Validação de potenciais biomarcadores proteómicos na Síndrome da Apneia Obstrutiva do Sono

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015A Síndrome da Apneia Obstrutiva do Sono (SAOS) é um distúrbio respiratório muito prevalente na população em geral, com graves repercussões na saúde e na qualidade de vida dos doentes. A Proteómica tem vindo a ser aplicada no estudo da SAOS na pesquisa de novos biomarcadores de diagnóstico, prognóstico, monitorização e/ou alvo terapêutico para esta doença. Estudos preliminares realizados no Laboratório de Proteómica do INSARJ revelaram a existência de proteínas diferencialmente moduladas em glóbulos vermelhos (GVs) de doentes com SAOS comparados com roncadores simples. Algumas destas proteínas, como a catalase (CAT) e a peroxiredoxina 2 (Prx2), estão envolvidas em processos antioxidantes, e este mecanismo parece estar significativamente comprometido nos doentes com SAOS. O principal objectivo deste trabalho foi a validação da CAT e da Prx2 por técnicas ortogonais à Proteómica, como o western blotting (WB) e/ou por estudos de cinética enzimática em amostras de GVs de doentes com SAOS, antes e após seis meses de tratamento CPAP (Continuous Positive Airway Pressure), e em indivíduos roncadores simples como controlos. Dois tipos de amostras de GVs foram avaliadas por doente, amostra recolhida no final da tarde antes do exame de polissonografia – PSG (denominadas de “Noite”) e amostra recolhida na manhã seguinte ao exame (denominadas de “Manhã”). Após tratamento CPAP, apenas as amostras de GVs “Manhã” foram preparadas e, portanto, consideradas no estudo. O estudo da CAT, por cinética enzimática, revelou que a actividade da CAT é significativamente menor nos GVs “Manhã” em comparação com as amostras “Noite”, sejam de doentes SAOS ou de controlos (Anova p<0,05). Contudo, esta diminuição da actividade da CAT durante a manhã em relação à noite estava significativamente (T Student, p<0,05) mais acentuada nos doentes com SAOS do que nos controlos. Após seis meses de tratamento com CPAP, observou-se um aumento muito significativo da actividade da CAT nos GVs “Manhã”. Surpreendentemente, constatou-se por WB, não haver diferenças quantitativas e significativas dos níveis de expressão da CAT entre os grupos de doentes/condições analisados, e tão pouco havia, nos doentes SAOS, antes e após o tratamento CPAP. Estes resultados sugerem que a modulação da atividade da CAT nos GVs está essencialmente associada a modificações pós-traducionais da proteína, o que corrobora com os resultados obtidos pela Proteómica, que identificou oito proteoformas para a CAT nestas células. A avaliação do estado redox/oligomérico da Prx2 nos GVs por WB em SDS-PAGE não reduzido, utilizando anticorpos específicos para a Prx2 e para formas hiperoxidadas da Prx (PrxSO2/3), revelou que as formas monoméricas e diméricas da Prx2 estavam significativamente hiperoxidadas nos GVs “Manhã” ou “Noite” de doentes com SAOS. O tratamento CPAP diminui esta hiperoxidação nos monómeros e dímeros da Prx2. Contudo, as formas multiméricas da Prx2 hiperoxidadas, descritas com actividade chaperone/transdução de sinal associadas à protecção celular, surgiram quase que exclusivamente nos doentes SAOS após CPAP. A forma dissulfídica oxidada/hiperoxidada da Prx2 está associada a inativação da sua actividade peroxidática, e por conseguinte, a estado de stresse oxidativo. A conversão entre as diferentes funções da Prx2, ou seja, entre a actividade peroxidática antioxidante e a atividade chaperone/transdução de sinal, é regulada pelo seu estado redox/oligomérico na célula. Em resumo, os resultados deste trabalho validaram os resultados anteriormente obtidos por Proteómica, indicando nos GVs de doentes com SAOS, existência de desregulação no seu sistema redox-homeostase envolvendo a CAT e a Prx2. Este trabalho propõe ainda, a CAT e a Prx2, como fortes candidatos a biomarcadores de gravidade e/ou de monitorização do tratamento CPAP. Contudo, serão necessários mais estudos para consolidar este resultados e contribuir para uma melhor compreensão acerca do diagnóstico, monitorização e tratamento para a SAOS.The Obstructive Sleep Apnea (OSA) is a breathing disorder very prevalent in the general population, with serious effects on health and quality of life of the patients. The Proteomics has been applied to the study of the OSA, in order to identify new biomarkers of diagnosis, prognosis, monitoring and/or as therapeutic target for this disease. Preliminary studies at Proteomics Laboratory of INSARJ revealed the existence of proteins differentially modulated in red blood cells (RBCs) of OSA patients compared with simple snorers (controls). Some of these proteins such as catalase (CAT) and peroxiredoxin 2 (Prx2), are involved in antioxidants processes, which seem to be significantly compromised in OSA patients. The main objective of this work was the validation of these proteins by orthogonal technologies to Proteomics, such as western blotting (WB) and/or kinetic enzyme assay on RBCs samples from OSA patients, before and after six months of treatment CPAP (Continuous Positive Airway Pressure), and from simple snorers as controls. Two types of RBCs samples were evaluated per patient, sample taken late in the afternoon and before polysomnography (PSG) (denominated here "Night") and sample collected in the morning after the examination ("Morning"). After CPAP treatment, only "Morning" RBC samples were prepared and therefore considered in this study. The kinetic enzyme assay showed that CAT activity is significantly higher in RBC "Morning" compared to the "Night" samples, whatever were OSA or control (Anova p<0.05). However, this decrease in “Morning” CAT activity compared to “Night” was even greater in OSA RBC samples than in controls ones (Student's T p <0.05). After six months of CPAP treatment, there was a very significant increase in CAT activity in this "Morning" RBC OSA samples. Surprisingly, no significant differences were observed in the CAT expression level among the patient’s group/condition analysed. These results suggest that the modulation of CAT activity in RBCs is mainly associated with post-translational modifications, which supports the previous proteomics data identifying eight proteoforms for CAT in these cells. The evaluation of redox/oligomeric study of the Prx2 in RBCs by WB in SDS-PAGE, non-reduced, using antibodies specific for Prx2 and hyperoxidized forms of Prx (PrxSO2/3) demonstrated that the monomeric and dimeric form of Prx2 were significantly hyperoxidized in “Morning” or “Night” OSA RBCs. The CPAP treatment reduced this hyperoxidation observed on monomers and dimers of Prx2. However, hyperoxidized multimeric forms of Prx2, described with chaperone activity, appeared almost exclusively in OSA patients after CPAP. The disulfide oxidized/hiperoxidized forms of Prx2 are associated with the inactivation of its peroxidatic activity and therefore with the state of stress-oxidative. The conversion between the different functions of Prx2, i.e. between antioxidant peroxidatic activity and chaperone activity or transduction signaling, is regulated by its redox/oligomeric state in the cell. In summary, the results of this study validated the results previously obtained by proteomics, indicating that in OSA RBCs the redox- homeostasis system involving the CAT and Prx2 is dysregulated. This work also proposes CAT and Prx2 as a promising candidates biomarkers of severity and/or to monitor CPAP treatment. However, more studies will be needed to consolidate this results and contribute to a better understanding of OSA diagnosis and therapy monitoring

Obstructive sleep apnea associated with Diabetes mellitus Type 2: a proteomic study

Background: We previously showed that Obstructive sleep apnea (OSA), a common public health concern causing deleterious cardiometabolic dysfunction, induced proteomic alterations in red blood cells (RBC) such as changes in the redox-oligomeric state of peroxiredoxin 2 (PRDX2)1-2. Herein, we aimed to investigate whether OSA patients with Type 2 Diabetes Mellitus before and after positive airway pressure (PAP) treatment present similar changes in the RBC antioxidant protein PRDX2 to better understand the molecular basic mechanisms associated with OSA and OSA outcomes. Methods: RBC samples from control snorers (n=22 being 3 diabetics) and OSA patients before and after six month of PAP-treatment (n=29 being 8 diabetics) were analysed by non-reducing western blot using antibody against PRDX2 or PRDXSO2/3 to measure the total and overoxidized levels of monomeric/dimeric/multimeric forms of PRDX2. Results: We confirmed previously data by showing that in OSA RBC the overoxidation on the monomeric forms of PRDX2 was higher compared to controls. After PAP treatment, this overoxidation decreased followed by an increase of multimeric-overoxidized forms of PRDX2 described to be associated with chaperone protective function. In contrast, the level of PRDX2 monomers in RBC diabetic OSA, although higher abundant its overoxidation level was much lower than those observed in OSA without comorbidity and did not significant change after treatment. Moreover, the level of PAP-induced PRDX2-overoxidized-multimers was also lower in these diabetic OSA patients. The level of overoxidized monomeric/dimeric forms of PRDX2 correlated negatively with levels of insulin / triglycerides and HbA1C, respectively. After PAP, the level of (overoxidized) PRDX2SO2/3 multimers correlated positively with adrenaline levels. Conclusions: The redox/oligomeric state of RBC PRDX2 that is regulated by overoxidation of the active cysteines was differentially modulated in diabetic OSA patients compared to OSA without this comorbidity. PAP-induced overoxidized oligo forms of PRDX2 that is associated with chaperone protective function showed decreased in OSA patients with diabetes. The clinical impact of these findings needs further investigation and validation.Project partially supported by Harvard Medical School-Portugal Program (HMSP-ICJ/0022/2011), ToxOmics - Centre for Toxicogenomics and Human Health (FCT-UID/BIM/00009/2013).info:eu-repo/semantics/publishedVersio

Mastitis diagnosis in dairy goats through somatic cell counts and California mastitis test. Preliminary results

The aim of this work was to evaluate somatic cell count (SCC) and Californian mastitis test (CMT) reliability as methods to survey mastitis in Serrana goats. Microbiological diagnosis, SCC and CTM were performed on 2028 samples, collected from individual glands during a lactation period. According to results CMT (predictive negative value = 69.5%) may be used as a cheap and practical method for sub clinical mastitis survey in Serrana goats. Decision on SCC use will depend on additional research works, since its values were very high even for bacteriological negative samples.IPB-ESA,DRATM

Seroprevalence and risk factors associated with small ruminant lentivirus infection in the north-eastern of Portugal: preliminary results

The small ruminants lentiviruses (SRLVs) are a group of viruses responsible for Maedi-Visna (MV) in sheep and caprine arthritis encephalitis (CAE) in goats. These diseases may result in progressive and persistent infections that affect animal health and cause severe economic losses of production. In the north-eastern of Portugal, small ruminants farming have great economic and social importance. Typical farm uses traditional methods of animal production, carrying out year- round roaming grazing. Until now there is scarce information on the seroprevalence of SRLVs in Portugal, neither about risk factors to these type of farming system.info:eu-repo/semantics/publishedVersio

Evening and morning peroxiredoxin-2 redox/oligomeric state changes in obstructive sleep apnea red blood cells: Correlation with polysomnographic and metabolic parameters

We have examined the effects of Obstructive Sleep Apnea (OSA) on red blood cell (RBC) proteome variation at evening/morning day time to uncover new insights into OSA-induced RBC dysfunction that may lead to OSA manifestations. Dysregulated proteins mainly fall in the group of catalytic enzymes, stress response and redox regulators such as peroxiredoxin 2 (PRDX2). Validation assays confirmed that at morning the monomeric/dimeric forms of PRDX2 were more overoxidized in OSA RBC compared to evening samples. Six month of positive airway pressure (PAP) treatment decreased this overoxidation and generated multimeric overoxidized forms associated with chaperone/transduction signaling activity of PRDX2. Morning levels of overoxidized PRDX2 correlated with polysomnographic (PSG)-arousal index and metabolic parameters whereas the evening level of disulfide-linked dimer (associated with peroxidase activity of PRDX2) correlated with PSG parameters. After treatment, morning overoxidized multimer of PRDX2 negatively correlated with fasting glucose and dopamine levels. Overall, these data point toward severe oxidative stress and altered antioxidant homeostasis in OSA RBC occurring mainly at morning time but with consequences till evening. The beneficial effect of PAP involves modulation of the redox/oligomeric state of PRDX2, whose mechanism and associated chaperone/transduction signaling functions deserves further investigation. RBC PRDX2 is a promising candidate biomarker for OSA severity and treatment monitoring, warranting further investigation and validation.Project partially supported by Harvard Medical School-Portugal Program (HMSPICJ/0022/2011), ToxOmics - Centre for Toxicogenomics and Human Health (FCT-UID/BIM/00009/2013), FCT/Poly-Annual Funding Program and FEDER/Saúde XXI Program (Portugal) and postdoctoral fellowship (SFRH/BPD/43365/2008) of Fundação para a Ciência e a Tecnologia (FCT), Portugal.info:eu-repo/semantics/publishedVersio

Proteome profiling in obstructive sleep apnea severity and treatment response towards early diagnosis and prognosis prediction

Obstructive Sleep Apnea (OSA) syndrome is a common public health concern characterized by recurrent episodes of apneas and hypopneas during sleep. These obstructive events result in recurrent intermittent hypoxia and sleep fragmentation that can lead to metabolic and cardiovascular diseases. We recently demonstrated that OSA syndrome can cause alterations in the red blood cells (RBC) proteome that may be associated with OSA outcomes. Here we intend to investigate whether the positive airway pressure (PAP) treatment can revert/modulate these proteome alterations. RBCs from Snorers and patients with severe OSA before/after 6 months of PAP treatment (n=10/condition) were depleted of hemoglobin, analyzed by 2D-DIGE using Progenesis SameSpots v4.5. The differentially abundant proteins were identified by MALDI-MS and protein annotations acquired by DAVIDv6.8. Western blotting (WB) validation was performed for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (overoxidized) GAPDHSO3 on a new Cohort (n=59). Statistical analysis including correlation studies with peroxiredoxin 2 (PRDX2) redox-oligomeric forms and several clinical parameters was carried out using SPSS software. Ten protein spots exhibited significant differences (Anova p<0.05) among groups and were associated with cell death, protein oligomerization and response to stress. Three proteoforms of GAPDH were identified decreased in OSA RBC (Anova p<0.05). Six months of PAP treatment increased these GAPDH proteoforms to the control levels. By WB, we confirmed these data by showing that the decreased GAPDH monomeric/tetrameric forms in OSA were increased by PAP treatment. PAP also increased GAPDHSO3 tetramers. In OSA, GAPDH monomers and GAPDHSO3 tetramers correlated positively with the respiratory disturbance index or triglycerides and adrenalin, respectively. After PAP, GAPDHSO3 tetramers correlated positively with PAP-induced PRDX2SO2/3 decameric forms, described having chaperone activity in cell protection. OSA induces alterations in the redox/oligomeric state of GAPDH and PRDX2 that can be reverted/modulated by PAP treatment. The clinical significant of these findings needs further validation and investigation. Selected Reaction Monitoring (SRM) and bioinformatics – based tools, will be used to validate the obtained data. The same proteomics workflow strategy will be applied to investigate the plasma proteome in OSA and OSA response to therapyN/

Effects of positive airway pressure therapy on red blood cells in patients with obstructive sleep apnea

Introduction: Obstructive Sleep Apnea (OSA) syndrome is characterized by recurrent arousals from sleep and intermittent hypoxemia. We recently demonstrated that OSA can cause alterations in the red blood cells (RBC) proteome that may be associated with OSA outcomes. Here we intend to investigate whether the positive airway pressure (PAP) treatment can revert/modulate these proteome alterations.Project partially supported by Harvard Medical School-Portugal Program (HMSP-ICJ/0022/2011), ToxOmics - Centre for Toxicogenomics and Human Health (FCT-UID/BIM/00009/2013).N/

Effects of Positive Airway Pressure Therapy on Red Blood Cell Proteome from Patients with Obstructive Sleep Apnea

Introduction: Obstructive Sleep Apnea (OSA) syndrome, a common public health concern, is characterized by recurrent arousals from sleep and intermittent hypoxemia that can lead to metabolic and cardiovascular diseases. We recently demonstrated that OSA syndrome can cause alterations in the red blood cells (RBC) proteome that may be associated with OSA outcomes. Here we intend to investigate whether the positive airway pressure (PAP) treatment can revert/modulate these proteome alterations.info:eu-repo/semantics/draf

Diabetes mellitus tipo 2 (DMT2) associada a Sindrome de Apneia Obstrutiva do Sono (SAOS): um estudo proteómico

Introdução: A prevalência da SAOS é elevada em doentes com DMT2. O não tratamento da SAOS pode levar ao agravamento ou desenvolvimento da DMT2. Temos vindo a demonstrar que a SAOS altera o proteoma do glóbulo vermelho (GV). A SAOS aumenta a overoxidação da peroxirredoxina 2 (PRDX2) (enzima antioxidante), o que pode levar à desregulação da homeostasia do GV e ao desenvolvimento de doenças metabólicas. Após tratamento com ventilação não invasiva (PAP), esta overoxidação diminuiu seguida de um aumento de PRDX2 decamérica overoxidada com funções chaperone na proteção celular (Feliciano et al. 2017). No presente estudo, fomos investigar o estado redox/oligomérico da PRDX2 em doentes DMT2 com SAOS, antes/após PAP, para melhor compreender a interligação entre estas patologias. Material e métodos: Amostras de GVs de controles (n=22 sendo 3 DMT2) e doentes SAOS antes/após 6 meses de tratamento com PAP (n=29 sendo 8 DMT2) foram analisadas por western-blot não reduzido, com anticorpo para a PRDX2 e PRDXSO2/3 (overoxidada). Os grupos foram comparados estatisticamente e correlacionados com dados clínicos e bioquímicos. Resultados: Nos GVs de doentes DMT2/SAOS, o nível de monómeros da PRDX2 mostrou-se aumentado e diminuía após PAP. Contudo, o nível destes monómeros PRDXSO2/3 estava diminuído e não se alterou com o tratamento. Após PAP, o nível de decâmeros PRDX2SO2/3 foi também menor nestes doentes. Os níveis de monómeros PRDX2 e PRDX2SO2/3 correlacionaram-se negativamente com os níveis de insulina/triglicéridos e HbA1C, respetivamente. Após PAP, os níveis de decâmeros PRDX2SO2/3 correlacionou-se positivamente com os níveis de adrenalina. Conclusões: O estado redox/oligomérico da PRDX2 do GV é diferencialmente modulado nos doentes DTM2/SAOS em comparação com doentes SAOS. Decâmeros PRDXSO2/3 induzidos pelo tratamento e associadas à função protetora “chaperone” estão diminuídos em doentes DMT2/SAOS. O impacto clínico destas descobertas, necessita de mais investigação e validação.N/