Advertisement and society in graphic magazine: La Esfera (1920-1930)

In the first third of the 20th century, Spain entered into modernity thanks to the positive state of its economic, cultural and social domains after the First World War. The objective of this article is to understand the role of artistic advertisements in the transformation of Spanish society that occurred during the decade of the ‘20s. My study of the illustrated magazine La Esfera has revealed that illustrated advertisements spread fashion, fostered sports and outdoors life, created the habit of smoking among women and had an educational influence that was powerful enough to change hygienic practices among children.

A view, different proposals: Concept of gender and public policy

This article analyzes the meaning of “gender” as a category of analysis in public policy. The concept has been transferred from the feminist theories and this has meant that the United Nations and European Union have incorporated the inequality as a structural inequality and an issue justice. So, the feminist demands enter the political agenda as an integral project which is characterized by the adoption of the gender perspective and its application from a transversal methodology (“gender mainstreaming”). In this sense, the "gender ideology" is a new paradigm against the “patriarchal ideology”. Now, political actions should be articulated in a double movement of correction and promotion to achieve real equality in societies more democratic and ultimately more just.

The intertextual pilgrimage to Compostela in the Spanish nineteenth-century travel narratives written in French

Pilgrimage to Compostela was decreasing in the nineteenth century. This situation was still worse in France, where the number of pilgrims dwindled dramatically. In fact, there are not many travel narratives in this period, as no relevant French author showed any interest in this religious event. An analysis of these works reveals that the worship to Santiago was somehow considered by these authors a mere historical remnant with an aura of prestige. They allow almost no space for factual descriptions, and therefore used documentary sources to discuss the topic in their own texts. As a consequence, their knowledge of this universe became indirect and intertextual.

The Weapons of Mass Destruction and the Global Security Strategy of the EU

The proliferation of weapons of mass destruction (WMD), nuclear, biological and chemical (NBC) is one of the main security challenges facing the international community today. However the new Global Security Strategy of 2016 raises the question of non-proliferation of WMD only as an incidental matter, not addressing directly the threat, a fundamental threat in the regional and global security. This is a clear step backwards for the European common security.

von Hippel Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von Hippel-Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integration-transcription transition of the viral replication cycle.

Comradeship of Cock? Gay porn and the entrepreneurial voyeur

Thirty years of academic and critical scholarship on the subject of gay porn have born witness to significant changes not only in the kinds of porn produced for, and watched by, gay men, but in the modes of production and distribution of that porn, and the legal, economic and social contexts in which it has been made, sold/shared, and watched. Those thirty years have also seen a huge shift in the cultural and political position of gay men, especially in the US and UK, and other apparently ‘advanced’ democracies. Those thirty years of scholarship on the topic of gay porn have produced one striking consensus, which is that gay cultures are especially ‘pornified’: porn has arguably offered gay men not only homoerotic visibility, but a heritage culture and a radical aesthetic. However, neoliberal cultures have transformed the operation and meaning of sexuality, installing new standards of performativity and display, and new responsibilities attached to a ‘democratisation’ that offers women and men apparently expanded terms for articulating both their gender and their sexuality. Does gay porn still have the same urgency in this context? At the level of politics and cultural dissent, what’s ‘gay’ about gay porn now? This essay questions the extent to which processes of legal and social liberalization, and the emergence of networked and digital cultures, have foreclosed or expanded the apparently liberationary opportunities of gay porn. The essay attempts to map some of the political implications of the ‘pornification’ of gay culture on to ongoing debates about materiality, labour and the entrepreneurial subject by analyzing gay porn blogs.

Modification of aftertaste with a menthol mouthwash reduces food wanting, liking, and ad libitum intake of potato crisps

This research investigated the effect of modifying the aftertaste of potato crisps on (1) temporal sensory perception and (2) appetite using three mouthwash conditions (no mouthwash, a water mouthwash, and a menthol mouthwash). For the sensory study, 17 screened female subjects were trained on the Temporal Dominance of Sensations (TDS) methodology. Subjects undertook TDS to monitor all sensory attributes during the mastication of a 2 g crisp until swallowing (at 20s), then conducted the mouthwash, and then continued the TDS task to monitor aftertaste until 90s. For the appetite study, 36 subjects (18 male, 18 female) completed 100 mm Visual Analogue Scales (VAS) for desire, liking, hunger, and thirst, followed by an ad libitum eating task. For the VAS scales testing, subjects chewed and swallowed a 2 g crisp, and then immediately conducted the mouthwash before completing the VAS scales. For the ad libitum task, subjects were given 12 min to consume as many crisps as they desired on a plate (up to 50 g). Every three minutes they were required to conduct a mouthwash. TDS results showed that in comparison with no mouthwash, the water mouthwash significantly reduced aftertaste attributes such as savoury, salty, and fatty mouthcoating, and the menthol mouthwash significantly increased aftertaste attributes of cooling, minty, and tingly. The water mouthwash did not influence desire and liking of crisps, or hunger and thirst. The water mouthwash did not influence ad libitum intake of the crisps over a 12 min period. The menthol mouthwash significantly reduced desire and liking of the crisps, as well as hunger and thirst. Furthermore, the menthol mouthwash significantly reduced ad libitum crisp intake by 29% over the 12 min period.

The role of intracellular calcium perturbations in muscle damage and dysfunction in mouse models of muscular dystrophy

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. DMD is clinically characterized by severe, progressive and irreversible loss of muscle function, in which most patients lose the ability to walk by their early teens and die by their early 20’s. Impaired intracellular calcium (Ca2+) regulation and activation of cell degradation pathways have been proposed as key contributors to DMD disease progression. This dissertation research consists of three studies investigating the role of intracellular Ca2+ in skeletal muscle dysfunction in different mouse models of DMD. Study one evaluated the role of Ca2+-activated enzymes (proteases) that activate protein degradation in excitation-contraction (E-C) coupling failure following repeated contractions in mdx and dystrophin-utrophin null (mdx/utr-/-) mice. Single muscle fibers from mdx/utr-/- mice had greater E-C coupling failure following repeated contractions compared to fibers from mdx mice. Moreover, protease inhibition during these contractions was sufficient to attenuate E-C coupling failure in muscle fibers from both mdx and mdx/utr-/- mice. Study two evaluated the effects of overexpressing the Ca2+ buffering protein sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) in skeletal muscles from mdx and mdx/utr-/- mice. Overall, SERCA1 overexpression decreased muscle damage and protected the muscle from contraction-induced injury in mdx and mdx/utr-/- mice. In study three, the cellular mechanisms underlying the beneficial effects of SERCA1 overexpression in mdx and mdx/utr-/- mice were investigated. SERCA1 overexpression attenuated calpain activation in mdx muscle only, while partially attenuating the degradation of the calpain target desmin in mdx/utr-/- mice. Additionally, SERCA1 overexpression decreased the SERCA-inhibitory protein sarcolipin in mdx muscle but did not alter levels of Ca2+ regulatory proteins (parvalbumin and calsequestrin) in either dystrophic model. Lastly, SERCA1 overexpression blunted the increase in endoplasmic reticulum stress markers Grp78/BiP in mdx mice and C/EBP homologous protein (CHOP) in mdx and mdx/utr-/- mice. Overall, findings from the studies presented in this dissertation provide new insight into the role of Ca2+ in muscle dysfunction and damage in different dystrophic mouse models. Further, these findings support the overall strategy for improving intracellular Ca2+ control for the development of novel therapies for DMD.

TRIB2 in normal and malignant hematopoiesis - a transcription factor network

Hematopoiesis is the tightly controlled and complex process in which the entire blood system is formed and maintained by a rare pool of hematopoietic stem cells (HSCs), and its dysregulation results in the formation of leukaemia. TRIB2, a member of the Tribbles family of serine/threonine pseudokinases, has been implicated in a variety of cancers and is a potent murine oncogene that induces acute myeloid leukaemia (AML) in vivo via modulation of the essential myeloid transcription factor CCAAT-enhancer binding protein α (C/EBPα). C/EBPα, which is crucial for myeloid cell differentiation, is commonly dysregulated in a variety of cancers, including AML. Two isoforms of C/EBPα exist - the full-length p42 isoform, and the truncated oncogenic p30 isoform. TRIB2 has been shown to selectively degrade the p42 isoform of C/EBPα and induce p30 expression in AML. In this study, overexpression of the p30 isoform in a bone marrow transplant (BMT) leads to perturbation of myelopoiesis, and in the presence of physiological levels of p42, this oncogene exhibited weak transformative ability. It was also shown by BMT that despite their degradative relationship, expression of C/EBPα was essential for TRIB2 mediated leukaemia. A conditional mouse model was used to demonstrate that oncogenic p30 cooperates with TRIB2 to reduce disease latency, only in the presence of p42. At the molecular level, a ubiquitination assay was used to show that TRIB2 degrades p42 by K48-mediated proteasomal ubiquitination and was unable to ubiquitinate p30. Mutation of a critical lysine residue in the C-terminus of C/EBPα abrogated TRIB2 mediated C/EBPα ubiquitination suggesting that this site, which is frequently mutated in AML, is the site at which TRIB2 mediates its degradative effects. The TRIB2-C/EBPα axis was effectively targeted by proteasome inhibition. AML is a very difficult disease to target therapeutically due to the extensive array of chromosomal translocations and genetic aberrations that contribute to the disease. The cell from which a specific leukaemia arises, or leukaemia initiating cell (LIC), can affect the phenotype and chemotherapeutic response of the resultant disease. The LIC has been elucidated for some common oncogenes but it is unknown for TRIB2. The data presented in this thesis investigate the ability of the oncogene TRIB2 to transform hematopoietic stem and progenitor cells in vitro and in vivo. TRIB2 overexpression conferred in vitro serially replating ability to all stem and progenitor cells studied. Upon transplantation, only TRIB2 overexpressing HSCs and granulocyte/macrophage progenitors (GMPs) resulted in the generation of leukaemia in vivo. TRIB2 induced a mature myeloid leukaemia from the GMP, and a mixed lineage leukaemia from the HSC. As such the role of TRIB2 in steady state hematopoiesis was also explored using a Trib2-/- mouse and it was determined that loss of Trib2 had no effect on lineage distribution in the hematopoietic compartment under steady-state conditions. The process of hematopoiesis is controlled by a host of lineage restricted transcription factors. Recently members of the Nuclear Factor 1 family of transcription factors (NFIA, NFIB, NFIC and NFIX) have been implicated in hematopoiesis. Little is known about the role of NFIX in lineage determination. Here we describe a novel role for NFIX in lineage fate determination. In human and murine datasets the expression of Nfix was shown to decrease as cells differentiated along the lymphoid pathway. NFIX overexpression resulted in enhanced myelopoiesis in vivo and in vitro and a block in B cell development at the pre-pro-B cell stage. Loss of NFIX resulted in disruption of myeloid and lymphoid differentiation in vivo. These effects on stem and progenitor cell fate correlated with changes in the expression levels of key transcription factors involved in hematopoietic differentiation including a 15-fold increase in Cebpa expression in Nfix overexpressing cells. The data presented support a role for NFIX as an important transcription factor influencing hematopoietic lineage specification. The identification of NFIX as a novel transcription factor influencing lineage determination will lead to further study of its role in hematopoiesis, and contribute to a better understanding of the process of differentiation. Elucidating the relationship between TRIB2 and C/EBPα not only impacts on our understanding of the pathophysiology of AML but is also relevant in other cancer types including lung and liver cancer. Thus in summary, the data presented in this thesis provide important insights into key areas which will facilitate the development of future therapeutic approaches in cancer treatment.

Estudio de factibilidad para la apertura de una sucursal de Pro-Lingua Asociados S.A. de C.V. en el Municipio de Santa Tecla, departamento de La Libertad.

El presente estudio de factibilidad surge ante la necesidad de ampliar los servicios que ofrece Pro-Lingua Asociados S.A. DE C.V. Con el propósito de dar cobertura a personas que buscan obtener nuevas competencias profesionales para aspirar a puestos de trabajo que ofrezcan mayor remuneración, accesibilidad geográfica para aquellos que tiene problemas de transporte y, además, una alternativa de centro de enseñanza de idioma ingles con cuotas al alcance de sus respectivos niveles de ingreso, es decir que no disponen de la opción de matricularse en una institución que requiera de una mayor inversión monetaria. En primera instancia la Junta Directiva de la institución manifestó que planifican realizar la apertura de una sucursal en la ciudad de Santa Tecla, departamento de La Libertad. Motivos por los que el grupo de investigación decidió realizar el estudio con los objetivos siguientes: El objetivo general: Elaborar un diagnóstico de la situación actual que permita conocer los beneficios y limitantes que presenta la apertura de la sucursal de Pro-Lingua Asociados S.A. de C.V. en el municipio de Santa Tecla, Departamento de La Libertad. Y sus objetivos específicos son: Identificar la existencia de demanda insatisfecha que cumpla con los requisitos para considerarse como segmento de mercado. Indagar sobre la operatividad de las instituciones enfocadas a la enseñanza del idioma inglés en la localidad bajo estudio, a fin de evaluar si se acepta o se rechaza la apertura de la sucursal. Realizar un estudio que muestre la necesidad de recursos humanos, monetarios, ubicación y tamaño de la infraestructura física, mobiliario y equipo, entre otros, que justifiquen la formulación del proyecto de inversión a proponer. Para el proyecto se utilizó la siguiente metodología: Se empleó el método científico por medio del análisis para separar y examinar los diversos componentes del estudio y la síntesis como una herramienta para relacionar los diferentes componentes y así identificar cuáles son los beneficios y limitantes del mismo; El tipo de investigación es correlacional y el diseño es no experimental ya que no se manipulan las variables del presente estudio. Las fuentes de investigación fueron: las primarias, la Población Ocupada, mayor de 18 años de edad, perteneciente al sector económico de clase media, residente del municipio de Santa Tecla y que devengue como mínimo un salario de $350.00 dólares mensuales en adelante, los administradores de los centros de enseñanza del idioma inglés ubicados en la zona geográfica mencionada y la Vice-Presidenta de la Junta Directiva de Pro-Lingua Asociados S.A. de C.V., y las secundarias son los datos recopilados en publicaciones de instituciones especializadas, libros, páginas web, leyes, entre otros, concernientes a la evaluación y formulación de proyectos de inversión Entre las técnicas e instrumentos utilizados están: la observación directa para obtener conocimientos sobre las instalaciones, la forma en que ellos brindan sus servicios a los clientes y como estos últimos lo perciben, la encuesta que estaba elaborada con un cuestionario de preguntas abiertas y cerradas, la entrevista que se llevó a cabo con la Gerente Comercial de un centro de enseñanza de idioma ingles en la localidad mencionada y la Vice-Presidenta de la Junta Directiva de Pro-Lingua Asociados S.A. de C.V. Por último, se concluyó en el desarrollo de la investigación que la mayoría de las personas que han recibido o no una preparación en el Idioma Inglés, están motivadas a continuar o iniciar con sus estudios en dicho lenguaje y además, manifestaron estar interesados en la apertura de una academia especializada en Santa Tecla; es por esta razón, que se recomienda diseñar programas publicitarios y servicios educativos que se distingan de los ofertados por la competencia a fin de motivar a las personas que han recibido formación previa como los que no la han efectuado.

Sistema informático para la gestión, administración, venta y control de acceso a eventos por medio de tickets de La Taquilla S.A. de C.V. (SIT)

El proyecto SIT es una iniciativa de modernización de los procesos de gestión, administración, venta y control de acceso a eventos por medio de tickets, en donde la centralización de los procesos está regida por el control mediante un sistema informático altamente estandarizado desde su análisis hasta la implementación, para proporcionar apoyo y una mejora sustancial en las actividades operativas y administrativas diarias de la empresa, dando como resultado la eficiencia en los procesos de adquisición de tickets, la disposición de información para productores y la administración de esta cadena

TRIB2 in human AML: a biological and clinical investigation

Acute myeloid leukemia (AML) involves the proliferation, abnormal survival and arrest of cells at a very early stage of myeloid cell differentiation. The biological and clinical heterogeneity of this disease complicates treatment and highlights the significance of understanding the underlying causes of AML, which may constitute potential therapeutic targets, as well as offer prognostic information. Tribbles homolog 2 (Trib2) is a potent murine oncogene capable of inducing transplantable AML with complete penetrance. The pathogenicity of Trib2 is attributed to its ability to induce proteasomal degradation of the full length isoform of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα p42). The role of TRIB2 in human AML cells, however, has not been systematically investigated or targeted. Across human cancers, TRIB2 oncogenic activity was found to be associated with its elevated expression. In the context of AML, TRIB2 overexpression was suggested to be associated with the large and heterogeneous subset of cytogenetically normal AML patients. Based upon the observation that overexpression of TRIB2 has a role in cellular transformation, the effect of modulating its expression in human AML was examined in a human AML cell line that expresses high levels of TRIB2, U937 cells. Specific suppression of TRIB2 led to impaired cell growth, as a consequence of both an increase in apoptosis and a decrease in cell proliferation. Consistent with these in vitro results, TRIB2 silencing strongly reduced progression of the U937 in vivo xenografts, accompanied by detection of a lower spleen weight when compared with mice transplanted with TRIB2- expressing control cells. Gene expression analysis suggested that TRIB2 modulates apoptosis and cell-cycle sensitivity by influencing the expression of a subset of genes known to have implications on these phenotypes. Furthermore, TRIB2 was found to be expressed in a significant subset of AML patient samples analysed. To investigate whether increased expression of this gene could be afforded prognostic significance, primary AML cells with dichotomized levels of TRIB2 transcripts were evaluated in terms of their xenoengraftment potential, an assay reported to correlate with disease aggressiveness observed in humans. A small cohort of analysed samples with higher TRIB2 expression did not associate with preferential leukaemic cell engraftment in highly immune-deficient mice, hence, not predicting for an adverse prognosis. However, further experiments including a larger cohort of well characterized AML patients would be needed to clarify TRIB2 significance in the diagnostic setting. Collectively, these data support a functional role for TRIB2 in the maintenance of the oncogenic properties of human AML cells and suggest TRIB2 can be considered a rational therapeutic target. Proteasome inhibition has emerged as an attractive target for the development of novel anti-cancer therapies and results from translational research and clinical trials support the idea that proteasome inhibitors should be considered in the treatment of AML. The present study argued that proteasome inhibition would effectively inhibit the function of TRIB2 by abrogating C/EBPα p42 protein degradation and that it would be an effective pharmacological targeting strategy in TRIB2-positive AMLs. Here, a number of cell models expressing high levels of TRIB2 were successfully targeted by treatment with proteasome inhibitors, as demonstrated by multiple measurements that included increased cytotoxicity, inhibition of clonogenic growth and anti-AML activity in vivo. Mechanistically, it was shown that block of the TRIB2 degradative function led to an increase of C/EBPα p42 and that response was specific to the TRIB2-C/EBPα axis. Specificity was addressed by a panel of experiments showing that U937 cells (express detectable levels of endogenous TRIB2 and C/EBPα) treated with the proteasome inhibitor bortezomib (Brtz) displayed a higher cytotoxic response upon TRIB2 overexpression and that ectopic expression of C/EBPα rescued cell death. Additionally, in C/EBPα-negative leukaemia cells, K562 and Kasumi 1, Brtz-induced toxicity was not increased following TRIB2 overexpression supporting the specificity of the compound on the TRIB2-C/EBPα axis. Together these findings provide pre-clinical evidence that TRIB2- expressing AML cells can be pharmacologically targeted with proteasome inhibition due, in part, to blockage of the TRIB2 proteolytic function on C/EBPα p42. A large body of evidence indicates that AML arises through the stepwise acquisition of genetic and epigenetic changes. Mass spectrometry data has identified an interaction between TRIB2 and the epigenetic regulator Protein Arginine Methyltransferase 5 (PRMT5). Following assessment of TRIB2‟s role in AML cell survival and effective targeting of the TRIB2-C/EBPα degradation pathway, a putative TRIB2/PRMT5 cooperation was investigated in order to gain a deeper understanding of the molecular network in which TRIB2 acts as a potent myeloid oncogene. First, a microarray data set was interrogated for PRMT5 expression levels and the primary enzyme responsible for symmetric dimethylation was found to be transcribed at significantly higher levels in AML patients when compared to healthy controls. Next, depletion of PRMT5 in the U937 cell line was shown to reduce the transformative phenotype in the high expressing TRIB2 AML cells, which suggests that PRMT5 and TRIB2 may cooperate to maintain the leukaemogenic potential. Importantly, PRMT5 was identified as a TRIB2-interacting protein by means of a protein tagging approach to purify TRIB2 complexes from 293T cells. These findings trigger further research aimed at understanding the underlying mechanism and the functional significance of this interplay. In summary, the present study provides experimental evidence that TRIB2 has an important oncogenic role in human AML maintenance and, importantly in such a molecularly heterogeneous disease, provides the rational basis to consider proteasome inhibition as an effective targeting strategy for AML patients with high TRIB2 expression. Finally, the identification of PRMT5 as a TRIB2-interacting protein opens a new level of regulation to consider in AML. This work may contribute to our further understanding and therapeutic strategies in acute leukaemias.

Stabilising suppressor of cytokine signalling 3 (SOCS3) protein levels to limit neointimal hyperplasia

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

Frammenti del sè: un caso clinico

Ad oggi, a fronte di un crescente interesse sociale, giuridico, medico e psicologico alle varie forme di abuso sessuale subite da minori e agli esiti a breve, medio e lungo termine che tali eventi possono esercitare sul benessere bio-psico-sociale dei sopravvissuti (Gulotta & Cutica, 2009), sembra, comunque, manifestarsi una insufficiente attenzione al tema del maltrattamento fisico (Di Blasio, 2000). Nel nostro contesto culturale gli episodi di aggressività fisica sono ancora facilmente rubricati nella categoria delle prassi genitoriali lecite, con un’implicita sottovalutazione dei rischi per la salute del bambino. Con questo non vogliamo sottendere che ogni singolo episodio di aggressività diretta del caregiver possa inserirsi tout court nella categoria dei maltrattamenti fisici; allo stesso tempo, riteniamo però necessario che, a livello sociale e sanitario, si presti attenzione ai possibili rischi per la salute della persona in caso di maltrattamenti fisici reiterati.

Protein quality and distribution determines anabolic signaling, cellular energetics, and body composition

Building and maintaining muscle is critical to the quality of life for adults and elderly. Physical activity and nutrition are important factors for long-term muscle health. In particular, dietary protein – including protein distribution and quality – are under-appreciated determinants of muscle health for adults. The most unequivocal evidence for the benefit of optimal dietary protein at individual meals is derived from studies of weight management. During the catabolic condition of weight loss, higher protein diets attenuate loss of lean tissue and partition weight loss to body fat when compared with commonly recommended high carbohydrate, low protein diets. Muscle protein turnover is a continuous process in which proteins are degraded, and replaced by newly synthesized proteins. Muscle growth occurs when protein synthesis exceeds protein degradation. Regulation of protein synthesis is complex, with multiple signals influencing this process. The mammalian target of rapamycin (mTORC1) pathway has been identified as a particularly important regulator of protein synthesis, via stimulation of translation initiation. Key regulatory points of translation initiation effected by mTORC1 include assembly of the eukaryotic initiation factor 4F (eIF4F) complex and phosphorylation of the 70 kilodalton ribosomal protein S6 kinase (S6K1). Assembly of the eIF4F initiation complex involves phosphorylation of the inhibitory eIF4E binding protein-1 (4E-BP1), which releases the initiation factor eIF4E and allows it to bind with eIF4G. Binding of eIF4E with eIF4G promotes preparation of the mRNA for binding to the 43S pre-initiation complex. Consumption of the amino acid leucine (Leu) is a key factor determining the anabolic response of muscle protein synthesis (MPS) and mTORC1 signaling to a meal. Research from this dissertation demonstrates that the peak activation of MPS following a complete meal is proportional to the Leu content of a meal and its ability to elevate plasma Leu. Leu has also been implicated as an inhibitor of muscle protein degradation (MPD). In particular, there is evidence suggesting that in muscle wasting conditions Leu supplementation attenuates expression of the ubiquitin-proteosome pathway, which is the primary mode of intracellular protein degradation. However, this is untested in healthy, physiological feeding models. Therefore, an experiment was performed to see if feeding isonitrogenous protein sources with different Leu contents to healthy adult rats would differentially impact ubiquitin-proteosome (protein degradation) outcomes; and if these outcomes are related to the meal responses of plasma Leu. Results showed that higher Leu diets were able to attenuate total proteasome content but had no effect on ubiquitin proteins. This research shows that dietary Leu determines postprandial muscle anabolism. In a parallel line of research, the effects of dietary Leu on changes in muscle mass overtime were investigated. Animals consuming higher Leu diets had larger gastrocnemius muscle weights; furthermore, gastrocnemius muscle weights were correlated with postprandial changes in MPS (r=0.471, P<0.01) and plasma Leu (r=0.400, P=0.01). These results show that the effect of Leu on ubiquitin-proteosome pathways is minimal for healthy adult rats consuming adequate diets. Thus, long-term changes in muscle mass observed in adult rats are likely due to the differences in MPS, rather than MPD. Factors determining the duration of Leu-stimulated MPS were further investigated. Despite continued elevations in plasma Leu and associated translation initiation factors (e.g., S6K1 and 4E-BP1), MPS returned to basal levels ~3 hours after a meal. However, administration of additional nutrients in the form of carbohydrate, Leu, or both ~2 hours after a meal was able to extend the elevation of MPS, in a time and dose dependent manner. This effect led to a novel discovery that decreases in translation elongation activity was associated with increases in activity of AMP kinase, a key cellular energy sensor. This research shows that the Leu density of dietary protein determines anabolic signaling, thereby affecting cellular energetics and body composition.