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<p>Among food allergies, peanut has attracted the most research attention because the allergy is typically lifelong, often severe and potentially fatal. Furthermore, other than epinephrine, there are no treatments available to date. A decade of research has provided a great deal of insight into the factors that promote and regulate the <em>development </em>of allergic responses. However, less in known about the factors involved in the <em>elicitation</em> of the most common and severe manifestation of peanut allergy, namely anaphylaxis. The research in this thesis centers on the investigation of cellular and molecular pathways leading to peanut-induced anaphylaxis (PIA) as well as potential therapeutic targets. Specifically presented are: i) the development and characterization of a mouse model of PIA (Chapter 2), ii) the role of molecules including histamine, leukotrienes (LT) and platelet-activating factor (PAF) (Chapter 3) and, iii) the relative contribution of mast cells, basophils and macrophages as well as IgE and IgG₁(Chapter 4). Our data show that oral sensitization to peanut in C57BL/6 mice generated local and systemic markers of type-2 immunity that was associated with robust and consistent clinical anaphylaxis following antigen challenge. In this context, concurrent blockade of PAF and histamine receptors markedly decreases the severity of these reactions. Moreover, they demonstrate that distinctive immune effector pathways involving activation of mast cells (via IgE and IgG₁) and macrophages (via IgG₁) cooperate to elicit a broad range of systemic reactions to peanut. These findings highlight that concomitant and progressive recruitment of immune-effector pathways leads to a full range of anaphylactic reactions and therefore, therapeutic strategies for PIA may need to target several pathways or, alternatively shared components within these pathways. Combination therapy blocking both PAF and histamine may represent such as a therapeutic approach.</p> / Doctor of Philosophy (Medical Science)

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

En Si Wang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 126-140 (2nd gp.)). / Acknowledgements --- p.i / Contents --- p.ii / Abstract --- p.iv / List of Acronyms and Abbreviations --- p.vi / introduction --- p.1 / Chapter I. --- "Platelet Activating Factor (PAF)´ؤPast, Present, and Future" --- p.1 / Chapter I-1. --- What is PAF? --- p.1 / Chapter I-2. --- Biochemistry of PAF --- p.2 / Chapter I-2-1. --- Metabolic Cycle of PAF --- p.3 / Chapter I-2-1-A. --- Biosynthesis of PAF --- p.4 / Chapter I-2-1 -B. --- Inactivation of PAF --- p.6 / Chapter I-2-2. --- Role of Endogenous PAF in Cell --- p.7 / Chapter I-3. --- Chemistry of PAF --- p.8 / Chapter I-4. --- Pathobiology of PAF --- p.9 / Chapter II. --- PAF Receptor --- p.10 / Chapter II-1. --- Presence and Characteristics of PAF Receptor --- p.10 / Chapter II-l-l. --- Solubilization of PAF Receptor --- p.10 / Chapter II-1-2. --- G-Protein Involvement --- p.11 / Chapter II-1-3. --- Species Differences --- p.11 / Chapter II-1-4. --- Multiple Conformational States of PAF Receptor --- p.12 / Chapter II-1-5. --- PAF Receptor Heterogeneity --- p.12 / Chapter II-2. --- Putative Conformation of PAF Membrane Binding Sites --- p.13 / Chapter II-3. --- Recent Progress in PAF Receptor Research --- p.15 / Chapter III. --- PAF Receptor Antagonist --- p.18 / Chapter III-1. --- Classification of PAF Antagonists --- p.18 / Chapter III-2. --- Inhibition Types of PAF Receptor Antagonists --- p.19 / Chapter III-2-1. --- Nonspecific Inhibition of the Effects of PAF --- p.21 / Chapter III-2-2. --- Specific Inhibition of PAF --- p.22 / Chapter III-3. --- Recent Progress in PAF Receptor Antagonist Research --- p.22 / Chapter IV. --- Pharmacology and Syntheses of Spiro-Ether Structural Units --- p.26 / Chapter IV-1. --- Natural Products Containing Spiro-Ether and Related Structural Units --- p.30 / Chapter IV-1-1. --- Labdane Diterpenoids Containing Spiro-Ether Structural Units --- p.30 / Chapter IV-1-2. --- Leucodrin and Related Derivatives --- p.32 / Chapter IV-2. --- Synthetic Methods of Spiro-Ethers and Related Derivatives --- p.34 / Chapter V. --- Aim of the Present Work --- p.45 / RESULTS AND DISCUSSION --- p.47 / Chapter I. --- Isolation and Structure Elucidation of Prehispanolone (1) and Preleoheterin (3) --- p.47 / Chapter I-1. --- Material and Isolation --- p.47 / Chapter I-2. --- Structure Elucidation of Prehispanolone (1) and Preleoheterin (3) --- p.47 / Chapter II. --- Synthesis of Model Compounds --- p.53 / Chapter II-l. --- "Synthesis of 2-Methyl-1,7-dioxaspiro[4.4]nonane (137)" --- p.53 / Chapter II-2. --- "Synthesis of 2,2-Dimethyl-l,7-dioxaspiro[4.4]nonane (139)" --- p.68 / Chapter II-3. --- "Synthesis of 2,2-Diphenyl-1,7-dioxaspiro[4.4]nonane (141) and 2,2-Diphenyl-l,7-dioxaspiro[4.4]non-8-ene (142)" --- p.72 / Chapter III. --- "Partial Synthesis of 13R, 14,15-Dihydroprehispanolone (5)，13S,14,15-Di- hydroprehispanolone (135) and prehispanolone (1)" --- p.76 / CONCLUSION --- p.89 / EXPERIMENTAL SECTION --- p.91 / REFERENCES --- p.126 / APPENDIX --- p.141

Um dos desafios recorrentes na prática clínica da Oncologia é o processo de repopulação, no qual células tumorais resistentes à terapia são capazes de proliferar e reconstituir o tumor. No entanto, os mecanismos envolvidos neste fenômeno ainda foram pouco explorados, sendo necessário melhor compreendê-los para evitar a falha terapêutica. Melanomas são bons modelos para estudar repopulação devido às baixas taxas de sobrevida livre de progressão e às altas taxas de resistência às terapias associadas a este tipo de tumor sólido. Sabe-se que a exposição de células tumorais a condições estressoras do microambiente, como hipóxia e hipóxia/reoxigenação, bem como ao próprio tratamento antitumoral, são pressões seletivas frequentemente encontradas em tumores sólidos que favorecem a resistência às terapias e a repopulação tumoral. Estudos prévios indicam que a sinalização mediada pelo Fator de Ativação de Plaquetas (PAF), um lipídio bioativo relacionado à diversas funções fisiológicas, e de seu receptor, PAFR, está associada com a resistência de células de melanoma aos tratamentos citotóxicos e com crescimento tumoral. Portanto, este trabalho teve como objetivo investigar o envolvimento da sinalização de PAFR frente às condições estressoras descritas acima no fenômeno de repopulação de melanomas. Os resultados de espectrometria de massa indicaram que hipóxia aumentou a geração de PAF nas linhagens de melanoma humano SKmel05 e A375, mas não na A375M, embora este aumento não tenha sido observado após a reoxigenação. Além disso, mostraram que SKmel37 exibiu os maiores níveis basais de PAF, aumentando substancialmente sua geração em diferentes tempos de exposição à hipóxia e hipóxia/reoxigenação. Investigamos também a geração de outros ligantes de PAFR, porém nenhum deles foi encontrado nas amostras. Os resultados de detecção de PAFR por Western Blot e/ou citometria de fluxo revelaram que os níveis proteicos não foram modulados por estas condições em nenhuma das linhagens e que, em termos basais, SKmel37 e SKmel05 apresentaram os maiores níveis. Estas linhagens foram, portanto, submetidas a ensaios de proliferação, os quais evidenciaram que frente ao tratamento com o antagonista de PAFR, WEB2086, em condições de hipóxia e hipóxia/reoxigenação, apenas as células SKmel37 tiveram sua proliferação reduzida e morfologia associada à morte. Ensaios de incorporação de iodeto de propídio indicaram que o tratamento destas células expostas à hipóxia/reoxigenação com WEB2086 levou a acúmulo em SG2M, morte celular e sensibilização à cisplatina. Além disso, imunofluorescência de cortes congelados de tumores induzidos com SKmel37 revelou que houve acúmulo de PAFR em áreas hipóxicas e em seu entorno. Em relação ao modelo de exposição à tratamentos antitumorais, por sua vez, curvas de concentração e tempo com Vemurafenib mostraram que SKmel37 e A375 foram resistentes à droga, ao passo que SKmel05 e UACC62 foram sensíveis. Além disso, WEB2086 potencializou o efeito de morte induzido por Vemurafenib nas linhagens sensíveis, mas não afetou as resistentes. Considerando os aspectos clínicos de resposta inicial com posterior desencadeamento de repopulação, seguimos com as linhagens sensíveis e verificamos por citometria que esta droga aumentou ROS em ambas as linhagens, mas só aumentou PAFR extracelular na SKmel05. O tratamento combinado potencializou a geração de ROS e levou a ativação de caspase3/7 apenas na SKmel05. Esta linhagem foi então submetida a ensaios clonogênicos cujos resultados mostraram que o tratamento com Vemurafenib reduziu o número de clones e que WEB2086 não potencializou este efeito. Assim, o conjunto de resultados apresentados evidencia que a sinalização de PAFR participa dos desfechos de sobrevivência frente à hipóxia/reoxigenação e/ou tratamentos antitumorais, podendo, de alguma forma, contribuir com a repopulação de melanomas / One of the recurrent challenges in the clinical practice of Oncology is the process of repopulation, in which therapy-resistant tumor cells can proliferate and reconstitute the tumor. However, the mechanisms involved in this phenomenon were still little explored. The understanding of these events is, therefore, needed to avoid therapeutic failure. Melanomas are good models for studying repopulation due to the low rates of progression-free survival and the high rates of resistance to therapies associated to this type of solid tumor. It is known that the exposure of tumor cells to microenvironmental stress conditions, such as hypoxia and hypoxia/reoxygenation, as well as the exposure to antitumor treatment itself, are selective pressures frequently found in solid tumors that favor therapy resistance and tumor repopulation. Previous studies have indicated that the signaling mediated by the Platelet Activation Factor (PAF), a bioactive lipid related to various physiological functions, and its receptor, PAFR, is associated with resistance of melanoma cells to cytotoxic treatments and with tumor growth. Therefore, the aim of this study was to investigate the involvement of PAFR signaling upon the adverse conditions described above in the phenomenon of melanoma repopulation. Mass spectrometry results indicated that hypoxia increased the generation of PAF in human melanoma cell lines SKmel05 and A375, but not in A375M, although this increase was not observed after reoxygenation. In addition, they showed that SKmel37 exhibited the highest PAF basal levels, whose generation increased substantially after different times of hypoxia and hypoxia/reoxygenation exposure. We also investigated the generation of other PAFR ligands, but none were found in the samples. The results of PAFR detection by Western Blot and/or flow cytometry revealed that protein levels were not modulated by these conditions in any of the cell lines and that, at baseline, SKmel37 and SKmel05 showed the highest levels. These lines were therefore submitted to proliferation assays, which showed that the treatment with the PAFR antagonist, WEB2086, under conditions of hypoxia and hypoxia/reoxygenation, led to proliferation reduction and death-associated morphology in SKmel37 cells only. Propidium iodide incorporation studies indicated that the treatment of these cells exposed to hypoxia/reoxygenation with WEB2086 led to accumulation in SG2M, cell death and cisplatin sensitization. In addition, immunofluorescence of frozen sections of SKmel37-induced tumors revealed that PAFR was found accumulated in hypoxic areas and its surroundings. Regarding the model of exposure to antitumor treatments, concentration and time curves with Vemurafenib showed that SKmel37 and A375 were resistant to the drug, whereas SKmel05 and UACC62 were sensitive. In addition, WEB2086 potentiated the effect of Vemurafenib-induced death on sensitive cell lines but did not affect the resistant ones. Considering the clinical aspects of initial response with subsequent repopulation triggering, we continued using the sensitive cell lines and we verified by cytometry that this drug increased ROS in both cell lines but only increased extracellular PAFR in SKmel05. The combined treatment potentiated the generation of ROS and led to the activation of caspase3/7 in SKmel05 only. This cell line was then submitted to clonogenic assays whose results showed that treatment with Vemurafenib reduced the number of clones and that WEB2086 did not potentiate this effect. Thus, the set of results presented highlights that PAFR signaling participates in the survival outcomes upon hypoxia/reoxygenation and/or antitumor treatments, and may, in some way, contribute to the repopulation of mel

Um dos desafios recorrentes na prática clínica da Oncologia é o processo de repopulação, no qual células tumorais resistentes à terapia são capazes de proliferar e reconstituir o tumor. No entanto, os mecanismos envolvidos neste fenômeno ainda foram pouco explorados, sendo necessário melhor compreendê-los para evitar a falha terapêutica. Melanomas são bons modelos para estudar repopulação devido às baixas taxas de sobrevida livre de progressão e às altas taxas de resistência às terapias associadas a este tipo de tumor sólido. Sabe-se que a exposição de células tumorais a condições estressoras do microambiente, como hipóxia e hipóxia/reoxigenação, bem como ao próprio tratamento antitumoral, são pressões seletivas frequentemente encontradas em tumores sólidos que favorecem a resistência às terapias e a repopulação tumoral. Estudos prévios indicam que a sinalização mediada pelo Fator de Ativação de Plaquetas (PAF), um lipídio bioativo relacionado à diversas funções fisiológicas, e de seu receptor, PAFR, está associada com a resistência de células de melanoma aos tratamentos citotóxicos e com crescimento tumoral. Portanto, este trabalho teve como objetivo investigar o envolvimento da sinalização de PAFR frente às condições estressoras descritas acima no fenômeno de repopulação de melanomas. Os resultados de espectrometria de massa indicaram que hipóxia aumentou a geração de PAF nas linhagens de melanoma humano SKmel05 e A375, mas não na A375M, embora este aumento não tenha sido observado após a reoxigenação. Além disso, mostraram que SKmel37 exibiu os maiores níveis basais de PAF, aumentando substancialmente sua geração em diferentes tempos de exposição à hipóxia e hipóxia/reoxigenação. Investigamos também a geração de outros ligantes de PAFR, porém nenhum deles foi encontrado nas amostras. Os resultados de detecção de PAFR por Western Blot e/ou citometria de fluxo revelaram que os níveis proteicos não foram modulados por estas condições em nenhuma das linhagens e que, em termos basais, SKmel37 e SKmel05 apresentaram os maiores níveis. Estas linhagens foram, portanto, submetidas a ensaios de proliferação, os quais evidenciaram que frente ao tratamento com o antagonista de PAFR, WEB2086, em condições de hipóxia e hipóxia/reoxigenação, apenas as células SKmel37 tiveram sua proliferação reduzida e morfologia associada à morte. Ensaios de incorporação de iodeto de propídio indicaram que o tratamento destas células expostas à hipóxia/reoxigenação com WEB2086 levou a acúmulo em SG2M, morte celular e sensibilização à cisplatina. Além disso, imunofluorescência de cortes congelados de tumores induzidos com SKmel37 revelou que houve acúmulo de PAFR em áreas hipóxicas e em seu entorno. Em relação ao modelo de exposição à tratamentos antitumorais, por sua vez, curvas de concentração e tempo com Vemurafenib mostraram que SKmel37 e A375 foram resistentes à droga, ao passo que SKmel05 e UACC62 foram sensíveis. Além disso, WEB2086 potencializou o efeito de morte induzido por Vemurafenib nas linhagens sensíveis, mas não afetou as resistentes. Considerando os aspectos clínicos de resposta inicial com posterior desencadeamento de repopulação, seguimos com as linhagens sensíveis e verificamos por citometria que esta droga aumentou ROS em ambas as linhagens, mas só aumentou PAFR extracelular na SKmel05. O tratamento combinado potencializou a geração de ROS e levou a ativação de caspase3/7 apenas na SKmel05. Esta linhagem foi então submetida a ensaios clonogênicos cujos resultados mostraram que o tratamento com Vemurafenib reduziu o número de clones e que WEB2086 não potencializou este efeito. Assim, o conjunto de resultados apresentados evidencia que a sinalização de PAFR participa dos desfechos de sobrevivência frente à hipóxia/reoxigenação e/ou tratamentos antitumorais, podendo, de alguma forma, contribuir com a repopulação de melanomas / One of the recurrent challenges in the clinical practice of Oncology is the process of repopulation, in which therapy-resistant tumor cells can proliferate and reconstitute the tumor. However, the mechanisms involved in this phenomenon were still little explored. The understanding of these events is, therefore, needed to avoid therapeutic failure. Melanomas are good models for studying repopulation due to the low rates of progression-free survival and the high rates of resistance to therapies associated to this type of solid tumor. It is known that the exposure of tumor cells to microenvironmental stress conditions, such as hypoxia and hypoxia/reoxygenation, as well as the exposure to antitumor treatment itself, are selective pressures frequently found in solid tumors that favor therapy resistance and tumor repopulation. Previous studies have indicated that the signaling mediated by the Platelet Activation Factor (PAF), a bioactive lipid related to various physiological functions, and its receptor, PAFR, is associated with resistance of melanoma cells to cytotoxic treatments and with tumor growth. Therefore, the aim of this study was to investigate the involvement of PAFR signaling upon the adverse conditions described above in the phenomenon of melanoma repopulation. Mass spectrometry results indicated that hypoxia increased the generation of PAF in human melanoma cell lines SKmel05 and A375, but not in A375M, although this increase was not observed after reoxygenation. In addition, they showed that SKmel37 exhibited the highest PAF basal levels, whose generation increased substantially after different times of hypoxia and hypoxia/reoxygenation exposure. We also investigated the generation of other PAFR ligands, but none were found in the samples. The results of PAFR detection by Western Blot and/or flow cytometry revealed that protein levels were not modulated by these conditions in any of the cell lines and that, at baseline, SKmel37 and SKmel05 showed the highest levels. These lines were therefore submitted to proliferation assays, which showed that the treatment with the PAFR antagonist, WEB2086, under conditions of hypoxia and hypoxia/reoxygenation, led to proliferation reduction and death-associated morphology in SKmel37 cells only. Propidium iodide incorporation studies indicated that the treatment of these cells exposed to hypoxia/reoxygenation with WEB2086 led to accumulation in SG2M, cell death and cisplatin sensitization. In addition, immunofluorescence of frozen sections of SKmel37-induced tumors revealed that PAFR was found accumulated in hypoxic areas and its surroundings. Regarding the model of exposure to antitumor treatments, concentration and time curves with Vemurafenib showed that SKmel37 and A375 were resistant to the drug, whereas SKmel05 and UACC62 were sensitive. In addition, WEB2086 potentiated the effect of Vemurafenib-induced death on sensitive cell lines but did not affect the resistant ones. Considering the clinical aspects of initial response with subsequent repopulation triggering, we continued using the sensitive cell lines and we verified by cytometry that this drug increased ROS in both cell lines but only increased extracellular PAFR in SKmel05. The combined treatment potentiated the generation of ROS and led to the activation of caspase3/7 in SKmel05 only. This cell line was then submitted to clonogenic assays whose results showed that treatment with Vemurafenib reduced the number of clones and that WEB2086 did not potentiate this effect. Thus, the set of results presented highlights that PAFR signaling participates in the survival outcomes upon hypoxia/reoxygenation and/or antitumor treatments, and may, in some way, contribute to the repopulation of mel

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.