A comparative study: long and short term effect of a nutrition sensitive approach to delay the progression of HIV to AIDS among people living with HIV (PLWH) in Nigeria

Background: Malnutrition has a negative impact on optimal immune function, thus increasing susceptibility to morbidity and mortality among HIV positive patients. Evidence indicates that the prevalence of macro and micronutrient deficiencies (particularly magnesium, selenium, zinc, and vitamin C) has a negative impact on optimal immune function, through the progressive depletion of CD4 T-lymphocyte cells, which thereby increases susceptibility to morbidity and mortality among PLWH. Objective: To assess the short and long term effects of a nutrition sensitive intervention to delay the progression of human immune-deficiency virus (HIV) to AIDS among people living with HIV in Abuja, Nigeria. Methods: A randomized control trial was carried out on 400 PLWH (adult, male and female of different religious background) in Nigeria between January and December 2012. Out of these 400 participants, 100 were randomly selected for the pilot study, which took place over six months (January to June, 2012). The participants in the pilot study overlapped to form part of the scale-up participants (n 400) monitored from June to December 2012. The comparative effect of daily 354.92 kcal/d optimized meals consumed for six and twelve months was ascertained through the nutritional status and biochemical indices of the study participants (n=100 pilot interventions), who were and were not taking the intervention meal. The meal consisted of: Glycine max 50g (Soya bean); Pennisetum americanum 20g (Millet); Moringa oleifera 15g (Moringa); Daucus carota spp. sativa 15g (Carrot). Results: At the end of sixth month intervention, mean CD4 cell count (cell/mm3) for Pre-ART and ART Test groups increased by 6.31% and 12.12% respectively. Mean mid upper arm circumference (MUAC) for Pre-ART and ART Test groups increased by 2.72% and 2.52% within the same period (n 400). Comparatively, participants who overlapped from pilot to scale-up intervention (long term use, n 100) were assessed for 12 months. Mean CD4 cell count (cell/mm3) for Pre-ART and ART test groups increased by 2.21% and 12.14%. Mean MUAC for Pre-ART and ART test groups increased by 2.08% and 3.95% respectively. Moreover, student’s t-test analysis suggests a strong association between the intervention meal, MUAC, and CD4 count on long term use of optimized meal in the group of participants being treated with antiretroviral therapy (ART) (P<0.05). Conclusion: Although the achieved results take the form of specific technology, it suggests that a prolong consumption of the intervention meal will be suitable to sustain the gained improvements in the anthropometric and biochemical indices of PLWHIV in Nigeria.

Projecting Changes in the Distribution and Productivity of Living Marine Resources: A Critical Review of the Suite of Modeling Approaches used in the Large European Project VECTORS

We review and compare four broad categories of spatially-explicit modelling approaches currently used to understand and project changes in the distribution and productivity of living marine resources including: 1) statistical species distribution models, 2) physiology-based, biophysical models of single life stages or the whole life cycle of species, 3) food web models, and 4) end-to-end models. Single pressures are rare and, in the future, models must be able to examine multiple factors affecting living marine resources such as interactions between: i) climate-driven changes in temperature regimes and acidification, ii) reductions in water quality due to eutrophication, iii) the introduction of alien invasive species, and/or iv) (over-)exploitation by fisheries. Statistical (correlative) approaches can be used to detect historical patterns which may not be relevant in the future. Advancing predictive capacity of changes in distribution and productivity of living marine resources requires explicit modelling of biological and physical mechanisms. New formulations are needed which (depending on the question) will need to strive for more realism in ecophysiology and behaviour of individuals, life history strategies of species, as well as trophodynamic interactions occurring at different spatial scales. Coupling existing models (e.g. physical, biological, economic) is one avenue that has proven successful. However, fundamental advancements are needed to address key issues such as the adaptive capacity of species/groups and ecosystems. The continued development of end-to-end models (e.g., physics to fish to human sectors) will be critical if we hope to assess how multiple pressures may interact to cause changes in living marine resources including the ecological and economic costs and trade-offs of different spatial management strategies. Given the strengths and weaknesses of the various types of models reviewed here, confidence in projections of changes in the distribution and productivity of living marine resources will be increased by assessing model structural uncertainty through biological ensemble modelling.

Projecting Changes in the Distribution and Productivity of Living Marine Resources: A Critical Review of the Suite of Modeling Approaches used in the Large European Project VECTORS

We review and compare four broad categories of spatially-explicit modelling approaches currently used to understand and project changes in the distribution and productivity of living marine resources including: 1) statistical species distribution models, 2) physiology-based, biophysical models of single life stages or the whole life cycle of species, 3) food web models, and 4) end-to-end models. Single pressures are rare and, in the future, models must be able to examine multiple factors affecting living marine resources such as interactions between: i) climate-driven changes in temperature regimes and acidification, ii) reductions in water quality due to eutrophication, iii) the introduction of alien invasive species, and/or iv) (over-)exploitation by fisheries. Statistical (correlative) approaches can be used to detect historical patterns which may not be relevant in the future. Advancing predictive capacity of changes in distribution and productivity of living marine resources requires explicit modelling of biological and physical mechanisms. New formulations are needed which (depending on the question) will need to strive for more realism in ecophysiology and behaviour of individuals, life history strategies of species, as well as trophodynamic interactions occurring at different spatial scales. Coupling existing models (e.g. physical, biological, economic) is one avenue that has proven successful. However, fundamental advancements are needed to address key issues such as the adaptive capacity of species/groups and ecosystems. The continued development of end-to-end models (e.g., physics to fish to human sectors) will be critical if we hope to assess how multiple pressures may interact to cause changes in living marine resources including the ecological and economic costs and trade-offs of different spatial management strategies. Given the strengths and weaknesses of the various types of models reviewed here, confidence in projections of changes in the distribution and productivity of living marine resources will be increased by assessing model structural uncertainty through biological ensemble modelling.

The regulatory role of eNOS-derived nitric oxide on transcription in endothelial cells: Impact of S-nitrosylation on β-catenin signaling

Les cellules endothéliales forment une couche semi-perméable entre le sang et les organes. La prolifération, la migration et la polarisation des cellules endothéliales sont essentielles à la formation de nouveaux vaisseaux à partir de vaisseaux préexistants, soit l’angiogenèse. Le facteur de croissance de l’endothélium vasculaire (VEGF) peut activer la synthase endothéliale du monoxyde d’azote (eNOS) et induire la production de monoxyde d’azote (NO) nécessaire pour la régulation de la perméabilité vasculaire et l’angiogenèse. β- caténine est une composante essentielle du complexe des jonctions d’ancrage ainsi qu’un régulateur majeur de la voie de signalisation de Wnt/β-caténine dans laquelle elle se joint au facteur de transcription TCF/LEF et module l’expression de nombreux gènes, dont certains sont impliqués dans l’angiogenèse. La S-nitrosylation (SNO) est un mécanisme de régulation posttraductionnel des protéines par l’ajout d’un groupement nitroso au niveau de résidus cystéines. Le NO produit par eNOS peut induire la S-nitrosylation de la β−caténine au niveau des jonctions intercellulaires et moduler la perméabilité de l’endothélium. Il a d’ailleurs été montré que le NO peut contrôler l’expression génique par la transcription. Le but de cette thèse est d’établir le rôle du NO au sein de la transcription des cellules endothéliales, spécifiquement au niveau de l’activité de β-caténine. Le premier objectif était de déterminer si la SNO de la β-caténine affecte son activité transcriptionnelle. Nous avons montré que le NO inhibe l’activité transcriptionnelle de β- caténine ainsi que la prolifération des cellules endothéliales induites par l’activation de la voie Wnt/β-caténine. Il est intéressant de constater que le VEGF, qui induit la production de NO via eNOS, réprime l’expression de AXIN2 qui est un gène cible de Wnt s’exprimant suite à la i i stimulation par Wnt3a et ce, dépendamment de eNOS. Nous avons identifié que la cystéine 466 de la β-caténine est un résidu essentiel à la modulation répressive de son activité transcriptionnelle par le NO. Lorsqu’il est nitrosylé, ce résidu est responsable de la perturbation du complexe de transcription formé de β-caténine et TCF-4 ce qui inhibe la prolifération des cellules endothéliales induite par la stimulation par Wnt3a. Puisque le NO affecte la transcription, nous avons réalisé l’analyse du transcriptome afin d’obtenir une vue d’ensemble du rôle du NO dans l’activité transcriptionnelle des cellules endothéliales. L’analyse différentielle de l’expression des gènes de cellules endothéliales montre que la répression de eNOS par siRNA augmente l’expression de gènes impliqués au niveau de la polarisation tels que : PARD3A, PARD3B, PKCZ, CRB1 et TJ3. Cette analyse suggère que le NO peut réguler la polarisation des cellules et a permis d’identifier des gènes responsables de l’intégrité des cellules endothéliales et de la réponse immunitaire. De plus, l’analyse de voies de signalisation par KEGG montre que certains gènes modulés par l’ablation de eNOS sont enrichis dans de nombreuses voies de signalisation, notamment Ras et Notch qui sont importantes lors de la migration cellulaire et la différenciation des cellules de têtes et de tronc (tip/stalk). Le regroupement des gènes exprimés chez les cellules traitées au VEGF (déplétées de eNOS ou non) révèle que le NO peut affecter l’expression de gènes contribuant au processus angiogénique, dont l’attraction chimiotactique. Notre étude montre que le NO module la transcription des cellules endothéliales et régule l’expression des gènes impliqués dans l’angiogenèse et la fonction endothéliale.

Regenerative Cues of Myeloid Cells on Pancreatic Epithelium

Thesis (Ph.D.)--University of Washington, 2016-07

Shapes and Dynamics of Blood Cells in Poiseuille and Shear Flows

The dynamics, shape, deformation, and orientation of red blood cells in microcirculation affect the rheology, flow resistance and transport properties of whole blood. This leads to important correlations of cellular and continuum scales. Furthermore, the dynamics of RBCs subject to different flow conditions and vessel geometries is relevant for both fundamental research and biomedical applications (e.g drug delivery). In this thesis, the behaviour of RBCs is investigated for different flow conditions via computer simulations. We use a combination of two mesoscopic particle-based simulation techniques, dissipative particle dynamics and smoothed dissipative particle dynamics. We focus on the microcapillary scale of several μm. At this scale, blood cannot be considered at the continuum but has to be studied at the cellular level. The connection between cellular motion and overall blood rheology will be investigated. Red blood cells are modelled as viscoelastic objects interacting hydrodynamically with a viscous fluid environment. The properties of the membrane, such as resistance against bending or shearing, are set to correspond to experimental values. Furthermore, thermal fluctuations are considered via random forces. Analyses corresponding to light scattering measurements are performed in order to compare to experiments and suggest for which situations this method is suitable. Static light scattering by red blood cells characterises their shape and allows comparison to objects such as spheres or cylinders, whose scattering signals have analytical solutions, in contrast to those of red blood cells. Dynamic light scattering by red blood cells is studied concerning its suitability to detect and analyse motion, deformation and membrane fluctuations. Dynamic light scattering analysis is performed for both diffusing and flowing cells. We find that scattering signals depend on various cell properties, thus allowing to distinguish different cells. The scattering of diffusing cells allows to draw conclusions on their bending rigidity via the effective diffusion coefficient. The scattering of flowing cells allows to draw conclusions on the shear rate via the scattering amplitude correlation. In flow, a RBC shows different shapes and dynamic states, depending on conditions such as confinement, physiological/pathological state and cell age. Here, two essential flow conditions are studied: simple shear flow and tube flow. Simple shear flow as a basic flow condition is part of any more complex flow. The velocity profile is linear and shear stress is homogeneous. In simple shear flow, we find a sequence of different cell shapes by increasing the shear rate. With increasing shear rate, we find rolling cells with cup shapes, trilobe shapes and quadrulobe shapes. This agrees with recent experiments. Furthermore, the impact of the initial orientation on the dynamics is studied. To study crowding and collective effects, systems with higher haematocrit are set up. Tube flow is an idealised model for the flow through cylindric microvessels. Without cell, a parabolic flow profile prevails. A single red blood cell is placed into the tube and subject to a Poiseuille profile. In tube flow, we find different cell shapes and dynamics depending on confinement, shear rate and cell properties. For strong confinements and high shear rates, we find parachute-like shapes. Although not perfectly symmetric, they are adjusted to the flow profile and maintain a stationary shape and orientation. For weak confinements and low shear rates, we find tumbling slippers that rotate and moderately change their shape. For weak confinements and high shear rates, we find tank-treading slippers that oscillate in a limited range of inclination angles and strongly change their shape. For the lowest shear rates, we find cells performing a snaking motion. Due to cell properties and resultant deformations, all shapes differ from hitherto descriptions, such as steady tank-treading or symmetric parachutes. We introduce phase diagrams to identify flow regimes for the different shapes and dynamics. Changing cell properties, the regime borders in the phase diagrams change. In both flow types, both the viscosity contrast and the choice of stress-free shape are important. For in vitro experiments, the solvent viscosity has often been higher than the cytosol viscosity, leading to a different pattern of dynamics, such as steady tank-treading. The stress-free state of a RBC, which is the state at zero shear stress, is still controversial, and computer simulations enable direct comparisons of possible candidates in equivalent flow conditions.

Image-Guided Precision Manipulation of Cells and Nanoparticles in Microfluidics

Manipulation of single cells and particles is important to biology and nanotechnology. Our electrokinetic (EK) tweezers manipulate objects in simple microfluidic devices using gentle fluid and electric forces under vision-based feedback control. In this dissertation, I detail a user-friendly implementation of EK tweezers that allows users to select, position, and assemble cells and nanoparticles. This EK system was used to measure attachment forces between living breast cancer cells, trap single quantum dots with 45 nm accuracy, build nanophotonic circuits, and scan optical properties of nanowires. With a novel multi-layer microfluidic device, EK was also used to guide single microspheres along complex 3D trajectories. The schemes, software, and methods developed here can be used in many settings to precisely manipulate most visible objects, assemble objects into useful structures, and improve the function of lab-on-a-chip microfluidic systems.

Adherence to antiretroviral therapy and its determinants among persons living with HIV/AIDS in Bayelsa state, Nigeria

Background: A high level of adherence is required to achieve the desired outcomes of antiretroviral therapy. There is paucity of information about adherence to combined antiretroviral therapy in Bayelsa State of southern Nigeria. Objectives: The objectives of the study were to determine the level of adherence to combined antiretroviral therapy among the patients, evaluate the improvement in their immune status and identify reasons for sub-optimal adherence to therapy. Methods: The cross-sectional study involved administration of an adapted and pretested questionnaire to 601 consented patients attending the two tertiary health institutions in Bayesla State, Nigeria: The Federal Medical Centre, Yenagoa and the Niger-Delta University Teaching Hospital Okolobiri. The tool was divided into various sections such as socio-demographic data, HIV knowledge and adherence to combined antiretroviral therapy. Information on the patient's CD4+ T cells count was retrieved from their medical records. Adherence was assessed by asking patients to recall their intake of prescribed doses in the last fourteen days and subjects who had 95-100% of the prescribed antiretroviral drugs were considered adherent. Results: Three hundred and forty eight (57.9%) of the subjects were females and 253 (42.1%) were males. The majority of them, 557 (92.7%) have good knowledge of HIV and combined anti-retroviral therapy with a score of 70.0% and above. A larger proportion of the respondents, 441 (73.4%), had > 95% adherence. Some of the most important reasons giving for missing doses include, “simply forgot” 147 (24.5%), and “wanted to avoid the side-effects of drugs” 33(5.5%). There were remarkable improvements in the immune status of the subjects with an increment in the proportion of the subjects with CD4+ T cells count of greater than 350 cells/mm3 from 33 (5.5%) at therapy initiation to 338 (56.3%) at study period (p<0.0001). Conclusion: The adherence level of 73.4% was low which calls for intervention and improvement. The combined antiretroviral therapy has significantly improved the immune status of the majority of patients which must be sustained. “Simply forgot” was the most important reason for missing doses.

Dendritic Cells for Anomaly Detection

Artificial immune systems, more specifically the negative selection algorithm, have previously been applied to intrusion detection. The aim of this research is to develop an intrusion detection system based on a novel concept in immunology, the Danger Theory. Dendritic Cells (DCs) are antigen presenting cells and key to the activation of the human immune system. DCs perform the vital role of combining signals from the host tissue and correlate these signals with proteins known as antigens. In algorithmic terms, individual DCs perform multi-sensor data fusion based on time-windows. The whole population of DCs asynchronously correlates the fused signals with a secondary data stream. The behaviour of human DCs is abstracted to form the DC Algorithm (DCA), which is implemented using an immune inspired framework, libtissue. This system is used to detect context switching for a basic machine learning dataset and to detect outgoing portscans in real-time. Experimental results show a significant difference between an outgoing portscan and normal traffic.

Nitrate metabolism in the dinoflagellate Lingulodinium polyedrum

Les dinoflagellés sont des eucaryotes unicellulaires retrouvés dans la plupart des écosystèmes aquatiques du globe. Ces organismes amènent une contribution substantielle à la production primaire des océans, soit en tant que membre du phytoplancton, soit en tant que symbiontes des anthozoaires formant les récifs coralliens. Malheureusement, ce rôle écologique majeur est souvent négligé face à la capacité de certaines espèces de dinoflagellés à former des fleurs d'eau, parfois d'étendue et de durée spectaculaires. Ces floraisons d'algues, communément appelées "marées rouges", peuvent avoir de graves conséquences sur les écosystèmes côtiers, sur les industries de la pêche et du tourisme, ainsi que sur la santé humaine. Un des facteurs souvent corrélé avec la formation des fleurs d'eau est une augmentation dans la concentration de nutriments, notamment l’azote et le phosphore. Le nitrate est un des composants principaux retrouvés dans les eaux de ruissellement agricoles, mais également la forme d'azote bioaccessible la plus abondante dans les écosystèmes marins. Ainsi, l'agriculture humaine a contribué à magnifier significativement les problèmes associés aux marées rouges au niveau mondial. Cependant, la pollution ne peut pas expliquer à elle seule la formation et la persistance des fleurs d'eau, qui impliquent plusieurs facteurs biotiques et abiotiques. Il est particulièrement difficile d'évaluer l'importance relative qu'ont les ajouts de nitrate par rapport à ces autres facteurs, parce que le métabolisme du nitrate chez les dinoflagellés est largement méconnu. Le but principal de cette thèse vise à remédier à cette lacune. J'ai choisi Lingulodinium polyedrum comme modèle pour l'étude du métabolisme du nitrate, parce que ce dinoflagellé est facilement cultivable en laboratoire et qu'une étude transcriptomique a récemment fourni une liste de gènes pratiquement complète pour cette espèce. Il est également intéressant que certaines composantes moléculaires de la voie du nitrate chez cet organisme soient sous contrôle circadien. Ainsi, dans ce projet, j'ai utilisé des analyses physiologiques, biochimiques, transcriptomiques et bioinformatiques pour enrichir nos connaissances sur le métabolisme du nitrate des dinoflagellés et nous permettre de mieux apprécier le rôle de l'horloge circadienne dans la régulation de cette importante voie métabolique primaire. Je me suis tout d'abord penché sur les cas particuliers où des floraisons de dinoflagellés sont observées dans des conditions de carence en azote. Cette idée peut sembler contreintuitive, parce que l'ajout de nitrate plutôt que son épuisement dans le milieu est généralement associé aux floraisons d'algues. Cependant, j’ai découvert que lorsque du nitrate était ajouté à des cultures initialement carencées ou enrichies en azote, celles qui s'étaient acclimatées au stress d'azote arrivaient à survivre près de deux mois à haute densité cellulaire, alors que les cellules qui n'étaient pas acclimatées mourraient après deux semaines. En condition de carence d'azote sévère, les cellules arrivaient à survivre un peu plus de deux semaines et ce, en arrêtant leur cycle cellulaire et en diminuant leur activité photosynthétique. L’incapacité pour ces cellules carencées à synthétiser de nouveaux acides aminés dans un contexte où la photosynthèse était toujours active a mené à l’accumulation de carbone réduit sous forme de granules d’amidon et corps lipidiques. Curieusement, ces deux réserves de carbone se trouvaient à des pôles opposés de la cellule, suggérant un rôle fonctionnel à cette polarisation. La deuxième contribution de ma thèse fut d’identifier et de caractériser les premiers transporteurs de nitrate chez les dinoflagellés. J'ai découvert que Lingulodinium ne possédait que très peu de transporteurs comparativement à ce qui est observé chez les plantes et j'ai suggéré que seuls les membres de la famille des transporteurs de nitrate de haute affinité 2 (NRT2) étaient réellement impliqués dans le transport du nitrate. Le principal transporteur chez Lingulodinium était exprimé constitutivement, suggérant que l’acquisition du nitrate chez ce dinoflagellé se fondait majoritairement sur un système constitutif plutôt qu’inductible. Enfin, j'ai démontré que l'acquisition du nitrate chez Lingulodinium était régulée par la lumière et non par l'horloge circadienne, tel qu'il avait été proposé dans une étude antérieure. Finalement, j’ai utilisé une approche RNA-seq pour vérifier si certains transcrits de composantes impliquées dans le métabolisme du nitrate de Lingulodinium étaient sous contrôle circadien. Non seulement ai-je découvert qu’il n’y avait aucune variation journalière dans les niveaux des transcrits impliqués dans le métabolisme du nitrate, j’ai aussi constaté qu’il n’y avait aucune variation journalière pour n’importe quel ARN du transcriptome de Lingulodinium. Cette découverte a démontré que l’horloge de ce dinoflagellé n'avait pas besoin de transcription rythmique pour générer des rythmes physiologiques comme observé chez les autres eukaryotes.

The regulatory role of eNOS-derived nitric oxide on transcription in endothelial cells: Impact of S-nitrosylation on β-catenin signaling

Les cellules endothéliales forment une couche semi-perméable entre le sang et les organes. La prolifération, la migration et la polarisation des cellules endothéliales sont essentielles à la formation de nouveaux vaisseaux à partir de vaisseaux préexistants, soit l’angiogenèse. Le facteur de croissance de l’endothélium vasculaire (VEGF) peut activer la synthase endothéliale du monoxyde d’azote (eNOS) et induire la production de monoxyde d’azote (NO) nécessaire pour la régulation de la perméabilité vasculaire et l’angiogenèse. β- caténine est une composante essentielle du complexe des jonctions d’ancrage ainsi qu’un régulateur majeur de la voie de signalisation de Wnt/β-caténine dans laquelle elle se joint au facteur de transcription TCF/LEF et module l’expression de nombreux gènes, dont certains sont impliqués dans l’angiogenèse. La S-nitrosylation (SNO) est un mécanisme de régulation posttraductionnel des protéines par l’ajout d’un groupement nitroso au niveau de résidus cystéines. Le NO produit par eNOS peut induire la S-nitrosylation de la β−caténine au niveau des jonctions intercellulaires et moduler la perméabilité de l’endothélium. Il a d’ailleurs été montré que le NO peut contrôler l’expression génique par la transcription. Le but de cette thèse est d’établir le rôle du NO au sein de la transcription des cellules endothéliales, spécifiquement au niveau de l’activité de β-caténine. Le premier objectif était de déterminer si la SNO de la β-caténine affecte son activité transcriptionnelle. Nous avons montré que le NO inhibe l’activité transcriptionnelle de β- caténine ainsi que la prolifération des cellules endothéliales induites par l’activation de la voie Wnt/β-caténine. Il est intéressant de constater que le VEGF, qui induit la production de NO via eNOS, réprime l’expression de AXIN2 qui est un gène cible de Wnt s’exprimant suite à la i i stimulation par Wnt3a et ce, dépendamment de eNOS. Nous avons identifié que la cystéine 466 de la β-caténine est un résidu essentiel à la modulation répressive de son activité transcriptionnelle par le NO. Lorsqu’il est nitrosylé, ce résidu est responsable de la perturbation du complexe de transcription formé de β-caténine et TCF-4 ce qui inhibe la prolifération des cellules endothéliales induite par la stimulation par Wnt3a. Puisque le NO affecte la transcription, nous avons réalisé l’analyse du transcriptome afin d’obtenir une vue d’ensemble du rôle du NO dans l’activité transcriptionnelle des cellules endothéliales. L’analyse différentielle de l’expression des gènes de cellules endothéliales montre que la répression de eNOS par siRNA augmente l’expression de gènes impliqués au niveau de la polarisation tels que : PARD3A, PARD3B, PKCZ, CRB1 et TJ3. Cette analyse suggère que le NO peut réguler la polarisation des cellules et a permis d’identifier des gènes responsables de l’intégrité des cellules endothéliales et de la réponse immunitaire. De plus, l’analyse de voies de signalisation par KEGG montre que certains gènes modulés par l’ablation de eNOS sont enrichis dans de nombreuses voies de signalisation, notamment Ras et Notch qui sont importantes lors de la migration cellulaire et la différenciation des cellules de têtes et de tronc (tip/stalk). Le regroupement des gènes exprimés chez les cellules traitées au VEGF (déplétées de eNOS ou non) révèle que le NO peut affecter l’expression de gènes contribuant au processus angiogénique, dont l’attraction chimiotactique. Notre étude montre que le NO module la transcription des cellules endothéliales et régule l’expression des gènes impliqués dans l’angiogenèse et la fonction endothéliale.

A Catalytically Inactive Lys49 PLA2 Isoform from Bothrops jararacussu venom that Stimulates Insulin Secretion in Pancreatic Beta Cells

A new secretory phospholipase A2 (sPLA2) isoform from Bothrops jararacussu venom (BjVIII) has been characterized by causing platelet aggregation, an absent activity in BthTx-I, Prtx-I and PrTx-II sPLA2s. According to our results, BjVIII also enhances insulin release by the pancreatic beta cells. The complete amino acid sequence of the new isoform was determined by Edman degradation and de novo peptide sequencing. These analyses showed a G35K amino acid modification for BjVIII in comparison with BthTx-I, PrTx-I and Prtx-II, a structural difference that has been related to the conflicting biological activities among BjVIII and other Lys49 sPLA2s. The whole set of evidences collected in this work indicates that, besides the C-terminal region and B-wing of PLA2, the calcium binding loop in BjVIII should be considered as an important region, involved in the pharmacological effects of Lys49-sPLA2 isoforms from the Bothrops genus.

Inherited Photoreceptor Degeneration Causes the Death of Melanopsin-Positive Retinal Ganglion Cells and Increases Their Coexpression of Brn3a

Purpose: To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in P23H-1 rats, a rat model of inherited photoreceptor degeneration. Methods: At postnatal (P) times P30, P365, and P540, retinas from P23H dystrophic rats (line 1, rapid degeneration; and line 3, slow degeneration) and Sprague Dawley (SD) rats (control) were dissected as whole-mounts and immunodetected for melanopsin and/or Brn3a. The dendritic arborization of m+RGCs and the numbers of Brn3a+RGCs and m+RGCs were quantified and their retinal distribution and coexpression analyzed. Results: In SD rats, aging did not affect the population of Brn3a+RGCs or m+RGCs or the percentage that showed coexpression (0.27%). Young P23H-1 rats had a significantly lower number of Brn3a+RGCs and showed a further decline with age. The population of m+RGCs in young P23H-1 rats was similar to that found in SD rats and decreased by 22.6% and 28.2% at P365 and P540, respectively, similarly to the decrease of the Brn3a+RGCs. At these ages the m+RGCs showed a decrease of their dendritic arborization parameters, which was similar in both the P23H-1 and P23H-3 lines. The percentage of coexpression of Brn3a was, however, already significantly higher at P30 (3.31%) and increased significantly with age (10.65% at P540). Conclusions: Inherited photoreceptor degeneration was followed by secondary loss of Brn3a+RGCs and m+RGCs. Surviving m+RGCs showed decreased dendritic arborization parameters and increased coexpression of Brn3a and melanopsin, phenotypic and molecular changes that may represent an effort to resist degeneration and/or preferential survival of m+RGCs capable of synthesizing Brn3a.

Antitumor effect and mechanism of action of polysaccharides extracted from Polygonum perfoliatum L whole plant in human lung carcinoma A549 cell line

Purpose: To optimize the extraction conditions of polysaccharides from Polygonum perfoliatum L. (PSDP) and to evaluate their anti-tumor activities on A549 cell line. Methods: Extraction of PSDP was optimized using Box-Behnken design (BBD). Three factors of response surface methodology (RSM) including extraction time, ratio of water to raw material and number of extractions were employed to optimize the yield of PSDP. The cytotoxic effect of PSDP on human lung carcinoma A549 cell line was evaluated in vivo, while its effects on expressions of caspase3, caspase-9, Bcl-2 and Bax were determined by western blot assay. Result: BBD was significant and applicable to PSDP extraction. Based on the contour plots, response surface plots and variance analysis, it predicted that the optimum conditions for PSDP extraction were: 1.58 h (extraction time); 30.18 mL/g (ratio of water to raw material); and 2.02 (number of extractions). PSDP had significant inhibitory effect on the growth of A549 cells in a concentration- and timedependent manner (p < 0.05). After treatment with PSDP, caspase-3, caspase-9 and Bax were significantly up-regulated (p < 0.05), whereas Bcl-2 was down-regulated, all concentration-dependently. Conclusion: RSM analysis is an appropriate method to optimize PSDP extraction. The results also indicate that PSDP has significant anti-tumor effect against A549 cells, most likely via inducing mitochondria-mediated apoptosis.

Automatic Counting of Microglial Cells in Healthy and Glaucomatous Mouse Retinas

Proliferation of microglial cells has been considered a sign of glial activation and a hallmark of ongoing neurodegenerative diseases. Microglia activation is analyzed in animal models of different eye diseases. Numerous retinal samples are required for each of these studies to obtain relevant data of statistical significance. Because manual quantification of microglial cells is time consuming, the aim of this study was develop an algorithm for automatic identification of retinal microglia. Two groups of adult male Swiss mice were used: age-matched controls (naïve, n = 6) and mice subjected to unilateral laser-induced ocular hypertension (lasered; n = 9). In the latter group, both hypertensive eyes and contralateral untreated retinas were analyzed. Retinal whole mounts were immunostained with anti Iba-1 for detecting microglial cell populations. A new algorithm was developed in MATLAB for microglial quantification; it enabled the quantification of microglial cells in the inner and outer plexiform layers and evaluates the area of the retina occupied by Iba-1+ microglia in the nerve fiber-ganglion cell layer. The automatic method was applied to a set of 6,000 images. To validate the algorithm, mouse retinas were evaluated both manually and computationally; the program correctly assessed the number of cells (Pearson correlation R = 0.94 and R = 0.98 for the inner and outer plexiform layers respectively). Statistically significant differences in glial cell number were found between naïve, lasered eyes and contralateral eyes (P<0.05, naïve versus contralateral eyes; P<0.001, naïve versus lasered eyes and contralateral versus lasered eyes). The algorithm developed is a reliable and fast tool that can evaluate the number of microglial cells in naïve mouse retinas and in retinas exhibiting proliferation. The implementation of this new automatic method can enable faster quantification of microglial cells in retinal pathologies.