Characterizing the Role of the Previously Undescribed Protein Caskin2 in Vascular Biology

Maintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. Defects in vascular homeostasis contribute to numerous disorders of significant clinical impact including hypertension and atherosclerosis. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence and that loss of Caskin2 in mice results in elevated blood pressure at baseline. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. When adenovirally expressed in vitro, Caskin2 inhibits EC proliferation and migration but promotes survival during hypoxia and nutrient deprivation. Likewise, loss of Caskin2 in vivo promotes increased vascular branching and permeability in mouse and zebrafish models. Caskin2 knockout mice are born in normal Mendelian ratios and appear grossly normal during early adulthood. However, they have consistently elevated systolic and diastolic blood pressure at baseline and significant context-dependent abnormalities in systemic metabolism (e.g., body weight, fat deposition, and glucose homeostasis). Although the precise molecular mechanisms of these effects remain unclear, we have shown that Caskin2 interacts with several proteins known to have important roles in endothelial biology and cardiovascular disease including the serine/threonine phosphatase PP1, the endothelial receptor Tie1, and eNOS, which is a critical regulator of vascular homeostasis. Ongoing work seeks to further characterize the functions of Caskin2 and its mechanisms of action with a focus on how Caskin2-mediated regulation of endothelial phenotype relates to its systemic effects on cardiovascular and metabolic function.

Augmentation sélective de l'expression protéique et de l'ARNm de la synthase du monoxyde d'azote dans les régions vulnérables du cerveau chez les rats déficients en thiamine

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

Mega-epibenthos at Bouvet Island (South Atlantic)

Mega-epibenthic diversity was analysed using a seabed photography at four stations off Bouvet Island and one station at the Spiess Seamount in the South Atlantic. Surprisingly, the intermediate-scale diversity within the area of investigation was not lower compared to that on the Patagonian shelf and only moderately lower than that on the Antarctic continental shelf. This result is incompatible with Mac Arthur and Wilson's Island Biogeography Theory describing species richness as a function of immigration of new species into an area and its extension. The relatively high species number and the very small extension of the Bouvet shelf compared to the much larger continental shelves of the other two areas can be explained by long-range dispersal of marine benthic animals in the Antarctic Circumpolar Current and high habitat heterogeneity. The observed uncoupling of intermediate-scale from large-scale background species diversity on the Antarctic shelf raises the question whether in these benthic systems an upper capacity limit for diversity exists.

Sedimentology of a Middle Jurassic Base-of-Slope environment, Cutri Formation, Mallorca

The Cutri Formation’s, type location, exposed in the NW of Mallorca, Spain has previously been described by Álvaro et al., (1989) and further interpreted by Abbots (1989) unpublished PhD thesis as a base-of-slope carbonate apron. Incorporating new field and laboratory analysis this paper enhances this interpretation. From this analysis, it can be shown without reasonable doubt that the Cutri Formation was deposited in a carbonate base-of-slope environment on the palaeowindward side of a Mid-Jurassic Tethyan platform. Key evidence such as laterally extensive exposures, abundant deposits of calciturbidtes and debris flows amongst hemipelagic deposits strongly support this interpretation.

Revisiting the Late Jurassic-Early Cretaceous of the NW South Iberian Basin: new ages and sedimentary environments

The study of the Upper Jurassic-Lower Cretaceous deposits (Higueruelas, Villar del Arzobispo and Aldea de Cortés Formations) of the South Iberian Basin (NW Valencia, Spain) reveals new stratigraphic and sedimentological data, which have significant implications on the stratigraphic framework, depositional environments and age of these units. The Higueruelas Fm was deposited in a mid-inner carbonate platform where oncolitic bars migrated by the action of storms and where oncoid production progressively decreased towards the uppermost part of the unit. The overlying Villar del Arzobispo Fm has been traditionally interpreted as an inner platform-lagoon evolving into a tidal-flat. Here it is interpreted as an inner-carbonate platform affected by storms, where oolitic shoals protected a lagoon, which had siliciclastic inputs from the continent. The Aldea de Cortés Fm has been previously interpreted as a lagoon surrounded by tidal-flats and fluvial-deltaic plains. Here it is reinterpreted as a coastal wetland where siliciclastic muddy deposits interacted with shallow fresh to marine water bodies, aeolian dunes and continental siliciclastic inputs. The contact between the Higueruelas and Villar del Arzobispo Fms, classically defined as gradual, is also interpreted here as rapid. More importantly, the contact between the Villar del Arzobispo and Aldea de Cortés Fms, previously considered as unconformable, is here interpreted as gradual. The presence of Alveosepta in the Villar del Arzobispo Fm suggests that at least part of this unit is Kimmeridgian, unlike the previously assigned Late Tithonian-Middle Berriasian age. Consequently, the underlying Higueruelas Fm, previously considered Tithonian, should not be younger than Kimmeridgian. Accordingly, sedimentation of the Aldea de Cortés Fm, previously considered Valangian-Hauterivian, probably started during the Tithonian and it may be considered part of the regressive trend of the Late Jurassic-Early Cretaceous cycle. This is consistent with the dinosaur faunas, typically Jurassic, described in the Villar del Arzobispo and Aldea de Cortés Fms.

El Índice Térmico Costero Peruano (ITCP)

El Ecosistema de Afloramiento Peruano (EAP) es una de las zonas marinas con mayor productividad pesquera en el mundo y por su ubicación geográfica, es afectada por procesos físicos remotos, principalmente por la variabilidad climática interanual proveniente del océano Pacifico Ecuatorial, cuya señal dominante es El Niño y la Oscilación Sur (ENOS). Con el fin de evaluar los efectos de ENOS frente al Perú, se desarrolló el Índice Térmico Costero Peruano (ITCP) que representa el 87,7% de la variación total de las anomalías de la temperatura superficial del mar del EAP. Se analizó el periodo 1982-2014, detectándose 12 periodos con condiciones cálidas y 16 con condiciones frías. El ITCP tuvo una tendencia lineal, un componente de bajas frecuencias y un componente de ruido, con 1,5%, 94,5 % y 4 % de contribución a la varianza total, respectivamente. El ITCP presenta ventajas respecto a otros índices climáticos de la costa peruana, porque comprende el área representativa del Ecosistema de Afloramiento Peruano y porque captura la señal del afloramiento costero así como de los efectos térmicos de El Niño y La Niña.

Changes of Soluble CD40 Ligand in the Progression of Acute Myocardial Infarction Associate to Endothelial Nitric Oxide Synthase Polymorphisms and Vascular Endothelial Growth Factor But Not to Platelet CD62P Expression

Reported in vitro data implicated soluble CD40 ligand (sCD40L) in endothelial dysfunction and angiogenesis. However, whether sCD40L could exert that influence in endothelial dysfunction and angiogenesis after injury in acute myocardial infarction (AMI) patients remains unclear. In the present study, we evaluated the association of sCD40L with markers of platelet activation, endothelial, and vascular function during a recovery period early after AMI. To achieve this goal, the time changes of soluble, platelet-bound, and microparticle-bound CD40L levels over 1 month were assessed in AMI patients and correlated with endothelial nitric oxide synthase (eNOS) polymorphisms, vascular endothelial growth factor (VEGF) concentrations, and platelet expression of P-selectin (CD62P). The association of soluble form, platelet-bound, and microparticle-bound CD40L with CD62P expression on platelets, a marker of platelet activation, was also assessed to evaluate the role of CD40L in the thrombosis, whereas the association with eNOS and VEGF was to evaluate the role of CD40L in vascular dysfunction. This work shows for the first time that time changes of sCD40L over 1 month after myocardial infarct onset were associated with G894T eNOS polymorphism and with the VEGF concentrations, but not to the platelet CD62P expression. These results indicate that, in terms of AMI pathophysiology, the sCD40L cannot be consider just as being involved in thrombosis and inflammation but also as having a relevant role in vascular and endothelial dysfunction.

Phenobarbital reduces EEG amplitude and propagation of neonatal seizures but does not alter performance of automated seizure detection

Objective: Phenobarbital increases electroclinical uncoupling and our preliminary observations suggest it may also affect electrographic seizure morphology. This may alter the performance of a novel seizure detection algorithm (SDA) developed by our group. The objectives of this study were to compare the morphology of seizures before and after phenobarbital administration in neonates and to determine the effect of any changes on automated seizure detection rates. Methods: The EEGs of 18 term neonates with seizures both pre- and post-phenobarbital (524 seizures) administration were studied. Ten features of seizures were manually quantified and summary measures for each neonate were statistically compared between pre- and post-phenobarbital seizures. SDA seizure detection rates were also compared. Results: Post-phenobarbital seizures showed significantly lower amplitude (p < 0.001) and involved fewer EEG channels at the peak of seizure (p < 0.05). No other features or SDA detection rates showed a statistical difference. Conclusion: These findings show that phenobarbital reduces both the amplitude and propagation of seizures which may help to explain electroclinical uncoupling of seizures. The seizure detection rate of the algorithm was unaffected by these changes. Significance: The results suggest that users should not need to adjust the SDA sensitivity threshold after phenobarbital administration.

Phosphomimetic Mutation of the N-Terminal Lid of MDM2 Enhances the Polyubiquitination of p53 through Stimulation of E2-Ubiquitin Thioester Hydrolysis.

Mouse double minute 2 (MDM2) has a phosphorylation site within a lid motif at Ser17 whose phosphomimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue shift in the λmax of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its ubiquitination function, thus uncoupling increases in p53 binding from its E3 ubiquitin ligase function. However, the Asp17mutation inMDM2 stimulated its discharge of the UBCH5a-ubiquitin thioester adduct (UBCH5a is a ubiquitin-conjugating enzyme E2D 1 UBC4/5 homolog yeast). This stimulation of ubiquitin discharge fromE2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2: (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction ofMDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein–protein interaction with the E2-ubiquitin complex in the absence of substrate p53 that, in turn, increases hydrolysis of theE2-ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction.

High glucose mediates prooxidant and antioxidant enzyme activities in coronary endothelial cells

Objective: Excess levels of free radicals such as nitric oxide (NO) and superoxide anion (O2-)are associated with the pathogenesis of endothelial cell dysfunction in diabetes mellitus. This study was designed to investigate the underlying causes of oxidative stress in coronary microvascular endothelial cells (CMEC) exposed to hyperglycaemia. Methods: CMEC were cultured under normal (5.5 mmol/L) or high glucose (22 mmol/L)concentrations for 7 days. The activity and expression (protein level) of eNOS, iNOS, NAD(P)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutahione peroxidase (GPx) were investigated by specific activity assays and Western analyses,respectively while the effects of hyperglycaemia on nitrite and O2 - generation were investigated by Griess reaction and cytochrome C reduction assay, respectively. Results: Hyperglycaemia did not alter eNOS or iNOS protein expressions and overall nitrite generation, an index of NO production. However, it significantly reduced the levels of intracellular antioxidant glutathione by 50% (p<0.05) and increased the protein expressions and/or activities of p22-phox, a membrane-bound component of pro-oxidant NAD(P)H oxidase and antioxidant enzymes (p<0.05). Free radical-scavengers, namely, Tiron and MPG (0.1-1 mol/L) reduced hyperglycaemia-induced antioxidant enzyme activity and increased glutathione and nitrite generation to the levels observed in CMEC cultured in normoglycaemic medium (p<0.01). The differences in enzyme activity and expressions were independent of the increased osmolarity generated by high glucose levels as investigated by using equimolar concentrations of mannitol in parallel experiments. Conclusions: These results suggest that hyperglycaemia-induced oxidative stress may arise in CMEC as a result of enhanced prooxidant enzyme activity and diminished generation of 3 antioxidant glutathione. By increasing the antioxidant enzyme capacity CMEC may protect themselves against free radical-induced cell damage in diabetic conditions. The definitive version is available at http://www.blackwell-synergy.com

Nitric oxide synthase and NAD(P)H oxidase modulate coronary endothelial cell growth

Reactive oxygen species (ROS) including nitric oxide (NO) and superoxide anion (O2-) are associated with cell migration, proliferation and many growth-related diseases. The objective of this study was to determine whether there was a reciprocal relationship between rat coronary microvascular endothelial cell (CMEC) growth and activity/expressions (mRNA and protein) of endothelial NO synthase (eNOS) and NAD(P)H oxidase enzymes. Proliferating namely, 50% confluent CMEC possessed approximately three-fold increased activity and expression of both enzymes compared to 100% confluent cells. Treatment of CMEC with an inhibitor of eNOS (L-NAME, 100M) increased cell proliferation as assessed via three independent methods i.e. cell counting, determination of total cellular protein levels and [3H]thymidine incorporation. Similarly, treatment of CMEC with pyrogallol (0.3-3 mM), a superoxide anion (O2-)- generator, also increased CMEC growth while spermine NONOate (SpNO), a NO donor, significantly reduced cell growth. Co-incubation of CMEC with a cell permeable superoxide dismutase mimetic (Mn-III-tetrakis-4-benzoic acid-porphyrin; MnTBAP) plus either pyrogallol or NO did not alter cell number and DNA synthesis thereby dismissing the involvement of peroxynitrite (OONO-) in CMEC proliferation. Specific inhibitors of NAD(P)H oxidase but not other ROS-generating enzymes including cyclooxygenase and xanthine oxidase, attenuated cell growth. Transfection of CMEC with antisense p22-phox cDNA, a membrane-bound component of NAD(P)H oxidase, resulted in substantial reduction in [3H]thymidine incorporation, total cellular protein levels and expression of p22-phox protein. These data demonstrate a cross-talk between CMEC growth and eNOS and NAD(P)H oxidase enzyme activity and expression, thus suggesting that the regulation of these enzymes may be critical in preventing the initiation and/or progression of coronary atherosclerosis.

Desacoplamento mitocondrial e estresse oxidativo em espermatozoides suínos resfriados

A espécie suína (Sus scropha domesticus) possui relevância nos âmbitos da pesquisa, da xenotransplantação e da produção de carnes. O resfriamento de sêmen é capaz de reduzir o metabolismo celular e possibilitar o armazenamento dos gametas, sendo auxiliar durante práticas de reprodução assistida. Contudo, os espermatozoides suínos são sensíveis ao estresse oxidativo gerado durante o processo de resfriamento. O 2,4 dinitrofenol (DNP) poderia gerar o desacoplamento mitocondrial reduzindo o estresse oxidativo e prolongando indiretamente a viabilidade e capacidade fertilizante de espermatozoides suínos resfriados pela diminuição de espécies reativas de oxigênio. O objetivo do presente estudo foi avaliar os efeitos do desacoplamento mitocondrial induzido pelo DNP e os efeitos desse desacoplamento sobre as de fluidez e integridade de membrana plasmática, funcionalidade de mitocôndria, motilidade espermática, além dos parâmetros de estresse oxidativo de lipoperoxidação e produção de espécies reativas de oxigênio, durante o resfriamento a 17 °C de 24 até 96 horas. Utilizou-se 22 ejaculados expostos ao diluente Betsville Thawing Solution (BTS) (controle) e ao mesmo diluente acrescido das concentrações de 0,01 µM (T1); 0,1 µM (T2); 1,0 µM (T3) e 10 µM (T4) de DNP. Através do teste de Shapiro-Wilk as variáveis que não apresentaram normalidade, tiveram suas médias comparadas pelo teste de KruskalWallis. As análises estatísticas demonstraram que o DNP não se diferiu do controle independente do tempo de armazenamento, para nenhuma das variáveis analisadas. Possivelmente, a falta de ação sobre as mitocôndrias, ou seja, a não promoção do desacoplamento mitocondrial foi o motivo principal para a falta dos efeitos do DNP. Acredita-se que a utilização do DNP em temperaturas mais elevadas por estas promoverem aumento da fluidez de membrana e/ou o aumento das concentrações de DNP poderiam gerar efeitos significativos sobre as mitocôndrias e demais variáveis analisadas. Finalmente, o DNP nas concentrações testadas não se diferiu do controle em todas as variáveis analisadas independente do tempo de armazenamento.

Adaptação do paradigma orientado a notificações para desenvolvimento de sistemas fuzzy

This work proposes to adjust the Notification Oriented Paradigm (NOP) so that it provides support to fuzzy concepts. NOP is inspired by elements of imperative and declarative paradigms, seeking to solve some of the drawbacks of both. By decomposing an application into a network of smaller computational entities that are executed only when necessary, NOP eliminates the need to perform unnecessary computations and helps to achieve better logical-causal uncoupling, facilitating code reuse and application distribution over multiple processors or machines. In addition, NOP allows to express the logical-causal knowledge at a high level of abstraction, through rules in IF-THEN format. Fuzzy systems, in turn, perform logical inferences on causal knowledge bases (IF-THEN rules) that can deal with problems involving uncertainty. Since PON uses IF-THEN rules in an alternative way, reducing redundant evaluations and providing better decoupling, this research has been carried out to identify, propose and evaluate the necessary changes to be made on NOP allowing to be used in the development of fuzzy systems. After that, two fully usable materializations were created: a C++ framework, and a complete programming language (LingPONFuzzy) that provide support to fuzzy inference systems. From there study cases have been created and several tests cases were conducted, in order to validate the proposed solution. The test results have shown a significant reduction in the number of rules evaluated in comparison to a fuzzy system developed using conventional tools (frameworks), which could represent an improvement in performance of the applications.

Efeito do GQ-02, derivado de tiazolidinadionas, sobre a aquisição de fenótipo termogênico pelo adipócito in vivo e em cultura de células

Dissertação (mestrado)—Universidade de Brasília, Faculdade em Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016.

El papel de la angiogénesis en la patogenia del actinomicetoma por nocardia brasiliensis

Objetivo: Investigar el papel de la angiogénesis durante el desarrollo del actinomicetoma y sobre el actinomicetoma establecido en un modelo experimental. Material y métodos: se determinó si el fenofibrato inhibe el crecimiento de la bacteria mediante la técnica de microdilución en placa. Se administró una suspensión de fenofibrato vía orogástrica cada 24 h durante el establecimiento del actinomicetoma y sobre el actinomicetoma establecido en ratones BALB/c infectados con la cepa N. brasiliensis HUJEG-1 (ATCC 700358). El tratamiento en ambos casos tuvo una duración de 45 días, tiempo en el que se evaluaron las diferencias en inflamación y la evolución clínica de las lesiones, empleando un vernier y la ecuación elipsoidea. También se realizó el análisis histológico en el sitio de la infección mediante tinciones con hematoxilina y eosina. El proceso de angiogénesis fue evaluado a diferentes tiempos en la administración del tratamiento mediante inmunohistoquímica, buscando como marcadores de vasos sanguíneos las moléculas VEGF, eNOS, y LYVE-1, este último como un marcador indirecto del proceso angiogénico. Resultados: se comprobó que el fenofibrato no inhibe el crecimiento de la bacteria a la concentración correspondiente a EC50 en ratones. Las lesiones características de la enfermedad se desarrollaron en un menor número de ratones que recibieron el tratamiento de fenofibrato, durante el establecimiento del actinomicetoma y sobre el actinomicetoma establecido. Además, se observó una disminución en las medidas de inflamación y en la aparición de lesiones histológicas, así como una disminución en el número de vasos sanguíneos en los sitios de la infección. Conclusiones: La angiogénesis contribuye a la patogenia del actinomicetoma por N. brasiliensis; la disminución de la formación de vasos sanguíneos por el tratamiento con fenofibrato limita el grado de la lesión.