Os iso-α-ácidos são os principais constituintes responsáveis pelo sabor amargo da cerveja, sendo estes facilmente degradados durante o seu processo de envelhecimento ou exposição a radiação luminosa, em particular o diastereoisômero trans-. O radical 1-hidroxietila é o radical majoritário formado na cerveja durante o processo de envelhecimento. O presente trabalho descreve a reatividade dos iso-α-ácidos frente ao radical 1-hidroxetila através do uso da técnica de spin-trapping com detecção por espectroscopia de ressonância paramagnética de elétrons (RPE) e espectrometria de massas (ESI-(+)-MS/MS). Observou-se que ambos os diastereoisômeros cis- e trans-iso-α-ácidos são degradados na presença do radical 1-hidroxietila com constantes de velocidade aparentes de 1,8 108 e 9,2 109 L mol-1 s-1, respectivamente. A reatividade dos dihidro-iso-α-ácidos com o radical estudado foi similar à reatividade da mistura diastereoisomérica dos iso-α-ácidos, apresentando constante de velocidade aparente de 1,5 109 L mol-1 s-1. Os análogos tetrahidro-iso-α-ácidos não apresentaram reatividade para com o radical 1-hidroxietila, sugerindo os hidrogênios alílicos como sítio reacional. Adicionalmente, os cálculos ab initio por DFT demonstraram que os valores de BDE para os hidrogênios alílicos das cadeias laterais prenila e isohexenoila são equivalentes e, desta maneira, sugerindo a maior reatividade do diastereoisômero trans- a ser creditada a um fator entrópico, já que ambos os grupos estão no mesmo plano espacial. Os produtos de oxidação foram determinados por LC-ESI-MSn e verificou-se a formação dos hidroxi-allo-iso-α-ácidos, recentemente descritos na literatura. O conjunto de resultados obtidos possibilitou a proposta de mecanismo para processo de oxidação e perda dos ácidos amargos da cerveja, observado no envelhecimento da bebida. / The iso-α-acids are the main responsible constituents for the bitter taste of beer, they are easily degraded during the aging and light exposed process, specially the trans- diastereoisomer. The 1-hydroxyethyl radical is the major radical produced during the beer aging process. The present work describes the reactivity of iso-α-acids towards the 1-hydroxyethyl radical as probed by the spin-trapping technique and detected by electron paramagnetic resonance (EPR) and mass spectrometry (ESI-(+)-MS/MS). It was observed that both diastereoisomers cis- and trans-iso-α-acids are degraded in the presence of 1-hydroxyethyl radical with apparent rate constant of 1.8 108 e 9.2 109 L mol-1 s-1, respectively. The reactivity of dihydro-iso-α-acids towards the studied radical was similar to the reactivity of the iso-α-acids diastereoisomeric mixture, showing apparent rate Constant of 1.5 109 L mol-1 s-1. The tetrahydro-iso-α-acids analogues did not observed reactivity towards the 1-hydroxyethyl radical suggesting the allilic hydrogens as the reaction sites. In addition, the ab initio DFT calculations demonstrated that the BDE values for the allilic hydrogens of the prenyl and isohexenoyl side chains are equivalents and according to that suggesting the higher reactivity of the trans- diastereoisomer to be accounted to an entropic factor since both goups are in the same plane of the space. The oxidation products were determined by LC-ESI-MSn and its was verified the formation of hydroxyl-allo-iso-α-acids. The data colected allows a mechanism to be proposed for the oxidation process and loss of bitter acids of beer during the beverage aging.

A estrutura básica dos alcaloides indolizidínicos é formada por anéis bicíclicos de cinco e seis membros contendo um átomo de nitrogênio compartilhado na posição 4. Esse sistema de anéis possui grande destaque dentre os alcaloides, pois está presente em um grande número de compostos e apresenta um interessante perfil biológico. A ipalbidina, por exemplo, é um alcaloide indolizidínico com propriedades analgésicas e antioxidantes. Este composto possui estrutura química relativamente simples, entretanto, poucas são as rotas que apresentam sínteses curtas e divergentes, sendo apenas quatro delas enantiosseletivas. Assim, este trabalho de dissertação visa o estudo de uma nova estratégia sintética que permite a preparação da (+)-ipalbidina, bem como de outros alcaloides que possuem o sistema 4-azabiciclo[4.3.0]-non-3-eno com um substituinte fenólico na posição 3. Uma rota promitente para a síntese desses alcaloides (objetivo deste trabalho) é a obtenção do esqueleto indolizidínico a partir da reação de ciclização de uma α-clorocetona funcionalizada derivada do (S)-prolinal protegido (Boc e Cbz). As etapas chaves dessa estratégia são: uma reação de olefinação (Wittig), a preparação de α-clorocetonas, adição do grupo aril a α-clorocetona e a conversão destas no esqueleto indolizidínico por uma reação de ciclização. A α-clorocetona pode ser preparada com rendimentos globais de 56% (Cbz) e 81% (Boc) a partir do (S)-prolinal protegido em apenas 3 etapas: reação de olefinação, seguida de uma reação de redução da olefina obtida e a preparação da α-cloroacetona a partir do éster. A adição do grupo aril a α-clorocetona foi obtida tanto para o grupo Boc (40%) quanto para o grupo Cbz (42%). O α-cloroálcool protegido com Boc foi convertido no esqueleto indolizidínico por meio de uma reação \"one-pot\" de desproteção seguida de ciclização (80%). O produto de ciclização, por sua vez, foi convertido ao análogo inédito da (+)-ipalbidina, a (S)-desoxiipalbidina (30%). Essa estratégia levou a síntese da (S)-desoxiipalbidina em 6 etapas e com rendimento global de 8%. Cabe ressaltar que este tipo de abordagem utilizando α-clorocetonas nunca foi empregado na síntese de alcaloides indolizidínicos, sendo que esta estratégia também poderá ser aplicada a síntese total da (+)-ipalbidina e de outros alcaloides indolizidínicos tais como as fenantroindolizidinas. / The basic structure of indolizidine alkaloids is formed by a five and sixmembered bicyclic ring containing one nitrogen atom shared at the 4 position. This ring system has great prominence among the alkaloids, it is present in a large number of compounds and possess interesting biological profiles. Ipalbidine, for example, is an indolizidine alkaloid with analgesic and anti-oxidant properties. Although this compound has a relatively simple chemical structure, only four enantioselective synthesis are described for this compound. Thus, this dissertation aims to study a new synthetic strategy that allows the preparation of (+)-ipalbidine, as well as other alkaloids having the system 4- azabicyclo[4.3.0]non-3-ene with a phenolic substituent in position 3. A possible interesting route for the synthesis of these alkaloids is to obtain the indolizidine skeleton from a cyclization reaction using a functionalized α-chloroketone (derivative of protected (S)-prolinal (Boc and Cbz)). The key steps of this strategy are: an olefination reaction (Wittig), the preparation of α-chloroketones, addition of aryl group to the α-chloroketones and converting them into the indolizidine skeleton by a cyclization reaction. The α-chloroketones were prepared with overall yields varying from 56 % (Cbz) to 81% (Boc) starting from protected (S)-prolinal in just three steps: olefination reaction , followed by a reduction reaction of the obtained olefin and preparation of the α-chloroketone from an ester . The addition step of the aryl group to α-chloroketone was obtained for both Boc (40%) and Cbz (42%) groups. The Boc-protected α-chloroalcohol was converted to indolizidine skeleton through an \"one-pot\" deprotection reaction, followed by a cyclization reaction (80 %). The cyclization product, in turn, was converted to the novel (+)-ipalbidine analog, (S)-desoxyipalbidine (30 %). This strategy led to the synthesis of (S)-desoxyipalbidine in 6 steps and overall yield of 8 %. It is noteworthy that this type of approach using α-chloroketones was never employed in the synthesis of indolizidine alkaloids, and that strategy can be applied also to the total synthesis of (+)-ipalbidine and other indolizidine alkaloids such as phenanthroindolizidine.

by Shan Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 145-166). / Abstracts in English and Chinese. / Title --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xx / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- What are the general functions of cytokines? --- p.2 / Chapter 1.2 --- What is TNP-α? --- p.4 / Chapter 1.3 --- Actions of TNF-α --- p.5 / Chapter 1.4 --- General functions of TNF-α in astrocytes --- p.6 / Chapter 1.5 --- TNF-α receptors (TNF-Rs) --- p.8 / Chapter 1.6 --- Second messengers induced by TNP-α --- p.10 / Chapter 1.7 --- Glial Cells --- p.11 / Chapter 1.7.1 --- Oligodendroglia --- p.12 / Chapter 1.7.2 --- Brain Macrophages (Microglia) --- p.12 / Chapter 1.7.3 --- Astrocytes --- p.14 / Chapter 1.7.3.1 --- Functions of astrocytes --- p.15 / Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.20 / Chapter 1.9 --- β-Adrenergic receptors (β-ARs) --- p.21 / Chapter 1.9.1 --- The active functional unit: the receptor complex --- p.22 / Chapter 1.9.2 --- General functions and distribution of β-ARs --- p.22 / Chapter 1.10 --- Functions of β-ARs in astrocytes --- p.24 / Chapter 1.10.1 --- Regulations of astrogliosis by β-ARs --- p.24 / Chapter 1.10.1.1 --- β-ARs are expressed in normal optic nerves and up-regulated after nerve crush --- p.24 / Chapter 1.10.1.2 --- Injury-induced alterations in endogenous catecholamine leads to enhanced β-AR activation --- p.25 / Chapter 1.10.1.3 --- β-AR blockade suppresses glial scar formation --- p.25 / Chapter 1.10.1.4 --- β-AR agonists affect the proliferation of astrocytes in normal brain --- p.26 / Chapter 1.11 --- Manganese Superoxide Dismutase (MnSOD) --- p.27 / Chapter 1.11.1 --- MnSOD is the target gene of NF-kB --- p.29 / Chapter 1.11.2 --- Induction of MnSOD by proinflammatory cytokines in rat primary astrocytes --- p.29 / Chapter 1.11.3 --- SMase and ceramides induce MnSOD in various cell types --- p.30 / Chapter 1.12 --- Why do we use C6 glioma cells? --- p.31 / Chapter 1.13 --- Aims and Scopes of this project --- p.32 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Cell Line --- p.36 / Chapter 2.1.2 --- Cell Culture Reagents --- p.36 / Chapter 2.1.2.1 --- Complete Dulbecco´ةs modified Eagle medium (CDMEM) --- p.36 / Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.37 / Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.37 / Chapter 2.1.3 --- Recombinant cytokines --- p.38 / Chapter 2.1.4 --- Chemicals for signal transduction study --- p.38 / Chapter 2.1.4.1 --- Modulators of protein kinase C (PKC) --- p.38 / Chapter 2.1.4.2 --- Modulator of protein kinase A (PKA) --- p.39 / Chapter 2.1.4.3 --- β-Adrenergic agonist and antagonist --- p.39 / Chapter 2.1.5 --- Antibodies --- p.40 / Chapter 2.1.5.1 --- Anti-TNF-receptor type 1 (TNF-R1) antibody --- p.40 / Chapter 2.1.5.2 --- Anti-TNF-receptor type 2 (TNF-R2) antibody --- p.41 / Chapter 2.1.5.3 --- Anti-βi-adrenergic receptor (βl-AR) antibody --- p.42 / Chapter 2.1.5.4 --- Anti-β2-adrenergic receptor (β2-AR) antibody --- p.42 / Chapter 2.1.5.5 --- Antibody conjugates --- p.43 / Chapter 2.1.6 --- Reagents for RNA isolation --- p.43 / Chapter 2.1.7 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.43 / Chapter 2.1.8 --- Reagents for electrophoresis --- p.45 / Chapter 2.1.9 --- Reagents and buffers for Western blot --- p.45 / Chapter 2.1.10 --- Other chemicals and reagents --- p.47 / Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.47 / Chapter 2.3 --- RNA isolation --- p.48 / Chapter 2.3.1 --- Measurement of RNA yield --- p.49 / Chapter 2.4 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.5 --- Western blot analysis --- p.52 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- Effect of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.55 / Chapter 3.1.1 --- Effect of TNF-α on TNF-R1 and -R2 mRNA expression in C6 cells --- p.56 / Chapter 3.1.2 --- The signaling systems mediating TNP-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.1 --- The involvement of PKC in TNF-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.2 --- Effect of PMA on the TNF-R protein levels in C6 cells --- p.63 / Chapter 3.1.2.3 --- Effect of Ro31 on the TNF-α-induced TNF-R protein level in C6 cells --- p.65 / Chapter 3.1.2.4 --- Effect of PKA activator on the level of TNF-R2 mRNA in C6 cells --- p.67 / Chapter 3.2 --- Effect of TNP-α on the expression of β1- and β2-adrenergic receptors (β1- and β2-ARs) in C6 glioma cells --- p.69 / Chapter 3.2.1 --- Effect of TNF-α on β1- and β2-ARs mRNA expression in C6 cells --- p.70 / Chapter 3.2.2 --- The signaling systems mediating TNF-α-induced β1- and β2-AR expression in C6 cells --- p.70 / Chapter 3.2.2.1 --- The involvement of PKC mechanism between TNF-α and β-ARs in C6 cells --- p.71 / Chapter 3.2.2.2 --- Effect of PMA on the β1- and β2-ARs protein level in C6 cells --- p.76 / Chapter 3.2.2.3 --- Effect of Ro31 on the TNF-α-induced β1- and β2-AR protein levels in C6 cells --- p.78 / Chapter 3.2.2.4 --- Effect of dbcAMP on the levels of βl- and β2-ARs mRNA in C6 cells --- p.80 / Chapter 3.3 --- Relationship between TN1F-R2 and β-adrenergic mechanism in C6 cells --- p.82 / Chapter 3.3.1 --- Effects of isproterenol and propranolol on endogenous TNF-α mRNA levels in C6 cells --- p.82 / Chapter 3.3.2 --- Effects of isoproterenol and propranolol on TNF-R2 mRNA levels in C6 cells --- p.83 / Chapter 3.3.3 --- Effects of β1-agonist and antagonist on endogenous TNF-α mRNA expression in C6 cells --- p.87 / Chapter 3.3.4 --- Effects of β1-agonist and antagonist on TNF-R2 mRNA expression in C6 cells --- p.91 / Chapter 3.3.5 --- Effects of β2-agonist and antagonist on endogenous TNF-α mRNA in C6 cells --- p.93 / Chapter 3.3.6 --- Effects of β2-agonist and antagonist on TNF-R2 mRNA in C6 cells --- p.100 / Chapter 3.4 --- Effect ofTNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.102 / Chapter 3.4.1 --- Effect ofTNF-α on NF-kB (p50) mRNA expression in C6 cells --- p.106 / Chapter 3.4.2 --- Effect of β-agonist and antagonist on NF-kB (p50) mRNA expression in C6 cells --- p.108 / Chapter 3.4.3 --- Effect of PMA and Ro31 on the levels of NF-kB mRNA in C6 cells --- p.109 / Chapter 3.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.111 / Chapter 3.5.1 --- Effects of TNF-α on MnSOD and Cu-ZnSOD mRNAs expression in C6 cells --- p.114 / Chapter 3.5.2 --- Effects of β-agonist and β-antagonist on MnSOD mRNA expression in C6 cells --- p.115 / Chapter 3.5.3 --- Effects of PKC activator and inhibitor on the levels of MnSOD mRNA in C6 cells --- p.117 / Chapter Chapter 4 --- DISCUSSION AND CONCLUSION / Chapter 4.1 --- Effects of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.122 / Chapter 4.2 --- Effects of TNF-a on the expression of β1- and β2-adrenergic receptors (β1 and β2-ARs) in C6 glioma cells --- p.126 / Chapter 4.3 --- Relationship between TNF-α and β-adrenergic mechanism in C6 cells --- p.128 / Chapter 4.4 --- Effects of TNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.131 / Chapter 4.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.133 / Chapter 4.6 --- Possible sources of β-agonists --- p.136 / Chapter 4.7 --- Conclusions --- p.137 / Appendix A --- p.143 / References --- p.145

by Ko Samuel. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 209-227). / Abstract also in Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstract --- p.vii / Abstract in Chinese --- p.x / List of Figures --- p.xiii / List of Tables --- p.xx / Publication --- p.xxi / Contents --- p.xxii / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- Tumor Necrosis Factor --- p.2 / Chapter 1.1.1 --- History of Tumor Necrosis Factor --- p.2 / Chapter 1.1.2 --- TNF Subtypes and Their Purification --- p.3 / Chapter 1.1.3 --- Release of TNF --- p.9 / Chapter 1.1.4 --- Biological Actions of TNF --- p.9 / Chapter 1.2 --- Tumor Necrosis Factor Receptor --- p.11 / Chapter 1.2.1 --- Purification of TNF Receptor --- p.11 / Chapter 1.2.2 --- Regulation of TNF Receptor --- p.14 / Chapter 1.2.3 --- "Functions of TNF Receptor 1,Receptor 2 and Soluble TNF Receptors" --- p.15 / Chapter 1.3 --- Possible Signal Transductions of Tumor Necrosis Factor-Alpha --- p.17 / Chapter 1.3.1 --- Activation of Phospholipase A2 Cascade --- p.18 / Chapter 1.3.2 --- Activation of Phospho lipase C Pathway --- p.19 / Chapter 1.3.3 --- Activation of Sphingomyelin Pathway --- p.20 / Chapter 1.3.4 --- Activation of Protein Kinase --- p.22 / Chapter 1.3.5 --- Activation of the Cascade of Death Domain --- p.23 / Chapter 1.4 --- Induction of Both Necrosis and Apoptosis by Tumor Necrosis Factor-Alpha --- p.25 / Chapter 1.4.1 --- Apoptosis Versus Necrosis --- p.25 / Chapter 1.4.2 --- TNF Can Induce Both Apoptosis and Necrosis --- p.27 / Chapter 1.5 --- Possible Mechanisms of Tumor Necrosis Factor-Alpha- Mediated Cytotoxicity --- p.27 / Chapter 1.5.1 --- Release of Reactive Oxygen Species --- p.28 / Chapter 1.5.2 --- Release of Intracellular Calcium --- p.31 / Chapter 1.5.3 --- Miscellaneous Mechanisms --- p.36 / Chapter 1.6 --- Objective of Studies --- p.37 / Chapter Chapter 2. --- Materials and Methods --- p.39 / Chapter 2.1 --- Materials --- p.40 / Chapter 2.1.1 --- Buffer --- p.40 / Chapter 2.1.2 --- Culture Media --- p.45 / Chapter 2.1.3 --- Chemicals --- p.46 / Chapter 2.1.4 --- Culture of Cells --- p.49 / Chapter 2.1.4.1 --- "Tumor Necrosis Factor-Alpha-Sensitive Cell Line, L929" --- p.49 / Chapter 2.1.4.2 --- "Tumor Necrosis Factor-Alpha-Resistant Cell Line, rL929, rL929-l IE and rL929-4F" --- p.50 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Agarose Gel Electrophoresis --- p.50 / Chapter 2.2.2 --- Cytotoxicity Assay --- p.52 / Chapter 2.2.3 --- Confocal Laser Scanning Microscopy --- p.53 / Chapter 2.2.4 --- Flow Cytometry --- p.57 / Chapter Chapter 3. --- Results --- p.65 / Chapter 3.1 --- Induction of Apoptosis in Tumor Necrosis Factor-Alpha- Treated L929 Cell --- p.66 / Chapter 3.1.1 --- Introduction --- p.66 / Chapter 3.1.2 --- TNF Induced DNA Fragmentation in L929 Cells --- p.67 / Chapter 3.2 --- Effect of Tumor Necrosis Factor-Alpha on Cell Cycle --- p.73 / Chapter 3.2.1 --- Introduction --- p.73 / Chapter 3.2.2 --- Effect of TNF on Cell Cycle --- p.75 / Chapter 3.3 --- Release of Reactive Oxygen Species in Tumor Necrosis Factor-Alpha Treatment --- p.79 / Chapter 3.3.1 --- Introduction --- p.79 / Chapter 3.3.2 --- Release of Reactive Oxygen Species in TNF- Treated L929 Cells is Time Dependent --- p.81 / Chapter 3.3.3 --- Effect of Antioxidants on TNF-Mediated Cytotoxicity --- p.93 / Chapter 3.3.4 --- Effect of Mitochondrial Inhibitors on TNF-Mediated Cytotoxicity --- p.96 / Chapter 3.4 --- The Role of Calcium in Tumor Necrosis Factor-Alpha Treatment --- p.112 / Chapter 3.4.1 --- Introduction --- p.112 / Chapter 3.4.2 --- Release of Intracellular Calcium in TNF-Treated L929 Cells --- p.113 / Chapter 3.4.3 --- Effect of Calcium-Inducing Agents on TNF-Treated L929Cells --- p.127 / Chapter 3.5 --- Relationship between Reactive Oxygen Species and Calcium in Tumor Necrosis Factor-Alpha-Mediated Cytotoxicity --- p.133 / Chapter 3.5.1 --- Introduction --- p.133 / Chapter 3.5.2 --- Effect of Intracellular Calcium Chelator on TNF- Mediated ROS Release and Cytotoxicity --- p.133 / Chapter 3.5.3 --- Effect of Mitochondrial Calcium on TNF-Mediated ROS Release and Cytotoxicity --- p.147 / Chapter 3.6 --- Effect of Tumor Necrosis Factor-Alpha on pH --- p.162 / Chapter 3.6.1 --- Introduction --- p.162 / Chapter 3.6.2 --- Effect of TNF on pH --- p.162 / Chapter 3.7 --- Effect of Tumor Necrosis Factor-Alpha on Mitochondrial Membrane Potential --- p.165 / Chapter 3.7.1 --- Introduction --- p.165 / Chapter 3.7.2 --- Effect of TNF and Some Drugs on Mitochondrial Membrane Potential --- p.165 / Chapter 3.8 --- "Comparison of Effects of Tumor Necrosis Factor-Alpha on Susceptible Cell Line, L929 and Resistant Cell Line, rL929, rL929-11E and rL929-4F" --- p.169 / Chapter 3.8.1 --- Introduction --- p.169 / Chapter 3.8.2 --- Effect of TNF on the Cytotoxicity of Resistant Cell Lines --- p.170 / Chapter 3.8.3 --- Effect of TNF on the Release of ROS in Resistant Cell Lines --- p.170 / Chapter 3.8.4 --- Effect of TNF on the Release of Calcium in Resistant Cell Lines --- p.178 / Chapter 3.8.5 --- Effect of TNF on Cell Cycle in Resistant Cell Lines --- p.185 / Chapter Chapter 4. --- General Discussion --- p.187 / Chapter 4.1 --- Tumor Necrosis Factor Induced Apoptosis in L929 Cells --- p.188 / Chapter 4.2 --- Tumor Necrosis Factor Increased the Release of Reactive Oxygen Species in L929 Cells --- p.189 / Chapter 4.3 --- Tumor Necrosis Factor Increased the Release of Calcium in L929 Cells --- p.194 / Chapter 4.4 --- Calcium Induced Reactive Oxygen Species Release in TNF- Treated L929 Cells --- p.197 / Chapter 4.5 --- Tumor Necrosis Factor Did Not Change the pH and Mitochondrial Membrane Potential in TNF-Treated L929 Cells --- p.198 / Chapter 4.6 --- Tumor Necrosis Factor Did Not Increase the Release of Reactive Oxygen Species or Calcium in Resistant Cell Lines --- p.201 / Chapter Chapter 5. --- Future Perspective --- p.204 / Chapter 5.1 --- The Relationship Between Tumor Necrosis Factor and Cytochrome c --- p.205 / Chapter 5.2 --- The Relationship Between Tumor Necrosis Factor and Mitochondrial DNA Damage --- p.206 / Chapter 5.3 --- Clinical studies with Tumor Necrosis Factor --- p.206 / References --- p.208

In certain neurodegenerative diseases such Dementia with Lewy Bodies (DLB), it is hypothesised that misfolded α-synuclein (α-syn) and β-amyloid both contribute to pathology. α-Syn and β-amyloid have been suggested to synergistically promote one another’s accumulation and aggregation, but the mechanisms are unknown. β-Amyloid is generated from β-/γ-secretase-mediated processing of the amyloid precursor protein (APP). This study investigated how α-syn overexpression in cells affects β-amyloid production from APP, using multiplex assays, luciferase reporter assays, and western blotting. Wildtype α-syn expression induces β-amyloid generation from APP in SH-SY5Y human neuroblastoma cells, and similar changes to APP processing occur in another neuronal cell model. Dominant-negative overexpression of α-syn mutants revealed that disrupting the N-terminal domain can increase APP amyloidogenic processing. Secretase enzymes that perform APP processing were next investigated. γ-Secretase activity, measured by a luciferase reporter, was not increased by α-syn overexpression. A higher ratio of β- to α-secretase processing was hypothesised, which led to expression and activity studies of the major β- and α-secretases, BACE1 and ADAM10 respectively. It was shown that the BACE1 protein expression is post-transcriptionally upregulated in α-syn cells, with increased APP cleavage in cells. ADAM10 protein expression is transcriptionally suppressed in wild-type α-syn cells, reducing total levels of catalytically active enzyme. However the change in ADAM10-mediated APP processing may be negligible since, critically, plasma membrane expression of ADAM10 appears to be maintained. To aid understanding of the mechanism that connects α-syn to APP processing, BACE1 expression was used in pharmacological studies of cell stress signalling. This approach revealed that in α-syn cells BACE1 lysosomal and/or proteasomal degradation may be disturbed. Additionally, BACE1 expression is induced by translational de-repression mediated by eIF2α ser-51 phosphorylation, which was increased in α-syn cells. Although preliminary, the data suggests a role for oxidative stress mediating the increased BACE1 expression in wild-type α-syn cells.

L'endothelium vasculaire constitue la principale barrière entre le sang et les tissus régulant le passage de macromolécules et de cellules circulantes. Longtemps considéré comme une monocouche passive, l'endothélium joue d'importants rôles dans la régulation de la pression sanguine, de l'hémostase, des réponses immunitaires et inflammatoires. L'adhérence cellule/cellule est initiée dans l'endothélium vasculaire par des interactions homophiliques entre molécules de VE-cadhérine (= jonctions adhérentes). La dynamique de la jonction et du cytosquelette est importante pour le remodelage des jonctions intercellulaires qui a lieu au cours l'angiogenèse, de la vasculogenèse et lors de la réparation de l'endothélium. C'est pourquoi la détermination des mécanismes moléculaires sous-jacents est indispensable à la comprehension de phénomènes physiopathologiques (angiogenèse et progression tumorales, inflammation...). D'après la littérature, la protéine EPLIN intervient dans la formation du complexe E-cadhérine/alpha-caténine/EPLIN et stabilise l'actine corticale. Actuellement décrite comme spécifique des modèles épithéliaux, EPLIN peut-elle intervenir dans la liaison du complexe à base de VE-cadhérine au cytosquelette d'actine? De plus, il paraît essentiel de comprendre le rôle de cette protéine dans les cellules car son expression est fortement diminuée dans la plupart des cancers alors qu'à l'inverse sa surexpression bloque la prolifération cellulaire. / The endothelium forms the main barrier regulating the passage of macromolecules and circulating cells between the blood and tissue. Historically viewed as a passive vascular lining, vascular endothelium plays important roles in the regulation of vascular pressure, hemostasis, immune and inflammatory responses. In vascular endothelium, cell/cell adhesion is mediated by homophilic interactions of VE-cadherin molecules (= adherens junctions). Cytoskeleton and junction dynamics are important for intercellular junctions remodelling that occurs during angiogenesis, vasculogenesis and endothelium repair. So, determining the underlying molecular mechanisms is essential for the comprehension of pathologic phenomena such as angiogenesis, tumor progression or inflammation. We learn from the literature that EPLIN is involved in E-cadherin/α-catenin/EPLIN complex formation and cortical actin stabilization. Usually described as a protein specific of epithelial models, we wondered if EPLIN is able to link VE-cadherin complex to actin cytoskeleton. Furthermore, it seems essential to understand its cellular role since it is downregulated in many cancers while in contrast its overexpression blocks cell proliferation.

Ce travail de thèse s’intéresse à l’altération des verres nucléaires, et plus précisément à la durabilité du verre de référence SON68, dans un système complexe simulant les conditions du stockage géologique profond. L’approche repose sur une complexification progressive du système afin de mieux comprendre les mécanismes régissant l’altération du verre irradiant au contact de l’eau du site (eau du Callovo-Oxfordien) et en présence de fer simulant le surconteneur. Dans un premier temps, l’étude se focalise sur l’effet de l’eau souterraine sur l’altération du verre de référence non radioactif dans le régime de vitesse résiduelle à 70°C. Les résultats montrent que le magnésium présent dans l’eau contribue, au-dessus d’un pH seuil, à former des phyllosilicates magnésiens au détriment du gel passivant, ce qui induit une augmentation de l’altération du verre. Cette étude confirme des travaux antérieurs et apportent de nouvelles données à 70°C. Par la suite, une source de fer est introduite dans le système (verre non radioactif – eau du COx) afin de simuler la présence du surconteneur. Les expériences sont menées en milieu anoxique sur des monolithes de verre et de fer, mis en regard et séparés d’environ 80 µm, puis immergés dans l’eau du COx à 70 et 90 °C. Dans ces conditions, il est observé que la température n’induit qu’un faible impact sur les cinétiques et les réactions qui contrôlent l’altération du verre. La corrosion du fer conduit principalement à la précipitation de sidérite (carbonate de fer au degré d’oxydation II) et d’aragonite (carbonate de calcium). Par ailleurs, une altération du verre plus importante en présence de fer est observée. La cause la plus probable est la précipitation de silicates de fer, qui, comme les silicates magnésiens, se forment au détriment du gel passivant. Ces phases ont été identifiées principalement au degré d’oxydation III, indiquant ainsi une oxydation du fer proche du verre. Dans ce cas, la précipitation de silicates magnésiens est inhibée et seuls des silicates de fer précipitent. Enfin, l’influence de l’irradiation sur le comportement du verre a été étudiée. La radiolyse de l’eau devant produire des espèces oxydantes comme H2O2 à l’interface verre/eau, une première étude s’est focalisée sur l’altération du verre dans l’eau du COx en présence de H2O2 dans le régime de vitesse résiduelle. Les résultats montrent que la décomposition de cette espèce à la surface des grains de verre semble avoir un impact local en favorisant la précipitation de silicates magnésiens et, par conséquent, en augmentant l’altération du verre. Cet effet est immédiat et ne se produit que très localement sur les grains en surface du réacteur. Mais, globalement, les données montrent que cette espèce n’a pas un impact significatif sur l’altération du verre dans le régime cinétique résiduel et pour la géométrie considérée. Ce travail s’achève par la réalisation de deux expériences d’altération d’un verre radioactif, dopé en 238Pu, et altéré dans l’eau du COx à 70°C en présence de fer, en suivant une géométrie similaire à celle conduite sur le verre non radioactif. Les caractéristiques du verre permettent de s’intéresser à l’effet du débit de dose α. Par rapport aux témoins non radioactifs, une plus forte oxydation du fer est mise en évidence, avec principalement des précipités de goethite et de ferrihydrite, oxydes de fer au degré d’oxydation III. Cette précipitation de phases oxydées peut s’expliquer par la réaction entre H2O2 et Fe qui favoriserait l’oxydation du Fe2+ en Fe3+. Sur la base des analyses de solution et des premières observations par MEB, il semble que les épaisseurs de verre altéré soient globalement similaires en présence d’un champ d’irradiation  et dans le milieu non radioactif témoin. Néanmoins, des caractérisations complémentaires du solide devront être réalisées afin de mieux comprendre les processus limitants. / This work intends to provide a better understanding of the alteration of R7T7-type nuclear waste glass in a complex environment such as the deep geological repository. This approach aims to progressively complexify the glass leaching environment in order to better understand the mechanisms involved on the alteration of a radioactive glass in contact with the Callovo-Oxfordian groundwater (COx groundwater) and an iron source, simulating the glass carbon steel overpack. First, the study focusses on the impact of groundwater on the glass alteration under the residual rate regime at 70°C. Results show that magnesium present in the Callovo-Oxfordian groundwater contributes, over a pH threshold, to the precipitation of magnesium phyllosilicates consuming the passivating gel and thus increasing the glass alteration. This work confirms previous studies and brings new data at 70 °C. Thereafter, an iron source was introduced in the leaching environment (glass – COx groundwater) in order to simulate the carbon steel overpack. The experiments were performed in an anoxic environment with a glass monolith and an iron foil, facing each other with an 80 µm gap, and then immerged in the COx groundwater at 70 and 90 °C. On these conditions, a weak impact of the temperature was perceived on the kinetics and the reactions controlling glass alteration. The iron corrosion led mainly to the precipitation of siderite (an Fe2+-carbonate) and aragonite (a calcium carbonate). Furthermore, an increase of glass alteration was observed while using an iron foil. The main cause explaining this phenomenon is the formation of iron silicates, as seen with magnesium silicates, precipitating at the expense of the passivating gel. These phases were identified mainly at the oxidation state III, indicating an iron oxidation near the glass. It should be noted that in this case, magnesium precipitation is suppressed and only iron silicates precipitate. Finally, the influence of irradiation on glass alteration was studied. Water radiolysis induces the production of oxidizing species such as H2O2 at the glass/water interface. Therefore, a first study focused on glass alteration in the COx groundwater in the presence of H2O2 under the residual rate regime. Results showed that this specie decomposition was higher at the surface of the glass grains and seemed to have a very local impact, favouring the magnesium silicate precipitation and the enhancement of glass alteration. This prompt effect was only observed locally on grains at the surface of the reactor. However, globally, the data showed that H2O2 does not have a significant impact on glass alteration in this residual rate regime and this geometry. At last, this work came to experiments using a radioactive glass, doped with 238Pu, altered in the COx groundwater at 70 °C in the presence of an iron source, and using the same geometry than the one conducted with the non-radioactive glass. The glass characteristics allowed us to focus only on the α-dose rate effect. Comparing with the non-radioactive reference, a higher iron oxidation was noticed, with the precipitation of iron oxides at the oxidation state III, such as goethite and ferrihydrite. This can be explained by the reaction between H2O2 and iron, favouring the oxidation of Fe2+ to Fe3+. From solution analysis and the first SEM observations, it seems that globally, the glass alteration thickness is the same regardless the α-irradiation field. Nevertheless, supplementary data on solid characterisations must be performed in order to understand and be conclusive about the mechanisms involved on the glass alteration mechanisms under these conditions.

Tsang Sai Leong. / Thesis submitted in: December 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 96-104). / Abstracts in English and Chinese. / Abstract (in Chinese) --- p.(i) / Abstract --- p.(iii) / Table of contents --- p.(v) / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- The interferon --- p.1 / Chapter 1.1.1 --- About type I IFN --- p.1 / Chapter 1.1.2 --- IFN α/β receptor and signal transduction --- p.3 / Chapter 1.1.3 --- IFN induction --- p.3 / Chapter 1.1.4 --- Functions --- p.4 / Chapter 1.1.5 --- MuIFN α subtypes --- p.8 / Chapter 1.1.6 --- Gene expression --- p.9 / Chapter 1.2 --- Aim of study: Functions and gene expression --- p.9 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.15 / Chapter 2.1.1 --- "Cell line, bacterial strain, virus strain and plasmid vector" --- p.15 / Chapter 2.1.2 --- Chemicals --- p.20 / Chapter 2.1.3 --- "Culture media, buffer and other solutions" --- p.20 / Chapter 2.1.4 --- Reagents and nucleic acids --- p.21 / Chapter 2.1.5 --- Reaction kits --- p.22 / Chapter 2.1.6 --- Solutions --- p.22 / Chapter 2.1.7 --- Major equipments --- p.24 / Chapter 2.1.8 --- Primers used --- p.24 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- "Cloning of MuIFN αl2, MuIFN αl and MuIFN α4 from L929 genomic DNA and their subcloning into pEGFP-Nl mammalian expression vector" --- p.26 / Chapter 2.2.1.1 --- PCR of MuIFN αl2 --- p.26 / Chapter 2.2.1.2 --- Gel purification of MuIFN αl2 PCR product --- p.26 / Chapter 2.2.1.3 --- Ligation of MuIFN αl2 PCR product into pGEM-T vector --- p.26 / Chapter 2.2.1.4 --- Sequencing of clones which were positive in PCR screening --- p.26 / Chapter 2.2.1.5 --- Subcloning of the gene from pGEM-T vector to pEGFP-Nl --- p.28 / Chapter 2.2.1.6 --- Construction of expression vectors for MuIFN αl and MuIFN a4 gene --- p.28 / Chapter 2.2.2 --- Preparation ofplasmid DNA --- p.29 / Chapter 2.2.3 --- Preparation of cell culture medium --- p.30 / Chapter 2.2.4 --- Production of recombinant MuIFN α (rMuIFN α) --- p.30 / Chapter 2.2.5 --- Production of native MuIFN α by polyI：polyC induction --- p.31 / Chapter 2.2.6 --- Influenza A virus strain A/NWS/33 preparation and titration --- p.31 / Chapter 2.2.7 --- Virus infection in Influenza A virus challenge assay --- p.32 / Chapter 2.2.8 --- Cell culture techniques --- p.32 / Chapter 2.2.9 --- "MTT cell proliferation assay of JCS cell line, for measuring MuIFN α anti-proliferation activity" --- p.33 / Chapter 2.2.10 --- Quantitative analysis of MuIFN α --- p.34 / Chapter 2.2.11 --- Flow cytofluorometric analysis of cell cycle of MuIFN α treated JCS cells by propidium iodide staining --- p.34 / Chapter 2.2.12 --- FACS study on the effect of MuIFN α on MHC-I up-regulation in JCS cells --- p.35 / Chapter 2.2.13 --- FACS study on the effect of MuIFN α on MHC-I up-regulation on primary macrophages from Balb/c mice --- p.35 / Chapter 2.2.14 --- Anti-viral activity by transfection of MuIFN α gene --- p.36 / Chapter 2.2.15 --- Sequencing of MuIFN al2 coding region from genomic DNA of L929 and JCS cell lines --- p.37 / Chapter 2.2.16 --- "RNA extraction from L929 cell lines, with or without Influenza A virus infection or polyI:polyC induction" --- p.37 / Chapter 2.2.17 --- RNA extraction from tissues of Balb/c mouse --- p.38 / Chapter 2.2.18 --- Reverse transcription --- p.39 / Chapter 2.2.19 --- Polymerase Chain Reaction (PCR) --- p.39 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Overview --- p.40 / Chapter 3.2 --- "Subcloning of MuIFN α 12, MuIFN αl and MuIFN α4 coding sequences into the pEGFP-Nl vector" --- p.40 / Chapter 3.3 --- The growth inhibitory effect of different MuIFN α subtypes on murine myeloid leukemia cell line JCS --- p.41 / Chapter 3.4 --- Quantitation of MuIFN α subtype samples --- p.50 / Chapter 3.5 --- Cell cycle analysis of MuIFN α treated JCS cells --- p.50 / Chapter 3.6 --- FACS analysis of the effect of different MuIFN α subtypes on MHC-I expression in JCS cell line --- p.57 / Chapter 3.7 --- FACS analysis of the effect of different MuIFN α subtypes on MHC-I expression in primary macrophages in Balb/c mice --- p.65 / Chapter 3.8 --- Effect of MuIFN α subtype transgenes on L929 cells challenged with Influenza A virus --- p.72 / Chapter 3.9 --- Sequencing of MuIFN αl2 coding region from genomic DNA of L929 and JCS cell line --- p.78 / Chapter 3.10 --- "MuIFN αl2 expression in untreated, Influenza A virus infected or polyl:polyC induced L929 cells" --- p.78 / Chapter 3.11 --- Detection of MuIFN α12 transcripts in tissues of the 8-10 week untreated Balb/c mice --- p.85 / Chapter Chapter 4 --- Discussion --- p.89 / Chapter 4.1 --- Overview --- p.89 / Chapter 4.2 --- rMuIFN α 12 has anti-proliferative and apoptotic effects on JCS cell line --- p.89 / Chapter 4.3 --- "Up-regulation of MHC-I expression in JCS cells and primary macrophages by rMuIFN αl2, rMuIFN αl, rMuIFN α4 and mixed type I IFN" --- p.91 / Chapter 4.4. --- Transfection of MuIFN α12 gene could induce anti-viral state in L929 cell line --- p.91 / Chapter 4.5 --- Gene regulation of MuIFN al2 in L929 cells infected with Influenza A virus or induced by polyI:polyC --- p.92 / Chapter 4.6 --- Gene expression of MuIFN αl2 in different tissues of Balb/c mice --- p.94 / Conclusion --- p.95 / Reference List --- p.96 / List of figures: / Fig. 1.1 The 3D structure of recombinant human interferon alpha (HuIFN α) subtype 2B --- p.11 / Fig. 1.2 Current model of lFN induction --- p.12 / Fig. 1.3 Activation of RNase L --- p.13 / Fig. 2.1 Graphical map of plasmid vector pEGFP-Nl --- p.17

Le premier objectif de cette thèse était d’améliorer l’utilisation et la compréhension des outils de mesure de la déformabilité du globule rouge (GR) dans la drépanocytose (Etudes 1 et 2). L’étude 1 a montré l’importance de la standardisation des mesures de déformabilité par ektacytométrie chez les enfants drépanocytaires. Au cours de l’étude 2, les propriétés des GR ont été modifiées et la variation des courbes de déformabilité érythrocytaire « classique » (index d’élongation en fonction de la contrainte de cisaillement en milieu isotonique) a été comparée aux résultats d’osmoscan (mesure de la déformabilité érythrocytaire en gradient osmolaire à contrainte de cisaillement fixe), méthode de référence pour étudier les anomalies de la membrane du GR. Ainsi, les variations de déformabilité érythrocytaire au-delà de 3 Pa sont affectées à la fois par la viscosité interne du GR et par des modifications de la surface cellulaire (rapport surface/volume) alors que les modifications de l’élasticité membranaire affectent la déformabilité érythrocytaire quelles que soient les forces de cisaillements utilisées (faibles, modérées ou hautes). Le deuxième objectif de cette thèse était d’apporter des éléments supplémentaires sur l’implication des facteurs génétiques, des paramètres hémorhéologiques et du niveau de stress oxydant sur la survenue des complications vaso-occlusives chez les patients atteints de syndrome drépanocytaire majeur (Etudes 3 à 6). La mise en commun des résultats d’hémorhéologie obtenus sur 165 patients de notre cohorte lyonnaise et 240 patients de la cohorte guadeloupéenne a permis de montrer que la rhéologie du GR chez les patients drépanocytaires était dépendante de l’âge. Ainsi, la viscosité sanguine augmente avec l’âge pour atteindre un plateau vers 30 ans alors que la déformabilité érythrocytaire diminue avec l’âge (Etude 3). Ces modifications participent vraisemblablement à l’apparition de complications chroniques chez l'adulte drépanocytaire. Les études 4 et 5 ont été réalisées sur la cohorte pédiatrique lyonnaise. Au cours de ces 2 études, nous avons étudié l’influence sur la rhéologie du sang et la survenue de crises vaso-occlusives (CVO) des facteurs génétiques (alpha-thalassémie, déficit en Glucose-6-Phosphate Déshydrogénase (G6PD) et haplotypes S) d’une part (Etude 4) et du niveau de stress oxydant et nitrosatif d’autre part (Etude 5). L’alpha-thalassémie augmente la déformabilité des GR et l’agrégation érythrocytaire. Ces 2 phénomènes pourraient participer à augmenter le risque de CVO. De plus, l’alpha-thalassémie, en diminuant l’hémolyse, diminuerait le niveau de stress oxydant, élément majeur impliqué dans la physiopathologie de la drépanocytose. Enfin, l’étude 6 a montré que la rhéologie sanguine des patients Sbêta+ était quasi-identique à celle des sujets sains AA mais que les patients les plus sévères pourraient avoir un déficit en monoxyde d’azote circulant. En conclusion, mon travail de thèse contribue à une meilleure compréhension de la physiopathologie de la drépanocytose / The first goal of this thesis (Study 1 and 2) was to improve the use and the comprehension of tools for red blood cell (RBC) deformability measurements in sickle cell disease (SCD). The first study showed the importance of standardization of RBC deformability measurements by ektacytometry in SCD children. In the study 2, the RBC proprieties was modified and the variation of « classic » RBC deformability curve (elongation index as a function of the shear stress in isotonic medium) was compared to osmoscan results (elongation index in hyperosmolar gradient and constant shear stress), the gold standard for RBC membrane defect studies. Thus, the modifications of RBC deformability curve above 3 Pa were affected by RBC internal viscosity and cellular surface modification (and thus surface/volume ratio) while membran elasticity modifications affected RBC deformability whatever the shear stress (low, moderate or high). The second goal of this thesis was to study the effects of genetic modifiers, hemorheological parameters and oxidative stress level on vaso-occlusive complications (VOC) in SCD (Study 3 to 6). Hemorheological parameters were measured on 165 patients from Lyon and 240 patients from Gwada and the results showed that blood viscosity increased until the age of 30 and RBC deformability decreased with age (Study 3). This modifications probably play role in the chronic complications of SCD adult patients. The studies 4 and 5 were conducted on SCD children. We studied the effects of genetic modifiers (alpha-thalassemia, glucose-6-phospho-deshydrogenase deficiency and S haplotypes ; study 3) and nitro-oxidative stress level (study 5). Alpha-thalassemia increase RBC deformability and RBC aggregation. This phenomenon could contribute to increase VOC. Moreover, alpha-thalassemia decreased hemolysis and thus oxidative stress, a major component of SCD physiopathology. Then the study 6 showed that Sbeta+ patient hemorheology was quite the same of AA ubjects but the more severe patients could have a defect in circulating nitric oxide. To conclude, my thesis contribute to a better understanding of SCD physiopathology

O estradiol (E2) é requerido para a luteólise de fêmeas bovinas e injeções de E2 estimulam a liberação de prostaglandina F2α (PGF2α). É possível que o E2 estimule a síntese e/ou a atividade de moléculas envolvidas na cascata geradora de PGF2α, como enzimas e receptores para outros ligantes. O cálcio é um conhecido cofator para a proteína quinase C e fosfolipase A2, enzimas envolvidas na produção PGF2α. O objetivo geral desse estudo foi investigar os mecanismos endócrinos e moleculares estimulados pelo E2 durante a luteólise. No experimento 1, vacas holandesas não lactantes foram tratadas com 3mg de E2 nos dias 13 (n=2), 15 (n=2), 17 (n=3) e 19 (n=5) do ciclo estral e a produção de PGFM (metabólito plasmático da PGF2α) mensurada por radioimunoensaio. Concluiu-se que a administração de E2 no 17º dia do ciclo estral constitui um modelo experimental adequado para determinar os mecanismos envolvidos na síntese de PGF2α. O experimento 2, foi realizado para investigar o papel do E2 na cascata produtora de PGF2α. Novilhas cruzadas de corte, cíclicas, não lactantes, foram pareadas no dia 17 de um ciclo estral sincronizado, injetadas com 0 (n=6) ou 3mg de E2 (n=7) e abatidas 2 horas após. Explantes endometriais foram tratados com os seguintes estimuladores da síntese de PGF2α: ionóforo de cálcio (CI), melitina ou ocitocina. Explantes foram incubados em quadruplicata e amostras de meio foram coletadas imediatamente e 60 minutos após o início da cultura. As concentrações de PGF2α foram mensuradas por radioimunoensaio. Explantes endometriais tratados in vitro com CI tiveram um incremento na síntese de PGF2α de 48,41% quando foram previamente tratados com o E2 (P≤0,01). No experimento 3, células endometriais bovinas (células BEND) foram tratadas com 0, 10-7, 10-6 ou 10-5M CI por 12h em triplicata, em três experimentos independentes. As concentrações de 10-6 e 10-5M de CI estimularam a produção de PGF2α em comparação às outras concentrações (P≤0,05). No experimento 4, células BEND receberam 0 ou 10-13M E2 e 0 ou 10-6M CI em um arranjo fatorial 2 x 2, durante 12h em triplicata, em três experimentos independentes. A produção de PGF2α foi de 33,1; 32,5; 92,4 e 145,6 (EPM: 21,8) pg/mL para células não tratadas, tratadas com E2, CI e E2 + CI, respectivamente. Houve uma tendência do tratamento com CI estimular a produção de PGF2α (P≤0,08), entretanto, na presença de E2 o CI estimulou significativamente a síntese de PGF2≤ (P≤0,01). Conclui-se que em fêmeas bovinas o E2 potencializou os efeitos do CI na síntese de PGF2α endometrial. Propõe-se que o E2 ativa a síntese de enzimas que, estimuladas pelo cálcio, atuam na síntese de PGF2α endometrial / Estradiol (E2) is required for luteolysis in bovine female and E2 injections stimulate prostaglandin F2α (PGF2α) release. It is possible that E2 stimulates synthesis and/or activity of molecules in the cascade of PGF2α production, such as enzymes and receptors to other ligands. Calcium is a known cofactor for protein kinase C and phospholipase A2, enzymes involved in PGF2α production. The main goal of this study was to investigate endocrine and molecular mechanisms stimulated by E2 during luteolysis. In experiment 1, non-lactating Holstein cows were treated with 3mg E2 on days 13 (n=2), 15 (n=2), 17 (n=3) or 19 (n=5) of the day estrous cycle and production PGFM (a PGF2α plasma metabolite) was evaluated by radioimmunassay. It was concluded that E2 administration on day 17 of the cycle was an adequate experimental model to determine mechanisms involved in endometrial PGF2α synthesis. Experiment 2 was designed in order to investigate the role of E2 in the enzymatic cascade of PGF2α production. Cyclic, cross-bred beef heifers were paired on day 17 of a synchronized estrous cycle, injected with 0 (n=6) or 3mg of E2 (n=7) and slaughtered after two hours. Endometrial explants were treated with stimulators of the cascade of PGF2α synthesis, i.e., calcium ionophore (CI), melittin or oxytocin. Explants were incubated in quadruplicate and medium samples were collected immediately and 60min after to begin culture. The concentrations PGF2α were measured by radioimmunoassay. Endometrial explants in vitro treatment with CI production the PGF2α was 48,41% higher in from cows treated with E2 (P≤0,01). In experiment 3, bovine endometrium cells (BEND cells) were treated with 0, 10-7, 10-6 or 10-5M CI for 12h in triplicate, in three independent experiments. The 10-6 and 10-5M concentrations of CI stimulated production of PGF2α in comparison to other concentrations (P≤0,05). In experiment 4, BEND cells received 0 or 10-13M E2 and 0 or 10-6M CI in a 2 x 2 factorial arrangement, for 12h in triplicate cultures in three independent experiments. Production of PGF2α was 33.1, 32.5, 92.4 and 145.6 (pooled SEM: 21.8) pg/mL for cells treated with nothing, E2, CI and E2 + CI, respectively. Treatment with CI alone tended to stimulate PGF2α production (P≤0,08), however, in the presence of E2, CI significantly stimulated PGF2α synthesis (P≤0,01). It was concluded that in bovine female the E2 improved the CI effects in endometrial PGF2α synthesis. It proposes that E2 active the enzyme synthesis that, calcium stimulated, actuate in endometrial PGF2α synthesis