Search

Supplementary Table from Reengineering Ponatinib to Minimize Cardiovascular Toxicity

Supplementary Figure from Reengineering Ponatinib to Minimize Cardiovascular Toxicity

Small molecule tyrosine kinase inhibitors (TKI) have revolutionized cancer treatment and greatly improved patient survival. However, life-threatening cardiotoxicity of many TKIs has become a major concern. Ponatinib (ICLUSIG) was developed as an inhibitor of the BCR-ABL oncogene and is among the most cardiotoxic of TKIs. Consequently, use of ponatinib is restricted to the treatment of tumors carrying T315I-mutated BCR-ABL, which occurs in chronic myeloid leukemia (CML) and confers resistance to first- and second-generation inhibitors such as imatinib and nilotinib. Through parallel screening of cardiovascular toxicity and antitumor efficacy assays, we engineered safer analogs of ponatinib that retained potency against T315I BCR-ABL kinase activity and suppressed T315I mutant CML tumor growth. The new compounds were substantially less toxic in human cardiac vasculogenesis and cardiomyocyte contractility assays in vitro. The compounds showed a larger therapeutic window in vivo, leading to regression of human T315I mutant CML xenografts without cardiotoxicity. Comparison of the kinase inhibition profiles of ponatinib and the new compounds suggested that ponatinib cardiotoxicity is mediated by a few kinases, some of which were previously unassociated with cardiovascular disease. Overall, the study develops an approach using complex phenotypic assays to reduce the high risk of cardiovascular toxicity that is prevalent among small molecule oncology therapeutics.Significance:Newly developed ponatinib analogs retain antitumor efficacy but elicit significantly decreased cardiotoxicity, representing a therapeutic opportunity for safer CML treatment.

Small molecule tyrosine kinase inhibitors (TKI) have revolutionized cancer treatment and greatly improved patient survival. However, life-threatening cardiotoxicity of many TKIs has become a major concern. Ponatinib (ICLUSIG) was developed as an inhibitor of the BCR-ABL oncogene and is among the most cardiotoxic of TKIs. Consequently, use of ponatinib is restricted to the treatment of tumors carrying T315I-mutated BCR-ABL, which occurs in chronic myeloid leukemia (CML) and confers resistance to first- and second-generation inhibitors such as imatinib and nilotinib. Through parallel screening of cardiovascular toxicity and antitumor efficacy assays, we engineered safer analogs of ponatinib that retained potency against T315I BCR-ABL kinase activity and suppressed T315I mutant CML tumor growth. The new compounds were substantially less toxic in human cardiac vasculogenesis and cardiomyocyte contractility assays in vitro. The compounds showed a larger therapeutic window in vivo, leading to regression of human T315I mutant CML xenografts without cardiotoxicity. Comparison of the kinase inhibition profiles of ponatinib and the new compounds suggested that ponatinib cardiotoxicity is mediated by a few kinases, some of which were previously unassociated with cardiovascular disease. Overall, the study develops an approach using complex phenotypic assays to reduce the high risk of cardiovascular toxicity that is prevalent among small molecule oncology therapeutics. Significance: Newly developed ponatinib analogs retain antitumor efficacy but elicit significantly decreased cardiotoxicity, representing a therapeutic opportunity for safer CML treatment.

Acetylcholine (ACh) is best known as a neurotransmitter and was the first such molecule identified. ACh signalling in the neuronal cholinergic system has long been known to regulate numerous biological processes (reviewed by Beckmann and Lips). In actuality, ACh is a ubiquitous signalling molecule that is produced by numerous non-neuronal cell types and even by some single-celled organisms. Within multicellular organisms, a non-neuronal cholinergic system that includes the immune system functions in parallel with the neuronal cholinergic system. Several immune cell types both respond to ACh signals and can directly produce ACh. Recent work from our laboratory has demonstrated that the capacity to produce ACh is an intrinsic property of T cells responding to viral infection, and that this ability to produce ACh is dependent upon IL-21 signalling to the T cells. Furthermore, during infection this immune-derived ACh is necessary for the T cells to migrate into infected tissues. In this review, we will discuss the various sources of ACh that are relevant during immune responses and describe how ACh acts on immune cells to influence their functions. We will also address the clinical implications of this fascinating aspect of immunity, focusing on ACh's role in the migration of T cells during infection and cancer.

Mestrado em Educação Pré-Escolar e em Ensino do 1ºCiclo do Ensino Básico Submitted by Dina Rocha (dir.biblioteca@ipsantarem.pt) on 2021-11-30T15:18:54Z No. of bitstreams: 1 Relatório Estágio - Cristiana Teodoro.pdf: 1361886 bytes, checksum: 2f6f113771e80450bc723e140e7fb045 (MD5) Made available in DSpace on 2021-11-30T15:18:54Z (GMT). No. of bitstreams: 1 Relatório Estágio - Cristiana Teodoro.pdf: 1361886 bytes, checksum: 2f6f113771e80450bc723e140e7fb045 (MD5) Previous issue date: 2021-09-08 N/A

Trabalho de projeto apresentado à Escola Superior de Comunicação Social como parte dos requisitos para obtenção de grau de mestre em Publicidade e Marketing. Submitted by Anabela Lopes (ateixeira@escs.ipl.pt) on 2021-10-19T08:46:30Z No. of bitstreams: 1 Projeto de mestrado - Ativação de marca Samsung members.pdf: 3465406 bytes, checksum: 6bb85f3293553578b36a1946530473cb (MD5) Made available in DSpace on 2021-10-19T08:46:30Z (GMT). No. of bitstreams: 1 Projeto de mestrado - Ativação de marca Samsung members.pdf: 3465406 bytes, checksum: 6bb85f3293553578b36a1946530473cb (MD5) Previous issue date: 2021-07-13 N/A

Introduction and Purpose of the Study: The advent of small molecule Tyrosine Kinase Inhibitors (TKIs) has revolutionized cancer treatment and greatly improved patient survival. However, the life-threatening cardiovascular toxicity of many TKIs present major concerns. Ponatinib is the most effective treatment for patients harboring the common T315I mutation of BCR-ABL that drives Chronic Myeloid Leukemia (CML) yet is one of the most cardiotoxic of TKIs. Ponatinib-induced events include myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. The aim of this study is to reengineer the chemical structure of ponatinib to minimize its adverse cardiovascular effects by using in vitro and in vivo models of cardiovascular toxicity. Experimental Procedures: We developed in vitro cardiovascular toxicity assays using human iPSC-cardiomyocytes (hiPSC-CMs) and human microvascular endothelial cells (HMVECs), and assessed anti-tumor efficacy in T315I mutated K562 CML cells. The cardiotoxicity was determined by impairment of cardiomyocyte contractility function, the vascular toxicity was defined by inability to form capillary-like networks in vasculogenesis assays. In vitro screening of ponatinib analogues probed chemical moieties responsible for its cardiovascular toxicity that were confirmed using mice xenograft mice models with human T315I mutant K562 cells. Summary of Unpublished Data: Compared to ponatinib, refined analogues showed markedly decreased adverse effects on HMVECs vasculogenesis and hiPSC-CMs contractility in vitro. The therapeutic window was increased in vivo, leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increasing serum levels of cardiac troponin. The kinase inhibition profiles of the “safe” analogues compared to ponatinib identified candidate mediators of ponatinib-induced toxicity. Validation by selective knockdown revealed likely mediators of cardiotoxicity, including FGFR. Conclusions: We succeeded in engineering a cardiovascular safe TKI that retained effective therapeutic properties against BCR-ABL T351 mutated form of CML, but with substantially decreased cardiovascular toxicity. Citation Format: Anna Pavlovna Hnatiuk, Arne A. Bruyneel, Dhanir Taylor, Mallesh Pandrala, Arpit Dheeraj, Wendy Li, Ricardo Serrano Fernandez, Dries A. Feyen, Michelle M. Wu, Prashila Amatya, Isabel Morgado, Volker Wiebking, Matthew H. Porteus, Sanjay Malhotra, Mark Mercola. Reengineering ponatinib to minimize cardiovascular toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB077.

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

Hereditary apolipoprotein A-I (apoA-I) amyloidosis is a life-threatening incurable genetic disorder whose molecular underpinnings are unclear. In this disease, variant apoA-I, the major structural and functional protein of high-density lipoprotein, is released in a free form, undergoes an α-helix to intermolecular cross-β-sheet conversion along with a proteolytic cleavage, and is deposited as amyloid fibrils in various organs, which can cause organ damage and death. Glu34Lys is the only known charge inversion mutation in apoA-I that causes human amyloidosis. To elucidate the structural underpinnings of the amyloidogenic behavior of Glu34Lys apoA-I, we generated its recombinant globular N-terminal domain (residues 1-184) and compared the conformation and dynamics of its lipid-free form with those of two other naturally occurring apoA-I variants, Phe71Tyr (amyloidogenic) and Leu159Arg (non-amyloidogenic). All variants showed reduced structural stability and altered aromatic residue packing. The greatest decrease in stability was observed in the non-amyloidogenic variant, suggesting that amyloid formation is driven by local structural perturbations at sensitive sites. Molecular dynamics simulations revealed local helical unfolding and suggested that transient opening of the Trp72 side chain induced mutation-dependent structural perturbations in a sensitive region, including the major amyloid hot spot residues Leu14-Leu22. We posit that a shift from the "closed" to the "open" orientation of the Trp72 side chain modulates structural protection of amyloid hot spots, suggesting a previously unknown early step in the protein misfolding pathway.

Cartilage acidic protein1 (CRTAC1) is an extracellular matrix protein of chondrogenic tissue in humans and its presence in bacteria indicate it is of ancient origin. Structural modeling of piscine CRTAC1 reveals it belongs to the large family of beta-propeller proteins that in mammals have been associated with diseases, including amyloid diseases such as Alzheimer's. In order to characterize the structure/function evolution of this new member of the beta-propeller family we exploited the unique characteristics of piscine duplicate genes Crtac1a and Crtac1b and compared their structural and biochemical modifications with human recombinant CRTAC1. We demonstrate that CRTAC1 has a beta-propeller structure that has been conserved during evolution and easily forms high molecular weight thermo-stable aggregates. We reveal for the first time the propensity of CRTAC1 to form amyloid-like structures, and hypothesize that the aggregating property of CRTAC1 may be related to its disease-association. We further contribute to the general understating of CRTAC1's and beta-propeller family evolution and function. Proteins 2017; 85:242-255. © 2016 Wiley Periodicals, Inc.

Na atual conjuntura económica, caracterizada por uma grande competitividade dos mercados e mudanças rápidas é necessário encontrar formas de fazer face à concorrência. O desempenho das organizações é em grande parte influenciado pelo desempenho do seu capital humano. Por isso as organizações devem apostar na motivação dos seus recursos humanos, desta forma serão mais eficientes, criativos, produtivos, competentes e capazes de fazer face aos desafios. Para isso é importante ter uma liderança eficaz e ajustada à realidade da organização que seja capaz de motivar, orientar e conduzir no sentido dos objetivos da organização. Pretende-se assim com este estudo perceber o impacto dos líderes na motivação dos colaboradores. Perceber se há diferenças significativas entre o Líder Atual o Líder Eficaz e ainda se esta diferença prediz positiva e significativamente a motivação dos inquiridos. Este estudo tem como objetivos perceber de que forma a liderança tem impacto na motivação dos colaboradores, perceber se há diferenças entre as caraterísticas do Líder atual e do Líder Eficaz e se esta diferença influência significativamente a motivação. Para dar resposta aos objetivos, será usada uma abordagem quantitativa, com recurso a um questionário. Este terá medidas de Motivação, sobre o Líder eficaz e sobre o Líder atual. Os resultados obtidos a partir dos questionários revelaram que líder atual prediz, positiva e significativamente, a motivação dos colaboradores e que existem diferenças significativas entre o líder atual e o líder eficaz, contudo não se verificou que a diferença entre líder eficaz e líder atual tenha influência significativa na motivação dos inquiridos.

Amyloid light chain (AL) amyloidosis results from tissue deposition of clonal light chains, most commonly produced by clonal plasma cells. AL amyloidosis is closely associated with multiple myeloma (MM), another disease which arises from clonal plasma cell proliferation. Here we aim to identify gene signatures to distinguish AL cardiomyopathy (AL-CM) risk in MM patients. We utilized publicly available data sets and applied Graph Cluster Perturbation approach to cluster the co-expression networks based on pathways in MM and AL-CM utilizing 225 samples from four datasets: GSE42955 study (12 dilated CM, 12 ischemic CM and 5 control LV human heart tissues), GSE95077 study (16 amiloride drug treated/untreated myeloma cell line samples), GSE24128 study (16 newly diagnosed AL with monoclonal plasma cell samples over- or under-expressing cyclin D1), and GSE6477 study (76 primary bone marrow samples from hyper-diploid myeloma patients and 80 non-hyperdiploid MM patients). From these data sets, the networks for extracellular matrix organization, immune system, innate immune system, metabolism, and neutrophil degranulation pathways for MM and amyloid CM were extracted. Summary: Ranking of the perturbed genes based on pathways similarity index in MM and AL resulted in a panel of genes: CD44, NRAS, GRAP2, CTLA4, GSN, CCND1, NFKB1, and IRF1 (p= Figure , the high hazard ratio of this gene cluster in MM with AL-CM (3.76; p=0.021) compared to MM without CM (0.96; p=0.032) suggests the potential of this gene panel to distinguish high or low risk groups in MM based on survival.

Background: Neonatal mammalian hearts have the unique characteristic to fully repair and regenerate following injury. This discovery has led to visionary endeavors to understand and subsequently reawaken regeneration processes in the human adult heart that are lost after birth, contributing to heart failure and death after ischemic insults. In utero exposure to tobacco smoke has detrimental effects on fetal development and growth. However, the consequences of prenatal exposure to cigarette smoke on murine neonate’s cardiac regeneration have never been explored. Methods: To study the impact of cigarette smoke during the entire pregnancy on cardiac regeneration in the offspring, plugged female wild type C57LB/6J mice were exposed either to cigarette smoke (n=6) or filtered air (control, n=6). Two days after birth, 5 pups from each litter were assigned to the two experimental groups: Myocardial infarction (MI) and sham. Detrimental effects of in utero exposure to tobacco smoke on the recovery of cardiac function following MI surgery were followed using two-dimensional speckle tracking echocardiography and strain imaging. To investigate the underlying mechanism, cardiac tissue of the infarct and remote zone was collected for non-coding RNA analysis. Results: In utero exposure to cigarette smoke significantly compromised cardiac regeneration in neonates. At both early and late time points in the phase of cardiac repair, hearts of neonates exposed to cigarette smoke during pregnancy showed a marked impairment of cardiac regenerative potential. In particular, ejection fraction, fractional area change and shortening, as well as left ventricular internal diameter during systole remained pathologically changed following MI insult in the smoked group until the very endpoint. Conclusions: Collectively, we here provide evidence that in utero exposure to cigarette smoke strongly compromises cardiac regeneration in the newborn offspring. This result reinforces smoking cessation during pregnancy. Additionally, understanding the changes in non-coding RNA expression in a setting of preserved versus disrupted repair of the heart might be an important first step towards the identification of key cellular processes in cardiac regeneration after injury.

Cardiac dysfunction is the most frequent cause of morbidity and mortality in amyloid light chain (AL) amyloidosis caused by a clonal immunoglobulin light chain (LC). Previously published transgenic animal models of AL amyloidosis have not recapitulated the key phenotype of cardiac dysfunction seen in AL amyloidosis, which has limited our understanding of the disease mechanisms in vivo, as well as the development of targeted AL therapeutics. We have developed a transgenic zebrafish model in which a λ LC derived from a patient with AL amyloidosis is conditionally expressed in the liver under the control of the Gal4 upstream activation sequence enhancer system. Circulating LC levels of 125 µg/ml in these transgenic zebrafish are comparable to median pathological serum LC levels. Functional analysis links abnormal contractile function with evidence of cellular and molecular proteotoxicity in the heart, including increased cell death and autophagy. However, despite pathological and functional phenotypes analogous to human AL, the lifespan of the transgenic fish is comparable to control fish without the expressed AL-LC transgene. Nuclear labeling experiments suggest increased cardiac proliferation in the transgenic fish, which can be counteracted by treatment with a small molecule proliferation inhibitor leading to increased zebrafish mortality because of cardiac apoptosis and functional deterioration. This transgenic zebrafish model provides a platform to study underlying AL disease mechanisms in vivo further.NEW & NOTEWORTHY Heart failure is a major cause of mortality in amyloid light (AL) amyloidosis, yet it has been difficult to model in animals. We report the generation of a transgenic zebrafish model for AL amyloidosis with pathological concentration of circulating human light chain protein that results in cardiac dysfunction. The light chain toxicity triggers regeneration in the zebrafish heart resulting in functional compensation early in life, but with age develops into cardiac dysfunction.

The mammalian kidney contributes significantly to glucose homeostasis through gluconeogenesis. Considering that stanniocalcin 1 (STC1) regulates ATP production, is synthesized and acts in different cell types of the nephron, the present study hypothesized that STC1 may be implicated in the regulation of gluconeogenesis in the vertebrate kidney. Human STC1 strongly reduced gluconeogenesis from C-14-glutamine in rat renal medulla (MD) slices but not in renal cortex (CX), nor from C-14-lactic acid. Total PEPCK activity was markedly reduced by hSTC1 in MD but not in CX. Pck2 (mitochondrial PEPCK isoform) was down-regulated by hSTC1 in MD but not in CX. In fish (Dicentrarchus labrax) kidney slices, both STC1-A and -B isoforms decreased gluconeogenesis from C-14-acid lactic, while STC1-A increased gluconeogenesis from C-14-glutamine. Overall, our results demonstrate a role for STC1 in the control of glucose synthesis via renal gluconeogenesis in mammals and suggest that it may have a similar role in teleost fishes. (C) 2015 Elsevier Ireland Ltd. All rights reserved. Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) Brazil; Foundation for Science and Technology of Portugal [PTDC/MAR/121279/2010]; bilateral programme CAPES (Brazil)/GRICES (Portugal) CAPES/GRICES [215/08] info:eu-repo/semantics/publishedVersion

The Portuguese oyster Crassostrea angulata was the main oyster species produced in Europe until the 1970s when mass mortalities almost led to its disappearance. At that time, mortality events were associated with detection of irido-like viruses and no record of herpes-like virus exists. Nowadays, C. angulata is only present in relatively few locations in the Iberian Peninsula, unlike in Asia where production is widespread. In 2011 and 2013, we observed mortality outbreaks of C. angulata adults collected in the Ria Formosa (Portugal) with a cumulative mortality of 47 and 59%, respectively, a few days after the oysters were transferred to indoor conditions. Ostreid herpesvirus 1 (OsHV-1) DNA was detected by PCR in all dying oysters analyzed and a high viral load was observed by real time PCR. Sequence analysis of ORFs 4, 42, 43 and 88 as well as a non-coding region near ORF4 revealed that the OsHV-1 genotype was the microvar (μvar) variant. No nucleotide sequence variation was observed in the regions analyzed in any of the 18 isolates. However, polymorphism was observed in a non-coding region (NC1/NC2 region) located between ORFs 49 and 50. Two genotypes in the NC1/NC2 region differing by several nucleotides were identified during the 2 mortality outbreaks and each genotype was unique to each mortality event. In addition, a third genotype in the NC1/NC2 region of OsHV-1 μvar was observed in asymptomatic adults of C. angulata collected in Carrera River (Spain). OsHV-1 DNA was not detected by PCR in any of the oysters collected in Sado and Mira estuaries. The results obtained in the present study suggest that the NC1/NC2 region may be a highly polymorphic region and hence of particular interest for molecular epidemiological studies. In light of the results of the present and previous studies on the genetic diversity and detection of OsHV-1 in C. angulata, we hypothesize that the virus was introduced recently in Europe from Asia, possibly with the introduction of C. gigas in the 1970s or soon after.

A tubular photocatalytic reactor of the immersion type for water purification from organic pollutants has been developed. Few important principles were used in the construction of the reactor, namely, a symmetrical and uniform light distribution with direct incidence of UV irradiation on the photocatalyst surface, a highly active mixing of contaminated water as a result of an air bubbling flux, that simultaneously supplying oxygen that is necessary for a photocatalytic reaction. The implemented highly active thin film photocatalyst was prepared by the Chemical Vapor Deposition (CVD) technology using titanium(isopropoxide) (TTIP) as a precursor. The factor K = Surface/Volume of this reactor is about 255 m(-1). Together with an effective mixing, it creates excellent contacts between the contaminants and the photocatalyst which is very favorable for water purification. The efficiency of this reactor was proven by the decomposition of some pharmaceutical drugs (Ibuprofen, Acetylsalicylic acid, Sulphanilamide, Paracetamol, Caffeine) and of some pesticides (Dimethoate, Azoxystrobin, Iprodione, Propizamid, Isoproturon, Fenarimol). The relation between the kinetic constants of photocatalysis and of photolysis is K-photcat/K-photolysis = 2 divided by 18. These results demonstrate the feasibility of the developed photoreactor for the degradation of recalcitrant organic pollutants, such as pharmaceuticals and pesticides in water. (C) 2016 Elsevier B.V. All rights reserved. Fundacao para Ciencia e Tecnologia (FCT), Portugal [PEST-OE/QUI/UI4023/2014] Centre of Marine Sciences (CCMAR), Portugal info:eu-repo/semantics/publishedVersion

Background: Information provided by health professionals to potential donors and recipients is essential for an autonomous and objective decision to make a living kidney donation. Objectives: To determine the characteristics of the information received by living kidney donors and recipients, to find out their socio-sanitary profile, their socio-demographics, financial and labor characteristics, health and the caregiving activity of these donors and recipients. Methods: Observational, descriptive and cross-sectional study of the population of living kidney donors and recipients from the University Hospitals Puerta del Mar (Cádiz), Virgen del Rocío (Seville), and the University Hospital Complex of Granada, between 08/04/2014 and 08/06/2015. Results and conclusions: According to the 40 living kidney donors and their 40 recipients surveyed, it is mainly nephrologists who make people aware and provide information about living kidney donation. Almost half of recipients require more information so the evaluation processes and pre-donation information should be updated. In general, the living kidney donor is female, aged 50, with primary/secondary education, lives with a partner and is related to the kidney recipient. Also, the living kidney donor is in paid employment, is overweight, perceives her health as very good or good, and does not smoke or drink alcohol. However, the typical living kidney recipient is male, aged 44 and has completed secondary school studies and vocational training. Furthermore, he does not work, perceives his health as good or regular, and he is an independent person for activities of daily living. Resumen: Antecedentes: La información suministrada por profesionales sanitarios a posibles donantes y receptores es fundamental para una decisión autónoma y objetiva de donar un riñón en vida. Objetivos: Conocer las características de la información que reciben los donantes y receptores renales de vivo, averiguando su perfil sociosanitario, sus características sociodemográficas, económico-laborales, de salud y la actividad cuidadora de dichos donantes y receptores. Métodos: Estudio observacional, descriptivo, transversal, de la población de donantes y receptores renales de vivo, de los Hospitales Universitarios Puerta del Mar (Cádiz), Virgen del Rocío (Sevilla) y Complejo Hospitalario Universitario de Granada, entre el 8 de abril de 2014 y el 8 de junio de 2015. Resultados y conclusiones: Según los 40 donantes y 40 receptores renales de vivo encuestados, los facultativos de nefrología son principalmente quienes dan a conocer e informan sobre la donación renal en vida. Casi la mitad de receptores demandan más información, por lo que se deberían actualizar los procesos de evaluación y de información antes de la donación. En general, el donante renal vivo es mujer, de 50 años, con estudios de Primaria/ESO, vive en pareja, está emparentado con el receptor del riñón, realiza un trabajo remunerado, tiene sobrepeso, percibe su salud como muy buena o buena, y no fuma ni consume alcohol. Sin embargo, el receptor renal tipo es hombre, con 44 años, tiene estudios de bachillerato/FP, no trabaja, percibe su salud como buena o regular, y son personas independientes para las actividades de la vida diaria. Keywords: Health profile, Tissue and organ procurement, Tissue donors, Donor selection, Kidney transplantation, Living donors, Information, Consumer health information, Palabras clave: Perfil de salud, Obtención de tejidos y órganos, Donantes de tejidos, Selección de donante, Trasplante de riñón, Donantes vivos, Información, Información de salud al consumidor

The kinetic mechanism of amyloid aggregation remains to be fully understood. Investigations into the species present in the different kinetic phases can assist our comprehension of amyloid diseases and further our understanding of the mechanism behind amyloid β (Aβ) (1–40) peptide aggregation. Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) have been used in combination to monitor Aβ(1–40) aggregation in vitro at both normal and higher than standard concentrations. The observed fibrillation behaviour deviates, in several respects, from standard concepts of the nucleation–polymerisation models and shows such features as concentration-dependent non-linear effects in the assembly mechanism. Aβ(1–40) fibrillation kinetics do not always follow conventional kinetic mechanisms and, specifically at high concentrations, intermediate structures become populated and secondary processes may further modify the fibrillation mechanism.

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA2 pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA2 sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA2s while metazoan representatives are similar with GIX PLA2 of the marine snail Conus magus. Members of GXIV and GIX PLA2s show modest overall sequence similarity (21-35%) but considerable motif conservation within the putative Ca(2+)-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA2s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA2 homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA2 deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA2s, two of which correspond to those localized in GXIV PLA2s. Phylogenetic analysis demonstrates that metazoan GIX PLA2s cluster separate from the bacterial and fungal GXIV PLA2s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA2 pfam06951. Duplicate PLA2 pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA2s and support the idea of the common evolutionary origin of GXIV and GIX PLA2 pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.

Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML MLL cells (with an MLL-ENL fusion and a constitutively active N-RAS) independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML MLL and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML MLL mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.

Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source of endogenous DNA damage in cancer, which is suppressed by ATR and CHK1 kinases. We here show that ES suffer from high endogenous levels of RS, rendering them particularly dependent on the ATR pathway. Accordingly, two independent ATR inhibitors show in vitro toxicity in ES cell lines as well as in vivo efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas. We would want to thank Enrique de Alava for providing ES lines. Work in O.F. laboratory was supported by Fundación Botín, by Banco Santander through its Santander Universities Global Division and by grants from MINECO (SAF2014-57791-REDC and SAF2014-59498-R), Fundació La Marato de TV3, Howard Hughes Medical Institute and the European Research Council (ERC-617840). The A.N. laboratory was supported by the Intramural Research Program of the NIH, the National Cancer Institute, the Center for Cancer Research, an Ellison Medical Foundation Senior Scholar in Aging, and the Alex Lemonade Stand Foundation Award. J.A. laboratory is supported by Asociación Pablo Ugarte, ASION-La Hucha de Tomás, Fundación La Sonrisa de Alex and Instituto de Salud Carlos III (PI12/00816 and Spanish Cancer Network RTICC RD12/0036/0027). A.L. laboratory was supported by the Danish National Research Foundation (DNRF115), Danish Council for Independent Research (Sapere Aude, DFF-Starting Grant 2014) and Danish Cancer Society (KBVU-2014). Sí

Cartilage acidic protein1 (CRTAC1) is an extracellular matrix protein of chondrogenic tissue in humans and its presence in bacteria indicate it is of ancient origin. Structural modeling of piscine CRTAC1 reveals it belongs to the large family of beta-propeller proteins that in mammals have been associated with diseases, including amyloid diseases such as Alzheimer's. In order to characterize the structure/function evolution of this new member of the beta-propeller family we exploited the unique characteristics of piscine duplicate genes Crtac1a and Crtac1b and compared their structural and biochemical modifications with human recombinant CRTAC1. We demonstrate that CRTAC1 has a beta-propeller structure that has been conserved during evolution and easily forms high molecular weight thermo-stable aggregates. We reveal for the first time the propensity of CRTAC1 to form amyloid-like structures, and hypothesize that the aggregating property of CRTAC1 may be related to its disease-association. We further contribute to the general understating of CRTAC1's and beta-propeller family evolution and function. Proteins 2017; 85:242-255. © 2016 Wiley Periodicals, Inc.

Objectives: The detailed structure of brain-derived Aβ amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aβ(1–40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aβ amyloid plaque formation.Methods: We have used hydrogen–deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aβ fibrils.Results and conclusions: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aβ peptide fold is common to Aβ fibrils.

We have studied tertiary contacts in protofibrils and mature fibrils of amyloid-β (Aβ) peptides using solid-state NMR spectroscopy. Although intraresidue contacts between Glu-22 and Ile-31 were found in Aβ protofibrils, these contacts were completely absent in mature Aβ fibrils. This is consistent with the current models of mature Aβ fibrils. As these intramolecular contacts have also been reported in Aβ oligomers, our measurements suggest that Aβ protofibrils are structurally more closely related to oligomers than to mature fibrils. This suggests that some structural alterations have to take place on the pathway from Aβ oligomers/protofibrils to mature fibrils, in agreement with a model that suggests a conversion of intramolecular hydrogen-bonded structures of Aβ oligomers to the intermolecular stabilized mature fibrils (Hoyer, W., Gronwall, C., Jonsson, A., Stahl, S., and Hard, T. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5099–5104). Background: Little tertiary structure information is available for the toxic intermediates in the amyloid-β (Aβ) fibrillation process. Results: Aβ protofibrils show tertiary contacts between Glu-22 and Ile-31, which are not present in mature fibrils. Conclusion: Aβ protofibrils share tertiary structure features with oligomers but not with mature fibrils. Significance: Aβ protofibrils must undergo a major structural reorientation in the development of mature Aβ fibrils.

We have investigated the site-specific backbone dynamics of mature amyloid β (Aβ) fibrils using solid-state NMR spectroscopy. Overall, the known β-sheet segments and the turn linking these two β-strands exhibit high order parameters between 0.8 and 0.95, suggesting low conformational flexibility. The first approximately eight N-terminal and the last C-terminal residues exhibit lower order parameters between ∼0.4 and 0.8. Interestingly, the order parameters increase again for the first two residues, Asp(1) and Ala(2), suggesting that the N terminus could carry some structural importance.

The self-assembly of β-amyloid (Aβ) peptide into highly ordered amyloid fibril structures represents one of the pathological hallmarks of Alzheimer's disease. This process leads to the transient stabilization of ordered or disordered intermediates, which are thought to act as the main pathogenic culprits in neurodegenerative amyloid disease. This review describes recent results from different biophysical techniques, ranging from structure determination to single-particle methods by which the outgrowth of individual fibrils can be followed, and it explains their contributions towards understanding the mechanism of assembly. Finally, we will outline emerging methods and molecules to specifically interfere with the assembly and pathogenic impact of Aβ peptide.

Amyloid immunotherapy has led to the rise of antibodies, which target amyloid fibrils or structural precursors of fibrils, based on their specific conformational properties. Recently, we reported the biotechnological generation of the B10 antibody fragment, which provides conformation-specific binding to amyloid fibrils. B10 strongly interacts with fibrils from Alzheimer's β amyloid (Aβ) peptide, while disaggregated Aβ peptide or Aβ oligomers are not explicitly recognized. B10 also enables poly-amyloid-specific binding and recognizes amyloid fibrils derived from different types of amyloidosis or different polypeptide chains. Based on our current data, however, we find that B10 does not recognize all tested amyloid fibrils and amyloid tissue deposits. It also does not specifically interact with intrinsically unfolded polypeptide chains or globular proteins even if the latter encompass high β-sheet content or β-solenoid domains. By contrast, B10 binds amyloid fibrils from d-amino acid or l-amino acid peptides and non-proteinaceous biopolymers with highly regular and anionic surface properties, such as heparin and DNA. These data establish that B10 binding does not depend on an amyloid-specific or protein-specific backbone structure. Instead, it involves the recognition of a highly regular and anionic surface pattern. This specificity mechanism is conserved in nature and occurs also within a group of natural amyloid receptors from the innate immune system, the pattern recognition receptors. Our data illuminate the structural diversity of naturally occurring amyloid scaffolds and enable the discrimination of distinct fibril populations in vitro and within diseased tissues.

Amyloid fibrils are naturally occurring polypeptide scaffolds with considerable importance for human health and disease. These supermolecular assemblies are β-sheet rich and characterized by a high structural order. Clinical diagnosis and emerging therapeutic strategies of amyloid-dependent diseases, such as Alzheimer's, rely on the specific recognition of amyloid structures by other molecules. Recently, we generated the B10 antibody fragment, which selectively binds to Alzheimer's Aβ(1-40) amyloid fibrils but does not explicitly recognize other protein conformers, such as oligomers and disaggregated Aβ peptide. B10 presents poly-amyloid specific binding and interacts with fibrillar structures consisting of different polypeptide chains. To determine the molecular basis behind its specificity, we have analyzed the molecular properties of B10 with a battery of biochemical and biophysical techniques, ranging from X-ray crystallography to chemical modification studies. We find that fibril recognition depends on positively charged residues within the B10 antigen binding site. Mutation of these basic residues into alanine potently impairs fibril binding, and reduced B10-fibril interactions are also observed when the fibril carboxyl groups are covalently masked by a chemical modification approach. These data imply that the B10 conformational specificity for amyloid fibrils depends upon specific electrostatic interactions with an acidic moiety, which is common to different amyloid fibrils.

Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.

Aggregation of amyloid-beta (Aβ) peptide is the major event underlying neuronal damage in Alzheimer's disease (AD). Specific lipids and their homeostasis play important roles in this and other neurodegenerative disorders. The complex interplay between the lipids and the generation, clearance or deposition of Aβ has been intensively investigated and is reviewed in this chapter. Membrane lipids can have an important influence on the biogenesis of Aβ from its precursor protein. In particular, increased cholesterol in the plasma membrane augments Aβ generation and shows a strong positive correlation with AD progression. Furthermore, apolipoprotein E, which transports cholesterol in the cerebrospinal fluid and is known to interact with Aβ or compete with it for the lipoprotein receptor binding, significantly influences Aβ clearance in an isoform-specific manner and is the major genetic risk factor for AD. Aβ is an amphiphilic peptide that interacts with various lipids, proteins and their assemblies, which can lead to variation in Aβ aggregation in vitro and in vivo. Upon interaction with the lipid raft components, such as cholesterol, gangliosides and phospholipids, Aβ can aggregate on the cell membrane and thereby disrupt it, perhaps by forming channel-like pores. This leads to perturbed cellular calcium homeostasis, suggesting that Aβ-lipid interactions at the cell membrane probably trigger the neurotoxic cascade in AD. Here, we overview the roles of specific lipids, lipid assemblies and apolipoprotein E in Aβ processing, clearance and aggregation, and discuss the contribution of these factors to the neurotoxicity in AD.

Aggregation of amyloid-beta (Aβ) peptide is the major event underlying neuronal damage in Alzheimer's disease (AD). Specific lipids and their homeostasis play important roles in this and other neurodegenerative disorders. The complex interplay between the lipids and the generation, clearance or deposition of Aβ has been intensively investigated and is reviewed in this chapter. Membrane lipids can have an important influence on the biogenesis of Aβ from its precursor protein. In particular, increased cholesterol in the plasma membrane augments Aβ generation and shows a strong positive correlation with AD progression. Furthermore, apolipoprotein E, which transports cholesterol in the cerebrospinal fluid and is known to interact with Aβ or compete with it for the lipoprotein receptor binding, significantly influences Aβ clearance in an isoform-specific manner and is the major genetic risk factor for AD. Aβ is an amphiphilic peptide that interacts with various lipids, proteins and their assemblies, which can lead to variation in Aβ aggregation in vitro and in vivo. Upon interaction with the lipid raft components, such as cholesterol, gangliosides and phospholipids, Aβ can aggregate on the cell membrane and thereby disrupt it, perhaps by forming channel-like pores. This leads to perturbed cellular calcium homeostasis, suggesting that Aβ-lipid interactions at the cell membrane probably trigger the neurotoxic cascade in AD. Here, we overview the roles of specific lipids, lipid assemblies and apolipoprotein E in Aβ processing, clearance and aggregation, and discuss the contribution of these factors to the neurotoxicity in AD.

Tese de mestrado, Psicologia (Secção de Psicologia Clínica e da Saúde, Núcleo de Psicoterapia Cognitiva-Comportamental e Integrativa), Universidade de Lisboa, Faculdade de Psicologia, 2015 Submitted by Biblioteca FPIE-ULisboa (bibliorul@fpie.ulisboa.pt) on 2016-03-22T11:58:29Z No. of bitstreams: 1 ulfpie047655_tm.pdf: 1438961 bytes, checksum: b783b8eb1257394ddb224a604ac35aea (MD5) Made available in DSpace on 2016-03-22T11:59:05Z (GMT). No. of bitstreams: 1 ulfpie047655_tm.pdf: 1438961 bytes, checksum: b783b8eb1257394ddb224a604ac35aea (MD5) Previous issue date: 2015

Resumen Objetivo Implementacion y valoracion de un programa de participacion familiar en los cuidados del paciente critico. Diseno Estudio experimental, longitudinal y prospectivo, de series temporales y grupo de control aleatorio con tres variables dependientes: paciente, cuidador principal y personal sanitario. Paciente: estado cognitivo del enfermo evaluado con Minimental State Examination Test. Cuidador principal: niveles de ansiedad estado de los cuidadores principales (STAI). Personal: opinion de las enfermeras sobre los cambios introducidos medidos mediante escala tipo Likert. Hipotesis El diseno y la implementacion de un programa de participacion familiar en los cuidados basicos del paciente critico, influyen positiva y significativamente en el enfermo, el familiar y los profesionales implicados en el. Se realizo un muestreo aleatorio sistematico hasta alcanzar el tamano muestral establecido. Resultados Muestra de 117 casos, 49 grupo control, 49 grupo experimental y 19 no validos. El test de STAI obtuvo una media en el grupo experimental de 23,57 frente a 31,22 del grupo control (p Conclusiones Disminucion significativa del grado de ansiedad. No se modifica el estado cognitivo en el grupo experimental frente al grupo control despues de la intervencion. Mejoria progresiva de las creencias del personal de enfermeria con respecto a la visita abierta.

Amyloid oligomers are nonfibrillar polypeptide aggregates linked to diseases, such as Alzheimer's and Parkinson's. Here we show that these aggregates possess a compact, quasi-crystalline architecture that presents significant nanoscale regularity. The amyloid oligomers are dynamic assemblies and are able to release their individual subunits. The small oligomeric size and spheroid shape confer diffusible characteristics, electrophoretic mobility, and the ability to enter hydrated gel matrices or cells. We finally showed that the amyloid oligomers can be labeled with both fluorescence agents and iron oxide nanoparticles and can target macrophage cells. Oligomer amyloids may provide a new biological nanomaterial for improved targeting, drug release, and medical imaging.

Este trabalho de projeto desenvolve-se na procura de respostas para o problema - Como promover o desenvolvimento profissional dos professores, através da intervenção do diretor? Este foi formulado pela investigadora, com base na reflexão sobre a sua experiência profissional na interação direta com um grupo específico - os professores do Agrupamento do qual é Diretora - que constituiu o caso deste estudo. A revisão da literatura constituiu a maior fonte de informação útil para resposta às questões de investigação. Construíram-se categorias de análise que foram alinhadas com as prioridades estabelecidas pelos professores desse Agrupamento numa sessão de trabalho realizada, no âmbito do projeto European Policy Network on School Leadership [EPNoSL], numa perspetiva de investigação-ação. As três primeiras prioridades nortearam a definição das áreas de atuação contempladas no plano de intervenção: direção estratégica para o ensino e a aprendizagem; cultura de colaboração e aprendizagem; formação contextualizada. Este projeto espelha o papel central do Diretor na negociação de uma visão para o Agrupamento, com foco na aprendizagem, bem como no apoio à construção de uma cultura de colaboração entre os docentes, facilitadora do seu desenvolvimento profissional contínuo e contextualizado, através de processos de reflexão sobre as suas práticas., This research project is developed in the search for answers to the problem - How to promote the professional development of teachers, through the intervention of the principal? This was formulated by the researcher, based on the reflection of his professional experience in direct interaction with a specific group - the teachers School Cluster which is the Principal - which was the case in this study. The literature review was the most useful source of information to answer the research questions. Were constructed categories of analysis that were aligned with the priorities established by this School Cluster teachers at a workshop held under the project European Policy Network on School Leadership [EPNoSL], a perspective of action research. The first three priorities guided the definition of the practice areas covered in the intervention plan: strategic direction for teaching and learning; culture of collaboration and learning, contextualized training. This project reflects the central role of the Principal in negotiating a vision for the School Cluster, focusing on learning, as well as support for building a culture of collaboration among teachers, facilitating their continuous professional development and contextualized through processes of reflection on their practices., Orientação : Ana Paula Silva