Identification of genetic risk factors for Behçet’s disease

Tese de doutoramento, Ciências Biomédicas (Genética), Universidade de Lisboa, Faculdade de Medicina, 2013Background: Behçet’s disease (BD) is a complex disorder characterized by a generalized vasculitis, whose pathophysiology remains unclear. The identification of genes involved in BD can help to elucidate the disease mechanisms and, ultimately, result in diagnostic and treatment advances.Objectives: To identify genetic risk factors implicated in BD susceptibility. Methods: We performed four independent studies: 1) Analysis of the role of the mitochondrial genome by testing the association of mitochondrial haplogroups and variants with BD risk in 615 Iranian BD cases and 434 controls; 2) Follow-up of IL10 and IL23RIL12RB2 associations, previously identified as BD risk factors, in 973 Iranian BD cases and 637 controls; 3) Gene expression profiling in 15 Portuguese BD cases and 14 controls and association testing of the differentially expressed genes in 976 Iranian BD cases and 839 controls; 4) A genome-wide association study for the Iranian population in DNA pools of 292 BD cases and 294 controls and replication of the association findings in 684 BD cases and 532 controls.Results: We identified a novel association of BD with the mitochondrial 12S rRNA gene (7.00E-03<P<3.80E-02); replicated the association of IL10 (P=2.53E-02) and IL23R-IL12RB2 loci (1.93E-06<P<1.78E-05) and identified the region upstream IL23R as the most associated one; identified EREG, AREG and NRG1 (members of the Neuregulin signalling) as downregulated in BD patients, found a novel association in the EREG-AREG locus (P=2.51E-02) and replicated three associations at NRG1 (6.61E-04<Pmeta<2.10E-03); and identified five coding variants at FUT2 associated with BD (2.97E-06<P<1.34E-04).Conclusions: During the course of this project we have uncover the mitochondrial genome,the neuregulin signaling and the FUT2 gene as novel players in BD susceptibility that may contribute to the abnormal immunological response observed in BD patients. We have further contributed to establish the IL10 and IL23R loci as worldwide risk factors for Behçet’s disease.Introdução: A doença de Behçet (DB) é uma doença complexa caracterizada por uma vasculite generalizada, cuja patofisiologia é ainda pouco conhecida. A identificação de genes envolvidos na DB pode ajudar a elucidar os mecanismos de doença levando a avanços a nível do diagnóstico e tratamento.Objetivos: Identificar factores de risco genético para a DB. Métodos: Realizou-se quatro estudos independentes: 1) Análise do papel do genoma mitocondrial, onde se testou a associação da DB com haplogrupos e variantes mitocondriais, em 615 casos e 434 controlos Iranianos; 2) Follow-up da associação dos genes IL10 e IL23RIL12RB, previamente identificados como factores de risco para a DB, em 973 casos e 637 controlos Iranianos; 3) Estudo de perfis génicos em 15 casos com DB e 14 controlos Portugueses e teste da associação dos genes diferencialmente expressos em 976 casos e 839 controlos Iranianos; 4) Estudo de associação no genoma inteiro (GWAS) para a DB na população Iraniana, em pools de ADN com 292 casos e 294 controlos, e replicação das associações identificadas em 684 casos e 532 controlos.Resultados: Identificou-se uma nova associação da DB com o gene mitocondrial 12S rRNA (7.00E-03<P<3.80E-02); Replicou-se a associação do IL10 (P=2.53E-02) e locus IL23R-IL12RB2 (1.93E-06>P<1.78E-05) e identificou-se a região regulatória do IL23R como a mais mais fortemente associada; Verificou-se a sub-expressão do EREG, AREG e NRG1 (pertencentes à via da Neuregulina) em pacientes com DB, a associação do locus EREG-AREG (P=2.51E-02) e replicou-se três associações no NRG1 (6.61E-04<Pmeta<2.10E-03); Identificou-se cinco variantes codificantes no FUT2 associadas com a DB (2.97E-06<P<1.34E-04). Conclusões: Durante o curso deste projecto, identificou-se o genoma mitocondrial, a via da Neuregulina e o FUT2 como novos intervenientes na suscetibilidade para a DB que podem contribuir para a resposta imunológica alterada observada nos pacientes com DB. Adicionalmente, contribui-se para estabelecer o IL10 e IL23R como fatores de risco mundiais para a DB

The antimicrobial activity of heterotrophic bacteria isolated from the marine sponge Erylus deficiens (Astrophorida, Geodiidae)

Interest in the study of marine sponges and their associated microbiome has increased both for ecological reasons and for their great biotechnological potential. In this work, heterotrophic bacteria associated with three specimens of the marine sponge Erylus deficiens, were isolated in pure culture, phylogenetically identified and screened for antimicrobial activity. The isolation of bacteria after an enrichment treatment in heterotrophic medium revealed diversity in bacterial composition with only Pseudoalteromonas being shared by two specimens. Of the 83 selected isolates, 58% belong to Proteobacteria, 23% to Actinobacteria and 19% to Firmicutes. Diffusion agar assays for bioactivity screening against four bacterial strains and one yeast, revealed that a high number of the isolated bacteria (68.7%) were active, particularly against Candida albicans and Vibrio anguillarum. Pseudoalteromonas, Microbacterium, and Proteus were the most bioactive genera. After this preliminary screening, the bioactive strains were further evaluated in liquid assays against C. albicans, Bacillus subtilis and Escherichia coli. Filtered culture medium and acetone extracts from three and 5 days-old cultures were assayed. High antifungal activity against C. albicans in both aqueous and acetone extracts as well as absence of activity against B. subtilis were confirmed. Higher levels of activity were obtained with the aqueous extracts when compared to the acetone extracts and differences were also observed between the 3 and 5 day-old extracts. Furthermore, a low number of active strains was observed against E. coli. Potential presence of type-I polyketide synthases (PKS-I) and non-ribosomal peptide synthetases (NRPSs) genes were detected in 17 and 30 isolates, respectively. The high levels of bioactivity and the likely presence of associated genes suggest that Erylus deficiens bacteria are potential sources of novel marine bioactive compounds

Biodiversity and characterization of Staphylococcus species isolated from a small manufacturing dairy plant in Portugal

The level and the diversity of the staphylococcal community occurring in the environment and dairy products of a small manufacturing dairy plant were investigated. Species identification was performed using different molecular methods, viz. Multiplex-PCR, amplified ribosomal DNA restriction analysis (ARDRA), and sodA gene sequencing. The main species encountered corresponded to Staphylococcus equorum (41 isolates, 39.0%), S. saprophyticus (28 isolates, 26.7%) and S. epidermidis (15 isolates, 14.3%). Additionally, low incidence of enterotoxin genes was obtained, with only 9 strains (8.6%) being positive for one or more toxin genes. With regard to antimicrobial resistance, 57.1% of the isolates showed at least resistance against one antibiotic, and 28.6% were multi-resistant, which might accomplish resistance for up to 6 antibiotics simultaneously. These results provided evidence that the presence of Staphylococcus species in dairy environment are mostly represented by S. equorum and S. saprophyticus, and illustrate that carrying antimicrobial resistance genes has become reasonably widespread in cheese and dairy environment.info:eu-repo/semantics/acceptedVersio

Apoptosis-associated microRNAs are modulated in mouse, rat and human neural differentiation

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRs or miRNAs) regulate several biological processes in the cell. However, evidence for miRNAs that control the differentiation program of specific neural cell types has been elusive. Recently, we have shown that apoptosis-associated factors, such as p53 and caspases participate in the differentiation process of mouse neural stem (NS) cells. To identify apoptosis-associated miRNAs that might play a role in neuronal development, we performed global miRNA expression profiling experiments in NS cells. Next, we characterized the expression of proapoptotic miRNAs, including miR-16, let-7a and miR-34a in distinct models of neural differentiation, including mouse embryonic stem cells, PC12 and NT2N cells. In addition, the expression of antiapoptotic miR-19a and 20a was also evaluated.</p> <p>Results</p> <p>The expression of miR-16, let-7a and miR-34a was consistently upregulated in neural differentiation models. In contrast, expression of miR-19a and miR-20a was downregulated in mouse NS cell differentiation. Importantly, differential expression of specific apoptosis-related miRNAs was not associated with increased cell death. Overexpression of miR-34a increased the proportion of postmitotic neurons of mouse NS cells.</p> <p>Conclusions</p> <p>In conclusion, the identification of miR-16, let-7a and miR-34a, whose expression patterns are conserved in mouse, rat and human neural differentiation, implicates these specific miRNAs in mammalian neuronal development. The results provide new insights into the regulation of neuronal differentiation by apoptosis-associated miRNAs.</p

Getting Value from Pulp and Paper Industry Wastes: On the Way to Sustainability and Circular Economy

The pulp and paper industry is recognized as a well-established sector, which throughout its process, generates a vast amount of waste streams with the capacity to be valorized. Typically, these residues are burned for energy purposes, but their use as substrates for biological processes could be a more efficient and sustainable alternative. With this aim, it is essential to identify and characterize each type of waste to determine its biotechnological potential. In this context, this research highlights possible alternatives with lower environmental impact and higher revenues. The bio-based pathway should be a promising alternative for the valorization of pulp and paper industry wastes, in particular for bioproduct production such as bioethanol, polyhydroxyalkanoates (PHA), and biogas. This article focuses on state of the art regarding the identification and characterization of these wastes, their main applied deconstruction technologies and the valorization pathways reported for the production of the abovementioned bioproductspublishe

Targeted therapy using phage technology: a computational and experimental breast cancer study

During the past two decades cancer biology knowledge has widely increased and shifted the paradigm of cancer treatment from nonspecific cytotoxic agents to selective, mechanism-based therapeutics. Initially, cancer drug design was focused on compounds that rapidly killed dividing cells. Though still used as the backbone of current treatments, these highly unspecific targeting drugs lead to significant toxicity for patients, narrowing the therapeutic index, and frequently lead to drug resistance. Therefore, cancer therapies are now based on cancer immunotherapy and targeted agents, whereas novel treatments are strategically combining both to improve clinical outcomes. Despite the nanotechnology advances dictating the development of targeted therapies in diverse classes of nano-based carriers, virus-based vectors still remain highly used due to its biocompatibility and specificity for the target. Particularly, bacteriophages are an interesting alternative ‘nanomedicine’ that can combine biological and chemical components into the same drug delivery system. The great potential of this novel platform for cancer therapy is the ability to genetically manipulate the virus-vector to display specific targeting moieties. Phage display technology, a general technique used for detecting interfaces of various types of interacting proteins outside of the immunological context, allows the target agents to locate the target (with an increased selection process for the specific binding – termed biopanning) and play their essential role inhibiting molecular pathways crucial for tumour growth and maintenance. Phage display specificity core is related with the binding of small peptides displayed at their coat or capsid proteins, enriched during biopanning. Bioinformatics plays an important role in testing and improving phage display libraries by effective epitope mapping, selecting from a large set of random peptides those with a high binding affinity to a target of interest. In this work we demonstrate the screening of a manually constructed 7-mer peptide library of M13KE phage particles against MDA-MB-231 and -435 cancer cell lines. Two peptides – TLATVEV and PRLNVSP – with high affinity for the referred cells were identified, respectively. Based on computationally predicted epitopes based on the peptides extracted from this library the linear peptide sequence was docked onto known membrane proteins from the used cell lines and peptides-proteins interactions were mapped. Umbrella sampling studies were performed to predict the binding affinity and to improve future rational design of binding peptides to these cancer cells

Enzymatic Potential of Filamentous Fungi as a Biological Pretreatment for Acidogenic Fermentation of Coffee Waste

This work was developed within the scope of the project CICECO-Aveiro Institute of Materials (UIDB/50011/2020, UIDP/50011/2020, and LA/P/0006/2020), and the Associate Laboratory for Green Chemistry-LAQV (UIDB/50006/2020 and UIDP/50006/2020). It was financed by national funds through the FCT/MCTES (PIDDAC) and, when appropriate, co-financed by FEDER under the PT2020 Partnership Agreement. Paulo C. Lemos acknowledges the support of FCT/MCTES for contract IF/01054/2014/CP1224/CT0005 and Joana Pereira thanks FCT/MCTES for her Ph.D. grant SFRH/BD/130003/2017.Spent coffee grounds (SCGs) are a promising substrate that can be valorized by biotechnological processes, such as for short-chain organic acid (SCOA) production, but their complex structure implies the application of a pretreatment step to increase their biodegradability. Physicochemical pretreatments are widely studied but have multiple drawbacks. An alternative is the application of biological pretreatments that include using fungi Trametes versicolor and Paecilomyces variotii that naturally can degrade complex substrates such as SCGs. This study intended to compare acidic and basic hydrolysis and supercritical CO 2 extraction with the application of these fungi. The highest concentration of SCOAs, 2.52 gCOD/L, was achieved after the acidification of SCGs pretreated with acid hydrolysis, but a very similar result, 2.44 gCOD/L, was obtained after submerged fermentation of SCGs by T. versicolor. This pretreatment also resulted in the best acidification degree, 48%, a very promising result compared to the 13% obtained with the control, untreated SCGs, highlighting the potential of biological pretreatments.publishersversionpublishe

Genetic Variants Underlying Risk of Intracranial Aneurysms: Insights from a GWAS in Portugal

Subarachnoid hemorrhage (SAH) is a life-threatening event that most frequently leads to severe disability and death. Its most frequent cause is the rupture of a saccular intracranial aneurysm (IA), which is a blood vessel dilation caused by disease or weakening of the vessel wall. Although the genetic contribution to IA is well established, to date no single gene has been unequivocally identified as responsible for IA formation or rupture. We aimed to identify IA susceptibility genes in the Portuguese population through a pool-based multistage genome-wide association study. Replicate pools were allelotyped in triplicate in a discovery dataset (100 IA cases and 92 gender-matched controls) using the Affymetrix Human SNP Array 6.0. Top SNPs (absolute value of the relative allele score difference between cases and controls |RASdiff|≥13.0%) were selected for technical validation by individual genotyping in the discovery dataset. From the 101 SNPs successfully genotyped, 99 SNPs were nominally associated with IA. Replication of technically validated SNPs was conducted in an independent replication dataset (100 Portuguese IA cases and 407 controls). rs4667622 (between UBR3 and MYO3B), rs6599001 (between SCN11A and WDR48), rs3932338 (214 kilobases downstream of PRDM9), and rs10943471 (96 kilobases upstream of HTR1B) were associated with IA (unadjusted allelic chi-square tests) in the datasets tested (discovery: 6.84E-04≤P≤1.92E-02, replication: 2.66E-04≤P≤2.28E-02, and combined datasets: 6.05E-05≤P≤5.50E-04). Additionally, we confirmed the known association with IA of rs1333040 at the 9p21.3 genomic region, thus validating our dataset. These novel findings in the Portuguese population warrant further replication in additional independent studies, and provide additional candidates to more comprehensively understand IA etiopathogenesis.FCT grant: (CMUP-ERI/TPE/0028/2013), FCT fellowships and research contracts (SFRH/BPD/35737/2007, SFRH/BPD/70008/2010, SFRH/BD/43895/2008 and Ciência and Investigator- FCT contracts)

A Role for Gene-Environment Interactions in Autism Spectrum Disorder Is Supported by Variants in Genes Regulating the Effects of Exposure to Xenobiotics

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9161282/Heritability estimates support the contribution of genetics and the environment to the etiology of Autism Spectrum Disorder (ASD), but a role for gene-environment interactions is insufficiently explored. Genes involved in detoxification pathways and physiological permeability barriers (e.g., blood-brain barrier, placenta and respiratory airways), which regulate the effects of exposure to xenobiotics during early stages of neurodevelopment when the immature brain is extremely vulnerable, may be particularly relevant in this context. Our objective was to identify genes involved in the regulation of xenobiotic detoxification or the function of physiological barriers (the XenoReg genes) presenting predicted damaging variants in subjects with ASD, and to understand their interaction patterns with ubiquitous xenobiotics previously implicated in this disorder. We defined a panel of 519 XenoReg genes through literature review and database queries. Large ASD datasets were inspected for in silico predicted damaging Single Nucleotide Variants (SNVs) (N = 2,674 subjects) or Copy Number Variants (CNVs) (N = 3,570 subjects) in XenoReg genes. We queried the Comparative Toxicogenomics Database (CTD) to identify interaction pairs between XenoReg genes and xenobiotics. The interrogation of ASD datasets for variants in the XenoReg gene panel identified 77 genes with high evidence for a role in ASD, according to pre-specified prioritization criteria. These include 47 genes encoding detoxification enzymes and 30 genes encoding proteins involved in physiological barrier function, among which 15 are previous reported candidates for ASD. The CTD query revealed 397 gene-environment interaction pairs between these XenoReg genes and 80% (48/60) of the analyzed xenobiotics. The top interacting genes and xenobiotics were, respectively, CYP1A2, ABCB1, ABCG2, GSTM1, and CYP2D6 and benzo-(a)-pyrene, valproic acid, bisphenol A, particulate matter, methylmercury, and perfluorinated compounds. Individuals carrying predicted damaging variants in high evidence XenoReg genes are likely to have less efficient detoxification systems or impaired physiological barriers. They can therefore be particularly susceptible to early life exposure to ubiquitous xenobiotics, which elicit neuropathological mechanisms in the immature brain, such as epigenetic changes, oxidative stress, neuroinflammation, hypoxic damage, and endocrine disruption. As exposure to environmental factors may be mitigated for individuals with risk variants, this work provides new perspectives to personalized prevention and health management policies for ASD.This work was supported by the Fundação para a Ciência e a Tecnologia (FCT), through funding to the project “Gene-environment interactions in Autism Spectrum Disorder” (Grant PTDC/MED-OUT/28937/2017) and to Research Center Grants UIDB/04046/2020 and UIDP/04046/2020 (to BioISI) and UIDB/00006/2020 (to Centro de Estatística e Aplicações da Universidade de Lisboa). This work used the European Grid Infrastructure (EGI) with the support of NCG-INGRID-PT/INCD (Portugal). This work was produced with the support of INCD funded by the FCT and FEDER under the project 01/SAICT/2016 n◦ 022153. JS, ARM, MA, and JV are fellows of the BioSys Ph.D Program and awardees of scholarships funded by FCT, with references: PD/BD/114386/2016, PD/BD/113773/2015, PD/BD/52485/2014, and PD/BD/131390/2017, respectively. AGP data was collected from patients genotyped in the context of Autism Genome Project (AGP), funded by NIMH, HRB, MRC, Autism Speaks, Hilibrand Foundation, Genome Canada, OGI, and CIHR. ASC data was collected from patients genotyped in the context of Autism Sequencing Consortium supported by NIH grants U01MH100233, U01MH100209, U01MH100229, and U01MH100239.info:eu-repo/semantics/publishedVersio

Peripheral axonal ensheathment is regulated by RalA GTPase and the exocyst complex

Funding This work was supported by H2020 Marie Skłodowska-Curie Actions [H2020- GA661543-Neuronal Trafficking to R.O.T.], Fundo Regional para a Ciência e Tecnologia [IF/00392/2013/CP1192/CT0002 to R.O.T.] and iNOVA4Health (UID/Multi/04462/2013) (co-funded by FCT-FEDER-PT2020).Axon ensheathment is fundamental for fast impulse conduction and the normal physiological functioning of the nervous system. Defects in axonal insulation lead to debilitating conditions, but, despite its importance, the molecular players responsible are poorly defined. Here, we identify RalA GTPase as a key player in axon ensheathment in Drosophila larval peripheral nerves. We demonstrate through genetic analysis that RalA action through the exocyst complex is required in wrapping glial cells to regulate their growth and development. We suggest that the RalA-exocyst pathway controls the targeting of secretory vesicles for membrane growth or for the secretion of a wrapping glia-derived factor that itself regulates growth. In summary, our findings provide a new molecular understanding of the process by which axons are ensheathed in vivo, a process that is crucial for normal neuronal function.publishersversionpublishe