxy- cholecalciferol" --- p.35 / Chapter 1.5 --- IN VITRO CULTURE OF FETAL RAT CALVARIAL OSTEOBLASTS --- p.37 / Chapter 1.6 --- AIM AND SCOPE OF THIS DISSERTATION --- p.39 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- BONE FRACTURE OPERATION --- p.42 / Chapter 2.1.1 --- Animals --- p.42 / Chapter 2.1.2 --- Blood Sampling and Preparation of Plasma Samples --- p.42 / Chapter 2.1.3 --- Bone Fracture Operation --- p.43 / Chapter 2.1.3.1 --- Reagents and Apparatus --- p.43 / Chapter 2.1.3.2 --- Procedures --- p.44 / Chapter 2.1.4 --- Radiography --- p.50 / Chapter 2.1.5 --- Removal of Tibiae --- p.51 / Chapter 2.1.6 --- Extraction of Callus ALP --- p.51 / Chapter 2.1.6.1 --- Reagent --- p.51 / Chapter 2.1.6.2 --- Homogenization of the Callus --- p.51 / Chapter 2.1.6.3 --- Extraction of ALP --- p.52 / Chapter 2.1.7 --- Assay for Bone-Specific ALP --- p.53 / Chapter 2.1.7.1 --- Reagents --- p.53 / Chapter 2.1.7.2 --- Procedures --- p.54 / Chapter 2.1.8 --- Normal Curve for Plasma Bone-Specific ALP in Rabbits --- p.56 / Chapter 2.1.9 --- The Effects of Tibial Fracture on the Plasma Level of Bone-Specific ALP in Rabbits --- p.56 / Chapter 2.1.10 --- Profile of Plasma Bone-Specific ALP upon a Fracture Healing --- p.57 / Chapter 2.1.11 --- Profile of Callus Bone-Specific ALP at Different Stages of Fracture Healing --- p.57 / Chapter 2.2 --- CLINICAL STUDIES OF PLASMA BONE-SPECIFIC ALP --- p.58 / Chapter 2.2.1 --- Patient Groups --- p.58 / Chapter 2.2.1.1 --- Normal Adults --- p.58 / Chapter 2.2.1.2 --- Fracture Group --- p.58 / Chapter 2.2.1.3 --- Tumor Group --- p.59 / Chapter 2.2.2 --- Assays for Plasma Bone-Specific ALP --- p.59 / Chapter 2.3 --- "IN VITRO CULTURES OF FETAL, RAT OSTEOBLASTS AND UMR-106 OSTEOSARCOMA cell line" --- p.60 / Chapter 2.3.1 --- Animals --- p.60 / Chapter 2.3.2 --- UMR-106 Cell Line --- p.60 / Chapter 2.3.3 --- General Reagents Used for Cell Culture --- p.60 / Chapter 2.3.4 --- Isolation of Calvarial Osteoblasts --- p.64 / Chapter 2.3.4.1 --- Tools and Reagents --- p.64 / Chapter 2.3.4.2 --- Procedures --- p.65 / Chapter 2.3.5 --- Storage of UMR-106 Cell Line --- p.67 / Chapter 2.3.6 --- Subculture of Confluent Monolayer --- p.68 / Chapter 2.3.6.1 --- Reagents --- p.68 / Chapter 2.3.6.2 --- Procedures --- p.69 / Chapter 2.3.7 --- Staining for Calcium Deposits --- p.69 / Chapter 2.3.7.1 --- Reagents --- p.70 / Chapter 2.3.7.2 --- Procedures --- p.70 / Chapter 2.3.8 --- Protein Determination --- p.71 / Chapter 2.3.8.1 --- Reagents --- p.71 / Chapter 2.3.8.2 --- Procedures --- p.71 / Chapter 2.3.9 --- Microdetermination of Inorganic Phosphate --- p.72 / Chapter 2.3.9.1 --- Reagents --- p.72 / Chapter 2.3.9.2 --- Procedures --- p.73 / Chapter 2.3.10 --- Determination of Calcium --- p.73 / Chapter 2.3.10.1 --- Reagent --- p.73 / Chapter 2.3.10.2 --- Procedures --- p.73 / Chapter 2.3.11 --- Extraction and Assay for Cellular ALP --- p.74 / Chapter 2.3.11.1 --- Reagents --- p.74 / Chapter 2.3.11.2 --- Procedures --- p.75 / Chapter 2.3.12 --- Cell Surface ALP Assay --- p.75 / Chapter 2.3.12.1 --- Reagents --- p.75 / Chapter 2.3.12.2 --- Procedures --- p.76 / Chapter 2.3.13 --- Extraction of Calcium Phosphate Deposits --- p.76 / Chapter 2.3.13.1 --- Reagent --- p.76 / Chapter 2.3.13.2 --- Procedures --- p.76 / Chapter 2.3.14 --- Collagen Synthesis Assay --- p.77 / Chapter 2.3.14.1 --- Reagents --- p.77 / Chapter 2.3.14.2 --- Procedures --- p.78 / Chapter CHAPTER THREE: --- EFFECTS OF TIBIAL FRACTURE ON THE LEVEL OF BONE-SPECIFIC ALKALINE PHOSPHATASE IN RABBITS / INTRODUCTION --- p.81 / results: / Chapter 3.1 --- normal curve for plasma bone-specific alp in rabbits --- p.82 / Chapter 3.2 --- THE EFFECTS OF TIBIAL FRACTURE ON THE PLASMA LEVEL OF BONE-SPECIFIC ALP IN RABBITS --- p.84 / Chapter 3.3 --- PROFILE OF THE PLASMA ALP LEVEL UPON HEALING OF TIBIAL FRACTURE --- p.86 / Chapter 3.4 --- RADIOGRAPHY --- p.89 / Chapter 3.5 --- PROFILE OF CALLUS BONE-SPECIFIC ALP ACTIVITY UPON HEALING OF TIBIAL FRACTURE --- p.93 / DISCUSSION --- p.95 / Chapter CHAPTER FOUR: --- CLINICAL STUDIES OF PLASMA BONE-SPECIFIC ALKALINE PHOSPHATASE / INTRODUCTION --- p.100 / RESULTS: / Chapter 4.1 --- NORMAL VALUES --- p.100 / Chapter 4.2 --- FRACTURE GROUP --- p.101 / Chapter 4.3 --- BONE TUMOR GROUP --- p.102 / DISCUSSION --- p.102 / Chapter CHAPTER FIVE: --- IN VITRO CULTURE OF FETAL RAT OSTEOBLASTS AND UMR-106 CELL LINE / INTRODUCTION --- p.105 / RESULTS: / Chapter 5.1 --- IN VITRO MINERALIZATION OF UMR-106 CELLS AND PRIMARY RC CELLS --- p.107 / Chapter 5.2 --- STUDY OF BONE-SPECIFIC ALP RELEASED INTO MEDIUM BY UMR-106 CELLS AND PRIMARY RC CELLS --- p.113 / Chapter 5.3 --- STUDY OF CELLULAR ALP ACTIVITIES AND CALCIUM PHOSPHATE DEPOSITS --- p.116 / Chapter 5.4 --- STUDIES OF CELLULAR ALP ACTIVITIES AND RELATIVE RATES OF COLLAGEN SYNTHESIS --- p.125 / DISCUSSION --- p.128 / Chapter CHAPTER SIX: --- GENERAL DISCUSSION --- p.136 / BIBLIOGRAPHY --- p.142 / APPENDIX --- p.156

A asfixia perinatal e sua mais grave complicação, a encefalopatia hipóxicoisquêmica (EHI) são causas de elevada mortalidade e de sequelas neurológicas no recém-nascido (RN). Evidencias de comprometimento cerebral podem ser detectadas logo na primeira semana de vida e o diagnóstico precoce é de grande importância para o tratamento e seguimento. OBJETIVOS: Verificar a prevalência de asfixia perinatal e de EHI; analisar a utilidade de quatro marcadores sanguíneos: transaminase glutâmica oxalacética (TGO), transaminase glutâmica pirúvica (TGP), desidrogenasse láctica (DHL) e Creatina Quinase MB (CK-MB) coletados ao nascimento, com 24 e 72 horas de vida, como sinalizadores de lesões cerebrais nos RN asfixiados; identificar a presença de alterações neurológicas clínicas pelo Escore de Sarnat e Sarnat (1976) com 24 e 72 horas e com 28 dias de vida e identificar e descrever a presença de lesões cerebrais detectadas pela ultrassonografia de crânio com 24 e 72 horas e com 28 dias de vida. MÉTODO: Estudo de coorte prospectivo no qual foram incluídos RN de termo que apresentaram asfixia perinatal pelo critério de Buonocore (presença ao nascimento de pelo menos dois das seguintes condições clínico-laboratoriais: acidose metabólica com níveis de pH 7,20, Boletim de Apgar no quinto minuto de vida < 6, necessidade de fração inspirada de oxigênio 0,40 para manter uma saturação de oxigênio de 86%). Para realização do pH e dos marcadores bioquímicos foi coletado sangue logo ao nascimento, com 24 e 72 horas de vida. O exame clínico segundo critérios de Sarnat e Sarnat foi realizado com 24 e 72 horas e com 28 dias de vida e a ultrassonografia de crânio nos mesmos momentos. RESULTADOS: De 2.989 nascidos vivos durante o período do estudo, 28 recém-nascidos apresentaram asfixia perinatal (1% do total de nascimentos) e a EHI foi evidenciada em 21,42%. A enzima CK-MB se mostrou um bom marcador de asfixia, pois todos os valores foram superiores a 5,10 ng/ml, e se correlacionaram positivamente com as alterações apresentadas no exame clínico de Sarnat e com a ultrassonografia transfontanela. Os valores das outras enzimas como TGO (24h), TGO e TGP (72h) também se correlacionaram positivamente com as alterações ultrassonográficas as quais mostraram alteração em 3,5% dos pacientes com 24 horas de vida, 25% com 72 horas e 28,6% com 28 dias. A ultrassonografia de crânio com 28 dias mostrou aumento estatisticamente significante, no percentual de resultados anormais quando comparado com o observado com 24h (p=0,039), apesar dos estágios de Sarnat terem melhorado, com maior número de pacientes com estágio I no decorrer dos 28 dias. CONCLUSOES: A prevalência de asfixia perinatal e da EHI estão dentro da faixa citada na literatura. O melhor marcador bioquímico nesta casuística foi a isoenzima CK-MB e se correlacionou com as alterações apresentadas no exame clínico de Sarnat e na USG de crânio. A curva ROC mostrou: os valores de CK-MB de 24 horas em relação à USG de crânio de 72 horas Sensibilidade de 85,7%, Especificidade de 85,7% e Acurácia de 85,7%. O escore clínico de Sarnat não se alterou depois de 72 horas e apresentou correlação com as alterações na USG de crânio e este método de imagem foi adequado para detecção de lesões crebrais precoces e com 28 dias de vida / Perinatal asphyxia and its most serious complication, hypoxic-ischemic encephalopathy (HIE) are causes of high mortality and neurological sequelae in the newborn (NB). Evidence of cerebral impairment can be detected in the first week of life and early diagnosis is very important for the treatment and follow-up. OBJECTIVES: To assess the prevalence of perinatal asphyxia and HIE; consider the usefulness of four blood markers: glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB) collected the birth, with 24 and 72 hours of life, as markers of brain damage in asphyxiated infants, to identify the presence of neurological clinical score by Sarnat and Sarnat (1976) with 24 and 72 hours and 28 days of life and describe the presence of brain lesions detected by cranial ultrasound with 24 and 72 hours and 28 days. METHOD: A prospective cohort study which included fullterm infants who had perinatal asphyxia by Buonocore criteria (presence at birth of at least two of the following clinical and laboratory conditions: metabolic acidosis with pH levels of the umbilical vein 7.20, Apgar score in the fifth minute of life <6, need for inspired oxygen fraction (FiO2) 0.40 to maintain an oxygen saturation of 86%). To perform the pH and biochemical markers of blood was collected at birth, 24 and 72 hours of life. Clinical examination according to criteria of Sarnat and Sarnat was performed with 24 and 72 hours and 28 days of life and cranial ultrasound was performed in the same time. RESULTS: Of 2989 live births during the study period, 28 had asphyxia (1% of total births) and HIE was found in 21.42%. The CK-MB showed that all above normal values (<5.10ng/ml) and correlated with the changes presented in the clinical examination of Sarnat and the USG transfontanelle. The values of other enzymes such as GOT (24h), GOT and GPT (72h) also correlated positively with the brain lesins detected by cranial ultrasound in 3.5% of patients with 24 hours of life, 25% at 72 hours and 28 6% after 28 days. Ultrasonography of brain at 28 days showed a statistically significant increase in the percentage of abnormal results when compared with that observed at 24h (p = 0.039), despite the Sarnat stages have improved, with larger numbers of patients with stage I during the 28 days. CONCLUSION: The prevalence of perinatal asphyxia and HIE is within the range cited in literature. The best biochemical marker in this series was the CK-MB and correlated with the changes presented in the clinical examination of Sarnat and ultrasound transfontanelle. The ROC curve showed: values of CK-MB of 24 hours and USG 72 hours sensitivity of 85.7%, specificity of 85.7% and accuracy of 85.7%. The clinical Sarnat score did not change after 72 hours and correlated with changes in ultrasound transfontanelle and this imaging method proved to be an appropiate study to detect brain lesions early and with 28 days of life

Objetivo: o objetivo deste estudo foi avaliar a prevalência de autoanticorpos e a dosagem da concentração de células Natural Killer (NK) no sangue periférico em pacientes com endometriose. Métodos: Entre dezembro de 2004 e dezembro de 2007 foram avaliadas 155 pacientes submetidas a videolaparoscopia, divididas em um grupo sem endometriose(n=55) e outro com endometriose (n=100). Foi coletada amostra de sangue periférico de todas as pacientes no momento da laparoscopia e nessa amostra foi realizada a quantificação do percentual de células NK em relação aos linfócitos periféricos (por citometria de fluxo), e a determinação dos seguintes auto-anticorpos: anticorpos antinucleares (ANA, por imunofluorescência indireta), anticorpos antitireoglobulina e antiperoxidase (anti-TG e anti-TPO, por eletroquimioluminescência), anticorpos anticardilipina e antifosfatidilserina (aCL e aPS IgG, IgM e IgA, todos por ensaio imunoenzimático). Além da presença de endometriose, essas pacientes também foram avaliadas quanto ao estadiamento, os locais de doença, relações com a fase do ciclo, e a classificação histológica dessa doença. Resultados: as pacientes com endometriose apresentaram percentual de células NK (média DP de 15,3 9,8%) superiores àquelas sem a doença (média DP de 10,6 5,8%), p<0,001. Quanto aos autoanticorpos, as portadoras de endometriose também apresentaram positividade para ANA mais frequentemente (33%) que as pacientes do grupo controle (12,7%), p=0,006. Quanto aos anti-TG, anti-TPO, anti-CL (IgG, IgM e IgA) e aPS ( IgG, IgM e IgA), não houve diferenças estatísticas quanto à sua positividade. As células NK também mostraram-se mais elevadas nas protadoras de endometriose em estádios avançados e naquelas com comprometimento de retossigmóide, grupo no qual encontramos o maior percentual de células NK com concentração média de 19,8 10,3%. Concentrações de células NK 12,5% podem ser usadas como marcadores de endometriose em retossigmóide, com sensibilidade de 73% e especificidade de 65%. Utilizando-se de um modelo estatístico de probabilidades, demonstramos que associação desse marcador (NK 12,5%) com a presença de sintomas como dor e/ou sangramento intestinal durante a menstruação nos possibilitou estimar uma probabilidade de comprometimento de retossigmóide de 60,4%. Conclusões: pacientes com endometriose apresentam maior concentração de células NK periféricas, além de maior prevalência de ANA positivo em relação àquelas sem endometriose. As células NK aumentam nas pacientes com endometriose predominantemente nos estádios avançados, com comprometimento de retossigmóide. Nesse sentido poderiam ser utilizadas como marcadores diagnósticos desse tipo de comprometimento da doença, principalmente se forem avaliadas em conjunto com os sintomas das pacientes / Objectives: The objective of this study was to evaluate the prevalence of autoantibodies and the percentage of natural killer (NK) cells in the peripheral blood of patients with endometriosis. Methods: Between December 2004 and December 2007, 155 patients submitted to videolaparoscopy were evaluated. Patients were divided into two groups: one group of women without endometriosis (n = 55) and another in which all the women had endometriosis (n = 100). Samples of peripheral blood were collected from all the patients at the time of laparoscopy and flow cytometry was used to determine the percentage of NK cells in relation to peripheral blood lymphocytes in these samples. In addition, the following autoantibodies were measured: antinuclear antibodies (ANA) by indirect immunofluorescence, anti-thyroglobulin and anti-thyroid peroxidase antibodies (anti-TG and anti-TPO) by electrochemiluminescence, and anticardiolipin and anti-phosphatidylserine antibodies (aCL and aPS IgG, IgM and IgA), all performed using immunoenzymatic assay. In addition to the presence of endometriosis, these patients were also evaluated with respect to staging, to the sites of the disease, any association with the phase of the menstrual cycle and the histological classification of the disease. Results: The patients with endometriosis had a higher percentage of NK cells (15.3 ± 9.8%; mean ± SD) compared to those without the disease (10.6 ± 5.8%; mean ± SD), (p<0.001). Evaluation of the autoantibodies showed that positivity for ANA was more common in the group of patients with endometriosis (33%) compared to the patients in the control group (12.7%), (p = 0.006). With respect to anti-TG, anti-TPO, aCL (IgG, IgM and IgA) and aPS (IgG, IgM and IgA), no statistically significant differences were found between the groups of patients with or without endometriosis. NK cell concentrations were also found to be higher in patients with advanced stages of endometriosis and in those in whom the rectosigmoid was affected by the disease, this being the group in which the highest percentage of NK cells was found, with mean concentrations of 19.8 ± 10.3%. NK cell concentrations 12.5% may be used as markers of endometriosis of the rectosigmoid, with sensitivity of 73% and specificity of 65%. Using a statistical model of probability, these findings showed that the association of this marker (NK 12.5%) with the presence of symptoms such as pain and/or intestinal bleeding during menstruation permitted an estimation to be made of a likelihood of 60.4% of rectosigmoid endometriosis. Conclusions: Patients with endometriosis have higher percentages of peripheral NK cells, as well as a greater prevalence of positive ANA compared to those without endometriosis. The concentration of peripheral NK cells increases in patients with endometriosis, predominantly in patients with advanced stages of the disease and those in whom the rectosigmoid is affected. Therefore, the concentration of NK cells in peripheral blood could be used as a diagnostic marker of this type of endometriosis, particularly when evaluated together with patients symptoms

INTRODUÇÃO: A Doença Hepática Gordurosa Não Alcoólica (DHGNA) é atualmente a forma mais comum de doença hepática. Sem fisiopatologia totalmente esclarecida, apresenta-se na forma de esteatose, até formas mais avançadas denominadas Esteatohepatite não alcoólica (EHNA). Citocinas como TNF-, são frequentemente mensuradas em EHNA, diferente da IL-6 e IL-10. MÉTODOS: Pacientes moderadamente obesos (n=80) com histologia apresentando esteatose (n=29) e EHNA (N=51). Níveis de citocinas séricas foram dosadas. O objetivo foi correlacionar a correlação das citocinas com esta população. RESULTADOS: Pacientes diabéticos tendem a ser mais associados com EHNA (52.5% vs 41.4%, P=0.015), sem diferença na idade, gênero ou IMC considerando esteatose. Para a população total, não houve diferenças significantes entre as citocinas, incluindo TNF- e IL-6. Em paciente diabéticos, todas as citocinas tenderam à diminuir na EHNA, especialmente IL-10(P= 0.000). A citocina IL-10 correlacionou-se com o índice HOMA (P=0.000) e outras variáveis de homeostase no diabetes, representando assim, um marcador importante nesta doença. CONCLUSÕES: 1) Em geral, ocorrem mudanças inconsistentes nas citocinas quando comparados os pacientes com esteatose.2) Em contraste, baixa regulação da IL-6 e IL- 10 foram persistentes em pacientes diabéticos com NASH. 3) Hipertensão Arterial não apresentou alteração nessas circunstâncias.4) IL-10 manteve forte correlação com os índices de metabolismo glicêmico. 5) TNF- não pode ser responsabilizado pelo dano hepático progressivo, pois seus valores não aumentaram na EHNA.6) Investigação da IL-10 e outras citocinas contra reguladoras são necessárias neste contexto e merecem mais estudos. / Background: Fatty liver disease is a problem of obesity in the morbid modality and even more so in nonbariatric candidates, who rely on clinical treatment only. TNF- has been frequently measured in steatohepatitis (NASH) with or without diabetes, but less in known about IL-6 and IL-10. Methods: Moderately obese patients (n=80) with histologically proven Steatosis (n=29) and NASH (n=51) were recruited. Serum levels of cytokines were documented along with clinical information. The aim was to identify the correlates of such biomolecules in a stable population.Results: Diabetes tended to be more associated with NASH (52.5% vs 41.4%, P=0.015), with no difference of age, gender or BMI regarding steatosis . For the entire population cytokine changes were not significant., including TNF- and IL-6. In diabetics only, all markers tended to diminish with NASH, especially IL-10 (P= 0.000). IL-10 correlated with HOMA index (P=0.000) and other variables of glucose homeostasis in diabetes, thus representing a major marker of the disease.Conclusions: 1)Generally inconsistent changes in pro- and antiinflammatory cytokines occurred when NASH was globally compared to steatosis. 2) In contrast, downregulation of IL-6 and IL-10 was perceived in diabetics with NASH. 3) Arterial hypertension did not play a role in these circumstances. 4) IL-10 maintained strong correlations with glucose metabolism indices. 5) TNF- could not be incriminated for progressive liver damage, as values failed to increase in NASH. 6) Investigations of IL-10 and other counterregulatory cytokines are lacking in this context and deserve further studies.

Objetivo: o objetivo deste estudo foi avaliar a prevalência de autoanticorpos e a dosagem da concentração de células Natural Killer (NK) no sangue periférico em pacientes com endometriose. Métodos: Entre dezembro de 2004 e dezembro de 2007 foram avaliadas 155 pacientes submetidas a videolaparoscopia, divididas em um grupo sem endometriose(n=55) e outro com endometriose (n=100). Foi coletada amostra de sangue periférico de todas as pacientes no momento da laparoscopia e nessa amostra foi realizada a quantificação do percentual de células NK em relação aos linfócitos periféricos (por citometria de fluxo), e a determinação dos seguintes auto-anticorpos: anticorpos antinucleares (ANA, por imunofluorescência indireta), anticorpos antitireoglobulina e antiperoxidase (anti-TG e anti-TPO, por eletroquimioluminescência), anticorpos anticardilipina e antifosfatidilserina (aCL e aPS IgG, IgM e IgA, todos por ensaio imunoenzimático). Além da presença de endometriose, essas pacientes também foram avaliadas quanto ao estadiamento, os locais de doença, relações com a fase do ciclo, e a classificação histológica dessa doença. Resultados: as pacientes com endometriose apresentaram percentual de células NK (média DP de 15,3 9,8%) superiores àquelas sem a doença (média DP de 10,6 5,8%), p<0,001. Quanto aos autoanticorpos, as portadoras de endometriose também apresentaram positividade para ANA mais frequentemente (33%) que as pacientes do grupo controle (12,7%), p=0,006. Quanto aos anti-TG, anti-TPO, anti-CL (IgG, IgM e IgA) e aPS ( IgG, IgM e IgA), não houve diferenças estatísticas quanto à sua positividade. As células NK também mostraram-se mais elevadas nas protadoras de endometriose em estádios avançados e naquelas com comprometimento de retossigmóide, grupo no qual encontramos o maior percentual de células NK com concentração média de 19,8 10,3%. Concentrações de células NK 12,5% podem ser usadas como marcadores de endometriose em retossigmóide, com sensibilidade de 73% e especificidade de 65%. Utilizando-se de um modelo estatístico de probabilidades, demonstramos que associação desse marcador (NK 12,5%) com a presença de sintomas como dor e/ou sangramento intestinal durante a menstruação nos possibilitou estimar uma probabilidade de comprometimento de retossigmóide de 60,4%. Conclusões: pacientes com endometriose apresentam maior concentração de células NK periféricas, além de maior prevalência de ANA positivo em relação àquelas sem endometriose. As células NK aumentam nas pacientes com endometriose predominantemente nos estádios avançados, com comprometimento de retossigmóide. Nesse sentido poderiam ser utilizadas como marcadores diagnósticos desse tipo de comprometimento da doença, principalmente se forem avaliadas em conjunto com os sintomas das pacientes / Objectives: The objective of this study was to evaluate the prevalence of autoantibodies and the percentage of natural killer (NK) cells in the peripheral blood of patients with endometriosis. Methods: Between December 2004 and December 2007, 155 patients submitted to videolaparoscopy were evaluated. Patients were divided into two groups: one group of women without endometriosis (n = 55) and another in which all the women had endometriosis (n = 100). Samples of peripheral blood were collected from all the patients at the time of laparoscopy and flow cytometry was used to determine the percentage of NK cells in relation to peripheral blood lymphocytes in these samples. In addition, the following autoantibodies were measured: antinuclear antibodies (ANA) by indirect immunofluorescence, anti-thyroglobulin and anti-thyroid peroxidase antibodies (anti-TG and anti-TPO) by electrochemiluminescence, and anticardiolipin and anti-phosphatidylserine antibodies (aCL and aPS IgG, IgM and IgA), all performed using immunoenzymatic assay. In addition to the presence of endometriosis, these patients were also evaluated with respect to staging, to the sites of the disease, any association with the phase of the menstrual cycle and the histological classification of the disease. Results: The patients with endometriosis had a higher percentage of NK cells (15.3 ± 9.8%; mean ± SD) compared to those without the disease (10.6 ± 5.8%; mean ± SD), (p<0.001). Evaluation of the autoantibodies showed that positivity for ANA was more common in the group of patients with endometriosis (33%) compared to the patients in the control group (12.7%), (p = 0.006). With respect to anti-TG, anti-TPO, aCL (IgG, IgM and IgA) and aPS (IgG, IgM and IgA), no statistically significant differences were found between the groups of patients with or without endometriosis. NK cell concentrations were also found to be higher in patients with advanced stages of endometriosis and in those in whom the rectosigmoid was affected by the disease, this being the group in which the highest percentage of NK cells was found, with mean concentrations of 19.8 ± 10.3%. NK cell concentrations 12.5% may be used as markers of endometriosis of the rectosigmoid, with sensitivity of 73% and specificity of 65%. Using a statistical model of probability, these findings showed that the association of this marker (NK 12.5%) with the presence of symptoms such as pain and/or intestinal bleeding during menstruation permitted an estimation to be made of a likelihood of 60.4% of rectosigmoid endometriosis. Conclusions: Patients with endometriosis have higher percentages of peripheral NK cells, as well as a greater prevalence of positive ANA compared to those without endometriosis. The concentration of peripheral NK cells increases in patients with endometriosis, predominantly in patients with advanced stages of the disease and those in whom the rectosigmoid is affected. Therefore, the concentration of NK cells in peripheral blood could be used as a diagnostic marker of this type of endometriosis, particularly when evaluated together with patients symptoms

Kong Kam-chuen, Ebenezer. / Thesis submitted in: July 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 131-149). / Abstracts in English and Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.ii / Table of Content --- p.iii -vii / Acknowledgement --- p.viii / List of Abbreviations --- p.ix -x / List of Tables --- p.xi / List of Figures --- p.xii -xiii / List of Appendices --- p.xiv -xv / Chapter CHAPTER 1 --- Review of Literature / Chapter 1.1 --- Peptidomic Research / Chapter 1.1.1 --- Proteomics and Genomics --- p.1-2 / Chapter 1.1.2 --- Peptidomics and Quantitative Profiling --- p.2-5 / Chapter 1.1.3 --- Proteomics and Peptidomics in Medical Research --- p.5-6 / Chapter 1.1.4 --- Application of Quantitative Peptidomic Profiling in Cancer Research --- p.6-8 / Chapter 1.2 --- Technologies for Peptidomic Studies and Limitations --- p.9-11 / Chapter 1.2.1 --- High Performance Liquid Chromatograph (HPLC) --- p.12 / Chapter 1.2.1.1 --- Basic Principle --- p.13-15 / Chapter 1.2.1.2 --- Application in Peptidomic / Proteomic Research --- p.16-17 / Chapter 1.2.2 --- Peptide Gel Electrophoresis --- p.18 -19 / Chapter 1.2.2.1 --- Basic Principle --- p.19-21 / Chapter 1.2.2.2 --- Application in Peptidomic / Proteomic Research --- p.21 -22 / Chapter 1.2.3 --- Capillary Electrophoresis (CE) --- p.23 -24 / Chapter 1.2.3.1 --- Basic Principle --- p.24-29 / Chapter 1.2.4 --- Mass Spectrometry --- p.30 / Chapter 1.2.4.1 --- Electro spray Ionization (ESI) Mass Spectrometry --- p.31 / Chapter 1.2.4.1.1 --- Basic Principle --- p.31-34 / Chapter 1.2.4.2 --- Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry --- p.35 / Chapter 1.2.4.2.1 --- Ionization of Sample --- p.35 -38 / Chapter 1.2.4.2.2 --- Time-of-Flight (TOF) Analyzer --- p.39-40 / Chapter 1.2.4.2.3 --- Application in Peptidomic / Proteomic Research --- p.40-42 / Chapter 1.2.4.2.4 --- Isotope-Coded Affinity Tags (ICAT®) --- p.42 -46 / Chapter 1.2.4.2.5 --- Limitations --- p.46 -48 / Chapter 1.2.4.3 --- Surface Enhanced Laser Desorption/Ionization (SELDI) Mass Spectrometry --- p.49 / Chapter 1.2.4.3.1 --- Basic Principle --- p.49 -51 / Chapter 1.2.4.3.2 --- Application in Peptidomic / Proteomic Research --- p.51-52 / Chapter 1.2.4.3.3 --- Retentate Chromatography (RC) --- p.53-54 / Chapter 1.2.4.4. --- Recent Advances in Application of MALDI Technologies --- p.55 -57 / Chapter CHAPTER 2 --- Objectives --- p.58 -59 / Chapter CHAPTER 3 --- Methodologies / Chapter 3.1 --- Method Development / Chapter 3.1.1 --- Nitrocellulose (NC) Preparation --- p.60 / Chapter 3.1.2 --- Matrix Chemicals Preparation --- p.60 / Chapter 3.1.3 --- Spotting Methods --- p.61 / Chapter 3.1.4 --- Standard Preparation --- p.61 -63 / Chapter 3.1.5 --- Data Collection and Analysis --- p.64 / Chapter 3.2 --- Identification of Distinguishing Features for HCC --- p.65 / Chapter 3.2.1 --- Classification Trees --- p.65 -66 / Chapter 3.2.2 --- Statistical Analysis --- p.66 / Chapter CHAPTER 4 --- Results / Chapter 4.1 --- Optimization of Spotting Methods in Protein Quantification --- p.67 -68 / Chapter 4.1.1 --- Detection of Low-Molecular Weight Proteins / Peptides --- p.68 -71 / Chapter 4.1.2 --- Detection of High-Molecular Weight Proteins --- p.71 -74 / Chapter 4.2 --- Assay Linearity of the Nitrocellulose-MALDI-TOF MS for Peptides with Different Masses --- p.75 -84 / Chapter 4.3 --- Accuracy of Mass Measurement --- p.84 -85 / Chapter 4.4 --- Applications in Quantitative Peptidomic Profiling of Serum --- p.86 -88 / Chapter 4.5 --- Application in Tumor Marker Discovery / Chapter 4.5.1 --- Identification of Peptidomic Features For Classification Between the HCC and CLD Patients by Classification Tree Analysis --- p.89 - 92 / Chapter 4.5.2 --- Serum Levels of the Diagnostic Peptides in the HCC and CLD Patient Groups --- p.93 / Chapter 4.5.3 --- Spearman's Rank Correlation Analysis of the Diagnostic Peptides and AFP --- p.93-101 / Chapter 4.5.4 --- Combined Use of the Diagnostic Peptides and AFP in the Diagnosis of HCC --- p.102-105 / Chapter CHAPTER 5 --- Discussion --- p.106-108 / Chapter 5.1 --- Evaluation of Different Matrix Chemical and Sample Spotting Techniques / Chapter 5.1.1 --- Effect of CHCA and SA --- p.109 / Chapter 5.1.2 --- Effect of Nitrocellulose in Peptide Ions Formation --- p.110-112 / Chapter 5.2 --- MS Automation and High-Throughput Sampling --- p.113 / Chapter 5.3 --- Reproducibility and Signal Quantitations --- p.114-115 / Chapter 5.4 --- Peptidomics: The Study of Entire Peptidome --- p.116 / Chapter 5.5 --- Serum Peptides --- p.117-118 / Chapter 5.6 --- Application of Peptidomics to Discover Markers for HCC / Chapter 5.6.1 --- Hepatocellular Carcinoma --- p.119 / Chapter 5.6.2 --- Causes of HCC --- p.119-120 / Chapter 5.6.3 --- HCC Tumor Markers --- p.120-122 / Chapter 5.6.4 --- HCC Tumor Markers Identified in the Current Studies --- p.122-124 / Chapter 5.7 --- "Role of MALDI-TOF MS, SELDI-TOF MS and 2-DE in Peptidomics" --- p.125-128 / Chapter CHAPTER 6 --- Conclusion --- p.129-130 / Chapter CHAPTER 7 --- References --- p.131-140 / Chapter CHAPTER 8 --- Original Data --- p.150 / Chapter CHAPTER 9 --- Appendices --- p.151-167

1. Study on improving the diagnostic accuracy of treatment-naive nasopharyngeal carcinoma. / 2. Study on diagnostic accuracy of EBV-DNA on recurrent nasopharyngeal carcinoma. / 3. Studies on EBV-DNA as a screening tool for nasopharyngeal carcinoma. Part 1. To define the detection rate of NPC and the false-positive rate of IgA-VCA in an IgA-VCA-based screening problem, and to define the specificity of IgA-EA in IgA-VCA-positive screenees. Part 2. To define the specificity of EBV-DNA in IgA-VCA-positive screenees. Part 3. To define the sensitivity of IgA-EA, and EBV-DNA in IgA-positive NPC patients. / 4. Studies on pre-therapy prognostication of nasopharyngeal carcinoma Study Part 1. Objective. To assess the role of EBV-DNA in pre-therapy prognostication of early-stage NPC. / Background. The specific association between nasopharyngeal carcinoma (NPC) and the Epstein-Barr virus (EBV) had been exploited to develop a spectrum of EBV-antibodies-based blood markers. Among these markers, the Immunoglobulin A antibody against the viral capsid antigen (IgA-VCA) of the EBV has been the most popularly employed marker to assist diagnosis of NPC. There is however a relative paucity of data on the application of blood markers for screening, for detection of relapse, and for prognostification of patient cohorts managed in present-day therapy oncology protocols. Peripheral blood EBV-DNA, measured by quantitative polymerase chain reaction assay, is a newly-developed marker, and represents a prototype model of a nuclei acid-based, as opposed to antibody-based, EBV tumor marker for NPC. The present thesis describes the translation of this basic scientific advance into clinical applications, through several prospective and retrospective studies that address the diagnosis of treatment-naive NPC, the detection of recurrent NPC, the screening of individuals at risk of NPC, the pre-therapy prognostication for NPC to guide for choice of therapy. The role of integration of conventional markers and EBV-DNA in clinical applications was also examined. / Study Part 2. Objectives. To assess whether incorporation of EBV-DNA data to TNM staging improves prognostic discrimination of patients subsets within individual cancer stage, to assess if EBV-DNA is an independent prognostic factor for survival after ontological therapy. (Abstract shortened by UMI.) / Leung Sing-fai. / "February 2005." / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3695. / Thesis (M.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references. / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

Introdução: A evolução dos marcadores sorológicos da hepatite B em pacientes com hepatite B crônica coinfectados com o vírus da imunodeficiência humana (HIV) tem sido pouco documentada. Objetivos: O objetivo deste estudo foi descrever a evolução dos marcadores sorológicos AgHBe e AgHBs, com ênfase na avaliação da frequência de perda definitiva ou transitória desses marcadores, neste grupo de pacientes. Buscamos, também, comparar as variáveis clínicas e demográficas desses pacientes segundo a evolução desses marcadores sorológicos. Pacientes e métodos: A população de estudo foi composta por pacientes atendidos em um ambulatório de referência para atendimento a pacientes infectados pelo HIV em São Paulo, Brasil. Todos os pacientes selecionados eram portadores de HIV e de hepatite B crônica. Foram incluídos nesse estudo pacientes AgHBs positivos, com confirmação da presença desse marcador em, no mínimo, duas sorologias consecutivas, com intervalo mínimo de seis meses entre elas. Variáveis clínicas foram coletadas: idade, sexo, fator de exposição ao HIV/VHB, contagem de células T CD4+, carga viral do HIV, níveis de alaninoaminotransferase (ALT), uso de terapia antirretroviral, incluindo lamivudina, tenofovir ou outras drogas com ação anti-VHB. Resultados: Entre 2.242 pacientes HIV positivos encontrados, foram identificados 105 (4,68%) pacientes com hepatite B crônica. O tempo de seguimento variou de 06 meses a 20,5 anos e o número de coletas variou de 2 a 18 por paciente no período. A maioria dos pacientes era do sexo masculino (97%) e 43,9% (46/105) tinha história de uma ou mais infecções oportunistas. Todos os pacientes tiveram terapia antirretroviral iniciada durante o seguimento. Entre os pacientes com hepatite B crônica, 58% (61/105) eram AgHBe positivos na primeira avaliação. Entre eles, 15% (16/105) apresentaram clareamento de AgHBs e 50% (8/16) dos que clarearam AgHBs apresentaram posterior reativação desse marcador durante a evolução clínica. Dentre os pacientes AgHBe positivos na primeira sorologia, 57% (35/61) apresentaram clareamento desse marcador, sendo que 28,5% (10/35) dos que clarearam AgHBe voltaram a apresentar este marcador durante a evolução clínica. Conclusão: Observamos uma significativa taxa de clareamento e posterior reativação dos marcadores AgHBs e AgHBe no grupo de pacientes avaliados. Estes resultados sugerem que o monitoramento frequente desses marcadores sorológicos deveria ser recomendado / Introduction: Evolution of hepatitis B serological markers among HIV co-infected patients has rarely been documented. Objectives: The aim of this study was to describe the evolution of HBsAg and HBeAg serological markers, with emphasis on the frequency of transient or permanent loss of these markers, among this group of patients. It was also our objective to compare patients\' demographic and clinical variables according to the evolution of these serological markers. Patients and methods: The enrolled patients were selected from those registered at a HIV-Outpatient Clinic in Sao Paulo, Brazil. All included patients were diagnosed with HIV infection and chronic hepatitis B. HBsAg patients who underwent at least two repeated HBV serological testing during clinical follow up , with tests taken at least six months apart, were included in the analysis. Clinical information was collected: age, sex, patient history regarding HIV/HBV transmission, CD4 T+ cell count, HIV viral load, alanine amino transferase (ALT) level, and use of antiretroviral drugs including lamivudine, tenofovir or other anti-HBV drugs. Results: Among 2,242 HIV-positive patients 105 (4.68%) patients were identified with chronic hepatitis B. Follow-up time for these patients varied from 06 months to 20.5 years and the number of serological testing for each patient varied from 2 to 18 along this period. Most patients were male (97%) and 43.9% (46/105) had a history of one or more opportunistic infections. All patients had initiated antiretroviral medication during follow-up. Among patients with chronic hepatitis B, 58% (61/105) were HBeAg reagent at the first assessment. Also, fifteen percent of them (16/105) underwent HBsAg clearance and 50% (8/16) of those who initially lost HBsAg underwent HBsAg reactivation during clinical follow up. Among HBeAg positive patients in the first serology, 57% (35/61) lost this marker during clinical follow up, whereas 28.5% (10/35) of those who initially cleared this serological marker underwent HBeAg reactivation. Conclusion: A significant rate of changes of HBsAg and HBeAg was observed, during clinical follow up among this group of patients. These results suggest that periodic monitoring of HBV serological markers should be recommended