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ProteĆ³mica da Apneia Obstrutiva do Sono para a descoberta de biomarcadores
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Advisor(s)
Abstract(s)
[PT] A sĆndrome da apneia obstrutiva do sono (SAOS) Ć© um importante problema de
saĆŗde pĆŗblica, nĆ£o sĆ³ por comprometer o estilo de vida e bem-estar do doente, mas
tambƩm por ser um factor de risco para o desenvolvimento de doenƧas cardiovasculares,
metabĆ³licas e carcinogĆ©nicas. Apesar de grandes avanƧos nos mĆ©todos de diagnĆ³stico e
tratamento da SAOS, permanecem ainda por esclarecer os mecanismos moleculares que
levam ao seu desenvolvimento e Ć s patologias associadas.
A avaliaĆ§Ć£o do perfil proteĆ³mico do plasma sanguĆneo que possa estar associado Ć
SAOS, aos vĆ”rios estadios da doenƧa ou Ć resposta/eficĆ”cia do tratamento com pressĆ£o
positiva contĆnua das vias aĆ©reas (CPAP), foi o objectivo Ćŗltimo deste estudo. As proteĆnas
constituintes deste perfil, uma vez validadas, sĆ£o potenciais biomarcadores de
diagnĆ³stico, prognĆ³stico e/ou de monitorizaĆ§Ć£o terapĆŖutica da SAOS.
Numa primeira fase foi necessĆ”ria a optimizaĆ§Ć£o da preparaĆ§Ć£o de amostras de
plasma e sua anƔlise por electroforese bidimensional (2DE) em mini-gƩis, para futura
identificaĆ§Ć£o de proteĆnas diferencialmente expressas na doenƧa/condiĆ§Ć£o, por
espectrometria de massa. Este objectivo do projecto foi concluĆdo com sucesso, pois
mostrou que a utilizaĆ§Ć£o de 2DE em minigeis Ć© possĆvel em anĆ”lises futuras com uma boa
relaĆ§Ć£o custo-eficĆ”cia.
Em paralelo, investigou-se se o estado redox-oligomĆ©rico da proteĆna
peroxiredoxina 2 (Prx2) (um biomarcador de inflamaĆ§Ć£o/stress oxidativo), encontrava-se
alterado no plasma de doentes com SAOS e, se esta alteraĆ§Ć£o, era parcialmente revertida
apĆ³s tratamento CPAP, tal como vem sendo observado nos eritrĆ³citos destes doentes
(dados preliminares do laboratĆ³rio). A validaĆ§Ć£o em plasma dos dados de proteĆ³mica
obtidos em eritrĆ³citos, poderĆ” contribuir para uma melhor compreensĆ£o da relevĆ¢ncia do
papel da Prx2 na SAOS. Para isso, selecionaram-se amostras de plasma ānoiteā, recolhidas
na noite anterior ao diagnĆ³stico polissonogrĆ”fico e āmanhĆ£ā, recolhidas na manhĆ£
seguinte para cada indivĆduo roncador (controlo) e doente SAOS (ligeira, moderada e
grave). Para estes Ćŗltimos, foram tambĆ©m recolhidas amostras matinais āCPAPā, apĆ³s seis
meses de tratamento. As amostras de plasma foram analisadas por 1DE SDS-PAGE
reduzido e nĆ£o-reduzido (com ou sem agente redutor), seguido de western blotting com
anticorpo especĆfico para a Prx2 ou PrxSO2/3 (formas hiperoxidadas da Prx2). Os
resultados indicaram que as formas hiperoxidadas estavam aumentadas nos doentes
3
SAOS, mais significamente (pā¤0,05) Ć noite, em relaĆ§Ć£o aos indivĆduos controlo. O
tratamento CPAP pareceu reduzir esta hiperoxidaĆ§Ć£o. Estes resultados corroboram os
resultados obtidos nos eritrĆ³citos destes doentes. No entanto, formas da Prx2,
possivelmente com outras modificaƧƵes pĆ³s-traducionais, que nĆ£o a hiperoxidaĆ§Ć£o,
tambĆ©m mostraram uma tendĆŖncia para um aumento matinal nos doentes SAOS. Esta
tendĆŖncia foi significativamente (pā¤0.05) reduzida apĆ³s tratamento. Estes resultados,
embora preliminares, sĆ£o interessantes dado que a presenƧa desta proteĆna no meio
extra celular ser reconhecida como uma sinalizadora de stress/inflamaĆ§Ć£o.
Em suma, os dados obtidos sĆ£o motivadores para se prosseguir com a investigaĆ§Ć£o
no plasma, no desenvolvimento futuro de novas ferramentas de diagnĆ³stico, prognĆ³stico
e/ou monitorizaĆ§Ć£o terapĆŖutica da SAOS.
[EN] The obstructive sleep apnea (OSA) is a major public health problem, not only for compromising the lifestyle and the patient's well-being but also to be itself a risk factor for developing cardiovascular, metabolic and carcinogenic diseases. Despite great advances in diagnostic methods and treatment of OSA, still remain to be elucidated the molecular mechanisms that lead to its development and associted pathologies. The assessment of blood plasma proteomic profile that may be specifically associated with OSA, OSA severety or the response/efficacy of continuous positive airway pressure (CPAP) treatment, was the ultimate goal of this study. The proteins that constitute this profile, once validated, are potential biomarkers of diagnostic, prognostic and/or therapeutic monitoring of OSA. In a first phase, it was necessary to optimize the preparation of human plasma sample and its analysis by two-dimensional electrophoresis (2DE) on mini-gels, for future identification of differentially expressed proteins in the disease/condition by mass spectrometry. This objective was successfully completed, by showing that the use of 2DE minigels is possivel in future cost-effective analysis. In parallel, it was investigated wheather the redox-oligomeric state of the protein peroxiredoxin 2 (Prx2) (a biomarker of inflammation/oxidative stress) was changed in plasma of OSA patients and wheather this change was parcially reverted by CPAP treatment as it has been observed in erythrocytes of this patients. The validation of erythrocyte data in plasma, may contribute to a better understanding of the importance of the role of Prx2 in OSA. For this, ānightā and āmorningā plasma samples were selected, taken in the night before polysomnographic diagnosis, and in the next morning, respectively, from snorers (control) and OSA patients (mild, moderate or severe). For these last ones, morning samples "CPAP" were also collected, after six months of treatment. The samples were analyzed by reduced and non-reduced 1DE SDS-PAGE (with or without reducing agent), followed by western blotting with specific antibodys for Prx2 or PrxSO2/3 (hiperoxidized forms of Prx2). The results indicated that the hiperoxidized forms were increased in OSA patients plasma, most significantly (pā¤0.05) at night compared to the control ones. The treatment seemed to reduce this hyperoxidation. These results corroborate with the study conducted in erythrocytes. However, forms of Prx2, probably 5 with other post-translational modifications, which do not hyperoxidation, also showed a trend towards an morning increase in OSA patients. This tendency was significantly (pā¤0.05) reduced after treatment. These results, although preliminary, are interesting due to the presence of this protein in the extracellular medium and be recognized as a signal of stress/inflammation. In summary, the data are motivating to proceed with the investigation in plasma, in the future development of new diagnostic, prognostic and/or therapeutic monitoring of OSA.
[EN] The obstructive sleep apnea (OSA) is a major public health problem, not only for compromising the lifestyle and the patient's well-being but also to be itself a risk factor for developing cardiovascular, metabolic and carcinogenic diseases. Despite great advances in diagnostic methods and treatment of OSA, still remain to be elucidated the molecular mechanisms that lead to its development and associted pathologies. The assessment of blood plasma proteomic profile that may be specifically associated with OSA, OSA severety or the response/efficacy of continuous positive airway pressure (CPAP) treatment, was the ultimate goal of this study. The proteins that constitute this profile, once validated, are potential biomarkers of diagnostic, prognostic and/or therapeutic monitoring of OSA. In a first phase, it was necessary to optimize the preparation of human plasma sample and its analysis by two-dimensional electrophoresis (2DE) on mini-gels, for future identification of differentially expressed proteins in the disease/condition by mass spectrometry. This objective was successfully completed, by showing that the use of 2DE minigels is possivel in future cost-effective analysis. In parallel, it was investigated wheather the redox-oligomeric state of the protein peroxiredoxin 2 (Prx2) (a biomarker of inflammation/oxidative stress) was changed in plasma of OSA patients and wheather this change was parcially reverted by CPAP treatment as it has been observed in erythrocytes of this patients. The validation of erythrocyte data in plasma, may contribute to a better understanding of the importance of the role of Prx2 in OSA. For this, ānightā and āmorningā plasma samples were selected, taken in the night before polysomnographic diagnosis, and in the next morning, respectively, from snorers (control) and OSA patients (mild, moderate or severe). For these last ones, morning samples "CPAP" were also collected, after six months of treatment. The samples were analyzed by reduced and non-reduced 1DE SDS-PAGE (with or without reducing agent), followed by western blotting with specific antibodys for Prx2 or PrxSO2/3 (hiperoxidized forms of Prx2). The results indicated that the hiperoxidized forms were increased in OSA patients plasma, most significantly (pā¤0.05) at night compared to the control ones. The treatment seemed to reduce this hyperoxidation. These results corroborate with the study conducted in erythrocytes. However, forms of Prx2, probably 5 with other post-translational modifications, which do not hyperoxidation, also showed a trend towards an morning increase in OSA patients. This tendency was significantly (pā¤0.05) reduced after treatment. These results, although preliminary, are interesting due to the presence of this protein in the extracellular medium and be recognized as a signal of stress/inflammation. In summary, the data are motivating to proceed with the investigation in plasma, in the future development of new diagnostic, prognostic and/or therapeutic monitoring of OSA.
Description
DissertaĆ§Ć£o de mestrado em Biologia Humana e Ambiente, apresentada Ć Faculdade de CiĆŖncias da Universidade de Lisboa, 2015.
Trabalho de investigaĆ§Ć£o desenvolvido no LaboratĆ³rio de ProteĆ³mica do Instituto Ricardo Jorge.
Deborah Penque (orientadora), FĆ”tima Vaz (co-orientadora) - LaboratĆ³rio de ProteĆ³mica do Departamento de GenĆ©tica Humana do Instituto Nacional de SaĆŗde Doutor Ricardo Jorge I.P.; Maria Teresa Rebelo (orientadora interna) - Faculdade de CiĆŖncias da Universidade de Lisboa.
Trabalho de investigaĆ§Ć£o desenvolvido no LaboratĆ³rio de ProteĆ³mica do Instituto Ricardo Jorge.
Deborah Penque (orientadora), FĆ”tima Vaz (co-orientadora) - LaboratĆ³rio de ProteĆ³mica do Departamento de GenĆ©tica Humana do Instituto Nacional de SaĆŗde Doutor Ricardo Jorge I.P.; Maria Teresa Rebelo (orientadora interna) - Faculdade de CiĆŖncias da Universidade de Lisboa.
Keywords
Plasma Proteomica Apneia SĆndrome da apneia obstrutiva do sono SAOS ProteĆ³mica Biomarcadores PressĆ£o Positiva ContĆnua das Vias AĆ©reas DoenƧas GenĆ©ticas Proteomics Obstructive Sleep Apnea Biomarkers Continuous Positive Airway Pressure Genomica Funcional GenĆ³mica Funcional e Estrutural