The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia Type 3 pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Aging and Alzheimer's disease: Searching for novel molecular cues

Tese de Doutoramento em Ciências da Saúde

The effects of solvent composition on the affinity of a peptide towards hair keratin: experimental and molecular dynamics data

The study of the interaction between hair filaments and formulations or peptides is of utmost importance in fields like cosmetic research. Keratin intermediate filaments structure is not fully described, limiting the molecular dynamics (MD) studies in this field although its high potential to improve the area. We developed a computational model of a truncated protofibril, simulated its behavior in alcoholic based formulations and with one peptide. The simulations showed a strong interaction between the benzyl alcohol molecules of the formulations and the model, leading to the disorganization of the keratin chains, which regress with the removal of the alcohol molecules. This behavior can explain the increase of peptide uptake in hair shafts evidenced in fluorescence microscopy pictures. The model developed is valid to computationally reproduce the interaction between hair and alcoholic formulations and provide a robust base for new MD studies about hair properties. It is shown that the MD simulations can improve hair cosmetic research, improving the uptake of a compound of interest.

A Gß protein and the TupA Co-Regulator bind to protein kinase A Tpk2 to act as antagonistic molecular switches of fungal morphological changes

A Gß protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes

Sirtuins and proteolytic systems: implications for pathogenesis of synucleinopathies

Insoluble and fibrillar forms of a-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. a-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. a-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for a-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins's role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from a-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of a-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies.

To be or not to be a pseudogene: a molecular epidemiological approach to the mclx genes and its impact in tuberculosis

Tuberculosis presents a myriad of symptoms, progression routes and propagation patterns not yet fully understood. Whereas for a long time research has focused solely on the patient immunity and overall susceptibility, it is nowadays widely accepted that the genetic diversity of its causative agent, Mycobacterium tuberculosis, plays a key role in this dynamic. This study focuses on a particular family of genes, the mclxs (Mycobacterium cyclase/LuxR-like genes), which codify for a particular and nearly mycobacterial-exclusive combination of protein domains. mclxs genes were found to be pseudogenized by frameshift-causing insertion(s)/deletion(s) in a considerable number of M. tuberculosis complex strains and clinical isolates. To discern the functional implications of the pseudogenization, we have analysed the pattern of frameshift-causing mutations in a group of M. tuberculosis isolates while taking into account their microbial-, patient- and disease-related traits. Our logistic regression-based analyses have revealed disparate effects associated with the transcriptional inactivation of two mclx genes. In fact, mclx2 (Rv1358) pseudogenization appears to be primarily driven by the microbial phylogenetic background, being mainly related to the Euro-American (EAm) lineage; on the other hand, mclx3 (Rv2488c) presents a higher tendency for pseudogenization among isolates from patients born on the Western Pacific area, and from isolates causing extra-pulmonary infections. These results contribute to the overall knowledge on the biology of M. tuberculosis infection, whereas at the same time launch the necessary basis for the functional assessment of these so far overlooked genes.

Widespread environmental contamination with Mycobacterium tuberculosis complex revealed by a molecular detection protocol

Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70-0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.

Urothelial bladder cancer progression: lessons learned from the bench

Urothelial bladder carcinoma (UBC) is an intricate malignancy with a variable natural history and clinical behavior. Despite developments in diagnosis/prognosis refinement and treatment modalities, the recurrence rate is high, and progression from non-muscle to muscle invasive UBC commonly leads to metastasis. Moreover, patients with muscle-invasive or extra-vesical disease often fail the standard chemotherapy treatment, and overall survival rates are poor. Thus, UBC remains a challenge in the oncology field, representing an ideal candidate for research on biomarkers that could identify patients at increased risk of recurrence, progression, and chemo-refractoriness. However, progress toward personalized medicine has been hampered by the unique genetic complexity of UBC. Recent genome-wide expression and sequencing studies have brought new insights into its molecular features, pathogenesis and clinical diversity, revealing a landscape where classical pathology is intersected by the novel and heterogeneous molecular groups. Hence, it seems plausible to postulate that only an integrated signature of prognostic/predictive biomarkers inherent in different cancer hallmarks will reach clinical validation. In this review, we have summarized ours and others' research into novel putative biomarkers of progression and chemoresistance that encompass several hallmarks of cancer: tumor neovascularization, invasion and metastasis, and energy metabolism reprogramming of the tumor microenvironment.

Molecular regulation of flowering induction in Quercus suber

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepeneurship

The influence of kaolin application on key metabolic pathways associated with grape berry quality: a molecular and biochemical analysis

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Synthesis, antiangiogenesis evaluation and molecular docking studies of 1-Aryl-3-[(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase inhibitors

The synthesis and biological evaluation of novel 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 3, 4 and 5 as VEGFR-2 tyrosine kinase inhibitors, are reported. The 1-aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 4a-4h, with the arylurea in the meta position to the thioether, showed the lowest IC50 values in enzymatic assays (10-206 nM), the most potent compounds 4d-4h (IC50 10-28 nM) bearing hydrophobic groups (Me, F, CF3 and Cl) in the terminal phenyl ring. A convincing rationalization was achieved for the highest potent compounds 4 as type II VEGFR-2 inhibitors, based on the simultaneous presence of: (1) the thioether linker and (2) the arylurea moiety in the meta position. For compounds 4, significant inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) proliferation (BrdU assay), migration (wound-healing assay) and tube formation were observed at low concentrations. These compounds have also shown to increase apoptosis using the TUNEL assay. Immunostaining for total and phosphorylated (active) VEGFR-2 was performed by Western blotting. The phosphorylation of the receptor was significantly inhibited at 1.0 and 2.5 microM for the most promising compounds. Altogether, these findings point to an antiangiogenic effect in HUVECs.

Study of grapevine solute transporters involved in berry quality. A biochemical and molecular approach

Tese de Doutoramento em Biologia de Plantas

Development of folate-targeted liposomes for rheumatoid arthritis therapy

Tese de Doutoramento em Biologia Molecular e Ambiental (área de especialização em Biologia Celular e Saúde).

Molecular mass distribution of materials solubilized by xylanase treatment of Douglas-Fir kraft pulp

Irgazyme, a commercial xylanase preparation from Trichoderma longibrachiatum, and xylanase D a purified enzyme from Trichoderma harzianum E58 were tested for their ability to enhance peroxide bleaching of Douglas-fir (Pseudotsuga menziesii) kraft pulp. A treatment with Irgazyme caused a much larger increase in brightness than did xylanase D. A double xylanase treatment with Irgazyme, before and after peroxide bleaching, resulted in the highest final brightness. Alkaline extraction increased the brightness of Douglas-fir brownstock. Treatment with Irgazyme released more lignin and carbohydrates than did xylanase D. The molecular mass of the lignin extracted from Irgazyme-treated brownstock was much larger than that from the control pulp. The lignin-like macromolecules directly solubilized from peroxide bleached pulps were substantially larger than those solubilized from the brownstock, irrespective of whether they were produced during xylanase or control treatments. This indicates that different kinds of materials were solubilized when a xylanase treatment was applied at different points in the bleaching sequence and raises concerns about the role of lignin entrapment in the mechanism by which xylanase enhances peroxide bleaching.