Regulation of B Cell Gene Expression and Function by Ikaros, Helios and Bcl6

B lymphocytes constitute a key branch of adaptive immunity by providing specificity to recognize a vast variety of antigens by B cell antigen receptors (BCR) and secreted antibodies. Antigen recognition activates the cells and can produce antibody secreting plasma cells via germinal center reaction that leads to the maturation of antigen recognition affinity and switching of antibody effector class. The specificity of antigen recognition is achieved through a multistep developmental pathway that is organized by interplay of transcription factors and signals through BCR. Lymphoid malignancies arise from different stages of development in abnormal function of transcriptional regulation. To understand the B cell development and the function of B cells, a thorough understanding of the regulation of gene expression is important. The transcription factors of the Ikaros family and Bcl6 are frequently associated with lymphoma generation. The aim of this study was to reveal the targets of Ikaros, Helios and Bcl6 mediated gene regulation and to find out the function of Ikaros and Helios in B cells. This study uses gene targeted DT40 B cell lines and establishes a role for Ikaros family factors Ikaros and Helios in the regulation of BCR signaling that is important at developmental checkpoints, for cell survival and in activation. Ikaros and Helios had opposing roles in the regulation of BCR signals. Ikaros was found to directly repress the SHIP gene that encodes a signaling lipid-metabolizing enzyme, whereas Helios had activating effect on SHIP expression. The findings demonstrate a balancing function for these two Ikaros family transcription factors in the regulation of BCR signaling as well as in the regulation of gene expression. Bcl6 was found to repress plasma cell gene expression program while maintaining gene expression profile of B cells. Analysis of direct Bcl6 target genes suggested novel mechanisms for Bcl6-mediated suppression of plasma cell differentiation and promoting germinal center phenotype.

OSCE para Competências de Comunicação Clínica e Profissionalismo: Relato de Experiência e Meta-Avaliação

RESUMO A comunicação clínica e o profissionalismo estão entre as principais competências médicas e, portanto, devem ter sua avaliação garantida. Nesse contexto, o exame clínico objetivo estruturado (OSCE) tem papel fundamental. Objetivos Descrever as etapas de elaboração de um OSCE, bem como a avaliação da qualidade das estações e a percepção do estudante de Medicina sobre a sua realização. Método O estudo é composto pela realização de um OSCE com quatro estações por 16 estudantes de Medicina e pela análise da qualidade psicométrica e aplicação de um questionário de satisfação. Resultados Para os estudantes, o OSCE é o método que melhor avalia e ensina essas competências, ao passo que os testes de múltipla escolha estão no polo oposto quanto à avaliação. Em relação à qualidade múltipla das estações: duas se apresentaram com boa confiabilidade, uma se tornou satisfatória após adequação e uma se revelou inconsistente. Conclusão Mesmo bem avaliadas pelos estudantes, algumas estações apresentaram falhas. A análise do OSCE é fundamental para sua validade e mensurabilidade, em especial para o OSCE de alta aposta.

The Structural Basis for inorganic Pyrophosphatase Catalysis and Regulation

Inorganic pyrophosphatases (PPases) are essential enzymes for every living cell. PPases provide the necessary thermodynamic pull for many biosynthetic reactions by hydrolyzing pyrophosphate. There are two types of PPases: integral membrane-bound and soluble enzymes. The latter type is divided into two non-homologous protein families, I and II. Family I PPases are present in all kingdoms of life, whereas family II PPases are only found in prokaryotes, including archae. Family I PPases, particularly that from Saccharomyces cerevisiae, are among the most extensively characterized phosphoryl transfer enzymes. In the present study, we have solved the structures of wild-type and seven active site variants of S. cerevisiae PPase bound to its natural metal cofactor, magnesium ion. These structures have facilitated derivation of the complete enzyme reaction scheme for PPase, fulfilling structures of all the reaction intermediates. The main focus in this study was on a novel subfamily of family II PPases (CBSPPase) containing a large insert formed by two CBS domains and a DRTGG domain within the catalytic domain. The CBS domain (named after cystathionine beta-synthase in which it was initially identified) usually occurs as tandem pairs with two or four copies in many proteins in all kingdoms of life. The structure formed by a pair of CBS domains is also known as a Bateman domain. CBS domains function as regulatory units, with adenylate ligands as the main effectors. The DRTGG domain (designated based on its most conserved residues) occurs less frequently and only in prokaryotes. Often, the domain co-exists with CBS domains, but its function remains unknown. The key objective of the current study was to explore the structural rearrangements in the CBS domains induced by regulatory adenylate ligands and their functional consequences. Two CBS-PPases were investigated, one from Clostridium perfringens (cpCBS-PPase) containing both CBS and DRTGG domains in its regulatory region and the other from Moorella thermoacetica (mt CBS-PPase) lacking the DRTGG domain. We additionally constructed a separate regulatory region of cpCBS-PPase (cpCBS). Both full-length enzymes and cpCBS formed homodimers. Two structures of the regulatory region of cpCBS-PPase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate, were solved. The structures were significantly different, providing information on the structural pathway from bound adenylates to the interface between the regulatory and catalytic parts. To our knowledge, these are the first reported structures of a regulated CBS enzyme, which reveal large conformational changes upon regulator binding. The activator-bound structure was more open, consistent with the different thermostabilities of the activator- and inhibitor-bound forms of cpCBS-PPase. The results of the functional studies on wild-type and variant CBS-PPases provide support for inferences made on the basis of structural analyses. Moreover, these findings indicate that CBS-PPase activity is highly sensitive to adenine nucleotide distribution between AMP, ADP and ATP, and hence to the energy level of the cell. CBS-PPase activity is markedly inhibited at low energy levels, allowing PPi energy to be used for cell survival instead of being converted into heat.

The Influence of Diet and Microbes on Colonic Immune Regulation and their Implications on Type 1 Diabetes

Dietary and microbial factors are thought to contribute to the rapidly increasing prevalence of T1D in many countries worldwide. The impact of these factors on immune regulation and diabetes development in non-obese diabetic (NOD) mice are investigated in this thesis. Diabetes can be prevented in NOD mice through dietary manipulation. Diet affects the composition of intestinal microbiota, which may subsequently influence intestinal immune homeostasis. However, the specific effects of anti-diabetogenic diets on gut immunity and the explicit associations between intestinal immune disruption and type 1 diabetes onset remain unclear. The research presented herein demonstrates that newly weaned NOD mice suffer from a mild level of colitis, which shifts the colonic immune cell balance towards a proinflammatory status. Several aberrations can also be observed in the peritoneal B cells of NOD mice; an increase in activation marker expression, increased trafficking to the pancreatic lymph nodes and significantly higher antigen presenting cell (APC) efficiency towards insulin-specific T cells. A shift towards inflammation is likewise observed in the colon of germ-free NOD mice, but signs of peritoneal B cell activation are lacking in these mice. Remarkably, most of the abnormalities in the colon, peritoneal macrophages and the peritoneal B cell APC activity of NOD mice are abrogated when NOD mice are maintained on a diabetes-preventive, soy-based diet (ProSobee) from the time of weaning. Dietary and microbial factors hence have a significant impact on colonic immune regulation and peritoneal B cell activation and it is suggested that these factors influence diabetes development in NOD mice.

NADPH-Dependent Thioredoxin System In Regulation of Chloroplast Functions

Photosynthesis, the process in which carbon dioxide is converted into sugars using the energy of sunlight, is vital for heterotrophic life on Earth. In plants, photosynthesis takes place in specific organelles called chloroplasts. During chloroplast biogenesis, light is a prerequisite for the development of functional photosynthetic structures. In addition to photosynthesis, a number of other metabolic processes such as nitrogen assimilation, the biosynthesis of fatty acids, amino acids, vitamins, and hormones are localized to plant chloroplasts. The biosynthetic pathways in chloroplasts are tightly regulated, and especially the reduction/oxidation (redox) signals play important roles in controlling many developmental and metabolic processes in chloroplasts. Thioredoxins are universal regulatory proteins that mediate redox signals in chloroplasts. They are able to modify the structure and function of their target proteins by reduction of disulfide bonds. Oxidized thioredoxins are restored via the action of thioredoxin reductases. Two thioredoxin reductase systems exist in plant chloroplasts, the NADPHdependent thioredoxin reductase C (NTRC) and ferredoxin-thioredoxin reductase (FTR). The ferredoxin-thioredoxin system that is linked to photosynthetic light reactions is involved in light-activation of chloroplast proteins. NADPH can be produced via both the photosynthetic electron transfer reactions in light, and in darkness via the pentose phosphate pathway. These different pathways of NADPH production enable the regulation of diverse metabolic pathways in chloroplasts by the NADPH-dependent thioredoxin system. In this thesis, the role of NADPH-dependent thioredoxin system in the redox-control of chloroplast development and metabolism was studied by characterization of Arabidopsis thaliana T-DNA insertion lines of NTRC gene (ntrc) and by identification of chloroplast proteins regulated by NTRC. The ntrc plants showed the strongest visible phenotypes when grown under short 8-h photoperiod. This indicates that i) chloroplast NADPH-dependent thioredoxin system is non-redundant to ferredoxinthioredoxin system and that ii) NTRC particularly controls the chloroplast processes that are easily imbalanced in daily light/dark rhythms with short day and long night. I identified four processes and the redox-regulated proteins therein that are potentially regulated by NTRC; i) chloroplast development, ii) starch biosynthesis, iii) aromatic amino acid biosynthesis and iv) detoxification of H₂O₂. Such regulation can be achieved directly by modulating the redox state of intramolecular or intermolecular disulfide bridges of enzymes, or by protecting enzymes from oxidation in conjunction with 2-cysteine peroxiredoxins. This thesis work also demonstrated that the enzymatic antioxidant systems in chloroplasts, ascorbate peroxidases, superoxide dismutase and NTRC-dependent 2-cysteine peroxiredoxins are tightly linked up to prevent the detrimental accumulation of reactive oxygen species in plants.

Regulation of cell growth, death, and polarization by ERBB4

Regulation of cell growth, death, and polarization by ERBB4 ErbB4 is a member of the epidermal growth factor receptor (EGFR, ErbB) family. The other members are EGFR, ErbB2 and ErbB3. ErbB receptors are important regulators for example in cardiovascular, neural and breast development but control key cellular functions also in many adult tissues. Abnormal ErbB signaling has been shown to be involved in various illnesses such as cancers and heart diseases. Among the ErbBs, ErbB4 has been shown to have unique signaling characteristics. ErbB4 exists in four alternatively spliced isoforms that are expressed in a tissue-specific manner. Two of the isoforms can be cleaved by membrane proteases, resulting in release of soluble intracellular domains (ICD). Once released into the cytosol, the ICD is capable of translocating into the nucleus and participating in regulation of transcription. The functional differences and the tissue-specific expression patterns suggest isoformspecific roles for ErbB4 isoforms. However, the abilities of ErbB4 isoforms to differently regulate cellular functions were discovered only recently and are not well understood. This study aimed to determine the expression patterns of ErbB4 in normal and diseased tissue, and to define whether the cleavable and non-cleavable isoforms could regulate different target genes and therefore, cellular functions. In this study, a comprehensive ErbB4 expression pattern in several normal tissues, various cancers and non-neoplastic diseases was determined. In addition, the data demonstrated that the cleavable and non-cleavable ErbB4 isoforms could regulate different cellular functions and target genes. Finally, this study defined the cellular responses regulated by ErbB4 during kidney development.

The regulation and function of collagenase-3 (MMP-13) in cutaneous wound healing and squamous cell carcinoma

Matrix metalloproteinase-13 (MMP-13) is a potent proteolytic enzyme, whose expression has been previously associated with fetal bone development and postnatal bone remodeling and with adult gingival wound healing. MMP-13 is also known to be involved in the growth and invasion of various cancers including squamous cell carcinoma (SCC) of the skin. The aim of this study was to further elucidate the function and regulation of MMP-13 in wound repair and cancer. In this study, it was shown that fetal skin fibroblasts express MMP-13 in response to transforming growth factor-β in a p38 MAP kinase dependent manner. In addition, MMP-13 was found to be expressed in vivo by wound fibroblasts in human fetal skin grafted on SCID mice. Adenovirally delivered expression of MMP-13 enhanced collagen matrix contraction by fibroblasts in vitro in association with altered cytoskeletal structure, enhanced proliferation and survival. These results indicate that MMP-13 is involved in cell-mediated collagen matrix remodeling and suggest a role for MMP-13 in superior matrix remodeling and scarless healing of fetal skin wounds. Using an MMP-13 deficient mouse strain, it was shown that MMP-13 is essential for the normal development of experimental granulation tissue in mice. MMP-13 was implicated in the regulation of myofibroblast function and angiogenesis and the expression of genes involved in cellular proliferation and movement, immune response, angiogenesis and proteolysis. Finally, epidermal mitogen, keratinocyte growth factor (KGF) was shown to suppress the malignant properties of skin SCC cells by downregulating the expression of several target genes with potential cancer promoting properties, including MMP-13, and by reducing SCC cell invasion. These results provide evidence that MMP-13 potently regulates cell viability, myofibroblast function and angiogenesis associated with wound healing and cancer. In addition, fibroblasts expressing MMP-13 show high collagen reorganization capacity. Moreover, the results suggest that KGF mediates the anti-cancer effects on skin SCC

Tratamento cirúrgico da doença pilonidal: meta-análise dos principais procedimentos adotados mundialmente

OBJETIVO: Analisar e comparar os diversos procedimentos cirúrgicos descritos para o tratamento da doença pilonidal. MÉTODO: Foram selecionados 34 trabalhos publicados em revistas indexadas, totalizando 8698 doentes operados. Realizou-se meta-análise para comparação das sete principais técnicas cirúrgicas descritas na literatura, quanto aos resultados em relação à recidiva e ao tempo de cicatrização no pós-operatório. RESULTADOS: Do total de doentes estudados, houve recidiva em 230 doentes (2,6%). O tempo de cicatrização no pós-operatório foi significantemente maior no grupo de excisão sem sutura. As recidivas foram estatisticamente semelhantes nos métodos: excisão sem sutura, marsupialização, incisão e curetagem, excisão e retalho e técnica de Karidakys. Os métodos que apresentaram maior índice de recidiva (estatisticamente significante - p<0,001) foram: excisão e sutura primária e o método de Bascom. CONCLUSÕES: Conclui-se, por esse estudo, que os resultados em relação à recidiva são estatisticamente semelhantes em todos os métodos, com exceção da excisão e sutura primária e da técnica de Bascom, que apresentaram recidivas mais freqüentes. O tempo de cicatrização foi maior nos indivíduos operados pela técnica de excisão sem sutura primária.

A lei seca cumpriu sua meta em reduzir acidentes relacionados à ingestão excessiva de álcool?

OBJETIVO: avaliar se a Lei Seca cumpriu sua meta após três anos da promulgação. MÉTODOS: estudo retrospectivo dos pacientes com fraturas craniofaciais submetidos a tratamento cirúrgico em um hospital universitário, em dois períodos: antes (2005 a 2008) e após a implantação da lei (2008 a 2011). RESULTADOS:foram operados 265 pacientes (220 homens e 45 mulheres) nesse período sendo, 149 (56%) antes da lei e 116 (44%) após a lei, indicando redução no número de traumatismos (p=0,04). Houve predomínio da faixa etária entre 19 e 40 anos, em ambos os períodos. As principais causas dos traumas foram os acidentes automobilísticos, as agressões físicas e as quedas. O abuso de álcool foi identificado em 15,4% dos pacientes antes e 19% após a lei. A mandíbula e o complexo maxilozigomático foram os ossos mais acometidos. CONCLUSÃO:a redução no número de politraumatizados operados ficou aquém do esperado e almejado.

Conservative Treatment of Stress Urinary Incontinence: A Systematic Review with Meta-analysis of Randomized Controlled Trials

We performed a systematic review and meta-analysis of randomized controlled trials that studied the conservative management of stress urinary incontinence (SUI). There were 1058 results after the initial searches, from which 37 studies were eligible according to previously determined inclusion criteria. For the primary outcomes, pelvic floor muscle training (PFMT) was more efficacious than no treatment in improving incontinence-specific quality of life (QoL) scales (SMD = [1]1.24SDs; CI 95% = [1]1.77 to [1]0.71SDs). However, its effect on pad tests was imprecise. Combining biofeedback with PFMT had an uncertain effect on QoL (MD = [1]4.4 points; CI 95% = [1]16.69 to 7.89 points), but better results on the pad test, although with elevated heterogeneity (MD = 0.9g; 95%CI = 0.71 to 1,10g); group PFMT was not less efficacious than individual treatment, and home PFMT was not consistently worse than supervised PFMT. Both intravaginal and superficial electrical stimulation (IES and SES) were better than no treatment for QoL and pad test. Vaginal cones had mixed results. The association of IES with PFMT may improve the efficacy of the latter for QoL and pad test, but the results of individual studies were not consistent. Thus, there is evidence of the use of PFMT on the treatment of SUI, with and without biofeedback.

Integration of existing military capability models into the Comprehensive Capability Meta-model

Abstract—This paper discusses existing military capability models and proposes a comprehensive capability meta-model (CCMM) which unites the existing capability models into an integrated and hierarchical whole. The Zachman Framework for Enterprise Architecture is used as a structure for the CCMM. The CCMM takes into account the abstraction level, the primary area of application, stakeholders, intrinsic process, and life cycle considerations of each existing capability model, and shows how the models relate to each other. The validity of the CCMM was verified through a survey of subject matter experts. The results suggest that the CCMM is of practical value to various capability stakeholders in many ways, such as helping to improve communication between the different capability communities.

Professional sports and gambling regulation in Finland - Examining the institutional factors of gambling affecting professional sport clubs in Finland

This study examines how to institutional environment of gambling is currently in motion both in Europe and Finland. Furthermore, it examines the criticism by Finnish professional sport clubs directed towards the national gambling monopolies, especially Veikkaus Oy. This criticism addresses the acclaimed issue of low or non-existing sponsorship funds coming to the clubs despite the clubs’ duties to promote Veikkaus Oy in their stadiums etc. In essence the main research objective was to examine the interaction and institutional environments of both Finnish professional sport clubs and gambling regulation. This was done through three sub-objectives: 1) to analyze professional sport as business and its institutional environment 2) to analyze the institutions of gambling in their current state and their potential future 3) to evaluate the potential impact of an institutional change in gambling legislation to the professional sport clubs The findings from Finland were then compared to those of Denmark where an institutional change had occurred in gambling regulation. Empirical data was collected through multiple interviews. Interviewees represented sport clubs (7), sport association (1), sport league (1), Finnish monopoly representatives (2), commercial gambling providers (1), Danish monopoly system representatives (1), Danish sport club (1). In addition a vast amount of secondary data (e.g. Green and white books by EU, court decisions, a variety of studies etc.). Theoretically this study combines the aspects of institutional theory with the theory of professional sports as business. This proved to be a rather new approach and no published literature was found to have done specifically this. The findings of this study are twofold, on the European level it is clear that the momentum if towards a more liberated gambling market while Finland is at the moment trying to go the opposite direction and uphold its monopoly. From the sport club’s level the findings suggest that currently sport clubs do not directly benefit from the funds originated from Veikkaus Oy as these funds are more or less used on the association/league levels. However, the clubs themselves are also lacking in self-criticism as they are lacking in clear sponsorship packages/programs which Veikkaus Oy might be interested in participating. If liberation of the gambling market would occur it is highly possible that that the largest clubs in football and ice-hockey would be the main beneficiaries while smaller clubs and sports could possibly be worse off than currently. These interpretations were well supported by the findings from Denmark.