Faculdade de Medicina e Ciências Biomédicas
Permanent URI for this community
Browse
Browsing Faculdade de Medicina e Ciências Biomédicas by Field of Science and Technology (FOS) "Ciências Médicas::Ciências da Saúde"
Now showing 1 - 10 of 39
Results Per Page
Sort Options
- Androgen receptor expression in triple-negative breast cancerPublication . Rodrigues, Filipa Nascimento; Braga, SofiaWorldwide, 3 million the women are diagnosed with breast cancer which is the most common malignancy in female gender. Breast Cancer is a heterogeneous disease and clinically it is evaluated using three markers: the estrogen receptor (ER), the progesterone receptor (PR) and the epidermal growth factor receptor type 2 (HER2). The tumors that do not express any of these receptors are called triple-negative breast cancers (TNBC). TNBC represent approximately 20% of BC. These tumors constitute an important clinical challenge, as they do not respond to endocrine treatment and to anti HER2 directed therapy. As a group they harbor an aggressive clinical phenotype with early development of visceral metastases and a poor long-term prognosis. The only systemic treatment for TNBC is chemotherapy. Numerous experiments and trials have been done to try to understand what is targetable in TNBC and if so, if the clinical trials achieve their endpoints. Today, the trend in clinical practice is individualized treatment based on molecular biology markers of tumor and patient. Lehmann et. al. attempted to characterize TNBC and put forward a microarray signature that divides these tumors in 6 groups, according to differential gene expression profiles: basal-like 1 (BL1); basal-like 2 (BL2); immunomodulatory (IM); mesenchymal (M); mesenchymal stem–like (MSL) and luminal androgen receptor (LAR). Androgen receptor is member of the steroid hormone receptor family, expressed in more than 70% of breast cancers and has been implicated in breast cancer pathogenesis. The role of AR is of particular interest in patients with TNBC. In this project, immunohistochemistry was used to characterize the AR expression in a consecutive cohort of TNBC cases from Hospital CUF Lisboa. The immunohistochemical panels of markers used to characterize this subgroup of breast cancer, along with AR, were the CK 5/6, p-cadherin, PHH3 and EGFR in an attempt to further characterize TNBC subtypes. Of all the TNBC cases, 56% (28/50) were CK5/6, 0; 22% (11/50) were CK5/6, 1+; 12% (6/50) were CK5/6, 2+; and 10% (5/50) were CK5/6, 3+. In 96% (48/50) the expression of p-cadherin was positive while only 4% (2/50) was negative. Regarding EGFR, 38% (19/50) were EGFR, 0; 26% (13/50) were EGFR, 1+; 24% (12/50) were EGFR, 2+; and 12% (6/50) were EGFR, 3+. The expression of AR was considered positive in 62% of cases of TNBC. We have known from the literature that AR status is a significant independent prognostic factor, although we cannot show this in this sample. In this study was not possible to implement the Lehmann classification because it was not possible to separate the different TNBC by Immunohistochemical staining into 6 different categories of the Lehmann classification. The functional role of AR in breast cancer remains unclear, further exploration of this area could expand the repertoire of potential treatments for patients with AR+ TNBC.
- Avaliação da expressão de PD-L1 e de subpopulações linfocitárias em cancro da mamaPublication . Neto, Cátia Sofia Maltez; Braga, Sofia Azambuja; Borralho, PaulaO cancro da mama é atualmente o cancro mais comum entre as mulheres, com uma incidência anual de 14,9 milhões de novos casos no mundo e estima-se que esse valor continue a aumentar. Com o avanço da tecnologia têm existido melhorias significativas na taxa de sobrevivência dos doentes, provavelmente devido aos avanços nos tratamentos e à deteção precoce dos tumores com a melhoria dos programas de rastreio. Os subtipos de cancro da mama e a presença de tumour infiltrating lymphocytes (TILs), têm sido fatores importantes para o prognóstico e tratamento dos doentes. O ligando da proteína celular de morte programada 1 (PD-L1) tem sido também indicado como biomarcador preditivo de resposta ao tratamento. O objetivo deste estudo foi avaliar a expressão de PD-L1 e das subpopulações linfocitárias CD3, CD4, CD8 e CD20 em doentes diagnosticadas com carcinoma da mama e relacionar com os respetivos dados clínico-patológicos. Foi utilizada a técnica de citometria de fluxo para a escolha das doentes que possuíam TILs e recorremos à técnica de imunohistoquímica para uma avaliação da marcação linfocitária intratumoral e peritumoral das diferentes subpopulações linfocitárias. Foi possível concluir com este estudo que existem mais TILs na zona adjacente ao tumor do que no seu interior e que o PD-L1 está associado a tumores com graus mais agressivos. Conseguimos comprovar ainda que existiu uma maior marcação linfocitária nos subtipos RE positivo, Her2 negativo, ou seja nos subtipos de cancro da mama menos agressivos, e podemos assim concluir que uma maior marcação de TILs está correlacionada com um melhor prognóstico para a doente.
- Characterisation of PRKRA and WDR45 gene function, involved in Parkinson's diseasePublication . Bordone, Marie Catherine; Lewis, Patrick; Araújo, InêsThe PRKRA gene is situated on human chromosome 2p. It plays an important role in the regulation of gene expression in interferon–treated and virus–infected animal cells and is also implicated in the control of cell growth, proliferation and differentiation. The downstream target of this activation is a stress response protein involved in the Protein Kinase R (PKR) signalling pathway that mobilizes somatic cell death programs. It has been shown that aggregates of phosphorylated PKR are increased in the hippocampus of patients with Parkinson’s disease. The P222L mutation in this gene is associated to dystonia-parkinsonism syndrome (DYT16) although the mechanism underlying this disorder is not yet understood. BPAN (Beta propeller associatied neurodegeneration) disorder is caused by mutations in the WDR45 gene, which results in loss of function, and presents with some dystonia-parkinsonism features. This gene is situated on chromosome X (Xp11.23) and the mutations that occur are de novo, suggesting an atypical X-linked pattern disorder. WDR45 (WIPI4) belongs to the WD-40 family and is characterised by a seven-bladed β structural shape. Its ortholog in yeast is the Atg18, known to be involved in autophagy, so it is thought that WDR45 acts in the early steps of the autophagy cascade as a regulator of the ATG9A marked vesicles that transiently localize to the autophagosome formation site and induce autophagosome formation. WDR45 mutations primarily affect the brain, despite expression of the gene in several human tissues, suggesting that autophagy plays an important role in the brain. To date, WDR45 loss of function has been thought to cause impairments in autophagy, leading to a neurodevelopment and neurodegenerative phenotype. The main goals of this thesis, were to analyse if the PKR pathway was altered by overexpressing PRKRA wild-type and mutated in the HT1080 cell line and to investigate the impact of overexpressing WDR45 in H4, HEK and SHSY5 cell lines, in order to possibly provide insights with regard to the mechanisms that are underlie BPAN, DYT16 and Parkinson’s disease. These goals were performed by western blots and analyzing well known hallmarks of autophagy such as LC3 and p62. Immunocytochemistry analysis was also performed to investigate the localisation of WDR45 within the cell as well if the autophagy was induced, in standard and induced autophagy conditions.
- Characterization of bone pathologies in the ins2+/akita mouse, a new model for diabetes insulindependent: contribution for a better understanding of the disease in humansPublication . Carvalho, Filipe Ricardo Pires de; Cancela, Leonor; Gavaia, Paulo J.In the past 30 years developed and developing countries faced significant lifestyle changes that made the incidence of diabetes mellitus to reach pandemic proportion worldwide. This increase, seen in both types of the disease, made diabetes mellitus one of the leading causes of morbidity and mortality of our times. Bone is one t of several organs that are affected by diabetes mellitus, being morbidity caused by bone fractures highly correlated to diabetic bone. Alterations in the constituents of bone, microarchitecture changes and bone loss have been pointed as the main reasons for its fragility. This work aimed to contribute to identify the molecular players or the functional changes affecting bone and caused by diabetes mellitus. In chapter 2, using as animal model of type 1 diabetes mellitus the Ins2+/akita mouse, we identified bone growth retardation related to growth plate impairment. These changes were associated to reduced expression of Igf1 and increased expression of cartilage degradation enzymes like Adams-5. We also identified severe microarchitecture changes caused by reduced bone formation and resorption that could be explained by leptin deficiency and/or decreased insulin signaling. In chapter 3, we concluded that paricalcitol (vitamin D analog) and cinacalcet (calcimimetic), two drugs used for the treatment of secondary hyperthyroidism, have beneficial effects in fin regeneration and mineralization in a zebrafish model of diabetes. These results could be explained by the downregulation of pthr suggesting reduced signaling of parathyroid hormone, that is a potent activator of bone remodeling, and increased expression of runx2, indicating increased osteoblast differentiation. Increased expression of the two zebrafish insulin genes, insa and insb, could be observed, suggesting that both drugs promote an increase in insulin signaling. In chapter 4, we suggest that in humans, the insulin paralog gene, INS-IGF2, does not have a redundant function with the insulin gene, in contrast with what is seen in mice and probably in zebrafish. We could also conclude that extrapancreatic expression of insulin is present in human, mouse and zebrafish. In humans this expression results mainly from expression of the ancestral gene while in mice and zebrafish it is due to expression of the insulin paralogues.
- Characterization of pro- and anti-angiogenic factors in models of diabetic retinopathyPublication . Alves, Liliana Teresa da Silva; Silva, Gabriela A.; Calado, Sofia M.Diabetic retinopathy (DR) is a microvascular complication associated with Diabetes mellitus (DM) and the leading cause of blindness in developed countries. Hyperglycemia and hypoxia are suggested to play essential pathophysiological role in the onset, progression and prognosis of DR. Moreover, hypoxia inducible factor-1 (HIF-1), which is stabilized under hypoxic conditions, acts as a transcription factor of several genes such as vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). It was also described that an imbalance between pigment epithelium-derived factor (PEDF), a potent angiogenic inhibitor, and VEGF, the major angiogenic stimulator, is present in patients that suffer of DR. However little is known about the role of this imbalance in DR progression. Therefore the goal of this study was to characterize the expression of GLUT1, pro-, and anti-angiogenic factors involved in the development and progression of DR, using in vitro and in vivo models of the disease. The present study demonstrates that in D407 cells, an immortalized retinal pigmented epithelium (RPE), the levels of the glucose transporter GLUT1 are increased in the response to hypoxia and hyperglycemia and this increase is more pronounced in the cell membrane fraction. Additional support to our in vitro results is the fact that GLUT1 expression levels are increased in Ins2Akita mice, a model of type 1 DM. Regarding PEDF expression in vitro, there was no significant difference between tested conditions, but we observed the tendency to decrease in hypoxic conditions. In vivo, we could see a significant decrease of PEDF expression in the retina of 6 month-old mice; but the key result is the marked decrease detected in the RPE cell layer. VEGF mRNA levels were increased in vitro and in vivo at 6 month-old animals, but this was not observed for the protein levels. At 12 months of age, mRNA levels were similar in WT and diabetic mice, but the protein levels are significantly decreased in diabetic mice, a potential consequence of the overall senescence observed for these animals. Taken together, these results suggest that the increase in GLUT1 and the decrease in PEDF expression levels are associated with the development of diabetic retinopathy.
- Characterization of the behavioural phenotype of calpain-knockout micePublication . Silva, Joana Andreia Joaquim da; Araújo, InêsAdult neurogenesis consists of the production of new neurons in specific brain regions, or neurogenic niches. The most relevant niches in rodents are the subventricular zone lining the lateral ventricles and the dentate gyrus of the hippocampus. Neurogenesis has been shown to influence cognitive function dependent on these regions. Evidence from the literature suggests that calpains are able to influence neurogenesis. However, little information is available regarding their participation in neurological functions. We have evaluated the involvement of calpains in cognitive and emotional behaviour by evaluating these functions in mice genetically modified to lack calpain 1, calpain 2, or both calpains. In this work, 12 week old calpain knockout mice for calpain 1, calpain 2, both, or wild type (WT) littermates were used. The mice were tested in the open field, object recognition, Morris water maze, contextual and cued fear conditioning, passive avoidance, elevated plus maze and forced swimming tests, to evaluate neurological function. Results from the elevated plus maze show that calpain2 knockout mice and double knockout mice present an anxious phenotype comparing with WT mice, suggesting that calpain 2, but not calpain 1, is involved in anxiety. Memory, learning, locomotor activity and exploratory behaviour as well as helplessness were similar in calpain knockout and WT mice. Neurogenesis in calpain knockout mice was also similar to WT mice. Overall, our work shows that calpain 2 is involved in anxiety and a clear phenotype was identified in calpain knockout mice regarding their involvement in memory and learning, which is agreement with previously published data using mice that lack the small regulatory subunit of calpains.
- Characterization of TRIB2-mediated resistance to pharmacological inhibition of MEKPublication . Henriques, Vanessa Mendes; Link, Wolfgang; Ferreira, BibianaChemoresistance and metastasis are the main reasons for treatment failure in melanoma patients. MAPK pathway is often hyperactivated in melanoma due to BRAF mutations. BRAF and MEK inhibitors revolutionized the standard-care of patients with advanced melanoma. Yet, patients develop resistance to these drugs very fast. Previous studies showed that Tribbles homolog 2 (TRIB2) is overexpressed in melanoma and confers resistance to chemotherapeutic and targeted drugs such as darcarbazin, PI3K and mTOR inhibitors. Furthermore, TRIB2 protein contains a MEK1 binding site. Taking this into account, we hypothesize that TRIB2 might confer resistance to MEK inhibition. In order to test our hypothesis, we generated isogenic melanoma cell lines with TRIB2 knockdown, using shRNA, and cells with TRIB2 depletion using CRISPR technique. Since the members of the Tribbles protein family might be functionally redundant and compensate for TRIB2 depletion, we decided to determine mRNA and protein levels of TRIB1, TRIB2 and TRIB3 using q-PCR and Western-Blot techniques, respectively, on a panel of melanoma and non-melanoma cell lines. We treated these isogenic cell lines with the MEK inhibitor Refametinib for 72h. The resistance was evaluated through cell death analysis, using cell counting based on trypan-blue and annexin V/ Propidium iodide staining. The isogenic cell lines were successfully established and determined that compensation of TRIB2 through TRIB1 or TRIB3 only plays a minor role. Importantly Refametinib treatment of melanoma cell lines with different levels of TRIB2 showed that cell death correlated with TRIB2 expression level suggesting that TRIB2 confers resistance to MEK inhibitors. Understanding the resistance mechanisms to the therapeutic agents can improve the outcomes of current therapies and contribute to the development of new therapeutic approaches.
- Constitutive OGG1 variant together with BRCA mutations display accelerated telomere shorteningPublication . Ferreira, Sofia Maria Morgadinho; Osório, Ana; Maia, Ana TeresaOsorio, Milne et al. (2014) reported a SNP, rs2304277, in the OGG1 gene, with evidence of potential association with increased ovarian cancer risk in BRCA1 germline mutation carriers (p=4.8x10-3). The protein OGG1 is a main player in the DNA BER pathway, responsible for recognizing and excising oxidized guanines (8-oxoG). In the literature, oxidative stress has been well characterized to have a natural site-specific preference for guanines, in which telomeric DNA is enriched due to several TTAGGG repeats. 8-oxoG is highly mutagenic and affects DNA replication, and at the telomere level it can impair the recruitment and affinity of the shelterin complex proteins. This complex caps and protects telomeres from aberrant chromosomal rearrangements. Not only, when not properly corrected, 8-oxoG can give rise to ssDNA breaks. Therefore, telomeres are more susceptible to this kind of damage, and disruption of the normal telomere function and length can result in carcinogenesis, as a consequence of genomic instability. Hence, given the role of OGG1 and telomeres composition, we aimed to explore whether the increase of cancer risk in the carriers of rs2304277 (together with a germline mutation in the BRCA gene), might be due to an altered OGG1 function, which could accelerate the telomere shortening, resulting from a weaker response upon oxidative stress harms. For the functional characterization of the polymorphism, the experimental approach was based on the evaluation of the OGG1 mRNA expression levels by qPCR and measurement of telomeres length by High Throughput Q-FISH of the different OGG1 genotypes and BRCA1/2 mutation status from peripheral blood samples. Very preliminary results, suggest that the variant leads to a decrease in OGG1 mRNA levels which, and together with a mutation in BRCA gene might contribute to an accelerated telomere shortening. These results might explain, in part, the increase of an individual’s lifetime risk of developing cancer when harbouring both genetic conditions.
- Contribution of the intracellular cholesterol levels for the expression of VEGF-C and VEGFR-3 in acute myeloid leukemiaPublication . Silva, Andreia Cristina Martins; Silva, GabrielaSome subsets of Acute Myeloid Leukemia (AML) cells express the Vascular Endothelial Receptor 3 (VEGFR-3), a receptor for the Vascular Endothelial Growth Factor-C (VEGF-C). The VEGFR-3/VEGF-C axis has been related to many cancers, including leukemia, due to the promotion of cell proliferation, chemotherapy resistance and tumor aggressiveness. At the molecular level VEGF-C signaling has been shown to induce the expression of the Bcl-2 anti-apoptotic factor. Cholesterol has been reported to modulate the expression of VEGFR’s in AML. Here, we hypothesize that intracellular cholesterol levels could modulate signaling through the VEGFR-3/VEGF-C axis, contributing towards increased leukemia aggressiveness. To test this hypothesis we either enriched or partially depleted cholesterol from the THP-1 and HEL cell lines with Methyl-β-Cyclodextrin + cholesterol complexes and Methyl-β-Cyclodextrin alone, respectively. It was seen that Methyl-β-Cyclodextrin compromises cell viability, so we tested concentrations of 0,2 mM. In these conditions we looked at VEGFR-3, VEGF-C and Bcl-2 expression by RQ-PCR and Western Blot; VEGFR-3 expression was also assessed by flow cytometry. Our preliminary results show that cholesterol enrichment of cells leads to a decrease of VEGFR-3 mRNA levels, while cholesterol depletion leads to an increase of VEGF-C mRNA levels. No differences in Bcl-2 have been detected. We are now confirming these results at the protein level. By flow cytometry VEGFR-3 expression didn’t show differences between conditions. Subsequent experiments will include in vitro functional assays to assess cell viability, proliferation and resistance to apoptosis. This work may contribute to better understand how VEGF-C signaling pathways are regulated in leukemia. As VEGF-C expression has been associated with resistance to chemotherapy by AML cells, this work may contribute to better understand how resistance is achieved.
- Development of non-viral vectors for gene therapy for pathologies of the retinaPublication . Oliveira, Ana V.; Silva, GabrielaThe success of gene therapy relies on efficient gene transfer and stable transgene expression. Our goal was to develop non-viral vectors optimized for retinal gene therapy with continued gene expression. Polymers, chitosan and hyaluronic acid or the modified polymers (thiolated chitosan and aminated hyaluronic acid) were chosen considering their biocompatibility and biodegradability to prepare several formulations. Vectors were formulated and characterized regarding their physical properties, biocompatibility and gene transfer efficiency in vitro on both retinal pigment epithelial and HEK293 cells and gene expression in the mouse retina. Our results show that our vectors exhibit size and surface charge consistent with gene delivery. They also present long-term stability in both storage and physiological conditions, remain stable after several freeze-thaw cycles and are capable of efficiently protecting DNA from nuclease degradation. Transfection studies show that transfection efficiency and transgene expression is affected by cell type, polymer molecular weight and mode of integrase delivery with the polyplexes. The incorporation of hyaluronic acid affected formulation stability, as expected, but it did not affect DNA loading and protection. The combination of chitosan and hyaluronic acid in polyplexes showed a significant improvement of transfection efficiency compared to chitosan-based vectors. In order to achieve sustained gene transfer vectors were combined with phiC31-integrase to promote transgene integration. The combined strategy of chitosan-based delivery and integrase demonstrate prolonged gene expression of both small (GFP, 1 Kb) and large genes (CEP290, 8Kb) several weeks post-transfection. In vivo sub-retinal administration of our vectors showed efficient transfection and sustained transgene expression in RPE cells at least 6 months post- injection. Our results indicate this chitosan-based approach may overcome size limitations found in commonly used adeno-associated viruses mediated gene transfer, while maintaining a high safety profile and prolonged, sustained gene expression, thus constituting an alternative for retinal gene delivery.