Faculdade de Medicina e Ciências Biomédicas
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Browsing Faculdade de Medicina e Ciências Biomédicas by Field of Science and Technology (FOS) "Ciências Médicas::Biotecnologia Médica"
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- Analysis of the transcriptional regulatory network: underlying heart developmentPublication . Machado, Rui Sotero Rodrigues; Futschik, Matthias E.; Bragança, JoséHeart development is a highly complex process with a series of precisely spatially and temporally ordered events on molecular level. To understand how these events are controlled and coordinated, it is necessary to study the underlying gene expression and its regulation. While many studies have been carried out in the examination of single genes and their expression patterns, comprehensive analyses of genome-wide expression profiles associated with cardiomyogenesis (i.e. the differentiation of stem cells into cardiomyocytes) are still rare. In fact, no study exists to date which compares and consolidates the publicly available genome-wide measurement for cardiomyogenesis. Such endeavour however is important, as it is well known that individual microarray studies can be seriously compromised by artefacts. In contrast, the combination of various expression studies, which was performed in my study, can lead to more reliable results and help elucidate the different aspects of heart development and repair. Furthermore, a brief study was performed regarding the potential risk of originating cancer or teratomas from stem cell therapy. Finally, I carried out a network-based analysis, to identify regulatory actions between genes, based on published interaction data. This type of analysis can also help to identify novel genes with a role in heart development and provide new valuable targets to future experimental laboratorial analysis. The combination of the multiple dataset is thus an important approach to gain better insights of the different heart development processes as well as regenerative medicine applied to the heart.
- Changes in components of the brain extracellular matrix after experimental ischemic strokePublication . Guerreiro, Carla Sofia de Jesus; Wieloch, Tadeusz; Araújo, Inês; Quattromani, Miriana JleniaStroke is the 3rd cause of death in the world. During stroke, there is a disruption in the blood supply to the brain leading to rapid loss of brain function. Ischemic strokes are caused by obstruction of the blood supply, while hemorrhagic strokes results from rupture of a blood vessel. Eight-five percent of the strokes are ischemic. The only treatment recommended for acute ischemic stroke is the recombinant tissue activator of plasminogen but only a few percentages of patients are eligible for rtPA administration. Approximately 30% of the ischemic stroke victims die and 30% become severely disabled, resulting in among others deficits in motor function in the contralateral musculature. Spontaneous recovery occurs during weeks to months following injury. There are many physiological and anatomical examples of cortical brain plasticity and one of the most potent modulators of cortical structure and function is behavioral experience. Functional recovery after stroke can be enhanced by physical training in stroke patients. In the animal settings, physical training can be accomplished by enriched environment (EE). EE refers to housing conditions, either home cages or exploratory chamber, that facilitate enhanced sensory, cognitive and motor stimulation relative to standard housing conditions. The extracellular matrix (ECM) is important in the regulation of brain plasticity but is also a potential hampering factor for recovery after stroke. It is known that EE affects chondroitin sulfate proteoglycans (CSPGs) present in ECM, leading to functional recovery. Matrix metalloproteinases (MMPs) are able to cleave ECM components. There are some evidences that beta-dystroglycan (β-DG) is a MMP-9 target. After the degradation of β-DG, there is a 30 kDa product. The aim of this work is to explore how EE affects β-DG and gelatinases over 1 week of recovery after experimental stroke, performed as photothrombosis (PT). We show that EE does not affect the infarct size and improves tactile/proprioceptive response to limb stimulation. We found that β-DG is mostly present in vessels across the brain cortex and animals housed in an EE had a higher degradation than STD animals when comparing to sham non-operated animals. β-DG can be related with changes in the ECM that leads to brain plasticity, promoting functional recovery after experimental stroke, possibly due to MMPs enzymatic activity.
- Evaluation of pEPI-1 and pEPito expression systems for gene transfer to the retinaPublication . Calado, Sofia de Amaral Melo; Silva, GabrielaO objectivo de estudo do nosso laboratório é o desenvolvimento de vectores não virais para a terapia génica ocular. O principal objectivo da terapia génica é a introdução de material genético no interior das células. Em teoria, o veículo ideal para a terapia génica é aquele que consegue penetrar eficientemente a membrana celular e libertar o material genético, sem desencadear uma resposta imunológica agressiva.
- Identification of novel molecular determinants of tissue mineralization in fishPublication . Rosa, Joana Alexandra Teixeira; Cancela, Leonor; Laizé, VincentThe identification of genes involved in signaling and regulatory pathways, and matrix formation is paramount to the better understanding of the complex mechanisms of bone formation and mineralization, and critical to the successful development of therapies for human skeletal disorders. To achieve this objective, in vitro cell systems derived from skeletal tissues and able to mineralize their extracellular matrix have been used to identify genes differentially expressed during mineralization and possibly new markers of bone and cartilage homeostasis. Using cell systems of fish origin and techniques such as suppression subtractive hybridization and microarray hybridization, three genes never associated with mechanisms of calcification were identified: the calcium binding protein S100-like, the short-chain dehydrogenase/reductase sdr-like and the betaine homocysteine S-methyltransferase bhmt3. Analysis of the spatial-temporal expression of these 3 genes by qPCR and in situ hybridization revealed: (1) the up-regulation of sdr-like transcript during in vitro mineralization of gilthead seabream cell lines and its specificity for calcified tissues and differentiating osteoblasts; (2) the up-regulation of S100-like and the down-regulation of bhmt3 during in vitro mineralization and the central role of both genes in cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish. While expression of S100-like and bhmt3 was restricted to calcified tissues, sdr-like transcript was also detected in soft tissues, in particular in tissues of the gastrointestinal tract. Functional analysis of gene promoters revealed the transcriptional regulation of the 3 genes by known regulators of osteoblast and chondrocyte differentiation/mineralization: RUNX2 and RAR (sdr-like), ETS1 (s100-like; bhmt3), SP1 and MEF2c (bhmt3). The evolutionary relationship of the different orthologs and paralogs identified within the scope of this work was also inferred from taxonomic and phylogenetic analyses and revealed novel protein subfamilies (S100-like and Sdr-like) and the explosive diversity of Bhmt family in particular fish groups (Neoteleostei). Altogether our results contribute with new data on SDR, S100 and BHMT proteins, evidencing for the first time the role for these three proteins in mechanisms of mineralization in fish and emphasized their potential as markers of mineralizing cartilage and bone in developing fish.
- Interferindo com o "checkpoint" mitótico para potenciar a ação de agentes anti-mitóticosPublication . Teixeira, Joana Filipa Tomás; Bousbaa, Hassan; Silva, Patrícia; Tavares, ÁlvaroAtualmente, várias terapias anti-cancro apresentamuma toxicidade associada e são confrontadas com resistências por parte de muitos cancros. Por exemplo,em quimioterapia vários cancros exibem resistência a agentes anti-mitóticos como o Taxol. Estes agentesinterferem com a formação ou a dinâmica dos microtúbulos, o que leva a uma paragem das células em mitose, devido à ativação do “checkpoint” mitótico, o mecanismo que permite a correta segregação dos cromossomas.No entanto as célulastumorais resistentes ao taxolconseguem contornar esta paragem e continuar a sua proliferação. Posto isto, o“checkpoint” mitóticotem sido sugerido como um potencial alvo no tratamento anti-cancro. Assim este trabalho teve comoobjetivo inicial a avaliação do potencial inibidor de pequenas moléculas contra uma proteína envolvida no checkpoint mitótico (BubR1). Os resultados obtidos mostraram que uma das pequenas moléculas analisadas, demonstrou ter uma atividade anti-proliferativa em células HeLa, demonstrando uma possível interferência com a função da proteína BubR1. O objetivo principal deste trabalho consistiu emsensibilizar células tumorais à ação do taxol, depletando proteínas envolvida no silenciamento do“checkpoint” mitótico (Spindly) e na saída da mitose (Cdc20). Os resultadosdemonstraram que a depleção das proteínas leva a uma paragem das células em mitose, mais eficiente após a depleção da proteína Spindly. Foi demonstrado também uma redução da viabilidade e da capacidade proliferativa das células,com uma reduçãomais acentuada apósa combinação da depleção com o Taxol. Assim este trabalho sugere que a depleção destas duas proteínas ajuda a sensibilizar as células à ação do taxol nas concentrações usadas em quimioterapia.
- Lymphotoxin-beta receptor and RANK signaling in TEL-JAK2-induced T-cell leukemiaPublication . Fernandes, Mónica Alexandra Teotónio; Santos, Nuno Rodrigues dosT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy that arises from the combination of genetic and epigenetic alterations in thymic T-cell precursors and extracellular signals provided by the microenvironment. It was previously found that RelB expression in non-hematopoietic stromal cells promoted T-cell leukemogenesis in the EμSRalpha-TEL-JAK2 transgenic (TJ2-Tg) mouse model. In thymic stromal cells, RelB is a transcription mediator of lymphotoxin-beta receptor (LTβR). Lymphotoxin-mediated activation of LTβR has been implicated in physiological crosstalk between T cells and lymphoid organ stromal cells, but also pathological processes, including carcinogenesis. Since its role in T-ALL has remained elusive, we aimed to determine whether LTβR signaling is activated and playing a role in TEL-JAK2-induced leukemogenesis. In TJ2-Tg thymic lymphomas, activation of LTα1β2-LTβR signaling axis was supported by LTβRencoding gene expression, while the genes encoding its cognate ligand, lymphotoxin (LT)-α and LTβ, were found to be expressed by leukemic T cells, in an NF-κB-dependent manner. LTα1β2 protein was detected at the surface of TJ2-Tg leukemic cells only upon ex vivo culture or mitogenic stimulation. Moreover, we found that cell-surface LTα1β2 is downmodulated in vivo, indicating ongoing signaling. Further supporting a role for lymphotoxin signaling, LTβR genetic deficiency delayed TEL-JAK2-induced leukemia onset, but the tumor load in lymphoid organs and leukemia cell surface phenotype were comparable in end-stage disease. In accordance, the detection of reduced proportions of malignant thymocytes in TJ2-Tg;Ltbr-/- mice with no signs of disease implicated LTβR in early stages of leukemia development. Together, these data indicate that T-ALL-derived lymphotoxin activates LTβR signaling in thymic stromal cells, promoting leukemogenesis. Importantly, lymphotoxin-encoding genes were expressed in T-ALL patient samples, indicating that these may be also involved in human disease. Thus, future studies should provide a better understanding on how cellular crosstalk mediated by the lymphotoxin-LTβR axis supports T-ALL and assess the utility of blocking LTβR signaling as a novel therapeutic approach.
- MEF2C: expression, regulation and interaction with target genes in health and diseasesPublication . Adrião, Andreia Lúcia Gonçalves; Cancela, Leonor; Grazina, Manuela; Conceição, NatérciaMEF2C is the first MEF2 family factor to be expressed during embryonic development. Zebrafish has been proven to be an excellent model for human genetic diseases and has two MEF2C orthologues, mef2ca and mef2cb. The involvement of MEF2C in different developmental processes was studied in zebrafish through expression analysis and usage of mutant lines as loss of function models. Alterations in this gene were related to cardiac defects and its haploinsufficiency was linked to a human disease (MRD20) whose patients show abnormal neuronal and craniofacial development. Recently this gene was reported to be involved in dementia conditions such as Alzheimer disease, Parkinson disease and amyotrophic lateral sclerosis. Our work aims to further validate zebrafish as an animal model to study MEF2C related pathologies focusing on craniofacial and neuronal issues. Our in silico analysis demonstrated a high conservation of mef2c pattern of chromosome localization, protein structure and sites of mRNA expression throughout evolution. We also identified two transcriptional start sites for mef2cb that were conserved through evolution. These are related to the occurrence of alternative promoters that appear to be differentially regulated by Sox9b, Sox10 and Runx2, three nuclear factors associated to craniofacial or neuronal development. Through the use of Mef2ca and Mef2cb loss of function mutant lines, we described the craniofacial phenotype resulting from the absence of both mef2c isoforms in zebrafish. We concluded that both orthologues are involved in cartilage, bone and brain development in zebrafish and we described the molecular profile of specific marker genes resulting from mef2c loss of function. Our results allow us to consider zebrafish as a valuable animal model for analysis concerning MEF2C related pathologies. To complement our work we performed a pilot study in which we describe for the first time an alteration in MEF2C gene in a patient with frontotemporal lobar degeneration, suggesting an association between MEF2C and this disease, a result that extends the involvement of this gene to a previously unsuspected human pathology.
- Molecular and functional analysis of DAND5 in human Congenital Heart Disease (CHD)Publication . Cristo, Fernando Jorge Pego; Belo, JoséThe majority of congenital heart disease (CHD) is sporadic, with a minority of cases associated with a known genetic abnormality. Combinations of geneticenvironmental factors are implicated in the etiology of the disease. Recently, several studies, using mostly animal models, unraveled that perturbations in the molecular processes that precede the beginning of heart development might also be at the origin of CHD. In fact, some of the most complex CHDs are found associated with laterality defects, a disorder resulting from abnormal Left-Right axis formation. In our laboratory, the identified mouse Cerberus-like2 (Cerl2 – human DAND5), a protein that inhibit Nodal signaling, prompt us to study cardiac and laterality diseases, since the generated Cerl2 KO mice display a wide range of laterality defects and CHD. Considering the high conservation of genetic pathways regulating cardiac development in mouse and human, the main objective of the present thesis was the study of human genes involved in the Nodal pathway, focusing mostly in DAND5, in a CHD and/or laterality defects patients cohort. The sequence analysis of DAND5 revealed two patients displaying the same p.R152H variant, resulting in a substantial decreased in the function of the protein. We propose that p.R152H acts as a risk allele for CHD and/or laterality defects. In addition, we found two alterations in NODAL, two alterations in PITX2C and one alteration in CFC1. We hypothesized that the NODAL p.H165R variant can act as a common modifier and the intronic variants in NODAL and PITX2C might cause alterations in the splicing pattern of the mRNA molecules. Moreover, we generated patient-specific iPSCs to understand the molecular mechanisms of disease behind the DAND5 nucleotide variant. Although we cannot make a clearly genotype-phenotype correlation, the variants here identified probably increase the disease susceptibility due to the resulting abnormal Nodal signaling. Because most of the patients presented more than one alteration, the cumulative effect of each variant within the pathway seems to enhance even more these risk. Therefore, the imbalance in dosage-sensitive Nodal signaling is a common denominator for laterality defects and associated CHDs.
- Nano and microparticles as carriers for alveolar macrophage targeting in pulmonary tuberculosis therapyPublication . Cunha, Ludmylla Costa; Grenha, AnaTuberculosis (TB) is a leading infectious cause of death worldwide, even though a vaccine and several effective antibiotics are available for its prevention and treatment. Global TB control is very difficult due to various factors, including late diagnosis and patient non-compliance to long-term treatments, which leads to a high incidence of extensive resistance to effective anti-TB drugs. Overall, there are significant challenges associated with conventional TB therapy, including (i) drug resistance and toxicity; (ii) patient non-compliance, given the long-term therapy and severe side effects; (iii) and drug-drug interactions, particularly with antiretroviral drugs in patients co-infected with TB and HIV. Thus, the situation has come to a point where the development of novel intervention strategies is urgently needed. In this context, the pulmonary delivery of anti-TB drugs is a promising approach to treat lung TB. The disease represents approximately 80% of total cases, and thus the lung has been explored as an effective route for the delivery of drugs. This strategy not only allows targeting the infected organ instantly, but it can also reduce the systemic adverse effects of the antibiotics, which are main reasons for patient non-compliance. However, pulmonary drug delivery faces some limitations related with the proper airway structure, local degradation of drugs, and mucociliary clearance. In order to overcome some of these limitations of lung delivery, drug microencapsulation appears as a potential approach. In this sense, this work aimed at producing inhalable microparticles that efficiently associate two anti-TB drugs, isoniazid (INH) and/or rifabutin (RFB), for an application in pulmonary TB therapy. Fucoidan (FUC) and chitosan (CS) were the biomaterials selected to compose the matrix of the carriers. FUC is a polysaccharide composed of fucose units that has been reported to be specifically recognised by surface receptors of alveolar macrophages (the host cells of Mycobacterium tuberculosis). Likewise, CS is a polysaccharide composed of N-acetylglucosamine and D-glucosamine residues, the former being also specifically recognised by macrophages, according to the literature. This recognition by macrophages is believed to potentiate phagocytosis. The first approach involved the production of nanoparticles and it was considered that subsequent microencapsulation of the nanocarriers would be necessary to overcome aerodynamic limitations of nanosized carriers and their ability to reach the alveolar zone. Nanoparticles were spontaneously obtained by complexing FUC with CS, resulting from several formulations (polymeric mass ratio varying from 4:1 to 1:4). The produced unloaded FUC/CS nanoparticles presented average size range of 159 – 266 nm, PdI ranging between 0.21 and 0.36, and zeta potential varying from -39 mV to +12 mV, following the alteration of the mass ratios. The ability of FUC/CS nanoparticles to associate anti-TB drugs was assessed, and tests initiated with the incorporation of RFB, which was associated to obtain final polymer/drug mass ratio of 10/1 (w/w). Several attempts were made unsuccessfully, and therefore the work continued using another production method. Nanoprecipitation technique was then used, resulting in FUC nanoparticles and CS nanoparticles with mean size of 500 nm and 700 nm, respectively. However, the obtained nanoparticles showed little uniformity in size, indicated by PdI values, varying between 0.55 and 0.83. Moreover, an optimal protocol to obtain FUC- and CS-based nanoparticles that efficiently encapsulate INH and RFB could not be established. Taking into consideration the unsuccessful nanoparticle production and time restraints to accomplish the aims of the PhD plan, it was decided to focus the study on the development of polymeric microparticles. Microparticles were produced by spray-drying, associating the model drugs (INH and RFB), either separately or in combination. FUC microparticles effectively associated INH (95%) and RFB (81%), separately. Likewise, FUC microparticles loaded with the two anti-TB drugs simultaneously were also successfully produced, demonstrating high drug association efficiency (97% for INH and 95% for RFB). All FUC-based microparticles evidenced favourable aerodynamic properties for deep lung delivery upon inhalation. Single drug-loaded FUC microparticles showed aerodynamic diameter (MMAD) in the range of 2.0–3.8 μm. Likewise, the dual drug-loaded dry powder presented aerodynamic diameter of 3.6–3.9 μm. Overall, the formulations evidenced no cytotoxic effects on human alveolar epithelium cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells at the highest tested concentration (1 mg/mL). Nonetheless, this dose is considered overestimated compared to that effectively observed in vivo. The produced FUC microparticles also exhibited a propensity to be captured by macrophages or macrophage-like cells (target cells) in a dose-dependent manner. Particularly, dual drug-loaded microparticles displayed ability to activate the target cells and, moreover, effectively inhibited mycobacterial growth in vitro, preserving the bactericidal activity of the drugs. In vivo lung administration (BALB/c mice) of unloaded FUC microparticles indicated, in a preliminary assay, that the carriers induced no allergic responses. CS microparticles also associated INH (90%) and RFB (97%) efficiently, in separate formulations, whereas dual drug-loaded formulation resulted in 93% association efficiency for INH and 99% for RFB. All formulations presented adequate properties for deep lung delivery, with aerodynamic diameters ranging between 2.5 and 4 μm. Absence of toxicity was observed in human alveolar epithelium (A549 cells) but, as observed for FUC carriers, the highest tested concentration of microparticles (1 mg/mL) decreased the viability of macrophage-differentiated THP-1 cells upon 24 h exposure. This dose is however believed to be overestimated, as aforementioned. CS microparticles further evidenced strong ability to be internalised by macrophage-like cells (percentage of phagocytosis up to 99.9%), regardless of the dose. Yet, dual drug-loaded carriers induced macrophage activation and effectively inhibited the growth of mycobacteria in vitro. Moreover, the biomaterial (CS) was well tolerated by BALB/c mice upon pulmonary administration of unloadead CS microparticles. Overall, the obtained data gave positive indications on the potential of the proposed systems for an application as inhalable tuberculosis therapy.
- Regulation of neurogenesis by calpains: revelance for post-injury brain repairPublication . Machado, Vanessa Mendes; Araújo, InêsNo cérebro adulto dos mamíferos, novos neurónios são continuamente adicionados em duas regiões neurogénicas principais: a zona subventricular, nas paredes dos ventrículos laterais, e a zona subgranular, no giro dentado do hipocampo. As células estaminais neurais presentes nestes nichos têm a capacidade de proliferar e dar origem a neuroblastos em migração, que acabam por se diferenciar em novos neurónios. Curiosamente, no evento de uma lesão cerebral, a proliferação das células estaminais neurais aumenta, possivelmente numa tentativa de reparação do dano cerebral. No entanto, esta reparação é limitada pela redução da migração celular ou pela diminuição da sobrevivência dos novos neurónios. Descobrir como aumentar a eficiência da neurogénese após lesão poderá ser portanto benéfico para a reparação cerebral. Por outro lado, o dano cerebral também pode activar diversas protéases, incluindo as calpaínas. As calpaínas são proteases ubíquas, sabendo-se que estão envolvidas na proliferação, migração e diferenciação celular em diversos sistemas. No cérebro, a inibição das calpaínas tem efeito neuroprotector, mas os seus efeitos na neurogénese permanecem ainda pouco conhecidos. A delecção do inibidor endógeno das calpaínas, a calpastatina, já anteriormente mostrou ser capaz de reduzir as primeiras fases da neurogénese in vivo. No presente trabalho, mostramos que a inibição das calpaínas é capaz de reverter este efeito, implicando-as ainda mais no processo. Uma vez que a inibição das calpaínas parece ser um alvo promissor para o aumento da neurogénese após lesão, avaliámos várias fases do processo neurogénico, bem como a recuperação das alterações do comportamento cognitivo após lesão no hipocampo induzida por crises epilépticas, em murganhos com sobrexpressão de calpastatina. Avaliámos ainda os efeitos da delecção das calpaínas 1 e 2 nas primeiras fases da neurogénese no hipocampo. Observámos que o padrão de aumento da neurogénese após lesão foi mantido em murganhos com sobrexpressão de calpastatina, apesar de alguma recuperação cognitiva indicar algum grau de neuroprotecção, que por si só poderá ter mascarado a influência da inibição das calpaínas na neurogénese. Murganhos com falta de calpaínas, no entanto, não apresentaram alterações das primeiras fases da neurogénese no hipocampo, sugerindo que as calpaínas apenas afectam este processo quando a sua actividade está acima de um certo limite, abaixo do qual a neurogénese não é afectada. Isto significa que a inibição das calpaínas poderá potencialmente ser útil para prevenir a redução da formação de novos neurónios após lesão, caso os níveis de actividade das calpaínas possam ser reduzidos abaixo de um certo limite.