Endothelium-derived intermedin/adrenomedullin-2 protects human ventricular cardiomyocytes from ischaemia-reoxygenation injury predominantly via the AM1 receptor

Application of intermedin/adrenomedullin-2 (IMD/AM-2) protects cultured human cardiac vascular cells and fibroblasts from oxidative stress and simulated ischaemia-reoxygenation injury (I-R), predominantly via adrenomedullin AM1 receptor involvement; similar protection had not been investigated previously in human cardiomyocytes (HCM). Expression of IMD, AM and their receptor components was studied in HCM. Receptor subtype involvement in protection by exogenous IMD against injury by simulated I-R was investigated using receptor component-specific siRNAs. Direct protection by endogenous IMD against HCM injury, both as an autocrine factor produced in HCM themselves and as a paracrine factor released from HCMEC co-cultured with HCM, was investigated using peptide-specific siRNA for IMD. IMD, AM and their receptor components (CLR, RAMPs1-3) were expressed in HCM. IMD 1 nmol L−1, applied either throughout ischaemia (3 h) and re-oxygenation (1 h) or during re-oxygenation (1 h) alone, attenuated HCM injury (P < 0.05); cell viabilities were 59% and 61% respectively vs. 39% in absence of IMD. Cytoskeletal disruption, protein carbonyl formation and caspase activity followed similar patterns. Pre-treatment (4 days) of HCM with CLR and RAMP2 siRNAs attenuated (P < 0.05) protection by exogenous IMD. Pre-treatment of HCMEC with IMD (and AM) siRNA augmented (P < 0.05) I-R injury: cell viabilities were 22% (and 32%) vs. 39% untreated HCMEC. Pre-treatment of HCM with IMD (and AM) siRNA did not augment HCM injury: cell viabilities were 37% (and 39%) vs. 39% untreated HCM. Co-culture with HCMEC conferred protection from injury on HCM; such protection was attenuated when HCMEC were pre-treated with IMD (but not AM) siRNA before co-culture. Although IMD is present in HCM, IMD derived from HCMEC and acting in a paracrine manner, predominantly via AM1 receptors, makes a marked contribution to cardiomyocyte protection by the endogenous peptide against acute I-R injury.

Hypofractionated radiotherapy versus conventionally fractionated radiotherapy for patients with intermediate-risk localised prostate cancer: 2-year patient-reported outcomes of the randomised, non-inferiority, phase 3 CHHiP trial

BACKGROUND: Patient-reported outcomes (PROs) might detect more toxic effects of radiotherapy than do clinician-reported outcomes. We did a quality of life (QoL) substudy to assess PROs up to 24 months after conventionally fractionated or hypofractionated radiotherapy in the Conventional or Hypofractionated High Dose Intensity Modulated Radiotherapy in Prostate Cancer (CHHiP) trial.

METHODS: The CHHiP trial is a randomised, non-inferiority phase 3 trial done in 71 centres, of which 57 UK hospitals took part in the QoL substudy. Men with localised prostate cancer who were undergoing radiotherapy were eligible for trial entry if they had histologically confirmed T1b-T3aN0M0 prostate cancer, an estimated risk of seminal vesicle involvement less than 30%, prostate-specific antigen concentration less than 30 ng/mL, and a WHO performance status of 0 or 1. Participants were randomly assigned (1:1:1) to receive a standard fractionation schedule of 74 Gy in 37 fractions or one of two hypofractionated schedules: 60 Gy in 20 fractions or 57 Gy in 19 fractions. Randomisation was done with computer-generated permuted block sizes of six and nine, stratified by centre and National Comprehensive Cancer Network (NCCN) risk group. Treatment allocation was not masked. UCLA Prostate Cancer Index (UCLA-PCI), including Short Form (SF)-36 and Functional Assessment of Cancer Therapy-Prostate (FACT-P), or Expanded Prostate Cancer Index Composite (EPIC) and SF-12 quality-of-life questionnaires were completed at baseline, pre-radiotherapy, 10 weeks post-radiotherapy, and 6, 12, 18, and 24 months post-radiotherapy. The CHHiP trial completed accrual on June 16, 2011, and the QoL substudy was closed to further recruitment on Nov 1, 2009. Analysis was on an intention-to-treat basis. The primary endpoint of the QoL substudy was overall bowel bother and comparisons between fractionation groups were done at 24 months post-radiotherapy. The CHHiP trial is registered with ISRCTN registry, number ISRCTN97182923.

FINDINGS: 2100 participants in the CHHiP trial consented to be included in the QoL substudy: 696 assigned to the 74 Gy schedule, 698 assigned to the 60 Gy schedule, and 706 assigned to the 57 Gy schedule. Of these individuals, 1659 (79%) provided data pre-radiotherapy and 1444 (69%) provided data at 24 months after radiotherapy. Median follow-up was 50·0 months (IQR 38·4-64·2) on April 9, 2014, which was the most recent follow-up measurement of all data collected before the QoL data were analysed in September, 2014. Comparison of 74 Gy in 37 fractions, 60 Gy in 20 fractions, and 57 Gy in 19 fractions groups at 2 years showed no overall bowel bother in 269 (66%), 266 (65%), and 282 (65%) men; very small bother in 92 (22%), 91 (22%), and 93 (21%) men; small bother in 26 (6%), 28 (7%), and 38 (9%) men; moderate bother in 19 (5%), 23 (6%), and 21 (5%) men, and severe bother in four (<1%), three (<1%) and three (<1%) men respectively (74 Gy vs 60 Gy, ptrend=0.64, 74 Gy vs 57 Gy, ptrend=0·59). We saw no differences between treatment groups in change of bowel bother score from baseline or pre-radiotherapy to 24 months.

INTERPRETATION: The incidence of patient-reported bowel symptoms was low and similar between patients in the 74 Gy control group and the hypofractionated groups up to 24 months after radiotherapy. If efficacy outcomes from CHHiP show non-inferiority for hypofractionated treatments, these findings will add to the growing evidence for moderately hypofractionated radiotherapy schedules becoming the standard treatment for localised prostate cancer.

FUNDING: Cancer Research UK, Department of Health, and the National Institute for Health Research Cancer Research Network.

LL-37 quantification in gingival crevicular fluid (GCF) from periodontitis patients

Background: LL-37, an anti-microbial peptide belonging to the cathelicidin family, derives its name from two N-terminal leucine residues and the 37 amino acids comprising the peptide. LL-37 is the only known cathelicidin to exist in humans. It exhibits both anti-bacterial and immunomodulatory properties. Objectives: In the current study, LL-37 was quantified in GCF from periodontitis patients. Previous studies have relied on qualitative results from Western blotting to detect LL-37 in GCF. This study aims to quantitatively determine LL-37 levels in GCF. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 sites in 12 patients presenting with advanced periodontitis. Plaque samples were analysed by QPCR for the presence or absence of the periodontopathic bacterium Porphyromonas gingivalis (P. gingivalis). The concentrations of LL-37 in patient samples pre- and post-treatment were deduced by indirect enzyme linked immunosorbent assay (ELISA). Results: Concentrations of LL-37 in samples varied between a minimum and maximum of 1 and 40 ng/ml. LL-37 levels were shown, pre-treatment, to be higher in deep pockets (6-9 mm) compared with shallower pockets (3-5 mm) and highest in those sites which were positive for P. gingivalis. Non-surgical therapy resulted in a significant improvement in clinical indices while expression levels of P. gingivalis were reduced. Following treatment, LL-37 levels in GCF decreased from an average of 6.5 ± 1 - 5.8 ± 1.2 ng/ml. The most interesting observation however was the reduction in LL-37 levels, from an average of 7 ± 1.3 – 2.5 ± 1.1 ng/ml in those sites where P. gingivalis infection was eradicated post-treatment. Conclusions: LL-37 levels are increased at sites showing advanced periodontal disease, reduce following treatment and appear to be linked to the presence of P. gingivalis. This study will further our knowledge of host defence in chronic diseases such as periodontitis.

The role of LL-37 in periodontal wound healing

Introduction: As a result of chronic inflammation during periodontal disease the junctional epithelium becomes micro-ulcerated. The inflammatory process is mediated by both bacterial and host cell products. Host defence peptides such as defensins, secretory leucocyte protease inhibitor (SLPI) and the sole human cathelicidin, LL-37, are secreted by both periodontal cells and neutrophils into gingival crevicular fluid (GCF). They have the ability to modulate the immune response in periodontitis and are thought to have a potential role in periodontal wound healing. Objectives: The aims of this study were to determine the role of LL-37 in the production of Interleukin (IL)-8, IL-6, hepatocyte growth factor (HGF) and basic-fibroblast growth factor (bFGF) by gingival fibroblasts. The role of LL-37 in modulating total matrix metalloproteinase (MMP) activity and expression of tissue inhibitors of metalloproteinase (TIMP)-1 and -2 by gingival fibroblasts was also investigated. Methods: Primary gingival fibroblasts were co-cultured with concentrations of LL-37 (1, 5 and 10µg/ml) for 24 hours and their supernatants tested for levels of IL-8 and IL-6, HGF, bFGF, TIMP-1 and TIMP-2 by ELISA. Rates of MMP turnover in the supernatants were tested by fluorogenic assay using fluorescence resonance energy transfer (FRET) peptide substrates. Cytotoxicity was measured by MTT assay. Statistical significance was measured using the independent t-test and p<0.05 was considered significant. Results: LL-37 significantly upregulated levels of IL-8, IL-6, HGF, bFGF and TIMP-1 (p<0.05) in a dose-dependent fashion. LL-37 significantly decreased the total MMP activity (p<0.05). None of the LL-37 concentrations tested were cytotoxic to gingival fibroblasts. Conclusion: These results indicate that LL-37 is involved in periodontal wound healing. LL-37 increased levels of proinflammatory cytokines and increased levels of growth factors involved in re-epithelialisation. LL-37 has the ability to regulate remodelling of the periodontium by controlling MMP overactivity both directly and by stimulating production of inhibitors by gingival fibroblasts.

LL-37 Degradation by GCF: A Role for Porphyromonas gingivalis Proteinases?

Background: LL-37, composed of 37 amino acid residues, is an innate host defence peptide of the cathelicidin family. It is expressed by neutrophils, monocytes and epithelial cells and exhibits both anti-bacterial and immunomodulatory properties. LL-37 is however prone to proteolytic degradation by proteinases, thus potentially limiting its inherent host defence properties in the inflammatory milieu. Objectives: The present study was designed to determine whether LL-37 was degraded by components of gingival crevicular fluid (GCF) from healthy subjects or those with periodontitis. In addition, we aimed to deduce whether degradation of the peptide was accelerated in GCF samples which were determined to be positive for the periodontopathic bacterium Porphyromonas gingivalis. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 individual sites in patients presenting with advanced periodontitis. In healthy subjects, GCF samples only were collected. Plaque samples were analysed by QPCR for the presence or absence of P. gingivalis. Pooled GCF samples from healthy sites; periodontitis sites which were P. gingivalis negative (Pg-); or periodontitis sites which were P. gingivalis positive (Pg+), were incubated with synthetic LL-37 for 0 – 180 min. The degradation products were then analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Results: LL-37 was not degraded when incubated with GCF from healthy subjects. In contrast, LL-37 was degraded after 30 min when incubated with Pg- GCF. However degradation of LL-37 was apparent after only 2 min incubation with Pg+ GCF and the parent molecule was almost completely degraded after 30 min. Conclusions: The rapid degradation of LL-37, particularly in Pg+ sites, highlights the limited role which this host defence peptide may play in the presence of biologically active proteinases. It also underscores a potent virulence mechanism of P. gingivalis used to circumvent innate host responses.

Maus-tratos entre iguais e percepção de suporte social: um estudo com alunos dos 2º e 3º ciclos do ensino básico

O bullying e o suporte social, apesar de conceptualmente distintos, interagem entre si. O bullying é um subgrupo do comportamento agressivo, que ocorre intencional e repetidamente, numa relação assimétrica de poder entre pares. O suporte social envolve o apoio recebido pelas fontes de suporte da rede social. O principal objetivo deste estudo é analisar a relação entre bullying e perceção de suporte social. O mesmo realizou-se numa escola básica de Évora, com 335 alunos dos 2° e 3° ciclos do ensino básico, sendo utilizados o Questionário de Violência Escolar e Isolamento Social e o Questionário de Percepção de Suporte Social. Os resultados obtidos indicam baixos níveis de incidência de comportamentos de bullying. Verificou-se que uma fraca perceção de suporte social associa-se a um maior envolvimento no bullying, o que aponta para a perceção de suporte social como um fator protetor para o envolvimento em situações de vitimação e de agressão; ABSTRACT: Bullying and social support, although conceptually distinct, interact between itself. Bullying is a sub-group of the aggressive behavior that occurs repeatedly and intentionally, in an asymmetrical power relationship between peers. Social Support involves the support received by the sources of social support networks. The aim of this study is to analise the relationship between bullying and perceived social support. The aim was done in a basic school of Évora, with 335 students of 2nd and 3th cycles of basic education. The used questionnaires had been the Questionnaire for School Violence and Social isolation and the Questionnaire for Perceived Social Support. The results indicate low levels of incidence of bullying behavior. lt was found that a low perception of social support is associated with an increased involvement in bullying, which points to the social support as a protective for involvement in situations of victimization and agression.

O sucesso/insucesso escolar na transição do 2º para o 3º Ciclo do Ensino Básico

Dissertação de mest., Psicologia da Educação - Necessidades Educativas Especiais, Faculdade de Ciências Humanas e Sociais, Universidade do Algarve, 2007

Diabetes Mellitus do tipo 2: influência da farmacogenómica na terapêutica oral

Dissertação de mest., Ciências Farmacêuticas, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Activities of an aqueous plant extract against Herpes simplex virus type 2 (HSV 2): role in virus replication and progeny yield

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2015

Acute hypoxia improves insulin sensitivity (SI2*) and β cell function in individuals with Type 2 Diabetes

Type 2 diabetes is a multifactorial metabolic disease characterized by defects in β-cells function, insulin sensitivity, glucose effectiveness and endogenous glucose production (1). It is widely accepted that insulin and exercise are potent stimuli for glucose transport (2). Acute exercise is known to promote glucose uptake in skeletal muscle via an intact contraction stimulated mechanism (3), while post-exercise improvements in glucose control are due to insulin-dependant mechanisms (2). Hypoxia is also known to promote glucose uptake in skeletal muscle using the contraction stimulated pathway. This has been shown to occur in vitro via an increase in β-cell function, however data in vivo is lacking. The aim of this study was to examine the effects of acute hypoxia with and without exercise on insulin sensitivity (SI2*), glucose effectiveness (SG2*) and β-cell function in individuals with type 2 diabetes. Following an overnight fast, six type 2 diabetics, afer giving informed written consent, completed 60 min of the following: 1) normoxic rest (Nor Rest); 2) hypoxic rest [Hy Rest; O2 = 14.6 (0.4)%]; 3) normoxic exercise (Nor Ex); 4) hypoxic exercise [Hy Ex; O2 = 14.6 (0.4)%]. Exercise trails were set at 90% of lactate threshold. Each condition was followed by a labelled intravenous glucose tolerance test (IVGTT) to provide estimations of SI2*, SG2* and β-cell function. Values are presented as means (SEM). Two-compartmental minimal model analysis showed SI2* to be higher following Hy Rest when comparisons were made with Nor Rest (P = 0.047). SI2* was also higher following Hy Ex [4.37 (0.48) x10-4 . min-1 (μU/ml)] compared to Nor Ex [3.24 (0.51) x10-4 . min-1 (μU/ml)] (P = 0.048). Acute insulin response to glucose (AIRg) was reduced following Hy Rest vs. Nor Rest (P = 0.014 - Table 1). This study demonstrated that 1) hypoxia has the ability to increase glucose disposal; 2) hypoxic-induced improvements in glucose tolerance in the 4 hr following exposure can be attributed to improvements in peripheral SI2*; 3) resting hypoxic exposure improves β-cell function and 4) exercise and hypoxia have an additive effect on SG2* in type 2 diabetics. These findings suggest a possible use for hypoxia both with and without exercise in the clinical treatment of type 2 diabetes.

Prática de ensino supervisionada no 1.º e no 2.º ciclo do ensino básico: a aprendizagem do conceito de área, com recurso a materiais manipuláveis

Relatório de Estágio apresentado à Escola Superior de Educação de Lisboa para obtenção de grau de mestre em Ensino do 1.º e 2.º ciclo do Ensino Básico

CuxSnSx+1 (x = 2, 3) thin films grown by sulfurization of metallic precursors deposited by dc magnetron sputtering

We report the results of the growth of Cu-Sn-S ternary chalcogenide compounds by sulfurization of dc magnetron sputtered metallic precursors. Tetragonal Cu2SnS3 forms for a maximum sulfurization temperature of 350 ºC. Cubic Cu2SnS3 is obtained at sulfurization temperatures above 400 ºC. These results are supported by XRD analysis and Raman spectroscopy measurements. The latter analysis shows peaks at 336 cm-1, 351 cm-1 for tetragonal Cu2SnS3, and 303 cm-1, 355 cm-1 for cubic Cu2SnS3. Optical analysis shows that this phase change lowers the band gap from 1.35 eV to 0.98 eV. At higher sulfurization temperatures increased loss of Sn is expected in the sulphide form. As a consequence, higher Cu content ternary compounds like Cu3SnS4 grow. In these conditions, XRD and Raman analysis only detected orthorhombic (Pmn21) phase (petrukite). This compound has Raman peaks at 318 cm-1, 348 cm-1 and 295 cm-1. For a sulfurization temperature of 450 ºC the samples present a multi-phase structure mainly composed by cubic Cu2SnS3 and orthorhombic (Pmn21) Cu3SnS4. For higher temperatures, the samples are single phase and constituted by orthorhombic (Pmn21) Cu3SnS4. Transmittance and reflectance measurements were used to estimate a band gap of 1.60 eV. For comparison we also include the results for Cu2ZnSnS4 obtained using similar growth conditions.

Prática de ensino supervisionadas do 1º e 2º ciclo do ensino básico:linguagem entre pares: código de grupo ou agressão?

Relatório de Estágio apresentado à Escola Superior de Educação de Lisboa para obtenção de grau de mestre em Ensino do 1.º e 2.º Ciclos do Ensino Básico

The neogene of Algarve: excursion 2

Second Congress R.C.A.N.S., Salamanca, pp.37-55