Co-expression of NPY and VIP in human dental pulp

Introduction: The regulation of pulpal haemodynamics in health and disease involves sympathetic and parasympathetic mechanisms in which both neuropeptide Y (NPY; a sympathetic vasoconstrictor) and vasoactive intestinal polypeptide (VIP; a parasympathetic vasodilator) may play potential pathophysiological roles. We have previously investigated the levels of NPY or VIP present in human dental pulp tissue and shown that their expression is up-regulated in caries induced pulpal inflammation. Objectives: The aim of this study was to investigate the potential correlation between NPY and VIP levels measured in the same dental pulp samples using radioimmunoassay (RIA). Methods: Pulp tissue was obtained from extracted teeth, classified as follows; healthy (n=22), moderately carious (n=20) and grossly carious (n=26). Samples were processed for RIA by boiling in acetic acid as previously described. The levels of NPY and VIP, measured by RIA, were expressed as ng/gram of pulp tissue. The nature of the relationship between NPY and VIP levels in human pulp tissue was tested by calculating Pearson's product moment correlation coefficient using the linear regression test. Results: Calculation of Pearson product moment correlation coefficient showed a significant negative correlation between NPY and VIP levels in pulp tissue samples from non-carious teeth (p = 0.02, r = -.48). This negative correlation in non-carious teeth changed to a significant positive correlation in carious teeth when the levels of NPY and VIP were compared (p = 0.03, r= 0.311). Conclusions: In non-carious teeth, the negative correlation between NPY and VIP levels is in keeping with the previously described modulatory influence of cholinergic nerves on sympathetic function which may be perturbed as caries develops.

Neuropeptides regulate expression of angiogenic growth factors in pulpal fibroblasts

In the dental pulp angiogenesis is crucial for tooth development and a prerequisite for successful repair following injury and inflammation. The role of neuropeptides in pulpal inflammation has been well documented but their role in the regulation of angiogenesis in the dental pulp has not been elucidated. Objectives: The aim was to profile the expression of angiogenic growth factors produced by pulp fibroblasts and to study the effects of neuropeptides on their expression. Methods: Human pulp fibroblasts derived from healthy molar teeth were stimulated with neuropeptides previously identified in dental pulp, namely, Substance P (SP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and calcitonin related gene peptide (CGRP) for 24 and 48 hrs. Simultaneous expression of ten growth factors was quantified using a novel human angiogenesis array (Ray Biotech, USA). Results: Pulp fibroblasts expressed human angiogenic growth factors, VEGF, bFGF, PDGF-BB, HGF, ANG2, HB-EGF, PIGF, angiogenin and leptin. Among the growth factors expressed VEGF, angiogenin and HGF were abundantly expressed compared to others. Neuropeptides induced variable effects on the expression of the angiogenic factors: CGRP potently up-regulated VEGF, bFGF, HGF and PIGF after 24 hr, while NPY tended to down regulate growth factors after 24 hr in culture but markedly up regulated ANG2, bFGF and leptin after 48 hr. SP down regulated expression of all angiogenic growth factors except for leptin, while VIP induced a small increase in expression of each growth factor, irrespective of time. Conclusion: Pulp fibroblasts express a range of angiogenic growth factors including angiogenin and leptin. Neuropeptides regulate the expression of these factors, suggesting an additional role for neuropeptides in the regulation of inflammation and healing in the dental pulp.
This work is supported by TC White Research Fund

Utensilagem óssea do paleolítico superior português

Dissertação mest., Arqueologia - Teoria e Métodos, Universidade do Algarve, 2007

Étude de l’enzyme UGT2B28 et son rôle dans le cancer de la prostate

Contexte: L’inactivation des androgènes est majoritairement régulée par des enzymes du métabolisme de la famille des UDP-glucuronosyltransferase (UGT). Ce procédé métabolique permet de contrôler la biodisponibilité des hormones stéroïdiennes systémiques et locales. Objectif : L’objectif était d’étudier la relation entre l’expression de l’enzyme UDP-glucuronosyltransferase 2B polypeptide 28 (UGT2B28), impliquée dans la biotransformation des hormones, avec les niveaux hormonaux circulants, et les caractéristiques clinico-pathologiques dans le cancer de la prostate (CaP). Conception et participants : Nous avons utilisé dans cette étude la technique d’immunohostochimie à grande échelle (tissue microarray) sur les tissus de 239 patients ayant un CaP localisé. L’étude des 51 patients additionnels ne possédant pas l’enzyme UGT2B28 dans leur génome, a été effectuée pour confirmer l’importance de cette enzyme sur les niveaux hormonaux circulants. Résultats : La surexpression de l’enzyme UGT2B28 a été associée à des niveaux d’antigène prostatique spécifique (APS) au diagnostic plus faibles, à un score de Gleason plus élevé, à des marges et statuts nodaux positifs, et fut associée de façon indépendante au risque de progression. La surexpression de l’enzyme fut également associée à des niveaux circulants de testostérone (T) et dihydrotestostérone (DHT) plus élevés. Les patients n’exprimant pas le gène UGT2B28 avaient des niveaux plus bas de T (19%), de DHT (17%), de métabolites glucuronidés (18-38%), et des niveaux plus élevés du précurseur surrénalien androsténédione (36%). Conclusion : L’enzyme UGT2B28 modifie les niveaux circulants de T et DHT, et sa surexpression est associée avec un CaP à plus haut grade. Notre étude a permis de découvrir un nouveau rôle d’UGT2B28, celui de régulateur de la stéroïdogenèse, et a souligné l’interconnexion entre les capacités de biotransformation hormonale des cellules cancéreuses, des niveaux hormonaux, des caractéristiques clinicopathologiques et du risque de progression.

Modulation of calcitonin amyloid formation by anionic lipid membranes

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2014

The N-terminal domain modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization

AMPA receptors are tetrameric glutamate-gated ion channels that mediate fast synaptic neurotransmission in mammalian brain. Their subunits contain a two-lobed N-terminal domain (NTD) that comprises over 40% of the mature polypeptide. The NTD is not obligatory for the assembly of tetrameric receptors, and its functional role is still unclear. By analyzing full-length and NTD-deleted GluA1-4 AMPA receptors expressed in HEK 293 cells, we found that the removal of the NTD leads to a significant reduction in receptor transport to the plasma membrane, a higher steady state-to-peak current ratio of glutamate responses, and strongly increased sensitivity to glutamate toxicity in cell culture. Further analyses showed that NTD-deleted receptors display both a slower onset of desensitization and a faster recovery from desensitization of agonist responses. Our results indicate that the NTD promotes the biosynthetic maturation of AMPA receptors and, for membrane-expressed channels, enhances the stability of the desensitized state. Moreover, these findings suggest that interactions of the NTD with extracellular/synaptic ligands may be able to fine-tune AMPA receptor-mediated responses, in analogy with the allosteric regulatory role demonstrated for the NTD of NMDA receptors.

Can power laws help us understand gene and proteome information?

Proteins are biochemical entities consisting of one or more blocks typically folded in a 3D pattern. Each block (a polypeptide) is a single linear sequence of amino acids that are biochemically bonded together. The amino acid sequence in a protein is defined by the sequence of a gene or several genes encoded in the DNA-based genetic code. This genetic code typically uses twenty amino acids, but in certain organisms the genetic code can also include two other amino acids. After linking the amino acids during protein synthesis, each amino acid becomes a residue in a protein, which is then chemically modified, ultimately changing and defining the protein function. In this study, the authors analyze the amino acid sequence using alignment-free methods, aiming to identify structural patterns in sets of proteins and in the proteome, without any other previous assumptions. The paper starts by analyzing amino acid sequence data by means of histograms using fixed length amino acid words (tuples). After creating the initial relative frequency histograms, they are transformed and processed in order to generate quantitative results for information extraction and graphical visualization. Selected samples from two reference datasets are used, and results reveal that the proposed method is able to generate relevant outputs in accordance with current scientific knowledge in domains like protein sequence/proteome analysis.

Spectroscopic characterization of a novel 2 /[4Fe /4S] ferredoxin isolated from Desulfovibrio desulfuricans ATCC 27774

Inorganica Chimica Acta 356 (2003) 215-221

Metal ions and protein folding: conformational and functional interplay

Dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Oriented Tailoring of Plastic Antibodies for Prostate Specific Antigen and Application of the Imprinted Material as Ionophore in Potentiometric Detection

Prostate Specific Antigen (PSA) is the biomarker of choice for screening prostate cancer throughout the population, with PSA values above 10 ng/mL pointing out a high probability of associated cancer1. According to the most recent World Health Organization (WHO) data, prostate cancer is the commonest form of cancer in men in Europe2. Early detection of prostate cancer is thus very important and is currently made by screening PSA in men over 45 years old, combined with other alterations in serum and urine parameters. PSA is a glycoprotein with a molecular mass of approximately 32 kDa consisting of one polypeptide chain, which is produced by the secretory epithelium of human prostate. Currently, the standard methods available for PSA screening are immunoassays like Enzyme-Linked Immunoabsorbent Assay (ELISA). These methods are highly sensitive and specific for the detection of PSA, but they require expensive laboratory facilities and high qualify personal resources. Other highly sensitive and specific methods for the detection of PSA have also become available and are in its majority immunobiosensors1,3-5, relying on antibodies. Less expensive methods producing quicker responses are thus needed, which may be achieved by synthesizing artificial antibodies by means of molecular imprinting techniques. These should also be coupled to simple and low cost devices, such as those of the potentiometric kind, one approach that has been proven successful6. Potentiometric sensors offer the advantage of selectivity and portability for use in point-of-care and have been widely recognized as potential analytical tools in this field. The inherent method is simple, precise, accurate and inexpensive regarding reagent consumption and equipment involved. Thus, this work proposes a new plastic antibody for PSA, designed over the surface of graphene layers extracted from graphite. Charged monomers were used to enable an oriented tailoring of the PSA rebinding sites. Uncharged monomers were used as control. These materials were used as ionophores in conventional solid-contact graphite electrodes. The obtained results showed that the imprinted materials displayed a selective response to PSA. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8X10-11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed smaller sensitivity, with average slopes of -24.8 mV/decade. The best sensors were successfully applied to the analysis of serum samples, with percentage recoveries of 106.5% and relatives errors of 6.5%.

Functional and molecular characterization of SR45, a plant-specific factor involved in sugar and stress signaling in Arabidopsis thaliana

Dissertation presented to obtain the Ph.D degree in Molecular Biology

GLUT2 and the incretin receptors are involved in glucose-induced incretin secretion.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretins secreted in response to oral glucose ingestion by intestinal L and K cells, respectively. The molecular mechanisms responsible for intestinal cell glucose sensing are unknown but could be related to those described for beta-cells, brain and hepatoportal sensors. We determined the role of GLUT2, GLP-1 or GIP receptors in glucose-induced incretins secretion, in the corresponding knockout mice. GLP-1 secretion was reduced in all mutant mice, while GIP secretion did not require GLUT2. Intestinal GLP-1 content was reduced only in GIP and GLUT2 receptors knockout mice suggesting that this impairment could contribute to the phenotype. Intestinal GIP content was similar in all mice studied. Furthermore, the impaired incretins secretion was associated with a reduced glucose-stimulated insulin secretion and an impaired glucose tolerance in all mice. In conclusion, both incretins secretion depends on mechanisms involving their own receptors and GLP-1 further requires GLUT2.

In vivo and in vitro effects of somatostatin and insulin on glucagon release in a human glucagonoma.

Inhibition of pancreatic glucagon secretion has been reported to be mediated by glucose, insulin and somatostatin. As no human pancreatic alpha-cell lines are available to study in vitro the relative importance of insulin and glucose in the control of pancreatic glucagon release, we investigated a patient presenting with a malignant glucagonoma who underwent surgical resection of the tumour. Functional somatostatin receptors were present as octreotide administration decreased basal glucagon and insulin secretion by 52 and 74%, respectively. The removed tumour was immunohistochemically positive for glucagon, chromogranin A and pancreatic polypeptide but negative for insulin, gastrin and somatostatin. The glucagonoma cells were also isolated and cultured in vitro. Incubation experiments revealed that change from high (10 mM) to low (1 mM) glucose concentration was unable to stimulate glucagon secretion. A dose-dependent inhibition of glucagon release by insulin was however, observed at low glucose concentration. These findings demonstrate that insulin could inhibit glucagon secretion in vitro in the absence of elevated glucose concentrations. These data suggest, as observed in vivo and in vitro in several animal studies, that glucopenia-induced glucagon secretion in humans is not mediated by a direct effect of low glucose on alpha-cells but possibly by a reduction of insulin-mediated alpha-cell suppression and/or an indirect neuronal stimulation of glucagon release.

Role of retroviral restriction factors in the interferon-α-mediated suppression of HIV-1 in vivo.

The antiviral potency of the cytokine IFN-α has been long appreciated but remains poorly understood. A number of studies have suggested that induction of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) and bone marrow stromal cell antigen 2 (BST-2/tetherin/CD317) retroviral restriction factors underlies the IFN-α-mediated suppression of HIV-1 replication in vitro. We sought to characterize the as-yet-undefined relationship between IFN-α treatment, retroviral restriction factors, and HIV-1 in vivo. APOBEC3G, APOBEC3F, and BST-2 expression levels were measured in HIV/hepatitis C virus (HCV)-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated IFN-α/ribavirin (IFN-α/riba) combination therapy. IFN-α/riba therapy decreased HIV-1 viral load by -0.921 (±0.858) log(10) copies/mL in HIV/HCV-coinfected patients. APOBEC3G/3F and BST-2 mRNA expression was significantly elevated during IFN-α/riba treatment in patient-derived CD4+ T cells (P < 0.04 and P < 0.008, paired Wilcoxon), and extent of BST-2 induction was correlated with reduction in HIV-1 viral load during treatment (P < 0.05, Pearson's r). APOBEC3 induction during treatment was correlated with degree of viral hypermutation (P < 0.03, Spearman's ρ), and evolution of the HIV-1 accessory protein viral protein U (Vpu) during IFN-α/riba treatment was suggestive of increased BST-2-mediated selection pressure. These data suggest that host restriction factors play a critical role in the antiretroviral capacity of IFN-α in vivo, and warrant investigation into therapeutic strategies that specifically enhance the expression of these intrinsic immune factors in HIV-1-infected individuals.

In vivo characterization of the circadian clock output gene usp2

Life on earth is subject to the repeated change between day and night periods. All organisms that undergo these alterations have to anticipate consequently the adaptation of their physiology and possess an endogenous periodicity of about 24 hours called circadian rhythm from the Latin circa (about) and diem (day). At the molecular level, virtually all cells of an organism possess a molecular clock which drives rhythmic gene expression and output functions. Besides altered rhythmicity in constant conditions, impaired clock function causes pathophysiological conditions such as diabetes or hypertension. These data unveil a part of the mechanisms underlying the well-described epidemiology of shift work and highlight the function of clock-driven regulatory mechanisms. The post-translational modification of proteins by the ubiquitin polypeptide is a central mechanism to regulate their stability and activity and is capital for clock function. Similarly to the majority of biological processes, it is reversible. Deubiquitylation is carried out by a wide variety of about ninety deubiquitylating enzymes and their function remains poorly understood, especially in vivo. This class of proteolytic enzymes is parted into five families including the Ubiquitin-Specific Proteases (USP), which is the most important with about sixty members. Among them, the Ubiquitin-Specific Protease 2 (Usp2) gene encodes two protein isoforms, USP2-45 and USP2-69. The first is ubiquitously expressed under the control of the circadian clock and displays all features of core clock genes or its closest outputs effectors. Additionally, Usp2-45 was also found to be induced by the mineralocorticoid hormone aldosterone and thought to participate in Na+ reabsorption and blood pressure regulation by Epithelial Na+ Channel ENaC in the kidneys. During my thesis, I aimed to characterize the role of Usp2 in vivo with respect to these two areas, by taking advantage of a total constitutive knockout mouse model. In the first project I aimed to validate the role of USP2-45 in Na+ homeostasis and blood pressure regulation by the kidneys. I found no significant alterations of diurnal Na+ homeostasis and blood pressure in these mice, indicating that Usp2 does not play a substantial role in this process. In urine analyses, we found that our Usp2-KO mice are actually hypercalciuric. In a second project, I aimed to understand the causes of this phenotype. I found that the observed hypercalciuria results essentially from intestinal hyperabsorption. These data reveal a new role for Usp2 as an output effector of the circadian clock in dietary Ca2+ metabolism in the intestine.