1,721,099 research outputs found
Changes in the proteome of Huh7 cells induced by transient expression of hepatitis D virus RNA and antigens
No full textHepatitis delta virus (HDV) infection of human hepatocytes infected with the hepatitis B virus (HBV) is associated with increased liver damage and risk of fulminant disease. Although considerable progress has been made towards the elucidation of the mechanisms of HDV replication and pathogenesis, little is still known about the host factors involved in the different steps of the replication cycle. Here, we made use of a proteomic approach to analyse the global alterations in protein expression that arise in human hepatocytes separately transfected with each of the HDV components. Huh7 cells were transiently transfected with plasmids that code for the small delta antigen (S-HDAg), large delta antigen (L-HDAg), genomic RNA (gRNA), and antigenomic RNA (agRNA), respectively. Total protein extracts were separated by 2-DE and differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 32 proteins known to be involved in different pathways namely nucleic acid metabolism, protein metabolism, transport, signal transduction, apoptosis, and cell growth. Moreover, the down regulation of hnRNP D, HSP105, and triosephosphate isomerase was further confirmed by Real time PCR
Proteome analysis of a human liver carcinoma cell line stably expressing hepatitis delta virus ribonucleoproteins
No full textHepatitis delta virus (HDV) infects human hepatocytes already infected with the hepatitis B virus increasing about ten fold the risk of cirrhosis and fulminant hepatitis. The lack of an appropriate cell culture system capable of supporting virus replication has so far impaired the detailed investigation of the HDV biology including the identification of host factors involved in pathogenesis. Here, we made use of a HDV cDNA stably transfected cell line, Huh7-D12, in a proteomic approach to identify the changes in the protein expression profiles in human liver cells that arise as a consequence of HDV replication. Total protein extracts from Huh7-D12 cells and of the corresponding non transfected human liver carcinoma cell line, Huh7, were separated by 2-DE. Differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 23 differentially expressed proteins of which 15 were down regulated and 8 up regulated in Huh7-D12 cells. These proteins were found to be involved in different cellular pathways. The down regulation of the histone H1-binding protein and of triosephosphate isomerase was confirmed by Real time PCR, and the up regulation of the La protein and lamin A/C was validated by western blot
COVID-19 Vaccination. A study on red blood cells (RBCs) and their impact on the immune system
Full text linkRed blood cells (RBCs) are emerging as regulators of the innate immune response by
interacting with inflammatory molecules, including cytokines/chemokines, nucleic acids, and
pathogens, thereby modulating immune responses.
This thesis investigated how RBCs impact immune responses after COVID-19
vaccination, focusing on (a) cytokine profiles in RBCs and their conditioned media (RBC-CM)
before and after vaccination; (b) RBC effects on peripheral blood mononuclear cells (PBMCs)
activity, especially T-cell expansion.
The initial phase optimized the ELISA assay for TNF-α, establishing Lysis Method 2 and
a 1000 μg total protein extract as optimal parameters. TNF-α presence was confirmed in both
RBCs and RBC-CM.
IL-1β, IL-6, IL-12, IL-15, and TNF-α cytokines were, then, analyzed in RBCs and RBC-
CM samples, from healthy subjects (n=8) across different time points (T0-T4) of COVID-19
vaccination (Biobank-COVID-19 vaccines). The results revealed that these proinflammatory
cytokines are bound to or can be released from cultured RBCs in varying amounts in response to
vaccination. Similar temporal modulation in plasma following COVID-19 vaccination has been
described for these cytokines, suggesting an interaction between plasma and RBC might exist in
response to vaccine-induced proinflammatory cytokine signaling.
The subsequent phase delves into RBC samples (T0-T4) and PBMC interactions in cell
culture. ELISA assays measuring IL-12, IL-15, TNF-α, and IFN-γ in culture medium showed that
RBCs presenting differential cytokine profiles in response to vaccination can in turn differentially
modulate PBMC cytokines secretion in vitro with potential downstream consequences in the
immune activity of these cells.
Preliminary flow cytometry results suggested a specific T-cell subpopulations expansion
in response to vaccination in the presence of RBCs.
This study provides initial insights into cytokine changes in RBCs post-vaccination and
their influence on PBMC cytokine response and T-cell subpopulation expansion. Further research
confirming these findings will be necessary to better understand the complex interplay that might
exist between RBCs and immune cells during vaccination-induced immunization.Os glóbulos vermelhos (GVs) têm surgido como reguladores da resposta imunitária inata,
interagindo com moléculas inflamatórias, incluindo citocinas/quimiocinas, ácidos nucleicos e
patógenos, influenciando as respostas imunológicas.
Neste estudo, investigámos o impacto dos GVs nas respostas imunitárias após a vacinação
COVID-19, focando-nos: (a) nos perfis de citocinas dos GVs e dos seus meios condicionados
(GV-CM) antes e após a vacinação; (b) nos efeitos dos GVs na atividade das células
mononucleares do sangue periférico (CMSP), especialmente na expansão das células T.
Na primeira fase, otimizamos a técnica ELISA para TNF-α nos GVs e nos GV-CMs,
estabelecendo parâmetros como o Método de Lise 2 e a quantidade de 1000 μg de proteína total
nos GVs. Presença de TNF-α foi confirmada em ambas as amostras.
IL-1β, IL-6, IL-12, IL-15 e TNF-α foram analisadas por ELISA, nas amostras de GV e
GV-CM de indivíduos saudáveis (n=8), em diferentes momentos (T0-T4) da vacinação COVID-
19 (Biobanco de Vacinas-COVID-19). Estas citocinas pró-inflamatórias mostraram estar ligadas
aos GVs ou a serem libertas em quantidades variáveis em resposta à vacinação. No plasma, esta
modulação temporal após a vacinação COVID-19 já foi observada, sugerindo uma possível
interação entre o mesmo e os GVs em resposta à sinalização pró-inflamatória induzida pela
vacina.
Posteriormente, avaliaram-se as interações entre as amostras de GV (T0-T4) e as CMSP
em cultura celular. A análise de IL-12, IL-15, TNF-α e IFN-γ no meio de cultura indicou que os
GVs com diferentes perfis de citocinas em resposta à vacinação podem modular a secreção de
citocinas pelas CMSP in vitro, afetando potencialmente a atividade imunitária dessas células.
Resultados preliminares da citometria de fluxo sugeriram uma expansão específica de
subpopulações de células T em resposta à vacinação na presença de GV.
Este estudo fornece uma perspetiva inicial sobre as alterações das citocinas nos GV após
a vacinação e o seu impacto na resposta das CMSP. No entanto, é necessário validar estas
descobertas e compreender melhor a complexa interação entre os GV e as células do sistema
imunitário durante a imunização induzida pela vacinação contra a COVID-19
Proteomic Biomarkers for Occupational Secondhand Smoke Exposure
No full textA Global Health Problem: Smoking kills more than 7 million people/year worldwide; More than 890,000 are deaths resulting from
exposure to Second Hand Smoke (SHS); In adults, SHS is associated to cardiovascular
and respiratory diseases, including coronary
heart disease and lung cancer.N/
Computational and validation approaches in proteomics discovery of disease biomarkers
No full textDissertação de mestrado em Biologia Humana e Ambiente, apresentada à Faculdade de Ciências da Universidade de Lisboa, 2019.Dissertação orientada por: Doutora Deborah Penque, PhD, Instituto Nacional de Saúde Doutorr Ricardo Jorge; Professora Doutora Deodália Dias, PhD, Universidade de Lisboa.O estudo em larga escala de proteínas, a proteómica, está a mudar amplamente a nossa compreensão das funções dos genes na era pós-genómica. Depois da revolução na genómica pelos métodos de sequenciação de ADN (ácido desoxirribonucleico), a proteómica tem vindo a aumentar o nosso conhecimento sobre a variabilidade, localização, função e vias metabólicas das proteínas na célula, tecido ou organismo. O desenvolvimento de biomarcadores, como uma componente relevante na tomada de decisões, tanto nos processos clínicos quanto no desenvolvimento de novos medicamentos, é uma área emergente onde a proteómica tem vindo a ganhar relevo. As tecnologias proteómicas permitem uma análise comparativa, qualitativa e quantitativa de milhares de proteínas de células/tecidos de doentes versus indivíduos controles, assim como, de doentes antes e depois de um determinado tratamento. As proteínas identificadas diferencialmente abundantes e/ou modificadas pós-traducionalmente na condição de doença ou em resposta a terapia, são possíveis candidatas a biomacardores destas condições. No entanto, a interpretação da enorme quantidade de dados proteómicos, gerados principalmente por experiências baseadas em espectrometria de massa (MS), requer suporte computacional para processamento e análise dos dados de forma efetiva e robusta. A Proteómica computacional estuda os métodos computacionais, algoritmos, bases de dados e metodologias utilizadas para processar, gerir, analisar e interpretar os dados produzidos em experiências proteómicas na identificação de potenciais biomarcadores. O Laboratório de Proteómica do INSA (Instituto Nacional de Saúde Doutor Ricardo Jorge), através da busca de biomarcadores proteómicos para compreensão de doenças tais como a Anemia das Células Falciformes (ACF), tem produzido grandes dados de MS que necessitam de análise computacional, sendo este o principal propósito deste projeto (ver abaixo objetivo deste estudo). A ACF, também denominada por drepanocitose ou anemia drepanocítica, é um distúrbio monogénico autossómico recessivo, clinicamente heterogéneo, caracterizado por episódios recorrentes de hemólise grave, vaso-oclusão e infecção. Vários modificadores genéticos e ambientais foram sugeridos para modular o início e o curso da ACF. Especificamente, os componentes vasculares da patologia (por exemplo, acidente vascular cerebral) foram submetidos a pesquisas intensivas e o uso de metodologias proteómicas promete oferecer novas percepções moleculares sobre a fisiopatologia da ACF. A mudança do estado estacionário para a crise ainda é em grande parte imprevisível. A fim de descobrir biomarcadores putativos para essa exacerbação, o laboratório do INSA analisou por proteómica de shotgun MS, amostras de plasma e glóbulos vermelhos (GV), de um grupo de pacientes com ACF em estado estacionário e em crise (episódio de vaso-oclusão). Objetivo do estudo: Este estudo teve como objetivo analisar os dados de shotgun MS gerados para a ACF através de plataformas de proteómica computacional de código aberto, nomeadamente o PatternLab e o MaxQuant , no sentido de identificar proteínas como possíveis candidatos a biomarcadores da ACF e em particular da ACF associada a vaso-oclusão. O PatternLab for Proteomics é um ambiente computacional integrado para análise de proteómica shotgun, formatando bancos de dados de sequências, que realizam a correspondência de espectro peptídico, filtrando estatisticamente e organizando dados por proteómica diferencial, exibindo resultados em formato de gráficos, realizando estudos orientados por similaridade com dados de sequenciação de novo, ajudando à compreensão do significado biológico dos dados à luz da Ontologia Genética (Gene Ontology). O MaxQuant é um conjunto de algoritmos, que inclui a detecção de picos e a pontuação de péptidos, realiza a calibração em massa e pesquisas em bancos de dados para identificação de proteínas, quantifica proteínas identificadas e fornece estatísticas resumidas. Para validar os achados proteómicos, algumas das proteínas identificadas diferencialmente na patologia por essas plataformas computacionais, foram selecionadas para validação (verificação) através de abordagens como Western blot. O Western blot é uma técnica bioquímica imunológica na qual uma mistura de proteínas é separada por gel 1DSDS-PAGE (Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis), transferida para uma membrana, posteriormente incubada com anticorpo específico contra a proteína de interesse. A reação, geralmente visualizada por quimioluminescência, pode ser quantificada por densitometria. De todas as 111 proteínas diferencialmente expressas identificadas (74 da fração citoplasmática e 37 da fração membranar) associadas ao evento de crise na ACF, a peroxiredoxina-2, a catalase, a Hsp70 e Hsp 90 foram validadas por Western blot. Destas 4 proteínas, apenas a peroxiredoxina-2 apresentou significância estatística. Das proteínas diferencialmente expressas que hipoteticamente podem estar associadas à ACF, um promissor biomarcador de crise, nomeadamente, a Voltagedependent anion-selective channel protein 1 (VDAC1) foi encontrada diminuída. Os resultados sugerem que episódios de vaso-oclusão em doentes com ACF podem estar associados à diminuição da VDAC1 nos glóbulos vermelhos. Os resultados deste projeto após apresentação e discussão podem contribuir para um melhor entendimento das vias moleculares associadas à ACF, bem como para a identificação de modulações proteicas específicas, como possíveis candidatos a biomarcadores para essas patologias.The Laboratory of Proteomics at INSA (Instituto Nacional de Saúde Doutor Ricardo Jorge) by searching biomarkers for Sickle-cell disease (SCD) has been produced considerable Mass Spectrometry (MS) data, needing computational analysis to process, manage, analyze and interpret the data to reveal relevant biomarkers. SCD is a clinically heterogeneous autosomal recessive monogenic disorder characterized by recurrent episodes of severe haemolysis, vaso-occlusion and infection. Several genetic and environmental modifiers have been suggested to modulate the onset and course of SCD. The vascular components of the pathology have been thus subjected to intensive research and the usage of proteomics methodologies promises to offer novel unbiased molecular insights into the pathophysiology of SCD. The objective of this project is to analyze by different bioinformatics tools the MS raw data that have been generated by INSA’s Lab in order to investigate biological/molecular mechanisms responsible for protein changes that might be related with the development of SCD. The most pertinent proteins identified by these computational approaches associated with those pathologies will be selected for further validation as candidate biomarkers by using western blot methods. From all the identified 111 differentially expressed proteins (74 cytoplasmatic fraction and 37 membrane fraction) associated with SCD crisis event, Peroxiredoxin-2, Catalase, 70-kDa Heat shock protein and Heat shock protein 90 were validated with Western blot. From these 4 proteins only Peroxiredoxin-2 showed statically significant. Of the differentially expressed proteins that hypothetically may be associated with SCD, a promise candidate biomarker of crisis namely the Voltage-dependent anion-selective channel protein 1 (VDAC1) was found decreased. Our results suggest that vaso-occlusion episodes in SCD patients may be associated with decreased VDAC1 in their RBCs. This study indicated that SCD patients at crisis-state are under oxidative stress and the proteins such as PRDX2 and VDAC1 are promising candidates biomarkers for SCD crisis-state. In summary, the main objective of this project is to contribute to a better understanding of the molecular mechanisms associated with these pathologies, as well as to discover new diagnostic, prognostic or monitoring biomarkers for these diseases, leading to the development of new methods that would increase the quality of life of these patients.N/
Heat-mediated enrichment of α-synuclein from cells and tissue for assessing post-translational modifications
No full textα-synuclein (α-syn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of α-syn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward α-syn PTMs. Here, by taking advantage of α-syn thermostability, we applied a method to achieve high enrichment of soluble α-syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% α-syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of α-syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies.
In this study, we describe a method for enriching alpha-synuclein (α-syn) from a variety of biological samples, from cultured cells to brain tissues. Enrichment of α-syn was achieved by heating samples, further facilitating the identification of specific post-translational modifications by immunoblot, or mass spectrometry-based techniques. This approach will contribute to the clarification of the role of α-syn PTMs in Parkinson's disease
Proteome profiling in obstructive sleep apnea severity and treatment response towards early diagnosis and prognosis prediction
No full textObstructive 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/
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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