Structural and functional characterization of the interactions of platelet derived chemokines CCL5, CXCL4 and CXCL4L1

Chemokines play an important role in the development of inflammation and in the recruitment of leukocytes from the vessel into the inflamed tissue. Activated platelets serve as a rich source of chemokines, e.g. CCL5, CXCL4 and its closely related variant CXCL4L1. The pro-inflammatory CCL5 is a known potent chemoattractant for monocytes and also responsible for its arrest on inflamed endothelium. Since this arrest is enhanced by the interaction of CCL5 with CXCL4, we aimed to determine the structural interaction of this CCL5-CXCL4 heterodimer in the first part of this thesis. For this purpose, CCL5, CXCL4 and CXCL4L1 were expressed recombinantly in E. coli by different techniques, CCL5 as inclusion bodies in the cytoplasm, CXCL4 and CXCL4L1 as soluble proteins in the periplasmic space. For structural analysis of the heterodimerization of the CCL5-CXCL4 complex, 15N-enriched CCL5 was expressed. Additionally, CXCL4L1 was chemically synthesized by the solid-phase peptide synthesis method. All generated proteins were purified by sequential steps with chromatographic methods using FPLC and HPLC. Chemical shift mapping of the 15N-CCL5 by the 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) and 3D modeling revealed that the N-termini were responsible for the formation of the CCL5-CXCL4 heterodimer. In the second part of this dissertation, a functional comparison between CXCL4 and CXCL4L1 was carried out to evaluate the differences of these closely related chemokines. This comparison revealed that the angiogenesis, as assessed by endothelial tube formation, was inhibited by CXCL4 as well as CXCL4L1, but only CXCL4L1 triggered undirected locomotion of endothelial cells (chemokinesis). The chemotactic response towards the growth factors VEGF and bFGF was attenuated by both variants but only CXCL4L1-induced chemokinesis was blocked by bFGF or VEGF. Endothelial cell proliferation was inhibited by CXCL4 but not by CXCL4L1, while both chemokines bound directly to VEGF and bFGF. Moreover, CXCL4 enhanced CCL5-induced monocyte arrest in vitro and as well in vivo, whereas CXCL4L1 had no effect. CXCL4L1 revealed lower affinity for CCL5 than CXCL4 as quantified by isothermal fluorescence titration. As evidenced by the reduction of the activated partial thromboplastin time, CXCL4L1 exhibited less heparin-neutralizing activity than CXCL4. In addition, activation of protein C by thrombin/thrombomodulin was less effectively accelerated by CXCL4L1 compared with CXCL4. It was also discovered that the C-terminal natural changes in CXCL4L1 compared to CXCL4 determine a different quaternary structure since CXCL4 predominantly exists as tetramer and CXCL4L1 as dimer under physiological conditions despite a comparable general positive charge. These structural differences are closely related to the different biological effects most prominently exemplified by the distinct effects in monocyte recruitment and angiogenesis. Summing up, CXCL4L1 may act angiostatically by causing random endothelial cell locomotion, disturbing directed migration towards angiogenic chemokines, serving as a homeostatic chemokine with a moderate structural distinction yet different functional profile from CXCL4

Genetic dissection of a stem cell niche: the case of the Drosophila ovary

11 páginas, 6 figuras, 2 tablas.-- El material suplementario referido en este artículo puede verse en www.interscience.wiley.com/jpages/1058-8388/suppmatIn this work, we demonstrate a powerful new tool for the manipulation of the stromal component of a well-established Drosophila stem cell niche. We have generated a bric-a-brac 1 (bab1)-Gal4 line that drives UAS expression in many somatic ovary cell types from early larval stages. Using this Gal4 line, we could effectively induce FLP/FRT-mediated recombination in the stromal cells of the ovarian germline stem cell niche. Mutant clones were observed in the developing ovary of larvae and pupae, including in somatic cell types that do not divide in the adult, such as the cap cells and the terminal filament cells. Exploiting the ability of bab1-Gal4 to generate large clones, we demonstrate that bab1-Gal4 is an effective tool for analyzing stem cell niche morphogenesis and cyst formation in the germarium. We have identified a novel requirement for engrailed in the correct organization of the terminal filaments. We also demonstrate an involvement for integrins in cyst formation and follicle cell encapsulation. Finally using bab1-Gal4 in conjunction with the Gal80 system, we show that while ectopic dpp expression from stromal cells is sufficient to induce hyperplastic stem cell growth, neither activation nor inactivation of the BMP pathway within stromal cells affects germline stem cell maintenance.Grant sponsor: Spanish Ministerio de Ciencia y Tecnología; Grant number: BMC2003-01512; Grant sponsor: Junta de Andalucía; Grant number: CVI-280.Peer reviewe

Identification of Matrix Metalloproteinase-12 as a Candidate Molecule for Prevention and Treatment of Cardiometabolic Disease

Abstract Obesity is strongly associated with metabolic syndrome, a combination of risk factors that predisposes to development of the cardiometabolic diseases: atherosclerotic cardiovascular disease and type 2 diabetes mellitus. Prevention of metabolic syndrome requires novel interventions to address this health challenge. The objective of this study was to identify candidate molecules for the prevention and treatment of insulin resistance and atherosclerosis, conditions that underlie type 2 diabetes mellitus and cardiovascular disease, respectively. We used an unbiased bioinformatics approach to identify molecules that are upregulated in both conditions by combining murine and human data from a microarray experiment and meta-analyses. We obtained a pool of 8 genes that were upregulated in all the databases analyzed. This included well-known and novel molecules involved in the pathophysiology of type 2 diabetes mellitus and cardiovascular disease. Notably, matrix metalloproteinase 12 (MMP12) was highly ranked in all analyses and was therefore chosen for further investigation. Analyses of visceral and subcutaneous white adipose tissue from obese compared with lean mice and humans convincingly confirmed the upregulation of MMP12 in obesity at the mRNA, protein and activity levels. In conclusion, by using this unbiased approach, an interesting pool of candidate molecules was identified, all of which have potential as targets in the treatment and prevention of cardiometabolic diseases

Учет расчетов с поставщиками в бюджетном учреждении

Выпускная квалификационная работа включает: 82 с. (без учета приложений), 25 таблиц, 8 рисунков, 39 источников и 13 приложений. Ключевые слова: казенное учреждение, бюджетный учет, учет расчетов с поставщиками, виды бюджетных учреждений, способы определения поставщиков. Объект исследования – Межмуниципальный отдел Министерства внутренних дел Российской Федерации «Асиновский» Управления Министерства внутренних дел Российской Федерации по Томской области. Цель работы – изучение особенностей организации учета расчетов с поставщиками в бюджетных учреждениях на примере МО МВД России «Асиновский».Final qualifying work include: 82. (excluding attachments), 25 tables, 8 figures, 39 13 sources and applications. Keywords: state institution, budgetary accounting, accounts payable, the types of budgetary institutions, methods of determination of suppliers. The object of study – Intermunicipal Department of the Ministry of internal Affairs of Russia "Asinovsky" of the Ministry of internal Affairs of the Russian Federation for Tomsk region. Purpose – to study the peculiarities of organization of accounting of calculations with suppliers in budgetary institutions on the example of MO the Ministry of internal Affairs of Russia "Asinovsky"