Recently, they are examined various means for activating the endogenous signalling pathways leading to increased resistance of the myocardium from ischemic/reperfusion (I/R) injury. One of them is the adaptation to chronic hypoxia, which has been shown to reduce the incidence and severity of ventricular arrhythmias, improves the recovery of postischemic contractile function of the heart and particularly reduces the extent of myocardial infarction. Since the function of the heart depends on the maintenance of membrane integrity of cardiomyocytes there are very important phospholipase A2 (PLA2) which are involved in the repair of cellular membranes. Also they are an important component of the protective signalling pathways because they cleave membrane phospholipids to produce lipid signalling molecules. Elucidate the role of PLA2 and the precise mechanism of action of signalling pathways leading to cardioprotection could be important for the prevention and treatment of cardiovascular diseases. Therefore, in this thesis we examined the influence of continuous normobaric hypoxia (CNH) to the relative representation of cardiac PLA2 (secretory - sPLA2IIA, calcium-independent - iPLA2, cytosolic - cPLA2α and its phosphorylated form - p-cPLA2α), and proteins involved in the activation and phosphorylation of...

Orientador: Marcos Roberto de Mattos Fontes / Resumo: A toxicidade do veneno das serpentes do gênero Bothrops é resultante da ação integrada de várias toxinas, entre elas as fosfolipases A2 (PLA2s). Entre o grupo das PLA2s, há as PLA2s ativas enzimaticamente e as PLA2s desprovidas de atividade enzimática, denominadas proteínas PLA2-like. Apesar de sua inatividade enzimática, as proteínas PLA2-like desses venenos possuem potente ação miotóxica local; efeito o qual o soro antiofídico não é capaz de neutralizar efetivamente. Tendo em vista as limitações da soroterapia, é necessário compreender como essas miotoxinas agem localmente. Uma abordagem eficaz é o estudo estrutural-funcional destas toxinas com potenciais inibidores ou ativadores. Portanto, o presente trabalho tem como objetivo estudar a relação estrutura-função da BnSP-7 e sua isoforma BnSP-6, duas miotoxinas PLA2-like do veneno de Bothrops pauloensis, através de sua interação com ácidos graxos. Deste modo, essas proteínas foram obtidas a partir do veneno bruto de B. pauloensis por cromatografia líquida de troca iônica seguida por cromatografia em fase reversa. As frações obtidas foram analisadas por espectrometria de massa para a confirmação de ambas as identidades, no entando não foi possível identifica-las. Foi observado por técnica de espectroscopia de dicroísmo circular a preservação das estruturas secundárias dessas toxinas (enovelamento referente à PLA2). Foram obtidos cristais da amostra purificada após 21 dias de incubação a 283 K e por esses dados cristalográfico... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The toxicity of Bothrops venom is the result of the integrated action of several toxins, including phospholipases A2 (PLA2s). Among the group of PLA2s, there are the enzymatically active PLA2s and the PLA2s devoid of enzymatic activity, called PLA2-like proteins. Despite their enzymatic inactivity, the PLA2-like proteins of these poisons have a potent local myotoxic action; an effect that the anti-oxyde serum is not able to neutralize effectively. Given the limitations of serotherapy, it is necessary to understand how these myotoxins act locally. An effective approach is the structural-functional study of these toxins with potential inhibitors or activators. Therefore, the present study aims to study the structure-function relationship of BnSP-7 and its BnSP-6 isoform, two PLA2-like myotoxins from Bothrops pauloensis venom, through their interaction with fatty acids. Thus, these proteins were obtained from the raw venom of B. pauloensis by ion exchange liquid chromatography followed by reverse phase chromatography. The fractions obtained were analyzed by mass spectrometry to confirm both identities, but it was not possible to identify them. The preservation of secondary structures of these toxins (entanglement related to PLA2) was observed by circular dichroism spectroscopy technique. Crystals were obtained from the purified sample after 21 days of incubation at 283 K and by these crystallographic data it was found that the sample had the BnSP-7 protein sequence. In addition,... (Complete abstract click electronic access below) / Mestre

Cardiovascular diseases, particularly acute myocardial infarction, are the leading causes of death in developed countries including the Czech Republic. One of the ways to increase cardiac resistance against acute ischemia/reperfusion (I/R) injury is adaptation to chronic hypoxia. However, changes at the molecular level associated with this adaptation have still not been fully explored. It is obvious that the myocardial function depends on maintaining membrane integrity and cellular homeostasis of cardiomyocytes. From this perspective, phospholipases A2 (PLA2) are the key enzymes that take part in the remodeling and repairing of the cell membranes. Moreover, PLA2 are also involved in generation of lipid signaling molecules - free long chain fatty acids (FA) and 2-lysophopholipids. In myocardium, members of three major PLA2 classes are present: cytosolic PLA2 (cPLA2), calcium-independent PLA2 (iPLA2) and secretoric PLA2 (sPLA2). This thesis aimed to determine the following in the left ventricular myocardium of adult male Wistar rats: 1) The effect of intermittent hypobaric hypoxia (IHH; 8 hours/day, 5 days/week, 5 weeks, ~ 7000 m) on the expression of total cPLA2α and its phosphorylated form (p-cPLA2α, Ser505 ), and further iPLA2 and sPLA2IIA, as well as signaling proteins activating cPLA2α enzyme...

Use of biochemical markers (Lipoprotein phospholipase A2 and hyaluronic acid) for laboratory diagnosis of metabolic and degenerative diseases of the musculoskeletal system Abstract Musculoskeketal disorders currently belong to the most common diseases. The presented work describes the use of biochemical markers (Lipoprotein phospholipase A2 and hyaluronic acid) for laboratory diagnosis of metabolic and degenerative diseases of the musculoskeletal system. Concentrations of LP-PLA2 were significantly elevated in the patients with bone resorption compared to the control group of healthy individuals. Serum levels of Lp-PLA2 also negatively correlated with decreased levels of serum osteocalcin in patients. HA concentrations in synovial fluid did not differ from published reference values in synovial fluid. Patients who underwent arthroscopy had significantly elevated synovial HA concentration than patients who underwent total knee endoprosthesis. HA positively correlates with osmotic pressure determined by examination of osmolality in synovial fluid. Lipoprotein phospholipase A2 concentrations are elevated in patients with bone density impairment. LpPLA2 concentrations correlate with the severity of bone density impairment expressed by the T score. Hyaluronic acid concentrations in patients with knee...

No description available.

Thromboxane A2 receptor (TP) has been shown to play important roles in multiple aspects of cancer development including regulation of tumor growth, survival and metastasis. Molecular mechanisms of TP mediated cancer cell invasion remain to be identified. TP agonist, I-BOP, significantly elevated several matrix metalloproteinases (MMPs) including MMP-1, MMP-3, MMP-9 and MMP-10 in A549 human lung adenocarcinoma cells overexpressing TPα (A549-TPα) or TPβ (A549-TPβ). Signaling pathways of I-BOP-induced MMP-1 expression were examined in further detail as a model system for MMPs induction. Signaling molecules involved in I-BOP-induced MMP-1 expression were identified by using specific inhibitors including small interfering (si)-RNAs of signaling molecules and promoter reporter assay. The results indicate that I-BOP-induced MMP-1 expression is mediated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)-activator protein-1(AP-1) and ERK-CCAAT/enhancer-binding protein β (C/EBPβ) pathways. I-BOP-induced cellular invasiveness of A549-TPα cells was blocked by, GM6001, a general inhibitor of MMPs. Knockdown of MMP-1 and MMP-9 by their respective siRNA partially reduced I-BOP-stimulated A549-TPα cells invasion suggesting that other MMPs induced by I-BOP were also involved. Furthermore, secreted MMP-1 in conditioned media from I-BOP-treated A549-TPα cells (CM-I-BOP) autocrinely induced monocyte chemoattractant protein-1 (MCP-1) expression. The induction of MCP-1 by MMP-1 in A549 cells was via activation of protease-activated receptor 2 (PAR2) instead of commonly assumed PAR1. This conclusion was reached from the following findings: (1) expression of MCP-1 induced by trypsin, a PAR2 agonist, was inhibited by a PAR2 antagonist. (2) expression of MCP-1 induced by MMP-1 and by CM-I-BOP was blocked by a PAR2 antagonist but not by other PAR antagonists; (3) expression of MCP-1 induced by MMP-1 and by CM-I-BOP was attenuated significantly by pretreatment of cells with PAR2-siRNA. Finally, MCP-1 also can be induced by direct activation of TP in a SP1 involved mechanism. CM-I-BOP enhanced MCP-1-dependent migration of RAW 264.7 macrophages. Co-culture of A549 cells with RAW 264.7 macrophages induced expression of MMPs, VEGF and MCP-1 genes, and increased the invasive potential in A549 cells. My studies provide molecular mechanisms by which TP-mediated cancer cell invasion and suggest that TP is a potential anti-cancer drug target.

Pseudomonas aeruginosa est une bactérie à Gram négatif ubiquitaire, pathogène opportuniste. Elle est la 3ème cause d’infections nosocomiales, notamment chez les immunodéprimés et les grands brûlés. Elle est aussi responsable de la mort de nombreux patients atteints de la mucoviscidose. Sa virulence est largement due à son aptitude à sécréter de nombreuses enzymes dégradatives et toxines, parmi lesquelles la protéine ExoU, sécrétée par le Système de Sécrétion de Type III. ExoU est une phospholipase, de la famille des « patatin-like proteins », dont l’activité est portée par une dyade catalytique Ser-Asp.Mon travail de thèse a permis d’identifier 4 homologues d’ExoU (PlpA, PlpB, PlpC et PlpD) dans le protéome de la souche PAO1 de P. aeruginosa (qui est dépourvu de cette protéine). En étudiant le mode de sécrétion de PlpD, nous avons découvert une nouvelle branche du Système de Sécrétion de Type V (SST5), le SST5d. La protéine représentant ce nouveau système possède un domaine C-terminal transporteur de type TpsB (SST5b), fusionné à un domaine N-terminal patatine sécrété dans le milieu extracellulaire (à l’image d’un autotransporteur, ou SST5a). Ce mode de sécrétion serait un mode dédié à la sécrétion de « patatin-like proteins », comme le suggèrent nos analyses phylogénétiques, à Nous avons en outre démontré que PlpD possède une activité lipase.L’autre protéine étudiée, PlpA, est également sécrétée, bien que nous n’ayons pu établir avec certitude sa voie de transport. Nous avons évalué le rôle de cette protéine lors de l’interaction de P. aeruginosa avec des cellules hôtes de type macrophages et cellules épithéliales. Nous avons observé que cette protéine confère une protection temporaire aux cellules infectées par P. aeruginosa. Ce retard semble être directement imputable à l’activité de la protéine, puisqu’il est dépendant de l’intégrité de la dyade catalytique putative de PlpA / Pseudomonas aeruginosa is an ubiquitous Gram negative bacteria, and efficient opportunistic pathogen. It is the third most common cause of nosocomial infections, most particularly within immunocompromized or burn patients. This pathogen is responsible for the death of numerous cystic fibrosis patients. Its virulence is due mainly to its capacity to secrete numerous degradative enzymes and toxins, among them, ExoU which is secreted via the Type III Secretion System. ExoU is a phospholipase of the patatin-like protein family, and its activity is based on a Ser-Asp catalytic dyad.During my thesis, we identify 4 ExoU homologs (PlpA, PlpB, PlpC, and PlpD) in the proteome of the P. aeruginosa PAO1 strain (this strain does not possess ExoU). Results obtained studying PlpD secretion led us to discover a new branch of the Type V Secretion System (T5SS), the T5dSS. PlpD is composed of a C-terminal TpsB-like transporter domain (like T5bSS), fused to a N-terminal patatin domain which is secreted into the extracellular medium (like autotransporters, or T5aSS). Our phylogenetic analysis suggests that this secretion pathway may be dedicated to the secretion of PLPs, like T5cSS, which secretes only adhesins. Moreover, we demonstrated that PlpD is a lipase.The other studied protein, PlpA, is also a secreted protein, but we still do not know which secretion system is involved in its secretion. We tested the role of PlpA during interaction of P. aeruginosa with host cells by carrying out infections of murin macrophages and epithelial cells. We observed a transitory protection of cells infected with P. aeruginosa. This protection seems to require an active PlpA protein as it is dependent on a intact catalytic dyad in this protein

<p>Predmet doktorske disertacije je analiza fonetskih i morfolo&scaron;kih gre&scaron;aka načinjenih prilikom učenja rumunskog jezika kao stranog jezika na Univerzitetu u Novom Sadu od &scaron;kolske 2010/11. do &scaron;kolske 2015/16. godine na nivoima A1 i A2. Korpus čine testovi zavr&scaron;nih ispita, kolokvijuma i usmenih delova ispita 159 studenata sa različitih studijskih grupa. Cilj rada je sistematizovanje gre&scaron;aka prema fonetskim i morfolo&scaron;kim nivoima i tipovima i opisivanje gre&scaron;aka prema uzrocima njihovog nastajanja.<br />Rad je podeljen na tri glavne celine. Prva celina sadrži predmet, cilj i zadatke istraživanja, zatim metode i tehnike istraživanja, kao i izvore za korpus. Druga celina započinje teorijskim okvirom, a nastavlja se pregledom postojeće relevantne literature o učenju stranog jezika, razmatranjem razloga za učenje stranog jezika, kao i uloge profesora u procesu učenja. Posebno su predstavljene gre&scaron;ke u učenju stranih jezika. Treća celina odnosi se na samu analizu dobijene građe, gre&scaron;aka i interpretaciju dobijenih rezultata. Ova celina podeljena je na dva veća poglavlja koja se bave fonetskom i morfolo&scaron;kom analizom gre&scaron;aka. U okviru poglavlja koje se bavi fonetskim gre&scaron;kama obrađene su sledeće oblasti: samoglasnici, diftonzi, triftonzi, alternacija samoglasnika, suglasnici, glasovne grupe ge, gi, ghe, ghi, ci, ce, chi, che, kao i alternacija suglasnika. U okviru drugog poglavlja, koje se bavi morfolo&scaron;kim gre&scaron;kama, obrađene su sledeće oblasti: pogre&scaron;na upotreba imenica, pogre&scaron;na upotreba određenog i neodređenog člana, gre&scaron;ke u upotrebi zamenica (lične zamenice u nominativu i akuzativu, povratno-refleksivne zamenice, pokazne zamenice), gre&scaron;ke u upotrebi prideva i gre&scaron;ke u upotrebi brojeva. U pomenutom poglavlju obrađena su i glagolska vremena (prezent, perfekat, futur I i kolokvijalni futur), kao i lični (konjunktiv i imperativ) i nelični glagolski načini (infinitiv i particip sa aktivnim značenjem). Na kraju poglavlja obrađeni su i predlozi (la i &icirc;n). Budući da su nastavni proces pratile promene koje se odnose na primenu modernih tehnologija u učenju stranog jezika i otklanjanju gre&scaron;aka, deo na&scaron;eg istraživanja je posvećen i tome.<br />Rezultati analize pokazali su da je najveći broj identifikovanih gre&scaron;aka zapravo na morfolo&scaron;kom nivou, ali da fonetske gre&scaron;ke predstavljaju prepreku u učenju rumunskog jezika. U najvećem broju primera ustanovili smo da su gre&scaron;ke unutarjezičkog karaktera, ali i da su studenti primenjivali strategije transfera iz maternjeg ili nekog drugog stranog jezika koji znaju. Namera nam je bila da ukažemo na koje gramatičke kategorije profesori, ali i studenti, moraju da obrate pažnju prilikom učenja rumunskog kao stranog jezika i koji su nedostaci nastavnog procesa. Rezultati istraživanja će se koristiti u nastavi rumunskog jezika kao stranog i<br />Fonetska i morfolo&scaron;ka analiza gre&scaron;aka u učenju<br />rumunskog jezika kao stranog jezika<br />mogu pomoći u kreiranju posebnog kurikuluma. Gre&scaron;ke se u dobroj meri mogu eliminisati kori&scaron;ćenjem posebnih metoda i vežbi, kao i inovacija u procesu nastave, jer je motivacija za učenje jezika mnogo veća.</p>

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Sterol regulatory element-binding proteins (SREBP) are transcription factors that regulate genes involved in lipid metabolism especially in the liver. Therefore, hepatic SREBP is significant regulator of systemic lipid metabolism. Evidence demonstrates that insulin and dietary unsaturated fatty acid (UFA) regulate SREBP1 expression and subsequent SREBP1-mediated gene transcription, events that in many instances result in modulation of systemic fatty acid and triglyceride (TG) homeostasis. A series of investigations was designed to uncover novel regulators of SREBP1. Dietary and exogenous addition of UFA has been shown to regulate SREBP function yet, an endogenous source of UFA capable of modulating SREBP remains elusive. Group VIA calcium-independent phospholipase A2 (iPLA2) releases UFA from the sn-2 position of glycerophospholipids. We hypothesized that iPLA2 provides UFA to suppress SREBP. iPLA2 overexpression and inhibition studies were implemented. iPLA2 inhibition increased SREBP1 expression, SREBP-mediated transcription and the expression of SREBP1 gene targets in vitro. In vivo overexpression of iPLA2 resulted in decreased expression of SREBP1 protein and plasma triglyceride. In contrast, iPLA2 overexpression attenuated SREBP1 expression, SREBP-mediated transcription and expression of SREBP1 targets genes. These data support the hypothesis that iPLA2 generates endogenous UFA that limit SREBP function. Use of a replication-deficient adenovirus 5 (Ad-5) expression vector in the iPLA2 study led to the unexpected observation of hepatic SREBP1 activation following Ad-5 infection. Because of this observation, we tested the hypothesis that replication-deficient Ad-5 might augment lipid synthesis in liver. We demonstrate that first generation Ad-5, a ubiquitous transgene expression vector, induces expression of SREBP1 and its target genes and leads to increases in fatty acid synthesis in vivo and in vitro. The phosphatidylinositol 3-kinase (PI3K) inhibitor, PX-866, suppressed Ad-5-induced SRBEP1 expression and hypertriglyceridemia implicating the PI3K/Akt pathway in Ad-5 activation of SREBP1. Use of PX-866 led to the discovery of a third mechanism of SREBP1 regulation. In vivo studies demonstrate that PX-866 modulates basal lipid metabolism in part through decreasing plasma TG, an increased trend toward decreased SREBP1 expression and a significant increase in plasma cholesterol. These studies characterize three distinct novel regulatory mechanisms of SREBP1.