by Ho Wai Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 207-243). / Abstracts in English and Chinese. / Title --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.viii / Table of Contents --- p.x / List of Abbreviations --- p.xviii / List of Figures --- p.xxiv / List of Tables --- p.xxix / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Traumatic brain injury --- p.1 / Chapter 1.2 --- Ceils of the nervous system: glia --- p.1 / Chapter 1.2.1 --- Astroglia - / Chapter 1.2.1.1 --- Molecular markers of astroglia --- p.3 / Chapter 1.2.1.2 --- Functions of astroglia --- p.3 / Chapter 1.2.2 --- Oligodendrocyte --- p.5 / Chapter 1.2.2.1 --- Molecular markers of oligodendrocyte --- p.6 / Chapter 1.2.2.2 --- Functions of oligodendrocyte --- p.6 / Chapter 1.2.3 --- Microglia --- p.7 / Chapter 1.2.3.1 --- Molecular markers of microglia --- p.7 / Chapter 1.2.3.2 --- Functions of microglia --- p.8 / Chapter 1.3 --- Cytokine and brain injury --- p.8 / Chapter 1.4 --- Tumor necrosis factor alpha (TNF-α) --- p.9 / Chapter 1.5 --- TNF-α receptor --- p.10 / Chapter 1.6 --- Biological activities of TNF-α --- p.11 / Chapter 1.7 --- Signaling mechanism --- p.13 / Chapter 1.7.1 --- Protein kinase C --- p.13 / Chapter 1.7.2 --- Protein kinase A --- p.14 / Chapter 1.7.3 --- p38 mitogen-activated protein kinase (p38 MAPK) --- p.15 / Chapter 1.7.3.1 --- Biological activities of p38 MAPK --- p.18 / Chapter 1.7.4 --- Inducible nitric oxide synthase (iNOS) --- p.20 / Chapter 1.7.5 --- cAMP responsive element binding protein (CREB) --- p.21 / Chapter 1.7.6 --- Transcription factor c-fos --- p.23 / Chapter 1.7.7 --- Nuclear factor kappa-B (NF-kB) --- p.24 / Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.26 / Chapter 1.9 --- β-adrenergic receptor (β-AR) --- p.28 / Chapter 1.9.1 --- Functions of β-AR in astrocytes --- p.29 / Chapter 1.10 --- Why do we use C6 glioma cell? --- p.31 / Chapter 1.11 --- Fluorescent differential display (FDD) --- p.34 / Chapter 1.12 --- Aims and Scopes of this project --- p.36 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Material --- p.40 / Chapter 2.1.1 --- Cell line --- p.40 / Chapter 2.1.2 --- Cell culture reagents --- p.40 / Chapter 2.1.2.1 --- Complete Dulbecco's modified Eagle medium (CDMEM) --- p.40 / Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.41 / Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.41 / Chapter 2.1.3 --- Recombinant cytokines --- p.41 / Chapter 2.1.4 --- Chemicals for signal transduction study --- p.42 / Chapter 2.1.4.1 --- Modulators of p38 mitogen-activated protein kinase (p38 MAPK) --- p.42 / Chapter 2.1.4.2 --- Modulators of protein kinase C (PKC) --- p.42 / Chapter 2.1.4.3 --- Modulators of protein kinase A (PKA) --- p.42 / Chapter 2.1.4.4 --- β-Adrenergic agonist and antagonist --- p.43 / Chapter 2.1.5 --- Antibodies --- p.44 / Chapter 2.1.5.1 --- Anti-p38 mitogen-activated protein kinase (p38 MAPK) antibody --- p.44 / Chapter 2.1.5.2 --- Anti-phosporylation p38 mitogen-activated protein kinase (p-p38 MAPK) antibody --- p.44 / Chapter 2.1.5.3 --- Antibody conjugates --- p.44 / Chapter 2.1.6 --- Reagents for RNA isolation --- p.45 / Chapter 2.1.7 --- Reagents for DNase I treatment --- p.45 / Chapter 2.1.8 --- Reagents for reverse transcription of mRNA and fluorescent PCR amplification --- p.45 / Chapter 2.1.9 --- Reagents for fluorescent differential display --- p.46 / Chapter 2.1.10 --- Materials for excision of differentially expressed cDNA fragments --- p.46 / Chapter 2.1.11 --- Reagents for reamplification of differentially expressed cDNA fragments --- p.46 / Chapter 2.1.12 --- Reagents for subcloning of reamplified cDNA fragments --- p.47 / Chapter 2.1.13 --- Reagents for purification of plasmid DNA from recombinant clones --- p.47 / Chapter 2.1.14 --- Reagents for DNA sequencing of differentially expressed cDNA fragments --- p.47 / Chapter 2.1.15 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.48 / Chapter 2.1.16 --- Reagents for electrophoresis --- p.50 / Chapter 2.1.17 --- Reagents and buffers for Western blot --- p.50 / Chapter 2.1.18 --- Other chemicals and reagents --- p.50 / Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.51 / Chapter 2.3 --- RNA isolation --- p.52 / Chapter 2.3.1 --- Measurement of RNA yield --- p.53 / Chapter 2.4 --- DNase I treatment --- p.53 / Chapter 2.5 --- Reverse transcription of mRNA and fluorescent PCR amplification --- p.54 / Chapter 2.6 --- Fluorescent differentia display --- p.55 / Chapter 2.7 --- Excision of differentially expressed cDNA fragments --- p.59 / Chapter 2.8 --- Reamplification of differentially expressed cDNA fragments --- p.59 / Chapter 2.9 --- Subcloning of reamplified cDNA fragments --- p.60 / Chapter 2.10 --- Purification of plasmid DNA from recombinant clones --- p.63 / Chapter 2.11 --- DNA sequencing of differentially expressed cDNA fragments --- p.64 / Chapter 2.12 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.66 / Chapter 2.13 --- Western bolt analysis --- p.67 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- DNase I treatment --- p.71 / Chapter 3.2 --- FDD RT-PCR and band excision --- p.71 / Chapter 3.3 --- Reamplification of excised cDNA fragments --- p.74 / Chapter 3.4 --- Subcloning of reamplified cDNA fragments --- p.77 / Chapter 3.5 --- DNA sequencing of subcloned cDNA fragments --- p.77 / Chapter 3.6 --- Confirmation of the differentially expressed cDNA fragments by RT-PCR and Western blotting --- p.84 / Chapter 3.6.1 --- Effects of TNF-α on p38a mitogen protein kinase (p38 α MAPK) --- p.84 / Chapter 3.6.2 --- Effects of TNF-α on p38 a MAPK and p-p38 α MAPK protein level --- p.86 / Chapter 3.7 --- Effects of TNF-α on p38 MAPK --- p.88 / Chapter 3.7.1 --- "Effects of TNF-α on p38 α, β,γ andδ MAPK" --- p.88 / Chapter 3.7.2 --- Role of TNF-receptor (TNF-R) subtype in the TNF-α-induced p3 8 MAPK expression in C6 cells --- p.89 / Chapter 3.7.3 --- The signaling system mediating TNF-α-induced p38 a MAPK expression in C6 cells --- p.92 / Chapter 3.7.3.1 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.92 / Chapter 3.7.3.2 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.98 / Chapter 3.7.4 --- The relationship between p38 MAPK and β-adrenergic mechanisms in C6 cells --- p.99 / Chapter 3.7.4.1 --- Effects of isoproterenol and propanol on p38 MAPK mRNA levels in C6 cells --- p.103 / Chapter 3.7.4.2 --- Effects of β1-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.106 / Chapter 3.7.4.3 --- Effects of β2-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.107 / Chapter 3.8 --- The relationship between p3 8 MAPK and inducible nitric oxide synthase (iNOS) expression --- p.113 / Chapter 3.8.1 --- Effects of TNF-α on the iNOS expression in C6 cells --- p.113 / Chapter 3.8.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced iNOS expression in C6 cells --- p.115 / Chapter 3.8.3 --- The signaling system mediating TNF-α-induced iNOS expression in C6 cells --- p.115 / Chapter 3.8.3.1 --- The involvement of p38 MAPK in the TNF-α-induced iNOS expression in C6 cells --- p.117 / Chapter 3.8.3.2 --- The involvement of PKA in the TNF-α-induced iNOS expression in C6 cells --- p.119 / Chapter 3.9 --- The relationship between p38 MAPK and cAMP-responsive element binding protein (CREB) expression --- p.120 / Chapter 3.9.1 --- Effects of TNF-α on the CREB expression in C6 cells --- p.120 / Chapter 3.9.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced CREB expression in C6 cells --- p.124 / Chapter 3.9.3 --- The signaling system mediating TNF-α-induced CREB expression in C6 cells --- p.126 / Chapter 3.9.3.1 --- The involvement of p38 MAPK in the TNF-α-induced CREB expression in C6 cells --- p.126 / Chapter 3.9.3.2 --- The involvement of PKC in the TNF-α-induced CREB expression in C6 cells --- p.128 / Chapter 3.9.3.3 --- The involvement of PKA in TNF-α-induced CREB expression in C6 cells --- p.129 / Chapter 3.9.4 --- The relationship between CREB and β-adrenergic mechanisms in C6 cells --- p.136 / Chapter 3.9.4.1 --- Effects of isoproterenol and propanol on CREB mRNA levels in C6 cells --- p.136 / Chapter 3.9.4.2 --- Effects of β1-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.139 / Chapter 3.9.4.3 --- Effects of (32-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.142 / Chapter 3.10 --- The relationship between p38 MAPK and transcription factor c-fos expression --- p.146 / Chapter 3.10.1 --- Effects of TNF-α on the c-fos expression in C6 cells --- p.146 / Chapter 3.10.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced c-fos expression in C6 cells --- p.146 / Chapter 3.10.3 --- The signaling system mediating TNF-α-induced c-fos expression in C6 cells --- p.149 / Chapter 3.10.3.1 --- The involvement of p38 MAPK in the TNF-α-induced c-fos expression in C6 cells --- p.149 / Chapter 3.10.3.2 --- The involvement of PKC in the TNF-α-induced c-fos expression in C6 cells --- p.151 / Chapter 3.10.3.3 --- The involvement of PKA in TNF-α-induced c-fos expression in C6 cells --- p.154 / Chapter 3.10.4 --- The relationship between c-fos and β-adrenergic mechanisms in C6 cells --- p.157 / Chapter 3.10.4.1 --- Effects of isoproterenol and propanolol on c-fos mRNA levels in C6 cells --- p.157 / Chapter 3.10.4.2 --- Effects of β1-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.160 / Chapter 3.10.4.3 --- Effects of β2-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.164 / Chapter 3.11 --- The relationship between p38 MAPK and transcription factor NF-kB expression --- p.168 / Chapter 3.11.1 --- Effects of TNF-α on the NF-kB expression in C6 cells --- p.168 / Chapter 3.11.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced NF-kB expression in C6 cells --- p.168 / Chapter 3.11.3 --- The signaling system mediating TNF-α-induced NF-kB expression in C6 cells --- p.171 / Chapter 3.11.3.1 --- The involvement of p38 MAPK in the TNF-α-induced NF-kB expression in C6 cells --- p.171 / Chapter 3.11.3.2 --- The involvement of PKC in the TNF-α-induced NF-kB expression in C6 cells --- p.173 / Chapter Chapter 4 --- DISCUSSION AND CONCLUSION / Chapter 4.1 --- Effects of tumor-necrosis factor-alpha (TNF-α) on C6 cell proliferations --- p.176 / Chapter 4.2 --- The Signaling System Involved in TNF-α-Induced p38 MAPK Expression in C6 cells --- p.178 / Chapter 4.3 --- The Signaling System Involved in TNF-α-Induced iNOS Expression in C6 cells --- p.184 / Chapter 4.4 --- The Signaling System Involved in TNF-α-Induced CREB Expression in C6 cells --- p.186 / Chapter 4.5 --- The Signaling System Involved in TNF-α-Induced c-fos Expressionin in C6 cells --- p.190 / Chapter 4.6 --- The Signaling System Involved in TNF-α-Induced NF-kB Expression in C6 cells --- p.193 / Chapter 4.7 --- Conclusions --- p.195 / Chapter 4.8 --- Possible application / References

by Lam Kai Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 156-165). / Abstract also in Chinese. / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Tumor Necrosis Factor-α in Cancer Treatment --- p.1 / Chapter 1.1.1 --- Historical Background --- p.1 / Chapter 1.1.2 --- Mechanisms of Action --- p.2 / Chapter 1.1.2.1 --- Production of Reactive oxidative Species / Chapter 1.1.2.2 --- Increase of Intracellular Free Calcium Concentration / Chapter 1.1.2.3 --- Activation of Ca2+/Mg2+-dependent Endonuclease / Chapter 1.1.2.4 --- Decrease of glucose uptake and Protein Synthesis / Chapter 1.1.2.5 --- Formation of Ion-permeable Channel / Chapter 1.1.2.6 --- Activation of Phospholipase / Chapter 1.1.2.7 --- Increase of S-phase Cells / Chapter 1.1.2.8 --- Immunomodulatory Effects / Chapter 1.1.3 --- Resistance of Cells to TNF-α --- p.7 / Chapter 1.1.4 --- Clinical Studies --- p.11 / Chapter 1.1.5 --- Side Effects --- p.12 / Chapter 1.2 --- Hyperthermia and Cancer Treatment --- p.14 / Chapter 1.2.1 --- Hyperthermic Agents --- p.15 / Chapter 1.2.2 --- Intrinsic Heat Sensitivity --- p.15 / Chapter 1.2.3 --- Mechanisms of Action --- p.17 / Chapter 1.2.3.1 --- Depolarization of Membrane Potential / Chapter 1.2.3.2 --- "Reduction of glucose transport and DNA, mRNA and Protein Synthesis" / Chapter 1.2.3.3 --- Decrease of Intracellular pH / Chapter 1.2.3.4 --- Calcium Imbalance / Chapter 1.2.3.5 --- Effect on Nucleolar Protein / Chapter 1.2.3.6 --- Apoptosis / Chapter 1.2.3.7 --- Induction of Autologous Tumor Killing / Chapter 1.2.3.8 --- "Blood Flow, Tumor Oxygenation and Vascular Damage" / Chapter 1.2.4 --- Clinical Studies --- p.20 / Chapter 1.3 --- Combined Treatment --- p.21 / Chapter 1.3.1 --- Combined Treatment with TNF-α and Fixed-temperature Hyperthermia --- p.22 / Chapter 1.3.2 --- Combined Treatment with TNF + Step-down Hyperthermia --- p.22 / Chapter 1.3.3 --- In Vivo Study --- p.23 / Chapter 1.3.4 --- Sequence of Treatment --- p.24 / Chapter 1.3.5 --- Proposed Mechanism of Synergism --- p.24 / Chapter 1.4 --- Objective of Study --- p.26 / Chapter 1.4.1 --- Sequence of Treatments --- p.26 / Chapter 1.4.2 --- Comparison of Treatments' Effectiveness --- p.27 / Chapter 1.4.3 --- Effect on Normal Cell --- p.27 / Chapter 1.4.4 --- Effect on Distribution of Cells in Cell Cycle Phases --- p.28 / Chapter 1.4.5 --- In Vivo Study --- p.28 / Chapter Chapter Two: --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.30 / Chapter 2.1.1 --- For Cell Culture --- p.30 / Chapter 2.1.2 --- In vitro Treatments --- p.31 / Chapter 2.1.3 --- DNA Electrophoresis --- p.31 / Chapter 2.1.4 --- Flow Cytometry --- p.32 / Chapter 2.2. --- Reagent Preparation --- p.33 / Chapter 2.2.1 --- Culture Media --- p.33 / Chapter 2.2.2 --- Human Recombinant Tumor Necrosis Factor alpha (rhTNF-α) --- p.33 / Chapter 2.2.3 --- Phosphate Buffered Saline (PBS) --- p.33 / Chapter 2.2.4 --- Lysis Buffer --- p.34 / Chapter 2.2.5 --- TE Buffer --- p.34 / Chapter 2.2.6 --- Proteinase K and Ribonuclease A (RNase A) --- p.34 / Chapter 2.2.7 --- 100 Base-Pair DNA Marker --- p.34 / Chapter 2.2.8 --- Propidium Iodide (PI) --- p.35 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Cell Culture --- p.35 / Chapter 2.3.1.1 --- Ehrlich Ascitic Tumor (EAT) and Human Leukemia (HL-60) / Chapter 2.3.1.2 --- Human Coronary Artery Endothelial Cells (HCAEC) / Chapter 2.3.2 --- In vitro Experiments --- p.36 / Chapter 2.3.3 --- Tumor Necrosis Factor Treatment --- p.37 / Chapter 2.3.4 --- Hyperthermia Treatments --- p.37 / Chapter 2.3.5 --- Cell Counting --- p.38 / Chapter 2.3.5.1 --- Trypan Blue Exclusion Assay / Chapter 2.3.5.2 --- Neutral Red Assay / Chapter 2.3.6 --- Determination of Additive or Synergistic Effect --- p.39 / Chapter 2.3.7 --- DNA Electrophoresis --- p.40 / Chapter 2.3.8 --- Flow Cytometry --- p.42 / Chapter 2.3.7.1 --- Preparation of Samples / Chapter 2.3.7.2 --- Flow Cytometry Acquisition / Chapter 2.3.7.3 --- Analysis / Chapter 2.3.9 --- In vivo Experiments --- p.44 / Chapter 2.3.8.1 --- Animal Strain / Chapter 2.3.8.2 --- Cell Line / Chapter 2.3.8.3 --- Tumor Necrosis Factor Treatment / Chapter 2.3.8.4 --- Hyperthermia Treatments / Chapter 2.3.8.5 --- Test of Body Temperature / Chapter 2.3.8.6 --- Cell Harvesting / Chapter Chapter Three: --- Result --- p.50 / Chapter 3.1 --- Optimal Sequence of Treatments --- p.50 / Chapter 3.1.1 --- Optimal Sequence of Treatments on Murine Ehrlich Ascitic Tumor (EAT) cells --- p.50 / Chapter 3.1.1.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.1.1.2 --- TNF + Step-down Hyperthermia2 / Chapter 3.1.1.3 --- TNF + Step-down Hyperthermia3 / Chapter 3.1.2 --- Optimal Sequence of Treatments on Human Leukemia cells HL-60 --- p.60 / Chapter 3.1.2.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.1.2.2 --- TNF + Step-Down Hyperthermia2 / Chapter 3.1.2.3 --- TNF + Step-Down Hyperthermia3 / Chapter 3.2 --- Comparison of Effectiveness of Treatments --- p.72 / Chapter 3.2.1 --- Effectiveness of Various treatments on EAT cells --- p.72 / Chapter 3.2.2 --- Synergistic Effect between rhTNF-α and Hyperthermia on EAT cells --- p.74 / Chapter 3.2.3 --- Decrease of Relative Growth and Viability of EAT with Time --- p.79 / Chapter 3.2.3.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.2.3.2 --- TNF + Step-down Hyperthermia2 / Chapter 3.2.3.3 --- TNF + Step-down Hyperthermia3 / Chapter 3.2.4 --- Comparison of Effectiveness of Various Treatments on HL-60 cells --- p.82 / Chapter 3.2.5 --- Synergistic Effect between rhTNF-α and Hyperthermia on HL-60 cells --- p.87 / Chapter 3.2.6 --- Change of Relative Growth and Viability of HL-60 with Time --- p.90 / Chapter 3.2.6.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.2.6.2 --- TNF + Step-down Hyperthermia2 / Chapter 3.2.6.3 --- TNF + Step-down hyperthermia3 / Chapter 3.3 --- Cell Death Pathway --- p.96 / Chapter 3.3.1 --- Experiments on Ehrlich Ascitic Tumor (EAT) Cells --- p.96 / Chapter 3.3.2 --- Experiments on Human Leukemia (HL-60) Cells --- p.100 / Chapter 3.4 --- Experiment on Normal Cell --- p.104 / Chapter 3.5 --- Effect of TNF + Fixed-temperature Hyperthermia on the Cell Cycle Progression --- p.107 / Chapter 3.5.1 --- Different Times of TNF Administration and Distribution of EAT cells in Cell cycle --- p.107 / Chapter 3.5.2 --- Different Times of TNF Administration and Distribution of HL-60 cells in Cell Cycle --- p.114 / Chapter 3.5.3 --- Shift of Cells Cycle after TNF Treatment --- p.120 / Chapter 3.5.3.1 --- Response of Ehrlich Ascitic Tumor Cells / Chapter 3.5.3.2 --- Response of Human leukemia Cells / Chapter 3.6 --- Effectiveness of Treatments in vivo: --- p.129 / Chapter 3.6.1 --- Dose-dependent Response --- p.129 / Chapter 3.6.2 --- Change of Body Temperature During Hyperthermia --- p.131 / Chapter 3.6.3 --- Comparison of Effectiveness of Various Treatments in vivo --- p.133 / Chapter 3.6.4 --- Synergistic Effect Between rhTNF-α and Hyperthermia in vivo --- p.135 / Chapter Chapter Four: --- Discussion --- p.138 / Chapter 4.1 --- Optimal Sequence of Treatments --- p.139 / Chapter 4.2 --- Comparison of Various Treatments --- p.143 / Chapter 4.3 --- Distribution of Cells in Cell Cycle Phases --- p.149 / Chapter 4.4 --- In vivo Study --- p.153 / Chapter Chapter Five: --- References --- p.156

Type I diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β cells in the pancreas. Destruction of the body's own proteins, cells, and tissues is precipitated by the dysfunction of cytokine production, protein modification, and signaling pathways in immune cell subtypes. Tumor Necrosis Factor α (TNFα) and its receptors Tumor Necrosis Factor 1 (TNFR1) also known as p55 and TNFRSF1A, and Tumor Necrosis Factor 2 (TNFR2) also known as P75 and TNFRSF1B play a crucial role in this autoimmune process. TNFα has been shown to stimulate cell death through TNFR1 signaling by the caspase system, while promoting cell survival through TNFR2 signaling using the Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B cells (NF-𝜅B) pathway. Recent findings show a defect in immuno-proteasomes found in autoreactive T cells in people with T1D. This defect causes improper signaling transduction when TNFα binds to TNFR2. The inability to save the cell by activating the NF-𝜅B pathway eventually leads instead to apoptosis using the caspase system. A decrease in TNFα or increase in soluble TNFα receptors might be an explanation for these autoreactive T cells to evade the host immune system, and allow them to cause destruction of the pancreas. We hypothesize that patients with T1D will show abnormal distribution of TNFα and its receptors at basal levels, as well as when stimulated with interleukins, cytokines, and bacteria such as interleukin-2 (IL-2), lipotechoic acid (LTA), granulocyte macrophage-colony stimulating factor (GM-CSF), and Bacillus Calmette-Guérin (BCG). To test this hypothesis, we obtained peripheral blood from T1D patients (n=102) and controls (n=89) and performed in vitro stimulation assays. After a 48-hour incubation, tissue culture supernatants were collected and analyzed for TNF and its receptors production by ELISA, as well as densities of cell membrane receptors by flow cytometry. The data from this study showed significant differences in basal levels of TNFα, TNFR1, and TNFR2 on both the membrane and in the serum between patients and controls. Patients contained a greater percentage of CD4, 8, and 14 - TNFR2 and not TNFR1 double positive cells than their healthy control counterparts. Patient's sera also contained higher levels of all three markers, sTNFα, sTNFR1, and sTNFR2 than the controls. However, no significant differences were found between patient and controls when stimulated with the various compounds listed above.

JUSTIFICATIVA E OBJETIVO: Muitos estudos reportaram o benefício dos métodos avançados de ressonância magnética (RM) para a avaliação da resposta ao tratamento do tumor cerebral, a fim de distinguir entre recidiva tumoral e radionecrose. No entanto, o tamanho da amostra em cada estudo é relativamente pequeno, o que torna difícil avaliar a validade externa. Nós realizamos uma revisão sistemática e metanálise de dados publicados afim de avaliar a acurácia dos métodos avançados de RM para diferenciação entre a recidiva e a radionecrose. Nosso objetivo foi determinar o valor diagnóstico da difusão (DWI), da perfusão Dynamic Susceptibility Contrast (PWI DSC), perfusão Dynamic Contrast Enhancement (PWI DCE) e espectroscopia (MRS). MATERIAL E MÉTODOS: A revisão sistemática incluiu todos os estudos que usaram métodos avançados de RM para detectar recidiva ou radionecrose em pacientes em segmento por tratamento radioterápico para tumor cerebral. A pequisa foi realizada nas bases de dados MEDLINE e Embase, das publicações até 31 de Julho de 2015. As sensibilidades e especificidades de cada estudo foram calculadas e a acurácia diagnóstica foi metanalisada, com um intervalo de confiança (IC) de 95%, em um modelo de efeito randômico. Foram realizados testes de heterogeneidade, sobre o efeito dos pontos de corte e modelos de metarregressão. Análises de subgrupos foram feitas com base em conjuntos de estudos com características homogêneas. RESULTADOS: 49 artigos foram incluídos na análise quantitativa, englobando um total de 1.508 pacientes. Cinco estudos avaliaram DWI, 32 avaliaram PWI e 21 avaliaram MRS. A sensibilidade (SEN) e especificidade (ESP) geral da DWI foi 81,0% (IC 95%: 71,0 a 89,0%) e 68,0% (IC 95%: 52,0 a 82,0%), respectivamente. A SEN e ESP da PWI DSC foi de 83,0% (IC 95%: 80,0 a 86,0%) e 81,0% (IC 95%: 76,0 a 85,0%) e da PWI DCE foi 76,0% (IC 95%: 66,0 a 85,0%) e 85,0% (IC 95%: 74,0 a 93,0%), respectivamente. A SEN e ESP da MRS foi de 76,0% (IC 95%: 71,0 a 80,0%) e 83,0% (IC 95%: 77,0 a 88,0%), respectivamente. O odds ratio diagnóstico (DOR) da DWI, PWI DSC, PWI DCE e MRS foi 14,83; 25,81; 14,45 e 27,39; respectivamente. O maior valor do DOR nos estudos com PWI DSC foi quando o ponto de corte do Volume Sanguíneo Cerebral relativo (rCBV) foi maior ou igual a 1,8 e o maior valor do DOR nos estudos com MRS foi quando o ponto de corte da relação Cho/Cr foi maior ou igual a 1,3. O valor do DOR nos estudos com MRS foi muito maior nos trabalhos realizados com equipamento de 3T (DOR = 40,07; IC 95%: 15,44 a 104,03) quando comparado aos trabalhos realizados em equipamento com 1,5T (18,69; IC95%: 8,32 a 42,02). CONCLUSÃO: Esta metanálise demonstrou que os métodos avançados de RM tem uma moderada e alta acurácia na diferenciação da recidiva tumoral da radionecrose usando os métodos de DWI, PWI DSC, PWI DCE e MRS. Algumas análises de subgrupos e testes de efeito do ponto de corte mostraram que alguns cenários têm uma tendência de melhor acurácia. / BACKGROUND AND PURPOSE: Several studies reported the benefit of magnetic resonance (MR) advanced methods for the treatment response of brain tumor assessment, for distinguishing tumor recurrence from radionecrosis in gliomas and other brain tumors. However, the sample size in each study is relatively small, which becomes difficult to draw conclusions about external validity. We performed a systematic review and meta-analysis of published data to evaluate the accuracy of the advanced MR methods for differentiating recurrence from radionecrosis. Our objective was to determine the diagnostic value of Diffusion (DWI), Dynamic Susceptibility Contrast Perfusion (PWI DSC), Dynamic Contrast Enhancement Perfusion (PWI DCE) and Spectroscopy (MRS), and compare the results between the methods MATERIALS AND METHODS: The systematic review included all studies that used MR advanced methods to detect recurrence or radionecrosis in patients followed by brain tumor radiotherapy. The databases selected were MEDLINE and Embase, for published data prior to July 31, 2015. The sensitivities and specificities of individual studies were calculated and the pooled diagnostic accuracies, with 95% confidence intervals (CI), were assessed under a random-effects model. It was also performed heterogeneity test, threshold effect test and meta-regression models for each MR method. A subgroup analysis was performed based on homogeneous subsets of the studies. RESULTS: 49 articles were included in the quantitative analysis, compromising 1,508 patients (919 with recurrence and 589 with radionecrosis). Five studies assessed DWI, 32 assessed PWI, and 21 assessed MRS. Overall sensitivity (SEN) and specificity (SPE) of DWI were 81.0% (95% CI: 71.0 to 89.0%) and 68.0% (95% CI: 52.0 to 82.0%), respectively. The SEN and SPE of PWI DSC were 83.0% (95% CI: 80.0 to 86.0%) and 81.0% (95% CI: 76.0 to 85.0%) and PWI DCE were 76.0% (95% CI: 66.0 to 85.0%) and 85.0% (95% CI: 74.0 to 93.0%), respectively. The SEN and SPE of MRS were 76.0% (95% CI: 71.0 to 80.0%) and 83.0% (95% CI: 77.0 to 88.0%), respectively. The overall diagnostic odds ratio (DOR) of DWI, PWI DSC, PWI DCE, and MRS were 14.83, 25.81, 14.45, and 27.39, respectively. The point with the highest DOR in the PWI DSC studies was when the relative Cerebral Blood Volume (rCBV) threshold was equal or higher than 1.8, and the point with the highest DOR in the MRS studies was when the Cho/Cr threshold was equal or higher than 1.3. The MRS DOR value is much higher in the 3T subgroup (40.07, 95% IC: 15.44 to 104.03), compared to the 1.5T subgroup (18.69, 95% CI: 8.32 to 42.02). CONCLUSIONS: This meta-analysis showed that MR advanced methods have moderate to high accuracy in differentiating tumor recurrence from radiation necrosis using DWI, PWI DSC, PWI DCE and MRS. Some subgroup analysis and threshold effect tests demonstrated subsets that have a better accuracy trend.

by Li Jian Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 157-181). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

A necrose pulpar pode ser comumente observada no paciente odontopediátrico que sofreu trauma dentário ou que apresenta profundas lesões de cárie dentária. Sabese que existe uma falta de consonância a cerca de quais sinais são utilizados para o diagnóstico da necrose pulpar em dentes decíduos, o que acarreta em uma literatura carente de estudos que integrem todas as informações a cerca desta patologia. Assim, o objetivo desta revisão sistemática foi realizar um levantamento para compilar e discutir os critérios descritos na literatura para realizar o diagnóstico de necrose pulpar em dentes decíduos. Esta revisão foi realizada seguindo as orientações do \"PRISMA Statement\". A busca para acessar os artigos existentes foi realizada até outubro de 2017 e a base de dados bibliográficos escolhida foi o PubMed. As palavras chaves utilizadas combinaram termos relacionados a crianças, dentição decídua e necrose pulpar. Dois examinadores, de maneira independente, analisaram os critérios de inclusão e de exclusão, sem restrição de idioma, e em casos de divergência um terceiro examinador, considerado expert no assunto, foi consultado para decisão final. Dessa maneira, foram encontrados 1823 artigos, dos quais 401 foram incluídos para leitura na íntegra, restando 59 (14,7%) que compreenderam os estudos de interesse para esta revisão sistemática. A concordância entre os examinadores foi calculada através do teste de Cohen\'s Kappa, resultando em K = 1,0 para a etapa de inclusão e K = 0,97 para a etapa de exclusão. Considerando o objetivo dos estudos encontrados, 49,1% eram estudos de tratamento, no qual a necrose pulpar era abordada como critério de inclusão dos participantes no estudo, seguido de 18,6% trabalhos de levantamento, sendo todos a respeito de trauma dentário. Foi observado que em apenas 13,5% dos artigos a necrose pulpar foi abordada como o desfecho do estudo. Em 95% dos estudos a definição do conceito sobre a necrose pulpar foi fornecida pelos próprios autores. No restante, quando a referência era citada, esta também apresentava a definição de necrose pulpar através dos próprios autores. A respeito da causa da necrose pulpar, foi encontrado que a maioria dos dentes decíduos a apresenta devido a lesões de cárie dental, sendo os molares decíduos os mais afetados. Observou-se que em relação aos critérios utilizados para realizar o diagnóstico da necrose pulpar, 44% dos estudos utilizam os critérios clínicos e radiográficos de maneira conjunta. Foi permitido calcular a ocorrência dos sinais clínicos utilizados em 27,1% dos estudos, dos sinais observados nos exames complementares em 50,8%, e da necrose pulpar em 15,2%. Além disso, permitiu-se identificar sinais e sintomas que foram relacionados com a presença da necrose pulpar em 23,7% dos estudos. Assim, é possível concluir que existe uma combinação de sinais clínicos e radiográficos para realizar o diagnóstico de necrose pulpar em dentes decíduos, destacando-se a presença da fístula em 57,6% dos estudos, e a presença de lesão periapical e/ou lesão de furca em 71,2%. Concluiu-se também que 76,2% dos estudos não utilizam testes pulpares. / Pulpal necrosis may be commonly observed in children that suffered dental trauma or severe caries lesions. It is known that there is a lack of agreement regarding which signs should be used to diagnose pulpal necrosis in primary teeth, leading to a literature in needy of studies that integrate all the information about this disease. Therefore, the aim of this systematic review was to perform a data collection to compile and discuss the criteria described in the literature to perform the diagnose of pulpal necrosis in primary teeth. This systematic review was conducted following the \"PRISMA Statement\". To access the literature a search was conducted through PubMed in order to identify papers published until October 2017. The search strategy combined words related to children, deciduous teeth and pulpal necrosis. Two reviewers, independently, identified the inclusion and exclusion criteria, with no language restriction, and in cases of disagreement a third examiner, considered an expert in the subject, was consulted for the final decision. Therefore, 1823 articles were identified, from them 401 were included for complete scanning, remaining 59 (14.7%) studies that fulfilled the eligibility criteria for this systematic review. The interexaminer agreement was measured through Cohen\'s Kappa Statistic, resulting in K = 1.0 for the inclusion criteria and K= 0.97 for the exclusion criteria. Regarding the aim of the studies that were found, 49.1% were treatment studies, where pulpal necrosis was accessed as inclusion criterion for the participants of the study, followed by 18.6% studies of investigation, all about dental trauma. It was observed that only in 13.5% of the papers pulpal necrosis was approached as the outcome of the study. In 95% of the studies, the definition about the concept of pulpal necrosis was given by the authors themselves. In the remaining studies, when the reference was quoted, it also reported the definition by the authors themselves. Considering the cause of pulpal necrosis, it was found that most of the deciduous teeth were affected by caries lesions and the primary molars were the most affected. It was also observed that related to the criteria for the diagnosis of pulpal necrosis, 44% studies apply clinical and radiographic criteria combined. It was possible to calculate the occurrence of the used clinical signs in 27.1% of the studies, of the signs observed in auxiliary exams in 50,8% and of the pulpal necrosis in 15.2%. Besides, it was possible to identify signs and symptoms that were related to the presence of pulpal necrosis in 23.7% of the studies. Hence, it is possible to conclude that there is a combination of clinical and radiographic signs to perform the diagnosis of pulpal necrosis in primary teeth, highlighting the presence of sinus tract in 57.6% of the studies and the presence of periapical/ interradicular lesion in 71.2%. It was also concluded that in 76.2% of the studies do not use pulp sensibility tests.

A desnutrição pode induzir lesão celular, comprometendo os mecanismos envolvidos de proliferação, diferenciação e morte celular. Estudos de nosso laboratório tem demonstrado, em modelo murino de desnutrição protéica e protéico-energética, hipoplasia medular com evidências histológicas de alteração da matriz extra celular. Nosso objetivo foi avaliar a eventual participação dos processos de apoptose, necrose e autofagia no desenvolvimento da hipoplasia medular observada nesse modelo. Para isso foram utilizados dois grupos de camundongos C57BL/6J machos, adultos, mantidos em gaioleiros metabólicos. O grupo controle (C) recebeu ração normoproteíca contendo 12% de proteína e o grupo desnutrido (D), alimentado com ração hipoprotéica contendo 2% de proteína. A fonte protéica utilizada foi a caseína. O período de indução da desnutrição foi cerca de cinco semanas, definido pela perda de 20 a 25 % de peso corpóreo por parte dos animais do grupo desnutrido. Após esse período, os animais de ambos os grupos foram anestesiados e realizada a coleta das amostras biológicas para avaliação nutricional e hematológica e coletadas células da medula óssea para avaliação da apoptose, necrose e autofagia. Para avaliação da apoptose e necrose as células foram duplamente marcadas com Annexina V, PI e caspase 3 que foram analisadas por citometria de fluxo . A expressão da protéina BCL-2 foi quantificada pela técnica de Western Blotting. A análise não demonstrou diferença estatística entre os grupos para esses parâmetros. Para avaliação da autofagia extraiu-se proteínas das células da medula óssea e avaliou-se a expressão das proteínas Akt e mTOR total e fosforilado , os complexos de mTor (Raptor, Rictor e Gβl) , Beclin-1 e LC3II. Os resultados demonstraram aumento significativo de mTOR total ,Raptor , Beclin-1 e LC3II e diminuição na fosforilação de mTOR nas células oriundas de animais desnutridos em relação ao grupo controle. A desnutrição não modificou a expressão de Akt total, porém houve diminuição da fosforilação de Akt e diminuição na expressão das proteínas Rictor e Gβl nas células analisadas. Como os processos apoptóticos e autofágicos podem ser de difícil detecção in vivo, também refizemos os experimentos in vitro, estimulando as células com compostos pró-apoptóticos (campotecina) e pró-autofágicos (tamoxifeno). Nesses experimentos observamos que, apenas quando estimulamos as células de animais desnutridos com camptotecina, as mesmas, no período de 12 horas apresentaram maior percentagem de apoptose inicial em relação a 0 horas , sugerindo que há um período em que as células desnutridas são sinalizadas para via apoptótica sendo mais susceptível ao estimulo. As células de animal desnutrido estimulado apresentaram após 12 horas aumento significativo da apoptose tardia em relação ao controle estimulado , indicando que nesse período há um aumento da apoptose tanto em processo inicial , tanto em processo tardio. Avaliamos a autofagia em uma cinética de 0, 2, 6, 18 e 24 horas in vitro e observamos aumento significativo da autofagia em células da medula óssea de animais desnutridos em 0 horas e após 18 horas de estímulo com tamoxifeno (20 µM) em relação ao respectivo controle, demonstrando que nesse período a autofagia começa a ser induzida através do estimulo mais facilmente do que o controle. Autofagia é um dos principais contribuintes para o metabolismo celular, fornecendo nutrientes quando os mesmos estão indisponíveis, e, portanto, no nosso modelo de desnutrição protéica a hipoplasia medular estaria em processo autofágico como mecanismo de reparo e sobrevivência. / Malnutrition can induce cell damage, compromising the mechanisms involved in proliferation, differentiation and cell death. Studies from our laboratory have demonstrated, in a murine model of protein malnutrition and protein-energy, marrow hypoplasia with histologic evidence of alteration of the extracellular matrix. Our objective was to evaluate the possible involvement of the processes of apoptosis, necrosis and autophagy in the development of bone marrow hypoplasia observed in this model. For this we used two groups of C57BL/6J adult male kept in metabolic gaioleiro. The control group (C) received normal protein diet containing 12% protein and undernourished group (D), fed low protein diet containing 2% protein. The protein source used was casein. The induction period of undernutrition was approximately five weeks, as defined by loss of 20 to 25% of body weight per part of group malnourished. After this period, the animals of both groups were anesthetized and held the collection of biological samples for nutritional assessment and hematology and bone marrow cells collected for evaluation of apoptosis, necrosis and autophagy. For assessment of apoptosis and necrosis of the cells were double labeled with Annexina V and PI caspase 3 were analyzed by flow cytometry. The expression of Bcl-2 was quantified by Western Blotting technique. The analysis revealed no statistical difference between the groups for these parameters. For evaluation of autophagy proteins extracted from bone marrow cells and evaluated the expression of proteins Akt and phosphorylated and total mTOR, complexes of mTOR (Raptor, and Rictor Gβl), Beclin-1 and LC3II. The results showed significant increase in overall mTOR, Raptor, and LC3II Beclin-1 and decreased phosphorylation of mTOR in cells derived from malnourished animals compared to the control group. Malnutrition did not modify the expression of Akt total, but decreased phosphorylation of Akt and decreased expression of the protein Rictor and Gβl cells analyzed. As apoptotic and autophagic processes can be difficult to detect in vivo, also redid the experiments in vitro, stimulating the cells with pro-apoptotic compounds (campotecina) and pro-autophagic (tamoxifen). In these experiments we observed that, when only stimulate cells with camptothecin malnourished, the same at 12 hours had a higher percentage of initial apoptosis compared to 0 hours, suggesting that there is a period in which cells are signaled to via malnourished being more susceptible to apoptotic stimuli. The animals starved cells stimulated after 12 h showed significant increase in apoptosis compared to control late stimulated, indicating that at that time there is an increase in apoptosis both in the initial process, both late process. Autophagy evaluated in kinetics of 0, 2, 6, 18 and 24 hours in vitro and observed a significant increase in autophagy in bone marrow cells of malnourished at 0 hours and after 18 hours stimulation with tamoxifen (20 microM) than the respective control, demonstrating that this period autophagy begins to be induced by stimulating more easily than the control. Autophagy is a major contributor to cellular metabolism, providing nutrients when they are unavailable, and therefore in our model of protein malnutrition in the marrow hypoplasia would autophagic process as a mechanism for survival and repair.

Steroid-associated osteonecrosis (SAON) accounts for a major fraction in non-traumatic osteonecosis (ON) and generally has poor prognosis even after surgical interventions. This suggests that prevention is one of the important intervention strategies for SAON. So far, there is lacking of proven prevention modalities for SAON. / Study I was to establish an alternative SAON model. Based on the proposed pathogenesis of SAON that the intravascular thrombosis and extravascular marrow fat deposition are the two important contributors to the development of ON lesion, lipopolysaccharides (LPS) could induce vascular dysfunction and even thrombosis, and methylprednisolone (MPS) could induce the adipogenesis of marrow mesenchymal stem cells (MSCs). They were accordingly used in a combination for ON induction in animals. / Study II was to investigate the effect of Herbal Epimedium-derived formula for prevention of ON using the validated SAON animal model. Efficacy of the herbal Epimedium-derived formula was assessed for prevention of SAON using the animal model. Thirty adult male rabbits were used in this study. The ON incidence was set as the end-point for evaluation of the prevention efficay. For the potential intervention targets, the intravascular thrombosis and extravascular marrow fat formation were evaluated hematologically and histopathologically. The vascular structure and function were evaluated by advanced bioimaging modalities of micro-CT and MRI. / Study III was to investigate the bioactive compound(s) from the herbal Epimedium-derived herbal formula for prevention of SAON. Phytochemical analysis identified seven compounds in this efficacy-proven formula, with icariin as the major compound accounting for more than 80% in weight. Icariin was therefore tested for its prevention efficacy using the SAON animal model. / Study IV was to investigate the underlying mechanism(s) of bioactive compound Icariin in effective prevention of SAON using in vitro cell models. As activation of endothelial cells and adipogenesis of MSCs are suggested to be the two major events involving in vascular dysfunction and marrow fat formation in SAON animal model, Icariin were accordingly hypothesized to be able to prevent activation of endothelial cells and inhibit adipogenesis of MSCs. / Summary. After summarizing the major findings of these four logically interrelated studies, it was able to conclude that Icariin was the identified bioactive compound from the herbal Epimedium-derived formula, which was able to reduce the SAON incidence with inhibition of intravascular thrombosis and extravascular marrow fat formation in an established rabbit model. The underlying mechanisms might be related to its effects on protection of endothelial cells activation and inhibition of MSCs adipogenesis (This can be summarized in the following picture). This study provides a new bioactive agent Icariin for SAON prevention and potential future clinical application. (Abstract shortened by UMI.) / The following research questions were addressed in the present study: (1) Is there an alternative SAON animal model? (Study I); (2) Whether the herbal Epimedium-derived formula is able to prevent SAON in this animal model? (Study II); (3) What is the bioactive compound(s) in this herbal Epimedium-derived formula? (Study III); (4) How does this bioactive compound prevent SAON? (Study IV) / Sheng, Hui. / Adviser: Ling Qin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3421. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / 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. / Abstracts in English and Chinese. / School code: 1307.

激素性骨壞死是由於經常使用脈衝性激素處理非骨科性問題引起的一種常見的骨科疾病。在組織病理學上，激素性骨壞死指骨死亡，血管內血栓閉塞和血管外骨髓脂肪沉積會引起缺血導致骨修復不足。上游的分子細胞病理學機制研究表明間充質幹細胞細胞池活性下降，成骨細胞凋亡和骨小梁基質退變導致的不充分修復是激素性骨壞死發生的重要因素。 / 間充質幹細胞是骨髓的基質組成部分，具有分化成多種細胞的潛能。最近的研究表明，激素性骨壞死可能是骨細胞和/或間質幹細胞病變引起的一種疾病。研究發現，在接受類固醇治療而發生骨壞死的病人中，骨髓間充質幹細胞活性下降和分化潛能發生改變。在骨髓細胞中，激素能夠誘導脂肪發生。盛輝等發現來源於激素性骨壞死兔子中的間充質幹細胞成脂分化增強，謝新薈等進一步發現發生激素性骨壞死的兔子，骨缺損修復延遲，這可能是由激素導致的間充質幹細胞潛能發生改變引起的。綜合以上研究表明，間充質幹細胞在骨壞死發生和修復過程中起著重要作用。我們之前報導過淫羊藿黃酮（EFs）的腸代謝產物淫羊藿素Icaritin通過抑制血栓的形成和脂肪沉澱預防激素性骨壞死。最近，我們把Icaritin整合到聚乳酸聚乙醇酸共聚物/磷酸三鈣（PLGA/TCP）支架材料中，形成PLGA/TCP/Icaritin複合支架材料。我們發現PLGA/TCP/Icaritin複合材料可以促進激素性骨壞死骨缺損的修復，肌肉移植發現PLGA/TCP/Icaritin也能促進新生血管的發生。我們也發現單純PLGA/TCP複合材料也能夠促進激素性骨壞死骨缺損的修復，但是潛在的機制尚不清楚。 / 骨是一個高度血管化的組織，依賴於血管和骨細胞密切的時空連結維持骨骼的完整性。因此，血管生成在骨骼發育和骨折修復過程中發揮著舉足輕重的作用。血管為骨的發育和再生提供氧氣，為基質輸送刺激間充質細胞特異性成骨的重要信號，另一方面，骨為血管生成輸送生長因數和細胞。 / 本論文分為以下四個主要部分： / 第一部分: 研究Icaritin對人源間充質幹細胞分化的作用及其機制。流式細胞分選鑒定結果表明我們使用的人源間充質幹細胞能夠特異表達間充質幹細胞表面標誌物。MTT實驗結果顯示Icaritin不影響間充質幹細胞的增殖；分化實驗表明Icaritin在沒有成骨誘導試劑存在的情況下無法影響間充質幹細胞的分化。在成骨誘導試劑存在的情況下，Icaritin促進間充質幹細胞成骨分化，抑制其成脂分化；即時螢光實時定量聚合酶鏈式擴增（RT-PCR）結果顯示Icaritin在間充質幹細胞分化過程中上調成骨基因的表達，下調成脂基因表達。進一步研發發現在成骨分化過程中，Icaritin能夠促進BMP2和beta-catenin 蛋白的表達，而BMP2抑制劑Noggin能夠能夠逆轉Icaritin促進的成骨發生。這些發現表明Icaritin能夠促進而非誘導間充質幹細胞的成骨分化，Icaritin調解間充質幹細胞成骨分化具有BMP2信號通路依賴性。 / 第二部分: 評估激素性骨壞死兔源間充質幹細胞的分化潛能及Icaritin 對異常分化的間充質幹細胞分化潛能的影響。結果表明Icaritin促進正常兔源間充質幹細胞的成骨分化，抑制其成脂分化。激素性骨壞死兔源間充質幹細胞的成骨分化潛能降低，成脂分化升高；而Icaritin能夠劑量依賴性地部分恢復降低的成骨分化潛能，抑制升高的成脂分化活性。激素性骨壞死兔源間充質幹細胞的增殖活性也下降但是不能被Icaritin恢復。Icaritin對激素性骨壞死兔源間充質幹細胞中下降的VEGF的表達無影響。這些發現顯示間充質幹細胞的分化潛能在激素性骨壞死發生過程中遭到破壞，但是能夠被Icaritin部分恢復。 / 第三部分: 評估Icaritin對體外成血管的影響。我們對Icaritin對人臍帶靜脈內皮細胞（HUVECs）的增殖、遷移、管狀結構形成及成血管相關基因的表達的影響進行了檢測。結果表明Icaritin不影響HUVECs的增殖、遷移和管狀結構的形成；RT-PCR結果顯示Icaritin對HUVECs中的VEGF, HIF1a, FGF2 and TGF-beta表達也沒有影響。這些發現表明Icaritin在體外並不能直接作用于血管生成。結果謝新薈和陳詩慧等人的體內研究結果可以推測在骨缺損修復過程中，Icaritin通過促進成骨間接促進血管生成。 / 第四部分: 主要研究Icaritin及複合生物材料在體外體內對間充質幹細胞歸巢的影響。結果表明Iaritin能夠促進間充質幹細胞的遷移並上調血管細胞黏附分子1（VCAM1）的表達。複合材料PLGA/TCP和PLGA/TCP/Icaritin在體外培養的條件下能夠募集間充質幹細胞到材料周圍及進入材料。間充質幹細胞體外用修飾性超順磁性氧化鐵（SPIO@SiO₂-NH₂）納米顆粒標記後，其分化潛能依然保留，增殖和潛能能力稍微下降。兔激素性骨壞死造模完成後，股骨遠端髓芯減壓壞死骨缺損手術，PLGA/TCP和PLGA/TCP/Icaritin複合材料植入缺損孔道，同時把SPIO@SiO₂-NH₂標記的間充質幹細胞注射到距離缺損區20毫米的骨髓腔內。結果顯示只有標記的間充質幹細胞植入而沒有材料植入時，缺損區被脂肪細胞充滿，並沒有標記的間充質幹細胞出現，而在缺損區附近和遠離缺損區的部位有標記的間充質幹細胞出現。同時植入PLGA/TCP複合材料和標記的間充質幹細胞時，標記的間充質幹細胞出現在缺損區的材料中，在缺損區附近沒有標記的間充質幹細胞出現，而在遠離缺損區的部位，有標記的間充質幹細胞出現。同時植入PLGA/TCP/Icaritin和標記的間充質幹細胞時，得到跟植入PLGA/TCP複合材料和標記的間充質幹細胞相似的結果，但是在缺損區域，SPIO陽性的間充質幹細胞數目在PLGA/TCP和PLGA/TCP/Icaritin組別中並未發現有顯著性差異。以上發現表明Icaritin和PLGA/TCP複合材料能夠在體外和體內促進間充質幹細胞的歸巢。 / 綜上所述，複合支架材料PLGA/TCP/Icaritin通過調節間充質幹細胞的歸巢和分化促進激素性骨壞死骨缺損的修復。Icaritin通過BMP2和Wnt/beta-catenin通路調解間充質幹細胞的成骨分化。這是首次研究發現Icaritin及PLGA/TCP支架材料影響骨缺損修復過程中幹細胞歸巢，但是分子細胞生物學機制還需要進一步的研究。 / Steroid-associated osteonecrosis (SAON) is a common orthopaedic problem as the pulsed steroids are frequently prescribed for the treatment of non-orthopaedic medical conditions. Histopathologically, SAON refers to death of bone. Intravascular thrombus occlusion and extravascular marrow lipid deposition cause ischemia, which leads to an inadequate repair of the bone. Recent study revealed upstream pathological mechanism at cellular and molecular level. The decrease in activity of mesenchymal stem cell (MSC) pool, apoptosis of osteocytes, and trabecular bone matrix degeneration may cause bone inadequate repair, a key pathological feature found in SAON. / MSCs are the stromal component of bone marrow (BM) and have the potential to differentiate into several cell types. Recent studies have suggested that SAON may be a disease of bone cells and/or MSCs. With corticosteroid therapy in patients, the MSCs activity decreased and differentiation potential changed. Steroids have been also shown to produce adipogenesis in bone-marrow cells. It has been found adipogenesis of MSCs from SAON rabbits elevated (Sheng et al., 2007a) and bone defect repair was delayed in rabbits with SAON (Xie et al., 2011), this may be caused by altered MSCs potentials. All these findings imply MSCs play a vital role in SAON development and bone defect repair. It had been reported that Icaritin, an intestinal metabolite of Epimedium-derived avonoids (EF) reduced SAON incidence with inhibition of both thrombosis and lipid deposition (Zhang et al., 2009a). More recently, we found integrating Icaritin into PLGA/TCP to form PLGA/TCP/Icaritin composite scaffold could promote SAON bone defect repair and more neovascularization formed in an intramuscular implantation model, and further found PLGA/TCP scaffold only also could promote SAON bone defect repair in rabbits (Wang et al., 2012a). But the underlying mechanism remains unclear. / Bone is a highly vascularized tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity. Angiogenesis thus plays a pivotal role in skeletal development and bone fracture repair. The vasculature supplies oxygen to developing and regenerating bone and also delivers critical signals to the stroma that stimulate MSC specification to promote bone formation and repair. On the other hand, bone also supplies growth factors and cells for angiogenesis. The content of this thesis is divided into the following four major parts: / Part I: to study the effect and molecular mechanism of Icaritin on the differentiation of human bone marrow-derived MSCs. Human MSC was identified first by flow cytometery and result showed our cultured human MSC expressed standard surface markers of MSCs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation ability of MSCs was not affected by Icaritin. Differentiation assay showed that without oseteogenic supplements (OS), Icaritin had no effect on osteogenic differentiation of MSCs. With presence of OS, Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs. Real- time polymerase chain reaction (RT-PCR) showed that Icaritin up-regulated osteoblastic marker genes expression during osteogenic differentiation of MSCs and inhibited adipogenic gene expression. Further studies showed that Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis. All these findings indicated Icaritin possessed osteopromotive but not osteoinductive potentials during the differentiation of MSCs. Icaritin regulated osteogenic differentiation of MSCs in BMP2 pathway dependent manner. / Part II: to evaluate the differentiation potential of MSCs derived from rabbit with SAON and the effect of Icaritin on the altered differentiation of MSCs. The results showed that Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs derived from normal rabbit. Osteogenic differentiation potential of mesenchymal stem cells derived from rabbit with SAON declined and Icaritin partly rescued the declined osteogenic differentiation potential in dose-dependent manner. Adipogenic differentiation potential of MSCs derived from rabbit with SAON enhanced while the enhanced adipogenesis could be depressed by Icaritin. The proliferation ability of MSCs derived from rabbit with SAON declined while could not be rescued by Icaritin. VEGF expression decreased in MSCs derived from rabbit with SAON but its expression could not be influenced by Icaritin. These findings showed that the differentiation potential of MSCs destroyed during SAON development and this potential could be partially restored by Icaritin. / Part III: to evaluate the in vitro angiogenic effect of Icaritin. The proliferation, migration and tube formation ability of human umbilical vein cells (HUVECs) were detected. The results showed that Icaritin did not affect HUVECs proliferation, migration and tube-like structure formation of HUVECs. Real time PCR showed that VEGF, HIF1a, FGF2 and TGF-beta expression in HUVECs was not changed when HUVECs were treated by Icaritin. These data indicated Icaritin did not directly impact angiogenesis in vitro. Combined with in vivo findings, we supposed Icaritin promoted angiogenesis through its enhanced osteogenesis during bone defect repair. / Part IV: to study Icaritin and scaffold impact on stem cell homing in vitro and in vivo. It was found Icaritin promoted the migration of rabbit MSCs and increased vascular cell adhesion molecule 1 (VCAM1) expression. Composite scaffolds PLGA/TCP and PLGA/TCP/Icaritin could recruit rabbit MSCs under in vitro culture condition. When labeled with SPIO@SiO₂-NH₂, the differentiation potential of rabbit MSCs retained while proliferation and migration ability of rabbit MSCs declined. Two weeks after SAON establishment, PLGA/TCP and PLGA/TCP/Icaritin scaffolds were implanted into the bone tunnel after core-decompression in initial necrotic bone defect in rabbits with SAON, immediately with SPIO@SiO₂-NH₂ labeled MSCs injected into bone marrow cavity locally. The results showed that without scaffold implantation, the tunnel was filled with fat cells and fibrotic tissues and there was no label MSC in the tunnel while there were more labeled cells appeared in bone marrow near the tunnel than far away the tunnel, with both PLGA/TCP and PLGA/TCP/Icaritin implantation, the labeled MSCs migrated into scaffold after its implantation into the bone tunnel while there was no labeled cell next to the tunnel but some were shown away from the tunnel. No significant difference was found in SPIO positive MSCs in bone tunnel between PLGA/TCP and PLGA/TCP/Icaritin group. The findings indicated that at least PLGA/TCP scaffold itself promoted MSCs homing in vitro and in vivo where the released icaritin could execute its osteopromotive effects. / In summary, the composite scaffold PLGA/TCP/Icaritin enhanced bone defect repair in rabbit with SAON by promoting homing and osteogenesis of MSCs. Icaritin promoted osteogenic differentiation of MSCs through BMP2 mediated signal pathway, such as Wnt/beta-catenin signal pathway. It is first time to report that PLGA/TCP scaffold promoted MSCs homing during bone defect repair, but underlying molecular and cellular mechanism need to be further studied. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yao, Dong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 137-158). / Abstract also in Chinese; some appendixes also in Chinese. / ACKNOWLEDGEMENTS --- p.i / TABLE OF CONTENTS --- p.iii / ABSTRACT (IN ENGLISH) --- p.x / ABSTRACT (IN CHINESE) --- p.xiv / FLOWCHART --- p.xviii / LIST OF PUBLICATIONS --- p.xix / LIST OF ABBREVIATIONS --- p.xxi / LIST OF FIGURES --- p.xxiv / Chapter CHAPTER 1: --- Introduction --- p.1 / Chapter 1 --- Osteonecrosis --- p.2 / Chapter 1.1. --- Etiology --- p.2 / Chapter 1.2. --- Anatomy of femoral head --- p.3 / Chapter 1.3. --- Pathogenesis --- p.4 / Chapter 1.3.1. --- Intraosseous hypertension (Compartment Syndrome of Bone --- p.4 / Chapter 1.3.2. --- Intraosseous hypertension (Compartment Syndrome of Bone) --- p.4 / Chapter 1.3.3. --- Coagulation --- p.5 / Chapter 1.4. --- Development stages of osteonecrosis --- p.5 / Chapter 2. --- Steroids-associated osteonecrosis --- p.11 / Chapter 2.1. --- Epidemiology --- p.12 / Chapter 2.2. --- Histopathology --- p.12 / Chapter 2.3. --- Etiopathogenesis --- p.13 / Chapter 2.3.1. --- Steroid and fat metabolism --- p.14 / Chapter 2.3.2. --- Steroid and endothelial cells --- p.15 / Chapter 2.3.3. --- Steroid and coagulation --- p.16 / Chapter 2.3.4. --- Steroid and angiogenesis --- p.17 / Chapter 2.4. --- Steroid and mesenchymal stem cells (MSCs) --- p.18 / Chapter 2.5. --- Treatment strategies for SAON --- p.18 / Chapter 2.5.1. --- Prevention --- p.19 / Chapter 2.5.2. --- Nonoperative treatment --- p.19 / Chapter 2.5.3. --- Operative treatment --- p.19 / Chapter 2.5.3.1. --- Core decompression strategy --- p.20 / Chapter 2.5.3.2. --- Tissue engineering approach --- p.22 / Chapter 3. --- Epimedium-derived flavonoids (EFs) --- p.22 / Chapter 3.1. --- Icaritin -Intestinal metabolism of EFs --- p.24 / Chapter 3.1.1. --- Anti-tumor activity --- p..25 / Chapter 3.1.2. --- Neuroprotective effects --- p.25 / Chapter 3.1.3. --- Embryonic stem cells differentiation --- p.25 / Chapter 3.1.4. --- Osteogenic differentiation --- p.26 / Chapter 4. --- Poly lactic-co-glycolic acid / tricalcium phosphate (PLGA/TCP) scaffold --- p.26 / Chapter 5. --- PLGA/TCP/Icaritin --- p.28 / Chapter 6. --- Hypothesis of this study --- p.28 / Chapter 7. --- Objective --- p.29 / Chapter CHAPTER 2: --- The effect of phytomolecule Icaritin on differentiation of human mesenchymal stem cells in vitro --- p.30 / Chapter 1. --- Introduction --- p.31 / Chapter 2. --- Material and Methods --- p.33 / Chapter 2.1. --- Ethics --- p.33 / Chapter 2.2. --- Reagents and cell culture --- p.33 / Chapter 2.3. --- Surface phenotypes of human BM-MSCs --- p.33 / Chapter 2.4. --- Osteogenic and adipogenic differentiation of human BM-MSCs treated with Icaritin --- p.34 / Chapter 2.5. --- MTT assay for proliferation of BM-MSCs --- p.34 / Chapter 2.6. --- ALP staining --- p.35 / Chapter 2.7. --- ALP activity assay --- p.35 / Chapter 2.8. --- Alizarin Red S staining --- p.35 / Chapter 2.9. --- Oil Red O staining --- p.35 / Chapter 2.10. --- Ribonucleic acid (RNA) isolation --- p.36 / Chapter 2.11. --- Reverse transcription --- p.36 / Chapter 2.12. --- Real time polymerase chain reaction (RT-PCR) --- p.37 / Chapter 2.13. --- Western blotting --- p.37 / Chapter 2.14. --- Osteogenetic analysis of human MSCs after the addition of BMP2 inhibitor Noggin --- p.39 / Chapter 2.15. --- Statistical analysis --- p.39 / Chapter 3. --- Results --- p.40 / Chapter 3.1. --- Characterization of surface phenotypes of human BM-MSCs --- p.40 / Chapter 3.2. --- Icaritin had no effect on human mesenchymal stem cells (MSCs) proliferation --- p..41 / Chapter 3.3. --- Icaritin promoted osteogenic differentiation of MSCs in presence of osteogenic supplement --- p.42 / Chapter 3.4. --- Icaritin enhanced mineralization in osteogenic differentiation of MSCs only in presence of osteogenic supplement --- p.44 / Chapter 3.5. --- Icaritin upregulated mRNA expression of osteoblastic marker genes during osteogenic differentiation of MSCs --- p.45 / Chapter 3.6. --- Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis --- p..48 / Chapter 3.7. --- Icaritin inhibited fat droplets formation during adipogenic differentiation of MSCs --- p.50 / Chapter 4. --- Discussion --- p.52 / Chapter 5. --- Conclusion --- p.56 / Chapter CHAPTER 3: --- Icaritin rescued abnormal differentiation potential of MSCs derived from rabbit with SAON --- p.57 / Chapter 1. --- Introduction --- p.58 / Chapter 2. --- Methods and materials --- p.59 / Chapter 2.1. --- SAON model establishment --- p.59 / Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.60 / Chapter 2.3. --- Osteogenic and adipogenic differentiation of rabbit BM-MSCs treated with Icaritin --- p.61 / Chapter 2.4. --- MTT Assay for Proliferation of BM-MSCs --- p.62 / Chapter 2.5. --- ALP Staining --- p.62 / Chapter 2.6. --- ALP Activity Assay --- p.62 / Chapter 2.7. --- Alizarin Red S Staining --- p.62 / Chapter 2.8. --- Oil Red O Staining --- p.63 / Chapter 2.9. --- RNA Isolation --- p.63 / Chapter 2.10. --- Reverse transcription --- p.64 / Chapter 2.11. --- Real time Polymerase chain reaction (RT-PCR) --- p.64 / Chapter 2.12. --- Western blotting performance --- p.65 / Chapter 2.13. --- Statistical analysis --- p.65 / Chapter 3. --- Results --- p.66 / Chapter 3.1. --- The osteogenic differentiation potential declined while adipogenic differentiation ability elevated of MSCs derived from SAON rabbits --- p.66 / Chapter 3.2. --- The dose-dependent effect of Icaritin on osteogenic differentiation enhancement of MSCs from normal and SAON rabbits --- p.68 / Chapter 3.3. --- Icaritin inhibited adipogenic differentiation of MSCs both derived from normal and SAON rabbits --- p..71 / Chapter 3.4. --- PPAR-γ and aP2 proteins expression increased in SAON rabbit while inhibited by Icaritin both in normal and SAON rabbit --- p.74 / Chapter 3.5. --- Proliferation ability of MSCs derived from SAON rabbit declined and Icaritin had no effect on proliferation both derived from normal and SAON rabbit --- p.75 / Chapter 3.6. --- Icaritin had no effect on the expression of VEGF which decreased in MSCs derived SAON --- p.76 / Chapter 4. --- Discussion --- p.76 / Chapter 5. --- Conclusion --- p.81 / Chapter CHAPTER 4: --- The effect of Icaritin on angiogenesis in vitro --- p.82 / Chapter 1. --- Introduction --- p.83 / Chapter 2. --- Material and Methods --- p.85 / Chapter 2.1. --- Cell culture --- p.85 / Chapter 2.2. --- Proliferation assay --- p.85 / Chapter 2.3. --- Scratch-wound healing assay --- p..86 / Chapter 2.4. --- Migration Assay --- p.86 / Chapter 2.5. --- In vitro Angiogenesis Assay --- p.87 / Chapter 2.6. --- RNA Isolation and Real-time PCR Performance --- p.87 / Chapter 2.7. --- Statistical Analysis --- p.88 / Chapter 3. --- Results --- p.88 / Chapter 3.1. --- Icaritin did not affect HUVECs migration --- p.88 / Chapter 3.2. --- Icaritin had no effect on tube formation on growth factors reduced Matrigel --- p.92 / Chapter 3.3. --- Icaritin had no effect on HUVECs proliferation --- p.94 / Chapter 3.4. --- Icaritin did not change the angiogenesis related gene expression --- p.95 / Chapter 4. --- Discussion --- p.96 / Chapter 5. --- Conclusion --- p.100 / Chapter CHAPTER 5: --- Effect of PLGA/TCP and PLGA/TCP/Icaritin composite scaffolds on stem cell homing during bone defect repair with SAON --- p.101 / Chapter 1. --- Introduction --- p.102 / Chapter 2. --- Material and Methods --- p.106 / Chapter 2.1. --- Preparation of porous PLGA/TCP/Icaritin composite scaffolds --- p.106 / Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.106 / Chapter 2.3. --- Wound healing assay --- p.107 / Chapter 2.4. --- In vitro MSCs recruitment assay of scaffolds --- p.107 / Chapter 2.5. --- MSCs labeling with SPIO@SiO2-NH2 nanoparticle --- p.108 / Chapter 2.6. --- Prussian blue staining --- p.108 / Chapter 2.7. --- MTT assay for SPIO@SiO2-NH2 labeled MSCs --- p.108 / Chapter 2.8. --- Osteogenic and adipogenic differentiation of SPIO@SiO2-NH2 labeled MSCs --- p.109 / Chapter 2.9. --- Real time PCR --- p.109 / Chapter 2.10. --- Animal model establishment --- p.109 / Chapter 2.11. --- Descriptive histology and histomorphometry --- p.110 / Chapter 2.12. --- In vivo magnetic resonance imaging (MRI) of nanoparticle-labeled MSCs --- p.112 / Chapter 2.13. --- Statistical analysis --- p.112 / Chapter 3. --- Results --- p.112 / Chapter 3.1. --- Icaritin promoted MSCs migration in vitro --- p.112 / Chapter 3.2. --- PLGA/TCP and PLGA/TCP/Icaritin recruited MSCs when incubated in vitro --- p.114 / Chapter 3.3. --- Stem cell potentials of MSC after SPIO@SiO2-NH2 labeling --- p.118 / Chapter 3.4. --- PLGA/TCP and PLGA/TCP/Icaritin promoted MSCs homing in vivo --- p.122 / Chapter 4. --- Discussion --- p.126 / Chapter 5. --- Conclusion --- p.136 / Chapter CHAPTER 6: --- Summary of the study and future research --- p.137 / Chapter 1. --- Summary of the study --- p.138 / Chapter 2. --- Limitations and further studies --- p.139 / APPENDIXES --- p.142 / REFERENCES --- p.147

Orientador: Fabio Ermínio Mingatto / Banca: Cézar Rangel Pestana / Banca: Guilherme de Paula Nogueira / Resumo: Abamectina (ABA), pertencente à família das avermectinas é utilizada mundialmente como parasiticida, porém a intoxicação por ABA pode prejudicar o funcionamento hepático. Existem substâncias que quando metabolizadas exercem atividade tóxica, afetando a função de importantes organelas como a mitocôndria, responsável por uma variedade de processos bioquímicos, como a produção de ATP e morte celular. O objetivo desse estudo foi caracterizar o mecanismo de toxicidade da ABA em hepatócitos isolados de ratos e avaliar se esse efeito é dependente do seu metabolismo. A toxicidade da ABA foi avaliada monitorando o consumo de oxigênio e o potencial de membrana mitocondrial, concentração intracelular de ATP, viabilidade celular por meio da liberação das enzimas ALT e AST, homeostase intracelular Ca2+, liberação de citocromo c, atividade da caspase 3 e morte celular por necrose. A ABA reduz a respiração celular, tanto em células energizadas com glutamato mais malato quanto com succinato. O metabolismo da ABA reduz sua toxicidade, uma vez que hepatócitos previamente incubados com proadifen apresentam maior sensibilidade ao composto, sendo isto observado pelo rápido decréscimo da formação do potencial de membrana mitocondrial acompanhado pelas reduções das concentrações de ATP, viabilidade celular e ruptura da homeostase intracelular de Ca2+ com estabelecimento de necrose. Nossos resultados indicam que a toxicidade da ABA diminui com a sua biotransformação, e sua ação tóxica está relacionada com a inibição da atividade mitocondrial, levando à diminuição da síntese de ATP seguida pela morte da célula / Abstract: Abamectin (ABA), which belongs to the family of avermectinas, used worldwide as a parasiticide, but the ABA poisoning can impair the functioning liver. There are substances which exert toxic activity when metabolized, thus affecting the function of important organelles such as mitochondria, responsible for a variety of biochemical processes, such as ATP production and cell death. The aim of this study was to characterize the mechanism of toxicity of ABA in isolated rat hepatocytes and to evaluate whether this effect is dependent on your metabolism. The toxicity of ABA was assessed by monitoring oxygen consumption and mitochondrial membrane potential, intracellular concentration of ATP, cell viability by releasing enzymes ALT and AST, homeostasis intracellular Ca2+, release of cytochrome c, activity of caspase 3 and cell death necrosis. The ABA reduces cellular respiration, both in cells energized with glutamate and succinate over malate. The metabolism of ABA reduces its toxicity, since hepatocytes pre-incubated with proadifen are more sensitive to the compound, this being observed by the formation of rapidly decreasing mitochondrial membrane potential accompanied by reductions in concentrations of ATP, cell viability and rupture of the intracellular homeostasis Ca2+ with the establishment of necrosis. Our results indicate that the toxicity decreases as the ABA its biotransformed and its toxic action is related to the inhibition of mitochondrial activity, leading to decreased synthesis of ATP followed by cell death / Mestre