Defining the role of NatC dependent N-terminal acetylation in Drosophila fertility

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Terminal interruption of relux source technique in the treatment of active venous ulcers

Introduction: The treatment for venous ulcers in most cases is unsatisfactory, with recurrences and poor healing. Objective: to evaluate adjuvant therapy in the treatment of active venous ulcers. Methods: We analyzed 20 patients with active venous ulcers attending the general Surgery outpatient clinic at the “Dr. José eleuterio gonzález” University Hospital from October 2012 to January 2013. they were randomly divided into 2 groups: group A (11 patients) underwent compression therapy and group B (9 patients) underwent compression therapy plus removal of the vein that gives terminal relux to the ulcer, guided by ultrasound (microphlebectomy). Patients were evaluated weekly (8 weeks). At each assessment, photographs and lesion measurements were taken and pain was evaluated using the visual analog scale. Results: No significant differences were found between the study groups in terms of age, weight, height, body mass index (BMi), ankle-brachial index, and baseline measurement of the ulcer (p>0.05). Group B showed a greater reduction in ulcer size and a statistically signiicant lower score on the visual analog pain scale (p<0.05) from the second and third week of treatment, respectively. Conclusions: the results obtained in patients with surgical procedure (group B) are consistent with the reported eficacy of chronic venous ulcer treatment with saphenectomy (conventional surgery), the difference is that in this study we used a minimally invasive procedure (microphlebectomy).

Reconversão de zonas portuárias para uso público. Estudo de caso: Terminal de contentores de Alcântara, Lisboa

Mestrado em Arquitectura Paisagista - Instituto Superior de Agronomia - UL

Galanin N-Terminal fragment (1-15) enhances the antidepressant effects of the 5-HT1A receptor agonist 8-OH-DPAT in the Forced Swimming Test.

Galanin and Galanin (1-15) [GAL(1-15)] are implicated in anxiety- and depression related behaviors. Moreover, Galanin modulates 5-HT1A receptor (5-HT1AR) function at autorreceptor and postsynaptic level in the brain. In this study, we have analysed the ability of GAL(1-15) to modulate the effects of the 8-OH-DPAT agonist in the Forced Swimming Test (FST). Groups of rats were assessed in the FST. In the first set of experiments, to evaluate the interactions of 8-OH-DPAT and GAL(1-15), rats received subcutaneously (s.c) the effective doses of 8-OH-DPAT (0.25mg/Kg) 60min before the test and intracerebroventricularly (icv) GAL(1-15)1nmol 15min before the tests alone or in combination. In the second set of experiments, groups of rats received s.c. 8-OH-DPAT (0.125mg/Kg), icv GAL(1-15) 1nmol and icv the GALR2 antagonist M871 3 nmol alone or in combination. The locomotor activity was analysed in the open field test. GAL(1-15) 1nmol enhanced the antidepressant-like effects mediated by the effective dose of the 8-OH-DPAT. GAL(1-15) significantly decreased the immobility (p<0.05) and climbing (p<0.05) and increased the swimming (p<0.01) behaviour induced by an effective dose of 8-OH-DPAT (0.25mg/Kg) in FST. Moreover, after coadministration of GAL(1-15) and threshold dose of 8-OH-DPAT (0.125mg/Kg) a significant decreased appeared in immobility (p<0.01) and climbing (p<0.01) and increased the swimming behavior (p<0.001) vs 8-OH-DPAT group. Moreover, M871 blocked completely this interaction. These results indicate that GAL(1-15) enhances the antidepressant effects induced by 8-OH-DPAT in the FST. These findings may give the basis for the development of novel therapeutic drugs. This study was supported by Junta de Andalucía CVI6476.

Galanin n-terminal gragment (1-15) modifies the 5-HT1A receptor against [H3]-8-OH-DPAT binding in the dorsal raphe and hippocampus of the rat

We have described that Galanin N-terminal fragment (1-15) [GAL(1-15)] is associated with depressive effects and also modulates the antidepressant effects induced by the 5-HT1A receptor (5-HT1AR) agonist 8-OH-DPAT. The aim of this study is to analyze the ability of GAL(1-15) to modulate 5-HT1AR at the autoreceptor and postsynaptic receptor level in rats by using quantitative autoradiography. We analyzed the effect of intracerebroventricular GAL(1-15)-3nmol (n=6) or aCSF (n=6), 10 minutes, 2 and 5 hours after the injection, on the binding characteristics of the 5-HT1AR agonist [H3]-8-OH-DPAT in sections of the Dorsal Raphe (DR) and Dorsal Hippocampus, specifically CA1 and Dentate Gyrus (DG). Student’s t-test was used to compare the experimental groups. GAL(1-15) produced a time-dependent effect on the binding of [H3]-8-OH-DPAT. In CA1 and DG, a significant increase in the KD and Bmax was observed, by 90%(p<0.05), at 10 minutes and 2 hours after injection. However, 5 hours after GAL(1-15) the only significant change remaining was the increase in Bmax at the DG. The coinjection of the GALR2 antagonist M871 blocked significantly the effects induced by GAL(1-15) in both areas. In DR, 2 hours after injection GAL(1-15) only produced a decrease in the Bmax by 20%(p<0.05). These results indicate that GAL(1-15) interacts with 5-HT1AR at the receptor level in DR and Dorsal Hippocampus. Therapeutic strategies based on these results could be developed for the treatment of depression disorders. This work has been supported by Junta de Andalucia CVI646 and Spanish Ministry of Economy PSI2013-44901-P.

Determinants Of Firm Terminal Value: The Perspective Of North American And European Financial Analysts

Company valuation models attempt to estimate the value of a company in two stages: (1) comprising of a period of explicit analysis and (2) based on unlimited production period of cash flows obtained through a mathematical approach of perpetuity, which is the terminal value. In general, these models, whether they belong to the Dividend Discount Model (DDM), the Discount Cash Flow (DCF), or RIM (Residual Income Models) group, discount one attribute (dividends, free cash flow, or results) to a given discount rate. This discount rate, obtained in most cases by the CAPM (Capital asset pricing model) or APT (Arbitrage pricing theory) allows including in the analysis the cost of invested capital based on the risk taking of the attributes. However, one cannot ignore that the second stage of valuation that is usually 53-80% of the company value (Berkman et al., 1998) and is loaded with uncertainties. In this context, particular attention is needed to estimate the value of this portion of the company, under penalty of the assessment producing a high level of error. Mindful of this concern, this study sought to collect the perception of European and North American financial analysts on the key features of the company that they believe contribute most to its value. For this feat, we used a survey with closed answers. From the analysis of 123 valid responses using factor analysis, the authors conclude that there is great importance attached (1) to the life expectancy of the company, (2) to liquidity and operating performance, (3) to innovation and ability to allocate resources to R&D, and (4) to management capacity and capital structure, in determining the value of a company or business in long term. These results contribute to our belief that we can formulate a model for valuating companies and businesses where the results to be obtained in the evaluations are as close as possible to those found in the stock market

Determinants of Terminal Value in the Evaluation of Companies: A Panel Data Approach to the Context of European Companies.

The uncertainty of the future of a firm has to be modelled and incorporated into the evaluation of companies outside their explicit period of analysis, i.e., in the continuing or terminal value considered within valuation models. However, there is a multiplicity of factors that influence the continuing value of businesses which are not currently being considered within valuation models. In fact, ignoring these factors may cause significant errors of judgment, which can lead models to values of goodwill or badwill, far from the substantial value of the inherent assets. Consequently, these results provided will be markedly different from market values. So, why not consider alternative models incorporating life expectancy of companies, as well as the influence of other attributes of the company in order to get a smoother adjustment between market price and valuation methods? This study aims to provide a contribution towards this area, having as its main objective the analysis of potential determinants of firm value in the long term. Using a sample of 714 listed companies, belonging to 15 European countries, and a panel data for the period between 1992 and 2011, our results show that continuing value cannot be regarded as the current value of a constant or growth perpetuity of a particular attribute of the company, but instead be according to a set of attributes such as free cash flow, net income, the average life expectancy of the company, investment in R&D, capabilities and quality of management, liquidity and financing structure.

The Terminal Value (TV) Performing in Firm Valuation: The Gap of Literature and Research Agenda

The uncertainty about the future of firms must be modeled and incorporated in the valuation of enterprises outside the explicit period of analysis, i.e., in the continuing or terminal value (TV). There is a multiplicity of factors that influence the TV of firms which are not being considered within current evaluation models. This aspect leads to the incurring of unrecoverable errors, thus leading to values of goodwill or bad will far away from the substantial value of intrinsic assets. As a consequence, the evaluation results will be presented markedly different from market values. There is no consensus in the scientific community about the method of computation of the TV as a forecast in an infinite horizon. The size of the terminal, or non-explicit period, assumed as infinite, is never called into question by scientific literature, or the probability of business bankruptcy. This paper aims to promote a study of the existing literature on the TV, to highlight the fragility of the evaluation models of companies that have been used by the academic community and by financial analysts, and to point out lines for future research to minimize these errors.

O valor terminal ou de continuidade, na avaliação de empresas

É um facto que a incerteza sobre o futuro das sociedades tem de ser modelada e incorporada na sua avaliação, fora do período explícito de análise, ou seja: nos valores de continuidade (VC), valor residual (VR) ou valor terminal (VT), considerados nos modelos de avaliação. Existem inúmeros fatores que influenciam o valor de continuidade das empresas e que não são, atualmente, considerados nos modelos de avaliação de empresas, destacando-se, entre os mais relevantes, a ausência de quaisquer referências à esperança média de vida das empresas. De facto, ao ignorarmos esses fatores, podemos incorrer em erros irreparáveis, conduzindo as avaliações a valores de goodwill ou badwill, muito longe do real valor substancial dos ativos, que lhes é intrínseco. Como consequência, os referidos resultados apresentar-se-ão vincadamente diferentes dos valores de mercado. Assim, porque não considerar modelos alternativos (incorporando nos mesmos a esperança de vida das empresas) e a influência de outros fatores, de forma a obter um ajustamento mais eficiente, no que respeita à forma de cálculo do valor da empresa? Este trabalho pretende fornecer um contributo neste domínio, tendo como primeiro objetivo (e para além da revisão da literatura existente sobre a matéria) a construção de uma tábua de mortalidade para as empresas portuguesas, que possa ser utilizada para eliminar ou, pelo menos, reduzir um dos principais problemas causadores de distorção dos atuais modelos de avaliação de empresas: a premissa de existência (ilimitada no tempo) de uma empresa. Com esse propósito, através da metodologia associada à construção de tábuas de mortalidade para os seres humanos, construímos uma tabela com a esperança média de vida associada às empresas portuguesas. Assim, usando uma base de dados (com cerca de 182.000 registos sobre falências, dissoluções e cessão de atividade em Portugal, desde 1900 até 2009), concluímos que, nos primeiros 5 anos, “morrem” 31% das empresas e que a esperança média de vida (à nascença) é de 12 anos. Estes resultados evidenciam a fragilidade dos modelos de avaliação de empresas, em que se estima o VT com uma perpetuidade. Após ficar patente que as empresas não têm uma esperança de vida infinita, preocupar-nos-emos em identificar quais os fatores responsáveis pela existência da empresa (no longo prazo), fatores esses que possam, porventura, justificar uma vida mais longa das sociedades. VI Nesse sentido, o segundo objetivo passou por identificar quais os fatores determinantes do valor terminal da empresa. Assim [utilizando uma amostra de 714 empresas cotadas, pertencentes a 15 países europeus e para um período compreendido entre 1992 e 2011, usando a metodologia GMM (Generalized method of moments), aplicada a dados em painel dinâmico], os resultados evidenciam que o valor de continuidade não pode ser considerado como o valor atual de uma perpetuidade constante (ou com crescimento) de um determinado atributo da empresa mas, sim, em função de um conjunto de atributos, como os free cash flows, os resultados líquidos, a esperança média de vida da empresa, o investimento em I&D, as capacidades e qualidade da gestão, a liquidez dos títulos e a estrutura de financiamento. Como terceiro objetivo (e mantendo a particular atenção na estimação do VT da empresa), procurou-se cruzar os resultados obtidos no estudo anterior com as perceções dos analistas Europeus e Estadunidenses acerca dos atributos da empresa que, na opinião destes, mais contribuem para o seu valor. Para o feito, recorreu-se a um inquérito, com respostas fechadas. Da análise das 123 respostas válidas, obtidas usando a análise fatorial, concluiuse serem determinantes do valor de uma empresa ou negócio os seguintes fatores: a esperança média de vida da empresa, a sua liquidez e desempenho operacional, a inovação e capacidade de afetação de recursos a I&D, as capacidades de gestão e a estrutura de capital, confirmando-se as conclusões até então obtidas. Por fim, fez-se um esforço no sentido de fornecer ao leitor uma nova aproximação teórica ao modelo Discounted CashFlow (DCF), tendo em conta as variáveis entretanto identificadas no nosso estudo. Estes resultados contribuem, a nosso ver, para que se possa caminhar no sentido da construção de um modelo de avaliação de empresas e negócios ainda mais apurado, em que os resultados obtidos nas avaliações se aproximem o mais possível dos verificados no mercado.

Supervivencia y mortalidad por tipo de terapia de reemplazo renal, en pacientes con enfermedad renal crónica terminal, en una cohorte de pacientes asegurados en Colombia

En Colombia se ha podido establecer que la incidencia y mortalidad de la Enfermedad Renal Crónica Terminal continúan en aumento en los últimos 6 años a pesar de las estrategias de intervención para prevención y control de la enfermedad implementadas nivel nacional. Este trabajo busca establecer la línea de base para la población asegurada en Colombia, frente a la supervivencia de pacientes en terapia de remplazo renal (TRR).

Isolation and characterization of plastid terminal oxidase gene from carrot and its relation to carotenoid accumulation

Carrot (Daucus carota L.) is a biennial plant that accumulates considerable amounts of carotenoid pigments in the storage root. To better understand the molecular mechanisms for carotenoid accumulation in developing storage roots, plastid terminal oxidase (PTOX) cDNA was isolated and selected for reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Present in photosynthetic species, PTOX is a plastid-located, nucleus encoded plastoquinone (PQ)-O2 oxidoreductase (plastioquinol oxidase). The enzyme is known to play a role as a cofactor for phytoene desaturase, and consequently plays a key role in the carotenoid biosynthesis pathway. A single PTOX gene was identified (DcPTOX) in carrot. DcPTOX encodes a putative protein with 366 amino acids that contains the typical structural features of PTOXs from higher plants. The expression of DcPTOX was analysed during the development of white, yellow, orange, red, and purple carrot roots, along with five genes known to be involved in the carotenoid biosynthesis pathway, PSY2, PDS, ZDS1, LCYB1, and LCYE. Expression analysis revealed the presence of DcPTOX transcripts in all cultivars, and an increase of transcripts during the time course of the experiment, with differential expression among cultivars in early stages of root growth. Our results demonstrated that DcPTOX showed a similar profile to that of other carotenoid biosynthetic genes with high correlation to all of them. The preponderant role of PSY in the biosynthesis of carotenoid pigments was also confirmed.

Raman spectroscopy of the molybdate minerals chillagite (tungsteinian wulfenite-I4), stolzite, scheelite, wolframite and wulfenite

Raman spectra of chillagite, wulfenite, stolzite, scheelite and wolframite were obtained at 298 and 77 K using a Raman microprobe in combination with a thermal stage. Chillagite is a solid solution of wulfenite and stolzite. The spectra of these molybdate minerals are orientation dependent. The band at 695 cm-1 is interpreted as an antisymmetric bridging mode associated with the tungstate chain. The bands at 790 and 881 cm-1 are associated with the antisymmetric and symmetric Ag modes of terminal WO2 whereas the origin of the 806 cm-1 band remains unclear. The 4(Eg) band was absent for scheelite. The bands at 353 and 401 cm-1 are assigned as either deformation modes or as r(Bg) and (Ag) modes of terminal WO2. The band at 462 cm-1 has an equivalent band in the infrared at 455 cm-1 assigned as as(Au) of the (W2O4)n chain. The band at 508 cm-1 is assigned as sym(Bg) of the (W2O4)n chain.

General practitioners’ experiences of the psychological aspects in the care of a dying patient

Objective: General practitioners (GPs) play an integral role in addressing the psychological needs of palliative care patients and their families. This qualitative study investigated psychosocial issues faced by GPs in the management of patients receiving palliative care and investigated the themes relevant to the psychosocial care of dying patients. Method: Fifteen general practitioners whose patient had been recently referred to the Mt. Olivet Palliative Home Care Services in Brisbane participated in an individual case review discussions guided by key questions within a semistructured format. These interviews focused on the psychosocial aspects of care and management of the referred patient, including aspects of the doctor/patient relationship, experience of delivering diagnosis and prognosis, addressing the psychological concerns of the patients' family, and the doctors' personal experiences, reactions, and responses. Qualitative analysis was conducted on the transcripts of these interviews. Results: The significant themes that emerged related to perceived barriers to exploration of emotional concerns, including spiritual issues, and the discussion of prognosis and dying, the perception of patients' responses/coping styles, and the GP's personal experience of the care (usually expressed in terms of identification with patient). Significance of results: The findings indicate the significant challenges facing clinicians in discussions with patients and families about death, to exploring the patient's emotional responses to terminal illness and spiritual concerns for the patient and family. These qualitative date indicate important tasks in the training and clinical support for doctors providing palliative care.

Biochemical characterization of Aprataxin, the protein deficient in Ataxia with Oculomotor Apraxia type 1

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.