http://hdl.handle.net/10400.22/986 Sat, 13 Oct 2018 12:49:30 GMT 2018-10-13T12:49:30Z http://hdl.handle.net/10400.22/6275 Título: Acetyl-L-Carnitine Prevents Methamphetamine-Induced Structural Damage on Endothelial Cells via ILK-Related MMP-9 Activity Autor: Fernandes, Sílvia; Salta, S.; Bravo, J.; Silva, A. P.; Summavielle, Teresa Resumo: Methamphetamine (METH) is a potent psychostimulant highly used worldwide. Recent studies evidenced the involvement of METH in the breakdown of the blood-brain-barrier (BBB) integrity leading to compromised function. The involvement of the matrix metalloproteinases (MMPs) in the degradation of the neurovascular matrix components and tight junctions (TJs) is one of the most recent findings in METH-induced toxicity. As BBB dysfunction is a pathological feature of many neurological conditions, unveiling new protective agents in this field is of major relevance. AcetylL-carnitine (ALC) has been described to protect the BBB function in different paradigms, but the mechanisms underling its action remain mostly unknown. Here, the immortalized bEnd.3 cell line was used to evaluate the neuroprotective features of ALC in METH-induced damage. Cells were exposed to ranging concentrations of METH, and the protective effect of ALC 1 mM was assessed 24 h after treatment. F-actin rearrangement, TJ expression and distribution, and MMPs activity were evaluated. Integrin-linked kinase (ILK) knockdown cells were used to assess role of ALC in ILK mediated METHtriggered MMPs’ activity. Our results show that METH led to disruption of the actin filaments concomitant with claudin-5 translocation to the cytoplasm. These events were mediated by MMP-9 activation in association with ILK overexpression. Pretreatment with ALC prevented METH-induced activation of MMP-9, preserving claudin-5 location and the structural arrangement of the actin filaments. The present results support the potential of ALC in preserving BBB integrity, highlighting ILK as a new target for the ALC therapeutic use. Wed, 03 Dec 2014 00:00:00 GMT http://hdl.handle.net/10400.22/6275 2014-12-03T00:00:00Z http://hdl.handle.net/10400.22/6222 Título: S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation Autor: Botelho, Hugo M.; Leal, Sónia S.; Cardoso, Isabel; Yanamandra, Kiran; Morozova-Roche, Ludmilla A.; Fritz, Günter; Gomes, Cláudio M. Resumo: S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/10400.22/6222 2012-01-01T00:00:00Z http://hdl.handle.net/10400.22/6221 Título: Methamphetamine promotes a-tubulin deacetylation in endothelial cells: The protective role of acetyl-L-carnitine Autor: Fernandes, Sílvia; Salta, S.; Summavielle, Teresa Resumo: Methamphetamine (METH) is a powerful psychostimulant drug used worldwide for its reinforcing properties. In addition to the classic long-lasting monoaminergic-disrupting effects extensively described in the literature, METH has been consistently reported to increase blood brain barrier (BBB) permeability, both in vivo and in vitro, as a result of tight junction and cytoskeleton disarrangement. Microtubules play a critical role in cell stability, which relies on post-translational modifications such as a-tubulin acetylation. As there is evidence that psychostimulants drugs modulate the expression of histone deacetylases (HDACs), we hypothesized that in endothelial cells METH-mediation of cytoplasmatic HDAC6 activity could affect tubulin acetylation and further contribute to BBB dysfunction. To validate our hypothesis, we exposed the bEnd.3 endothelial cells to increasing doses of METH and verified that itleads to an extensivea-tubulin deacetylation mediated by HDACs activation. Furthermore, since we recently reported that acetyl-L-carnitine (ALC), a natural occurring compound, prevents BBB structural loss in a context of METH exposure, we reasoned that ALC could also preserve the acetylation of microtubules under METH action. The present results confirm that ALC is able to prevent METH-induced deacetylation providing effective protection on microtubule acetylation. Although further investigation is still needed, HDACs regulation may become a new therapeutic target for ALC. Thu, 01 Jan 2015 00:00:00 GMT http://hdl.handle.net/10400.22/6221 2015-01-01T00:00:00Z http://hdl.handle.net/10400.22/6220 Título: SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3 Autor: Vieira, Filipa Quintela; Costa-Pinheiro, Pedro; Almeida-Rios, Diogo; Graça, Inês; Monteiro-Reis, Sara; Simões-Sousa, Susana; Carneiro, Isa; Sousa, Elsa Joana; Godinho, Maria Inês; Baltazar, Fátima; Henrique, Rui; Jerónimo, Carmen Resumo: Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa. Thu, 01 Jan 2015 00:00:00 GMT http://hdl.handle.net/10400.22/6220 2015-01-01T00:00:00Z http://hdl.handle.net/10400.22/6219 Título: MicroRNA-375 plays a dual role in prostate carcinogenesis Autor: Costa-Pinheiro, Pedro; Ramalho-Carvalho, João; Vieira, Filipa Quintela; Torres-Ferreira, Jorge; Oliveira, Jorge; Gonçalves, Céline S; Costa, Bruno M .; Henrique, Rui; Jerónimo, Carmen Resumo: Background: Prostate cancer (PCa), a highly incident and heterogeneous malignancy, mostly affects men from developed countries. Increased knowledge of the biological mechanisms underlying PCa onset and progression are critical for improved clinical management. MicroRNAs (miRNAs) deregulation is common in human cancers, and understanding how it impacts in PCa is of major importance. MiRNAs are mostly downregulated in cancer, although some are overexpressed, playing a critical role in tumor initiation and progression. We aimed to identify miRNAs overexpressed in PCa and subsequently determine its impact in tumorigenesis. Results: MicroRNA expression profiling in primary PCa and morphological normal prostate (MNPT) tissues identified 17 miRNAs significantly overexpressed in PCa. Expression of three miRNAs, not previously associated with PCa, was subsequently assessed in large independent sets of primary tumors, in which miR-182 and miR-375 were validated, but not miR-32. Significantly higher expression levels of miR-375 were depicted in patients with higher Gleason score and more advanced pathological stage, as well as with regional lymph nodes metastases. Forced expression of miR-375 in PC-3 cells, which display the lowest miR-375 levels among PCa cell lines, increased apoptosis and reduced invasion ability and cell viability. Intriguingly, in 22Rv1 cells, which displayed the highest miR-375 expression, knockdown experiments also attenuated the malignant phenotype. Gene ontology analysis implicated miR-375 in several key pathways deregulated in PCa, including cell cycle and cell differentiation. Moreover, CCND2 was identified as putative miR-375 target in PCa, confirmed by luciferase assay. Conclusions: A dual role for miR-375 in prostate cancer progression is suggested, highlighting the importance of cellular context on microRNA targeting. Thu, 01 Jan 2015 00:00:00 GMT http://hdl.handle.net/10400.22/6219 2015-01-01T00:00:00Z