IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes.

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

Survival response in the skin

Apoptosis is defined as a programmed cell death process operating in multicellular organisms in order to maintain proper homeostasis of tissues. Caspases are among the best characterized proteases to execute apoptosis although lately many studies have associated them with non-apoptotic functions. In the laboratory an antiapoptotic pathway relying on caspase-3 activation and RasGAP has been described in vitro. RasGAP bears two conserved caspase-3 cleavage sites. Under low stress conditions, RasGAP is first cleaved by low caspase-3 activity generating an N terminal fragment (fragment N) that induces a potent anti-apoptotic response mediated by the Ras/PI3K/Akt pathway. High levels of active caspase-3, associated with increased stress conditions, induce further cleavage of fragment N abrogating this anti-apoptotic response. In the present work I studied the functionality of fragment N-mediated protection in physiological conditions as well as the mechanism by which fragment N induces an anti-apoptotic response, with a focus on survivin, an inhibitor of apoptosis. During my work in the laboratory I found that mice lacking caspase-3 or unable to cleave RasGAP (KI mice) are deficient in Akt activation and more sensitive to apoptosis than wild-type mice in response to stress. This higher sensitivity to stress led to augmented tissue damage, highlighting the importance of this pathway in protection against low stress. In parallel I focused on the study of survivin expression in the skin in response to UV-B light and I found that survivin is induced in the cytoplasm of keratinocytes in response to stress where it may fulfill a cyto-protective role. However fragment N had no effect on survivin expression. In addition, cytoplasmic survivin was increased in keratinocytes exposed to UV-B light, whether RasGAP is cleaved (WT mice) or not (KI mice), indicating that survivin is not involved in fragment N mediated protection. Altogether these data indicate that fragment N is pivotal for cell protection against pathophysiologic damage and can encourage the development of therapies aimed to strengthen the resistance of cells against aggressive treatments. Importantly, this finding contributes to the characterization of how caspase-3 can be activated without inducing cell death, although further studies need to be conducted in order to completely characterize this pro-survival molecular mechanism.

Low density lipoprotein signaling and pancreatic beta cells protection

SummaryLow-density lipoproteins (LDLs) have an important physiological role in organism transporting cholesterol and other fatty substances to target tissues. However, elevated LDL levels in the blood are associated with the formation of arterial plaques and consequently atherosclerosis. It is therefore important to characterize the intracellular pathways induced upon LDL stimulation as they might be involved in the pathological properties of these lipoproteins. It has been previously found that LDL stimulation of mouse embryonic fibroblasts activates p38 mitogen activated protein kinases (MAPKs). This leads to cell spreading and increase in the wound healing capabilities of the cells. These two responses might occur within atherosclerotic plaques.The aim of this project is to reveal the missing links between LDL particle and activation of p38 MAPK kinase. As previously shown in our lab activation of p38 MAPK kinase by the LDL particles occur independently of classical LDL receptor (LDLR). In this study we have shown that scavenger receptor type Β class I (SR-BI) is responsible for the signal transduction from the LDLs to the p38 MAPK. We have also shown that Mitogen activated kinase kinases (MKKs) that can directly activate ρ 38 MAPK in these conditions are MKK3 and MKK6 but not MKK4. We have also tested some of the intermediate components of the pathway like Ras and PI3 kinase but found that they do not play a role.The data obtained in this study showed a part of molecular mechanism responsible for p38 MAPK activation and subsequent wound healing and can contribute to our knowledge on function of the fibroblasts in the development of the atherosclerotic plaques.Diabetes Mellitus is a condition caused by disordered metabolism of blood glucose level. It is one of the most commonly spread disease in the western world, with the incidence reaching 8% of population in United States. Two most common types of diabetes are type 1 and 2 that differs slightly in the mechanism of the development. However in the basis of both types lies the cell death of pancreatic beta cells. The aim of this work is to improve beta cells survival in different pathophysiological settings. This could be extrapolated to the conditions in which Diabetes develops in humans. We decided to use RasGAP- derived fragment Ν with its strong antiapoptotic effect in beta cells. In our lab we have demonstrated that in the mild stress conditions RasGAP can be cleaved by caspases at the position 455 producing two fragments, fragment Ν and fragment C. Fragment Ν exerts

Fatores associados à atenção domiciliária: subsídios à gestão do cuidado no âmbito do SUS

A identificação de variáveis associadas ao tipo de atenção domiciliária (AD) dos usuários do Sistema Único de Saúde (SUS) contribui para a gestão do cuidado na Rede de Atenção à Saúde (RAS). Objetiva-se identificar variáveis associadas ao tipo de AD dos usuários em Unidades Básicas de Saúde (UBS) selecionadas de Belo Horizonte. Estudo transversal em duas UBS com todos os usuários (n=114) em AD da área de abrangência. Utilizou-se a análise de regressão logística múltipla para seleção (stepwise) de variáveis significativas. Obteve-se maior comprometimento clínico dos usuários (OR=27,47), estado emocional triste (OR=24,36), risco para úlcera por pressão pela escala de Braden (OR=7,6) e a semidependência para as AVD pelo índice de Katz (OR=63,8) como fortemente associadas ao tipo de AD (p < 0,05). As variáveis fundadas no contexto social, familiar e clínico dos sujeitos subsidiam a abordagem integral e a tomada de decisão da equipe de saúde.

The Strategic Impact of Clinical Practice Guidelines in Nursing on the Managerial Function of Supervision

Clinical practice guidelines in nursing (CPG-N) are tools that allow the necessary knowledge that frequently remains specialist-internalised to be made explicit. These tools are a complement to risk adjustment systems (RAS), reinforcing their effectiveness and permitting a rationalisation of healthcare costs. This theoretical study defends the importance of building and using CPG-Ns as instruments to support the figure of the nursing supervisor in order to optimise the implementation of R&D and hospital quality strategies, enabling clinical excellence in nursing processes and cost-efficient reallocation of economic resources through their linear integration with SARs.

Biana: a software framework for compiling biological interactions and analyzing networks

Background: The analysis and usage of biological data is hindered by the spread of information across multiple repositories and the difficulties posed by different nomenclature systems and storage formats. In particular, there is an important need for data unification in the study and use of protein-protein interactions. Without good integration strategies, it is difficult to analyze the whole set of available data and its properties.Results: We introduce BIANA (Biologic Interactions and Network Analysis), a tool for biological information integration and network management. BIANA is a Python framework designed to achieve two major goals: i) the integration of multiple sources of biological information, including biological entities and their relationships, and ii) the management of biological information as a network where entities are nodes and relationships are edges. Moreover, BIANA uses properties of proteins and genes to infer latent biomolecular relationships by transferring edges to entities sharing similar properties. BIANA is also provided as a plugin for Cytoscape, which allows users to visualize and interactively manage the data. A web interface to BIANA providing basic functionalities is also available. The software can be downloaded under GNU GPL license from http://sbi.imim.es/web/BIANA.php.Conclusions: BIANA's approach to data unification solves many of the nomenclature issues common to systems dealing with biological data. BIANA can easily be extended to handle new specific data repositories and new specific data types. The unification protocol allows BIANA to be a flexible tool suitable for different user requirements: non-expert users can use a suggested unification protocol while expert users can define their own specific unification rules.

Impacte de les mutacions del gen kras en pacients afectes de càncer colorectal metastàtic i la seva relació amb els tractaments biològics associats a la quimioteràpia

El càncer colorectal és un dels càncers més comuns i amb major prevalença, presentant una supervivència als 5 anys per sota del 20% sense tractament. Existeixen estudis que demostren que les mutacions en k-ras, present en el 40-50% de càncers colorectals, s’han convertit en marcadors de pronòstic i en factors predictius de manca de resposta a fàrmacs biològics com Cetuximab® o Panitumumab®. Hem analitzat la mutació de KRAS en la nostra sèrie de 60 pacients afectes de càncer colorectal metastàtic candidats a primera línia de tractament. Aquesta mutació estava present en un 41.6%. En el moment del diagnòstic, presentaven malaltia metastàtica sincrònica el 56.6%. De tots ells, un 51.6% va rebre una combinació de quimioteràpia i anticòs monoclonal. En quant a la resposta obtinguda, el control de malaltia va arribar fins el 86.7% en el global de la sèrie. La supervivència lliure de progressió en la nostra sèrie va ser de 11.74 mesos. En pacients amb KRAS natural va ser de 12,59 mesos vs 11.25 mesos en pacients amb KRAS mutat sense observar diferències estadísticament significatives (p=0.958). No es van observar diferències significatives en funció del règim de quimioteràpia administrada, independentment de l’estat de KRAS. La supervivència global va ser de 32,16 mesos. En pacients amb KRAS natural va ser de 32,59 mesos vs. 28 mesos en pacients amb KRAS mutat sense observar diferències estadísticament significatives (p=0.915). La limitació que significa un número petit de pacients, dins una sèrie assistencial, ens ha de fer ser cautelosos en quant a la valoració dels resultats .

Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways.

BACKGROUND: Recent evidence indicates that zoledronate, a nitrogen-containing bisphosphonate used to treat conditions of increased bone resorption, may have anti-angiogenic activity. The endothelial cells signaling events modulated by zoledronate remain largely elusive. OBJECTIVES: The aim of this work was to identify signaling events suppressed by zoledronate in endothelial cells and responsible for some of its biological effects. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to zoledronate, isoprenoid analogs (i.e. farnesol and geranylgeraniol) and various inhibitors of signaling, and the effect on adhesion, survival, migration, actin cytoskeleton and signaling events characterized. RESULTS: Zoledronate reduced Ras prenylation, Ras and RhoA translocation to the membrane, and sustained ERK1/2 phosphorylation and tumor necrosis factor (TNF) induced JNK phosphorylation. Isoprenoid analogs attenuated zoledronate effects on HUVEC adhesion, actin stress fibers and focal adhesions, migration and survival. Isoprenoid analogs also restored Ras prenylation, RhoA translocation to the membrane, sustained FAK and ERK1/2 phosphorylation and prevented suppression of protein kinase B (PKB) and JNK phosphorylation in HUVEC exposed to TNF in the presence of zoledronate. Pharmacological inhibition of Rock, a RhoA target mediating actin fiber formation, phosphatidylinositol 3-kinase, an activator of PKB, MEK1/2, an activator of ERK1/2, and JNK, recapitulated individual zoledronate effects, consistent with the involvement of these molecules and pathways and their inhibition in the zoledronate effects. CONCLUSIONS: This work has demonstrated that zoledronate inhibits HUVEC adhesion, survival, migration and actin stress fiber formation by interfering with protein prenylation and has identified ERK1/2, JNK, Rock, FAK and PKB as kinases affected by zoledronate in a prenylation-dependent manner.

Molecular mechanisms of insulin exocytosis role of small monomeric GTP-ASES

SUMMARYInsulin secretion from pancreatic beta-cells is a fundamental condition for the maintenance of blood glucose levels. During the last decades, important components of the molecular machinery controlling hormone release have been characterized. My PhD thesis was dedicated to the study of new signaling pathways regulating insulin exocytosis and in particular to the role of small monomelic guanine triphosphatase or GTPases controlling the last events of hormone release.The first part of my thesis focused on Ras-like (Ral) RalA and RalB proteins. We investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed their impact on different steps of the insulin-secretory process. Our results have shown that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-IE cells by RNA interference led to a decrease in secretagogue-induced hormone release. The activation of the GTPase, followed by FRET imaging, is triggered by increases in intracellular Ca and cAMP. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane, detected by TIRF microscopy and with strong impairment in PLD1 activation in response to secretagogues. RalA was found to be activated by the exchange factor RalGDS, which regulates hormone secretion induced by secretagogues and the docking step of insulin-containing granules at the plasma membrane. In the second part of this work we have shown that a member of the Rab family, Rab37, is present on insulin-containing secretory granules of pancreatic beta-cells. In addition, our experiments have suggested that Rab37 is required to obtain an optimal insulin secretory response induced by secretogogues and is important for the docking step of insulin-containing granules at the plasma membrane.

Disseny i implementació de l'aplicació Protein-MetReS

El que s'ha dut a terme a estat l'anàlisi, disseny i implementació d'una aplicació, anomenada Protein-MetReS, integrada dins del projecte MetReS. Aquesta aplicació ha estat desenvolupada sota la tecnologia Java que, a través de servidors web i servidors localment instal·lats, a partir de la selecció d'un organisme realitza cerques sobre bases de dades que contenen estructures de proteïnes conegudes i, en el seu defecte, si aquestes no existeixen, crea el model tridimensional en base als homòlegs d'estructura coneguda, per després sotmetre'ls a un procés d'acoblament molecular, o més conegut, com a docking.

Síntesi de nous catecols amb aplicació en materials funcionals bioinspirats

Els catecols (compostos aromàtics amb dos hidroxils veïnals) són una unitat estructural present en la naturalesa de formes molt diverses. En el present treball es sintetitza el catecol amb una cadena alquílica saturada de 32 carbonis. L’objectiu és mimetitzar el comportament de les proteïnes segregades pels musclos que formen els filaments pels quals aquest mol·lusc pot adherir-se sobre diverses superfícies de manera hidrofòbica permanent i que li permet mantenir-se adherit tot i estar submergit en l’aigua del mar. Aquesta síntesi es realitza gràcies a l’obtenció d’un valuós intermedi tipus sal de fosfoni que permet introduïr la cadena hidrocarbonada a l’esquelet catecol.

Preferències metàl·liques de les metal·lotioneïnes de Tetrahymena thermophila: cinc isoformes i dos comportaments clarament oposats

Les metal·lotioneïnes constitueixen una superfamília de proteïnes amb unes característiques molt peculiars. Es troben presents en molts organismes, presentant sovint diferents isoformes. La presència de diferents isoformes dins un mateix organisme fa pensar que és possible que cadascuna d’elles presentin funcions diferents. Un cas paradigmàtic, que pot aportar informació són les MTs del protozou ciliat Tetrahymena thermophila, amb cinc isoformes que es mostren com un sistema ideal per estudiar els determinants d’especificitat metàl·lica de les MTs. L’ estudi de les propietats coordinants de les cinc isoformes de MT de Tetrahymena thermophila, servirà per aprofundir en la relació estructura/funció d’aquestes metal·lotioneïnes.

Qualitat microbiològica dels productes vegetals frescos mínimament processats i desenvolupament de tecnologies de bioconservació mitjançant bacteris de l'àcid làctic

Food safety today is conditiones by the implementation of new Tecnologies for food preservation based on mild treatments and mínimum process, Novel foods, severe restrictions in the toxicològic profile of chemical preservatives, And the drastic limitation /prohibition of antibiotics, and as a consequence, by the New emergint pathogens or by the increase of classical food-borne pathogens. Biopreservatives appear within this context with strong expectations, because thei are safe microorganisms of ten isolated from foods, chemical compounds of natural origin -antimicrobial peptides and proteins, botanical extracts, enzymes- and sintetic compounds based on natural structures, but less toxic and more eficients, like the amtimicrobial peptides. Among the microbial biopreservatives, lactic acid bacteria have shown great possibilities in the preservation of cured meat products, ready to eat fresh fruit and vegetables, as well as to decrease microbial spoilage in food by products before processing for valorization. Our laboratory has performed an extense survey of the microbiological quality of fresh fruit and vegetables, and of ready-to-eat products, and have detect low levels, but significant of Salmonella sp., E. coli and Listeria spp., including L.monocytogenes, in retail markets and Supermarkets of Catalonia. Due to this reason, we started a project consisting of Developing application of lactic acid bacteria (LAB) obtained from these products, as biopreservatives. LAB were abundant in ready-to-eat fresh fruits and vegetables, specially in germinated seeds. From these products we obtained strains of Leuconostoc, Lactobacillus and Weissella, producing bacteriocins and with a Significant activity of control of L.monocytogenes in fresh apple and cut salad. Ather strains were efective in the inhibition of fungal rot during postharvest caused by Penicillium expansum

Crescimento e acúmulo de crômio em alface cultivada em dois latossolos tratados com CrCl3 e resíduos de curtume

Resíduos sólidos de curtume e CrCl3 foram aplicados em dois solos, Latossolo Roxo eutrófico (LRe) unidade Ribeirão Preto e Latossolo Vermelho-Amarelo (LVA) unidade Laranja Azeda, que se diferenciaram, dentre outros atributos, pelo teor de manganês facilmente redutível. Os resíduos utilizados foram lodo do efluente de caleiro com concentração 0,06 g kg-1 de crômio (LCL) e um lodo do decantador primário (LCR), contendo 17,4 g kg-1 de crômio, ambos na matéria seca, aplicados em doses correspondentes a 10, 20 e 30 Mg ha-1 e 19, 38 e 57 Mg ha-1 (base seca), respectivamente, de acordo com o teor de nitrogênio total de cada um. O CrCl3 foi aplicado nas doses de 330, 660 e 990 kg ha-1 de Cr, equivalentes às doses do metal aplicadas na forma de lodo (LCR). Realizou-se o experimento em vasos alocados em casa de vegetação (blocos ao acaso), que foram monitorados quanto à formação de Cr6+, aos 1, 6, 14, 28, 54 e 86 dias da instalação. Após o 56° dia de incubação, foi transplantada uma muda de alface (Lactuca sativa L.) para cada vaso, cultivada por um período de trinta dias. A oxidação do Cr3+ a Cr6+ foi verificada apenas para o LRe nos tratamentos que receberam doses crescentes de CrCl3. A formação de Cr6+ teve máximo entre 0,72 e 1,16% do Cr3+ aplicado, após um dia de incubação, decrescendo com o tempo, não sendo detectada a sua presença, para nenhuma das doses, após o 54° dia. A aplicação dos resíduos elevou a condutividade elétrica do extrato de saturação (2:1) de 1,40 a 5,07 Ds m-1 e a RAS de 3,05 a 14,12, afetando o desenvolvimento da alface e causando a morte das plantas nas doses mais altas, sendo tais efeitos mais pronunciados no LVA. A concentração de crômio na parte aérea das plantas aumentou, nem sempre de forma proporcional, com o aumento das doses aplicadas na forma de lodo ou sal, com efeito mais acentuado para o LVA do que para o LRe. A aplicação de resíduos de curtume no experimento, para ambos os solos, mostrou-se mais limitante pelo seu conteúdo de sais do que pela presença de crômio.

Expression of an uncleavable N-terminal RasGAP fragment in insulin-secreting cells increases their resistance toward apoptotic stimuli without affecting their glucose-induced insulin secretion.

Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.