Variabilidade genética das glicoproteínas plaquetárias GPIba e GPIIIa e a sua possível associação com o risco trombótico em doentes com Diabetes mellitus tipo 2

Dissertação de mestrado em Genética MolecularAs patologias vasculares são a principal causa de mortalidade e morbilidade na Diabetes mellitus tipo 2. Uma vez que um endotélio vascular íntegro é fundamental para a homeostasia, eventos isquémicos são frequentes nos indivíduos com Diabetes mellitus tipo 2. Na isquemia arterial, a exposição do conteúdo da placa de ateroma leva à activação das plaquetas por intermédio das glicoproteínas existentes na superfície das mesmas. O objectivo principal deste estudo é verificar se a presença de polimorfismos nas glicoproteínas plaquetárias poderão estar associados com o aparecimento de isquemia arterial do membro inferior nos indivíduos com DM tipo 2. Para isso, estudou-se por PCRRFLP os SNPs HPA-2 e KOZAK na GPIbα, o PlA1/A2 na GPIIIa, e um VNTR na GPIbα. Verificou-se que existem diferenças estatisticamente significativas para o polimorfismo VNTR entre o grupo controlo e o grupo com DM tipo 2. O polimorfismo KOZAK parece estar associado ao aparecimento de isquemia arterial do membro inferior em indivíduos com DM tipo 2. Estes resultados sugerem que o polimorfismo KOZAK poderá contribuir para um aumento da probabilidade de desenvolver isquemia arterial do membro inferior em indivíduos com DM tipo 2. Os nossos resultados também evidenciam a importância do estudo dos polimorfismos plaquetários uma vez que podem contribuir para estabelecer um perfil genético de risco em indivíduos com DM tipo 2.Vascular diseases are the most important cause of mortality and morbidity in patients with type 2 diabetes mellitus. Once a functional endothelium is important for homeostasis, ischaemic events are frequent in patients with type 2 diabetes mellitus. In arterial ischaemia, an exposition to contents of aterome plaque leads to platelet activation by glycoproteins that exist in platelets. The main goal of this study is to search if the presence of platelet glycoproteins polymorphisms are associated with arterial ischaemia of lower limbs in type 2 diabetes mellitus patients. It was analyze the HPA-2 and KOZAK SNPs in GPIbα, the PlA1/A2 SNP in GPIIIa and a VNTR in GPIbα, by PCR-RFLP. There were statistically significant differences for VNTR polymorphism between control group and type 2 diabetes mellitus group. It seems that the KOZAK polymorphism is associated with the presence of arterial ischaemia of lower limbs in type 2 diabetes mellitus patients. These results suggest that KOZAK polymorphism could contribute for an increased probability of developing arterial ischaemia of lower limbs in type 2 diabetes mellitus patients. Our results also show the importance of the study of polymorphisms in platelets since it could establish a genetic risk profile in patients with type 2 diabetes mellitus.Centro de Investigação em Tecnologias da Saúde - Projecto (AL/12/2006/CESPU

Lysis buffer properties: influence on S. epidermidis biofilm proteome analysis

Besides being part of human commensal flora, S. epidermidis has the ability to colonize and form biofilms in artificial implants. Due to the particular characteristics of biofilms, conventional methods used to disrupt and lyses biofilms from Gram positive bacteria may include association between mechanical, enzymatic and chemical methods. Nevertheless, proteomic characterization is highly dependent of the extraction procedure. In order to characterize proteome from S. epidermidis biofilms grown in glucose excess, we used mechanical lysis (glass beads) associated with two distinct lysis solution with different charge characteristic detergents, namely, SDS (an ionic detergent) or CHAPS (a zwitterionic detergent). Protein extracted was separated by SDS-PAGE and identified by LC-MS/MS. SDS lysis buffer combined with glass-beads showed the highest number of identified proteins (332 proteins). With zwitterionic detergent extraction, most the identified proteins presented a lower GRAVY value (grand average of hydropathy) and a protein molecular weight under 30 KDa. In overall, this work evidence that SDS lysis buffer is the optimal protocol to proteome analysis of S. epidermidis biofilms

Dormancy within Staphylococcus epidermidis biofilms: an immunoproteomic characterization

Dormant bacteria within biofilms contribute to biofilm heterogeneity. Consequently, physiological heterogeneity of biofilms may influence host immune response and tolerance to antibiotics. Recently, we described an in vitro model to modulate dormancy in S. epidermidis biofilms. Here, we present a study based on immunoproteomics, where we compared the reactive profile of S. epidermidis biofilm proteins with prevented and induced dormancy, to human sera. A total of 19 immunoreactive proteins were identified by MALDI-TOF/TOF. Most of these proteins present molecular functions, such as catalytic activity and ion binding. CodY and GpmA proteins were more reactive to sera when biofilm dormancy was induced, while FtnA and ClpP were more reactive when dormancy was prevented. This is the first work identifying protein immunoreactivity differences between bacterial biofilms with induced or prevented dormancy. Considering the importance of dormancy within biofilms, further studies on these proteins may provide insights into the mechanisms related to dormancy and help improving current understanding on how dormancy affects the host immune response

Towards Staphylococcus epidermidis biofilm dormancy characterization

Book of Abstracts of CEB Annual Meeting 2017info:eu-repo/semantics/publishedVersio

Proteomic profile of dormancy within Staphylococcus epidermidis biofilms using iTRAQ and label-free strategies

Staphylococcus epidermidis is an important nosocomial bacterium among carriers of indwelling medical devices, since it has a strong ability to form biofilms. The presence of dormant bacteria within a biofilm is one of the factors that contribute to biofilm antibiotic tolerance and immune evasion. Here, we provide a detailed characterization of the quantitative proteomic profile of S. epidermidis biofilms with different proportions of dormant bacteria. A total of 427 and 409 proteins were identified by label-free and label-based quantitative methodologies, respectively. From these, 29 proteins were found to be differentially expressed between S. epidermidis biofilms with prevented and induced dormancy. Proteins overexpressed in S. epidermidis with prevented dormancy were associated with ribosome synthesis pathway, which reflects the metabolic state of dormant bacteria. In the opposite, underexpressed proteins were related to catalytic activity and ion binding, with involvement in purine, arginine, and proline metabolism. Additionally, GTPase activity seems to be enhanced in S. epidermidis biofilm with induced dormancy. The role of magnesium in dormancy modulation was further investigated with bioinformatics tool based in predicted interactions. The main molecular function of proteins, which strongly interact with magnesium, was nucleic acid binding. Different proteomic strategies allowed to obtain similar results and evidenced that prevented dormancy led to an expression of a markedly different repertoire of proteins in comparison to the one of dormant biofilms.This work was funded by Fundacao para a Ciencia e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009, FCOMP-01-0124-FEDER-014309, QOPNA research unit (project PEst-C/QUI/UI0062/2013), RNEM (National Mass Spectrometry Network), and CENTRO-07-ST24-FEDER-002034. The authors also thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013, the Project NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER, and the project RECI/EBB-EBI/0179/2012, FCOMP-01-0124-FEDER-027462. VC has an individual FCT fellowship (SFRH/BD/78235/2011). NC is an Investigator FCT

Characterization of an in vitro fed-batch model to obtain cells released from S. epidermidis biofilms

Both dynamic and fed-batch systems have been used for the study of biofilms. Dynamic systems, whose hallmark is the presence of continuous flow, have been considered the most appropriate for the study of the last stage of the biofilm lifecycle: biofilm disassembly. However, fed-batch is still the most used system in the biofilm research field. Hence, we have used a fed-batch system to collect cells released from Staphylococcus epidermidis biofilms, one of the most important etiological agents of medical device-associated biofilm infections. Herein, we showed that using this model it was possible to collect cells released from biofilms formed by 12 different S. epidermidis clinical and commensal isolates. In addition, our data indicated that biofilm disassembly occurred by both passive and active mechanisms, although the last occurred to a lesser extent. Moreover, it was observed that S. epidermidis biofilm-released cells presented higher tolerance to vancomycin and tetracycline, as well as a particular gene expression phenotype when compared with either biofilm or planktonic cells. Using this model, biofilm-released cells phenotype and their interaction with the host immune system could be studied in more detail, which could help providing significant insights into the pathophysiology of biofilm-related infections.European Union funds (FEDER/COMPETE) and by national funds (Fundação para a Ciência e a Tecnologia-FCT) under the project with reference FCOMP-01-0124-FEDER-041246 (EXPL/BIA-MIC/0101/2013). The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit and the project FCOMP-01-0124-FEDER-027462 (RECI/BBB-EBI/0179/2012); SFRH/BPD/99961/2014 and SFRH/BD/78235/201