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Caseous lymphadenitis (CLA) is a disease primarily in sheep and goats, caused by Corynebacterium pseudotuberculosis, characterized by abscess formation in external and/or internal lymph nodes and organs. Economic losses occur due to emaciation, reduced milk production and impaired growth in affected animals. At the Swedish Veterinary Agency, bacterial culture is used as a diagnostic method for detecting CLA, requiring puncture of clinical abscesses. This sampling method increases the risk for disease transmission and overlooks animals affected by internal abscesses. In this study, a commercially available enzyme-linked immunosorbent assay (ELISA)-kit, based on an indirect ELISA, was validated for detecting antibodies for CLA, aiming for a more cost-effective and safer method that also can detect animals with internal abscesses. Serum and milk samples were analyzed to investigate the concordance between the two sample types. A subset of serum and milk samples was sent to Norway for analysis to compare the ELISA results, and comparison with another laboratory's results was conducted. The ELISA-kit demonstrated high sensitivity (92% for serum, 100% for milk), crucial for controlling CLA at herd level, while the specificity was estimated at 68% for serum and 61% for milk. To avoid increased and unnecessary culling of animals, a confirmatory method should analyze positive individuals to increase the specificity. Pooled milk samples could reduce costs for herd-level analysis, followed by serum sampling for positive herds.

Tableau d'honneur de la Faculté des études supérieures et postdorales, 2015-2016 / Les acides gras polyinsaturés oméga-3 (AGPI n-3), plus spécifiquement l’acide eicosapentaénoïque (AEP) et l’acide docosahexaénoïque (ADH), abaissent le risque de maladies cardiovasculaires (MCV) en agissant sur différents facteurs de risque dont une diminution des triglycérides (TG) plasmatiques et de l’inflammation. Toutefois, une grande variabilité interindividuelle dans la réponse cardiométabolique à la supplémentation en AGPI n-3 est observée et elle serait en partie reliée à des facteurs génétiques. Les gènes du métabolisme des lipides, dont les phospholipases A2 (PLA2), ont été modulés suite à la supplémentation de 3 g d’AEP et d’ADH/jour pendant six semaines. Des effets de génotype*supplémentation ont été observés avec des variations des gènes des PLA2 sur les niveaux de TG et de protéine C-réactive (CRP). Les résultats suggèrent que des variations sur les gènes de PLA2 expliquent en partie la variabilité interindividuelle de la réponse des TG et de la CRP à la supplémentation en AGPI n-3. / Fish oil-derived long-chain omega-3 (n-3) polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), reduce the risk of cardiovascular disease by lowering plasma triglyceride (TG) and inflammation levels. However, a large inter-individual variability is observed, which could be explained by genetic factors. Genes involved in metabolic pathways of n-3 PUFA, including phospholipases A2 (PLA2) had changes in their expression in individuals who consumed 3 g/day of EPA and DHA for 6 weeks. Genotype by supplementation interaction effect on TG and C-reactive protein (CRP) levels were observed for PLA2 variations. These results suggest that variations in PLA2 genes may influence plasma TG and CRP levels during a supplementation with n-3 PUFA.

Les ligands orthostériques transmettent un signal complexe aux cellules en se fixant à l'intérieur de la pochette de liaison de leur récepteur cible. Le changement conformationel qui en résulte modifie l'efficacité du récepteur à recruter et activer les seconds messagers en amont des voies de signalisation, soit les protéines G hétérotrimériques dans le cas des récepteurs couplés aux protéines G (GPCRs). Ces variations entraînent une vaste gamme de modifications dans le milieu intracellulaire. Par exemple, l'activation de la protéine G q provoque entre autres l'activation de la phospholipase C? (PLC? ), la production d'inositol 1,4,5-trisphosphate (1P3), puis l'activation des protéines kinases C (PKC). L'activation de la protéine G s , pour sa part, stimule l'activité de l'adénylate cyclase (AC), ce qui entraîne la production d'AMPc et l'activation de la protéine kinase A (PKA). Un ligand n'influence pas nécessairement deux voies de signalisation indépendantes de façon similaire, ce qui lui confère la propriété de sélectivité fonctionnelle. Dans ce travail, nous avons caractérisé le profil pharmacologique de ligands orthostériques du récepteur FP de la PGF2? à l'aide d'un clone HEK-293-SL exprimant le récepteur FP de façon stable. La mesure de la production d'IP3 a permis d'évaluer la voie de la PLC alors que la mesure de la production d'AMP c a permis d'évaluer la voie de l'AC. Pour chacune d'entre elles, le fluprostenol s'est comporté comme un agoniste complet moins puissant que l'agoniste naturel. Le composé synthétique Al-8810 s'est comporté comme un agoniste partiel de la voie de la PLC, alors qu'il s'est avéré être un antagoniste de la voie de l'AC. Ces résultats démontrent que l'activité d'un ligand vis-à-vis un récepteur dépend du groupe d'effecteurs observé, ce qui illustre le concept de sélectivité fonctionnelle des ligands. L'étude des composés allostériques THG113 et THG113.824 démontre que ces derniers n'influencent pas la signalisation déclenchée en aval du récepteur FP par son agoniste naturel. Ces résultats suggèrent qu'ils agiraient comme antagoniste des effets de la PGF 2? par un mécanisme indépendant du récepteur FP. [Symboles non conformes]

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Both, the cytokine Tumor Necrosis Factor-α (TNF-α) and the enzyme cytosolic phospholipase A2 (cPLA2) are crucial driving forces in mediating the cellular inflammatory response and are involved in ischaemic injury. During an ischaemic insult, TNF-α is endogenously generated. Apart from the recognized effects of TNF- α, such as the induction of apoptosis, proliferation and differentiation, if present in low dosages, it also mediates cytoprotective effects. Upon activation, cPLA2 contributes to the ischaemic challenge with the generation of mediators of cellular injury and apoptosis. Upon stimulation, this calcium dependent enzyme translocates to the phospholipid compartment of the cell membrane and induces the hydrolysis of sn-2 ester bonds in phospholipids. It governs the release of free fatty acids and lysophospholipids and generates role players of inflammation. We suggest a role for cPLA2 in the TNF-α mediated cytoprotection, with a distinct phosphorylation and translocation pattern. Aims The involvement of cPLA2 in TNF-α mediated cytoprotection in the C2C12 murine skeletal muscle cell line in tolerance to ischaemia was examined. To investigate the nature of the cPLA2 phosphorylation pattern, the mitogen activated protein kinases (MAPKs) p38 and extracellular regulated kinase (ERK) as contributors to cPLA2 phosphorylation and activation, were examined at appropriate time points. To dissect out the cPLA2 interplay and dependencies with these MAPKs within the pathway context, the selective cPLA2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) was employed and its effect on cell viability was examined. Fluorescence microscopy was used to substantiate cPLA2 activation, by assessing its cellular distribution, translocation and cell organelle target preference, using co-localization and z-stack techniques. In addition, the induction of the apoptotic pathway through analysis of caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage was examined. The role of caspase-3 in cPLA2 turnover was addressed employing the caspase inhibitor, Z-DEVD-FMK. Methods Cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions, until 50%-70% confluent. Using DMEM supplemented with 1% horse serum, cell differentiation into myotubes was induced. Differentiated cells were preconditioned for 30 min classically, with 0.5 ng/ml TNF-α or the cPLA2 selective inhibitor AACOCF3 (10 μM) respectively. Followed by a 60 min washout period the cells were subjected to 8 hrs simulated ischaemia. Cellular viability; and cPLA2 phosphorylation- and translocation events were assessed using Western blots and advanced immunocytochemistry and imaging techniques. Results Preconditioning with TNF-α, ischaemic preconditioning; and the use of the cPLA2 inhibitor AACOCF3, attenuated the decrease is cell viability brough about by ischaemia. Western blot analysis indicates the induction of the apoptotic pathway with caspase-3 and PARP cleavage. A significantly reduced translocation of pcPLA2 to the nuclear region in the TNF-α preconditioned group compared to the ischaemic group, as reflected by reduced mean nuclear fluorescence intensity, was observed. A z-stack analysis confirmed that the nuclear and endonuclear region was the target organelle for cPLA2. 3-dimensional co-localazation analysis of pcPLA2 with the nuclear marker nucleoporin p62 mirrored these results. Discussion and conclusion Our results provide evidence that there is a role for cPLA2 in TNF-α mediated cytoprotection. Although we do not observe a differential activation pattern in terms of cPLA2 phosphorylation at various time points within the ischaemic event, and no differential inactivation of cPLA2 via caspase-mediated cPLA2 cleavage, we describe a differential cPLA2 translocation pattern, similar to that in IPC. Through inhibition of cPLA2 translocation, a functional cPLA2 inhibition might be achieved. This would imply inhibition of the inflammatory pathway and a subsequent reduction in the generation of inflammatory mediators. In addition we describe an effect of TNF-α preconditioning on the efficacy of the caspase inhibitor Z-DEVD-FMK. Our results shed light on the survival mechanisms employed by the ischaemically challenged cell in a setting of TNF-α mediated cytoprotection. This might lead to novel approaches in the context of inflammation treatment, through agents that control differential cPLA2 trafficking within the cell. / AFRIKAANSE OPSOMMING: Beide, die sitokien “Tumor Necrosis Factor-α (TNF-α)” en die ensiem, sitosoliese fosfolipase A2 (cPLA2) is uiters belangrike bemiddelaars van die sellulêre inflammatoriese respons en is verder ook betrokke by isgemiese selskade. TNF-α word endogeen gegenereer tydens ‘n isgemiese intervensie. Afgesien van ‘n verskeidenheid effekte, soos die inisiëring van apoptose, sel-proliferasie en - differensiasie, bemiddel dit ook selbeskermende meganismes indien dit in lae konsentrasies in die sel teenwoordig is. Na aktivering dra cPLA2 by tot die isgemiese intervensie deur die vorming van bemiddelaars van selskade en apoptose. Hierdie kalsium-afhanklike ensiem translokeer na die fosfolipied membraankomponent na stimulering en induseer die hidrolise van die sn-2 esterbinding in die fosfolipied. Die vrystelling van vry vetsure en lisofosfolipiede word sodoende bewerkstellig wat verder gemetaboliseer kan word tot inflammatoriese bemiddelaars. Ons stel voor dat cPLA2 ‘n rol in TNF-α bemiddelde selbeskerming speel en dat dit gepaardgaan met kenmerkende fosforilerings- en translokeringspatrone. Doelwitte Die rol van cPLA2 tydens TNF-α bemiddelde selbeskerming is in ‘n C2C12 skeletspiersellyn na blootstelling aan isgemie ondersoek. Die rol van die MAPKs, p38 en ERK, is ondersoek om vas te stel of hulle betrokke is by die aktivering van cPLA2. Die selektiewe cPLA2 inhibitor, AACOCF3, is gebruik om te bepaal of die fosforilering van MAPKs ook cPLA2-afhanklik is. Die sellulêre cPLA2 verspreiding, translokering en teiken selorganelle is ook ondersoek met behulp van fluoresensie mikroskopie deur gebruik te maak van ko-lokalisering en z-plaat tegnieke. Verder, is die indusering van die apoptotiese paaie ondersoek deur tegnieke wat kaspase- en PARP kliewing meet. Die kaspase inhibitor, Z-DEVD-FMK, is gebruik om vas te stel of kaspase-3 ‘n rol speel in cPLA2 kliewing in ons selmodel. Metodes Selle is gekweek in Dulbecco’s gemodifiseerde Eagles Medium (DMEM) waarby 10% fetale kalf serum (FBS) gevoeg is, en wat geïnkubeer is in 5% CO2 totdat dit 50%-70% konfluent was. Die selle is verder gedifferensieer in miobuise deur gebruik te maak van DMEM waarby 1% perdeserum gevoeg is. Gedifferensieerde selle is vir 30 min klassiek geprekondisioneer asook respektiewelik met 0.5 ng/ml TNF-α en die cPLA2 selektiewe inhibitor, AACOCF3 (10 μM). Na ‘n 60 minute uitwas periode is die selle blootgestel aan 8 h gesimuleerde isgemie. Sellulêre lewensvatbaarheid, cPLA2 fosforilering- and translokering is ondersoek deur onderskeidelik gebruik te maak van die “Western” klad metode en gesofistikeerde immunositochemiese beeld tegnieke. Resultate Prekondisionering met TNF-α, isgemiese prekondisionering asook inhibisie van as cPLA2 met die inhibitor, AACOCF3, het ‘n beduidende toename in sellewensvatbaarheid tot gevolg gehad. Daar is ook dmv die “Western” klad tegniek bewys dat apoptose geïduseer word deur middel van kaspase-3- en PARP kliewing. Daar is insiggewend minder translokasie van cPLA2 na die nukluêre fraksie in die isgemiese groep in vergelyking met die TNF-α geprekondisioneerde groep waargeneem (die gemiddelde nukluêre fluoreserende intensiteit is bepaal om voorafgaande feit te staaf). Die cPLA2 teiken organel is geverifieer as die nukleus en die endonukluêre gebied deur middel van z-plaat analises. Drie-dimensionele kolokaliserings analises van pcPLA2 met die nukluêre merker, nucleoporin p62 het hierdie resultate bevestig. Bespreking en Gevolgtrekking Ons resultate verskaf bewyse vir ‘n rol vir cPLA2 in TNF-α bemiddelde selbeskerming. Alhoewel daar nie ‘n differensiële aktiveringspatroon in terme van cPLA2 fosforilering tydens verskeie tydspunte in die isgemiese intervensie waargeneem is nie, en ook geen kaspase-3 bemiddelde kliewing van cPLA2 nie, word ‘n differensiële translokeringspatroon soorgelyk aan die isgemiese prekondisioneringsgroep, waargeneem. Funsksionele cPLA2 inhibisie kan dus moontlik bewerkstellig word deur inhibisie van cPLA2 translokasie. Die inflammatoriese respons kan dus moontlik so inhibieer word en die vorming van minder inflammatoriese bemiddelaars tot gevolg hê. Verder het TNF-α prekondisionering ook ‘n effek op die effektiwiteit van die kaspase-inhibitor, ZDEVD- FMK. Ons resultate werp ook lig op die meganismes wat deur selle onder isgemiese toestande uitgeoefen word tydens TNF-α bemiddelde selbeskerming. Hierdie resultate mag lei tot nuwe benaderings in die konteks van behandeling teen inflammasie deur gebruik te maak van middels wat cPLA2 translokering in die sel beheer.

Platelet-activating factor acetylhydrolase (PAFAH) is elevated in atherosclerosis and may play a role in pathogenesis of this disease. Molecular mechanisms regulating the expression of this lipoprotein-associated PLA2 are indistinct. Mildy oxidized low density lipoprotein (oxLDL) and monocytes (the primary source of PAFAH) are co-localized in early atheromas. Monocytes are activated by oxidized phospholipids (oxPL) in the oxLDL particle. We hypothesized that oxPL-activated monocytes are the source of increased levels of PAFAH in atherosclerosis. We found that PAFAH expression is significantly induced by OxPAPC and in particular long-chain fractions of oxPAPC in monocytes and cytokine-differentiated DC, but not cytokine-differentiated MO. Furthermore, spontaneously differentiated MO and DC from monocytes of non-periodontitis and aggressive periodontitis subjects, oxPAPC induced PAFAH in DC alone. 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphocholine (PEIPC) is a particularly bioactive component of long-chain oxPAPC fractions that binds the prostaglandin receptor subtypes DP1 and EP2. We revealed using selective agonists and antagonists of these receptors that DP1 and EP2 are required for the induction of PAFAH expression. OxPAPC stimulates IL-6 release from monocytes and this cytokine is required for oxPAPC-induced PAFAH expression. We next tested the hypothesis that oxPAPC did not induce PAFAH in MO because a key component of the signaling machinery was lacking. Flow cytometric and immunoblot analyses demonstrated that MO express very low levels of IL-6 receptor in comparison to DC and monocytes. Based on these observations, we propose that long-chain oxPL induce PAFAH expression by binding DP1 and/or EP2 and stimulating IL-6 production. These data strongly support the hypothesis that oxLDL-activated DC are the source of high PAFAH levels in atherosclerosis. Platelet activating factor (PAF) is the inflammatory phospholipids for which PAFAH is named. PAF has been shown by other investigators to induce the expression of PAFAH. In our physiologically relevant monocytes, PAF suppresses PAFAH transcription and expression. 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine (POVPC) is a short-chain oxPL that signals through the PAF receptor. Our preliminary data suggest that like PAF, POVPC suppresses PAFAH expression in monocytes. Further investigation into the effects of the short-chain oxPL are warranted. Our data support the hypothesies that oxPL-activated DC are the source of high PAFAH levels in atherosclerosis.

Os avanços tecnológicos vêm contribuindo cada vez mais nas áreas biológicas e científicas oferecendo ferramentas computacionais e sistemas específicos que em análise de dados in silico fornecem resultados rápidos e confiáveis. O presente projeto propõe o desenvolvimento de um portal na Internet para instalação e utilização de um banco de dados laboratoriais das principais serpentes brasileiras com seus respectivos venenos e antivenenos naturais, e a análise dos dados obtidos em ensaios farmacológicos, e bioquímicos. Utilizando a via de comunicação e interação mais simples atualmente, a Internet permite o compartilhamento de dados entre comunidades de pesquisadores, viabilizando recursos e tempo, além de permitir uma significante interação entre pesquisadores de todo o mundo, principalmente brasileira, na troca de informações e compartilhamento de dados. Dados elementares relacionados às serpentes foram armazenados no banco de dados, bem como as atividades tóxicas, farmacológicas e enzimáticas dos componentes dos venenos, e ainda, as aplicações biotecnológicas dos produtos que podem ser obtidos destes venenos, abrangendo ainda dados clínicos e valores estatísticos dos acidentes ofídicos. Aspectos bioquímicos dos ensaios realizados em laboratório permitiram a construção de uma ferramenta para análise comparativa de estruturas primárias de PLA2s, depositadas em bancos de dados internacionais. Além da interatividade entre pesquisadores, em Fóruns de Discussões, o sistema conta com listas dos principais artigos publicados em periódicos indexados, e devidamente atualizados periodicamente, com revisões bibliográficas. / Technological advances have been contributing, more and more, with biological and scientific areas, offering computational tools and specific systems which in silico data analysis supply reliable and fast results. The present project considers the development of an Internet portal for installation and use of laboratory data base for the main Brazilian serpents, with its respective venom and natural anti-venom, and the analysis of obtained data in pharmacological assays and biochemists. Using the easiest way of communication and interaction, the Internet allows sharing of data and information between communities of researchers around the world, especially for Brazilian researchers, making resources and time possible. Elementary data about serpents have been stored in the data base, as well as toxic, pharmacological and enzymatic activities of venom components, besides biotechnological applications of the products that can be obtained from these venom, enclosing clinical data and statistical values of ophidian accidents. Biochemists aspects of the assays carried through in laboratory allowed the construction of a comparative analysis tool for primary structures of PLA2s, deposited in international data bases. Beyond interactivity between researchers, in discussion forums, the system counts with lists of main articles published in indexed periodic, duly and constantly updated with bibliographical revisions.

O tratamento atual indicado para casos de envenenamentos por peçonhas é a administração intravenosa de antivenenos, produzidos através da hiperimunização de animais. Entretanto, os antivenenos disponíveis podem, algumas vezes, não proteger os pacientes e causar reações de hipersensibilidade. Fosfolipases A2 (PLA2), L-aminoácido oxidases (LAAO), metalo e serinoproteases são os principais componentes de peçonhas ofídicas e contribuem para a neurotoxicidade, hemorragia, hemólise, miotoxicidade, cardiotoxicidade e formação de edemas. Foram empregados ensaios para avaliar as atividades das enzimas presentes na peçonha de serpentes da espécie Lachesis muta rhombeata e aquele para atividade de protease foi otimizado. A tecnologia de Phage display foi empregada para a seleção de fagos-anticorpos capazes de reconhecer a peçonha bruta. Os fagos foram amplificados em Escherichia coli TG1 e usados para infectar E. coli HB2151, a qual produz fragmentos de anticorpos humanos solúveis. Estes foram purificados e utilizados em testes de inibição de alguns dos componentes tóxicos da peçonha. Os testes de atividade para PLA2, protease e Laminoácido oxidase foram padronizados com sucesso e as 3 proteínas mostraram elevada atividade enzimática. Após otimização, a quantidade de peçonha necessária para o ensaio de protease foi reduzida em 25 vezes. A massa molecular de PLA2 foi estimada em 17 kDa e as massas moleculares de proteases foram estimadas em 40, 35 e 24 kDa, através de zimogramas. O método de bio panning foi eficiente para a seleção de fagos-anticorpos contra a peçonha bruta. Diversos fragmentos de anticorpos foram purificados e incubados com a peçonha bruta para testar suas capacidades de neutralização sobre cada enzima. Cinco clones demonstraram-se hábeis em inibir a PLA2 através da inibição da hemólise. O clone 4E inibiu 100% da hemólise durante as duas horas de ensaio quando pré-incubado na proporção 2:1 (scFv:peçonha). Os clones 2C e 4E inibiram 100% durante uma hora quando pré-incubados na proporção 1:1 e os clones 2F e 9F inibiram a hemólise parcialmente. Outros testes serão conduzidos para a seleção de clones capazes de neutralizar as demais enzimas, os quais, juntamente com os clones já selecionados, serão analisados através de ensaios in vivo. Espera-se que eles possam contribuir para a construção de um novo antiveneno capaz de superar algumas das dificuldades associadas às técnicas de imunoterapia convencionais / The current treatment for animal envenoming is the intravenous administration of antivenoms, produced by animal hyperimmunization. Unfortunately, available antivenoms sometimes do not protect patients and may cause hypersensitivity reactions. Phospholipases A2 (PLA2), L-amino acid oxidases, metallo and serine proteases are considered the most important snake venom components and contribute to neurotoxicity, hemorrhage, hemolysis, myotoxicity, edematogeny and cardiotoxicity. Assays for evaluating the enzymes present in Lachesis muta rhombeata venom were developed and the protease one was optimized. Phage display technology was used to select phage antibodies able to recognize the crude venom. Phages were amplified in Escherichia coli TG1 and used to infect E. coli HB2151, which produces human antibody fragments. Inhibition tests aiming the neutralization of some toxic components of the venom were performed using purified antibody fragments. Activity assays for evaluating PLA2, protease and L-aminoacid oxidase were successfully performed and all enzymes showed high activity levels. The molecular mass of PLA2 was estimated in 17 kDa and the molecular mass of proteases were estimated in 40, 35 and 24 kDa, by zymography. After optimizing the conditions for proteolytic assay, it was possible to use 25 times less venom than it was necessary at first. The bio panning method was efficient for selecting specific phage antibodies against the crude venom. Several clones were selected to infect HB2151 and to produce soluble antibody fragments, which were purified and incubated with the venom to test their inhibition capacity over each enzyme. Five clones demonstrated ability to neutralize PLA2 by inhibiting hemolysis. The clone 4E could inhibit 100% of hemolysis for over 2 hours when preincubated at the ratio 2:1 (scFv:venom). Clones 2C and 4E could inhibit 100% for 1 hour when preincubated at the ratio 1:1 and clones 2F e 9F could inhibit partially. Other tests will be performed to select clones able to neutralize other enzymes and, together with the clones already selected, will be evaluated by in vivo experiments. It is expected that they may contribute to the construction of a potential new antivenom able to overcome some of the problems associated with conventional immunotherapy

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva que causa comprometimento cognitivo e demência. O diagnóstico é baseado em parâmetros clínicos, mas sua confirmação é post-mortem, após avaliação patológica durante a autópsia. Os tratamentos disponíveis para a DA são os inibidores da colinesterase (IChEs) e os antagonistas de receptores de N-metil-D-aspartato (NMDA), sendo que os IChEs compõe o principal grupo. Diversos estudos tem mostrado um efeito neuroprotetor dos IChEs, levando a alterações na patogênese da DA. Avaliar e mensurar essas alterações são papeis atribuídos aos biomarcadores. Neste sentido podemos destacar a fosfolipase A2 (PLA2), a principal responsável pelo metabolismo de fosfolípides de membrana, e que tem sido achada diminuída na DA, assim como a glicogênio sintase-quinase (GSK), responsável pela fosforilação da proteína Tau, que é um dos processos alterados na DA. O objetivo deste trabalho foi avaliar o efeito do tratamento com IChE sobre a atividade da PLA2 e expressão da GSK3B em plaquetas de 30 pacientes com DA após 3 e 6 meses de tratamento. Como grupo controle foram investigados 42 individuos idosos sem doença neurodegenerativa. Encontramos nos pacientes com DA antes do tratamento uma diminuição da atividade da iPLA2 quando comparada ao grupo controle. Após três e seis meses de tratamento a PLA2 aumentou, voltando ao nível dos controles. Os pacientes que apresentaram um aumento maior da iPLA2 apos 3 meses de tratamento apresentaram melhora cognitiva mais marcante após seis meses de tratamento, avaliado pelo CAMCOG. Apos 6 meses de tratamento encontramos um inativação da GSK3B, medida por um aumento em sua forma fosforilada. Nossos resultados sugerem que o donepezil apresenta propriedades modificadoras na doença de Alzheimer, e ainda que a medida da atividade da iPLA2 poderia ser usada como marcador de resposta terapêutica ao donepezil e, possivelmente, a outros IChEs, na doença de Alzheimer / Alzheimer\'s disease (AD) is a progressive neurodegenerative disorder that causes dementia and cognitive impairment. The Diagnosis is based on clinical parameters, but confirmation is post-mortem after pathologic evaluation during autopsy. The treatments available for AD are cholinesterase inhibitors (IChEs) and N-methyl-D-aspartate (NMDA) antagonists. The main group comprises the IChEs. Several studies have shown a neuroprotective effect of IChEs, leading to alterations in the pathogenesis of AD. Evaluate and measure these changes are assigned to biomarkers. In this regard we can highlight the phospholipase A2 (PLA2) the main enzyme in membrane phospholipids metabolism and that has been found decreased in AD as well as Glycogen Synthase kinase (GSK), a major responsible for tau phosphorylation which is one processes altered in AD. The objective of this study was to evaluate the effect of treatment with IChE on PLA2 activity and GSK3B expression in platelet of 30 AD patients after 3 and 6 months of treatment. The control group comprised 42 elderly individuals without neurodegenerative disease The results obtained were a decreased iPLA2 activity in patients with AD before treatment as compared to controls. After 3 and 6 months of treatment, we observed a significant increase in iPLA2 activity, restoring enzymatic activity similar to that observed among control. The patients who showed higher iPLA2 activity in the first three months were those showing cognitive improvement after six months of treatment, measured by CAMCOG. After 6 months of treatment a GSK3B inactivation were found, measured by an increase in its phosphorylated form. Our results suggest that donepezil present modifying properties in Alzheimer disease and that iPLA2 activity measurement could be used as a marker of therapeutic response to donepezil and possibly other IChEs in Alzheimer\'s disease

A Infecção pelo dengue afeta todas as regiões tropicais e subtropicais do globo, registra aproximadamente 390 milhões de casos por ano, e se destaca como um problema emergente de proporções crescentes, especialmente no Brasil, que atualmente responde por 60% dos casos no continente americano. Assim, ações para criar, adaptar e atender as condições para promover avanços na compreensão do processo de infecção pelo vírus em nível molecular pode ser de grande valor, tanto para enfrentar os desafios impostos pela conjuntura atual do dengue, quanto pela possibilidade de ampliar o conhecimento de mecanismos moleculares para outros vírus relacionados. Aqui, focando na proteína E do envelope do vírus do dengue, abordamos o problema da infecção viral sob os aspectos estruturais da proteína do envelope (E) quando enfrenta as condições ambientais distintas ao longo do caminho desde o trato digestivo do vetor exotérmico até a maquinaria celular do hospedeiro homeotérmico. Para este efeito, empregamos simulações de Dinâmica Molecular, avaliando e quantificando os processos de rearranjo conformacional do o domínio III isolado da proteína E dos quatro sorotipos de dengue, os quais são induzidos por alterações nos parâmetros termodinâmicos intensivos (pH 3, 5 e 7, T = 298K e 310K). Além disso, a configuração quaternária pentarradial de cinco domínios III é estudada, correlacionando flexibilidades específicas das regiões de loops a ajuste induzido a eventuais ligantes. Nós também verificamos a estabilidade estrutural do complexo DIII com o fragmento da região ligante de antígeno de um anticorpo monoclonal (Fab 1A1D-2), bem como as forças de interação entre a interface de ligação, identificando resíduos-chave. Além disso, os eventos relacionados com a interação da proteína E (tanto na forma monomérica quanto na dimérica) com o envelope lipídico viral são minuciosamente estudados, abrindo o caminho para um possível estudo centrado na formação de trímeros da proteína E, levando à fusão de membranas e subsequente inoculação do RNA viral no citoplasma. Finalmente, alternativas são propostas para a compreensão do mecanismo de ação da fosfolipase do veneno de Crotalus durissus terrificus - a cascavel sul-americana, que tem se mostrado eficaz na inibição in vitro da infecção pelo vírus do dengue. / Dengue infection burdens all tropical and subtropical regions of the globe, registers approximately 390 million cases annually, and stands out as an emerging problem of increasing proportions, especially in Brazil, which currently accounts for 60% of cases in the American continent. Thus, actions to create, adapt and meet conditions to promote advances in understanding the process of virus infection at the molecular level can be of great value both to meet the challenges posed by current conjuncture of dengue, and for the possibility to extend the knowledge of the molecular mechanisms to related viruses. Here, focusing on the E protein of the dengue virus envelope, we address the problem of viral infection under stereochemical aspects of the envelope (E) protein when it undergoes the distinct environmental conditions along the pathway from the exothermic vector\'s digestive tract to the homeothermic host\'s cell machinery. To this end, we employ molecular dynamics to assess and quantify the processes of conformational rearrangement of isolated domain III of the E protein of the four serotypes of dengue, as induced by changes on intensive thermodynamic parameters (pH 3, 5 and 7; T = 298K and 310K). Further, the 5-fold quaternary configuration of five domains III is studied, correlating specific loop regions flexibilities with induced fit with eventual ligands. We also check the structural stability and interaction forces between the interface of the complex DIII and the antigen binding fragment of a monoclonal antibody (Fab 1A1D-2), identifying key residues. Additionally events related to the interaction of the E protein (both as a monomer and as a dimer) with the viral membrane are thoroughly studied, paving the way for a possible study focused on the formation of trimers of E protein, leading to membrane fusion and subsequent inoculation of viral RNA in the cytoplasm. Finally, alternatives are proposed for understanding the mechanism of action of the phospholipase from the venom of Crotalus durissus terrificus - the South American rattlesnake, which has proven effective in vitro inhibition of Dengue virus infection.