Malaria is a serious disease that can lead to fatal consequences if not treated. It is mainly spread via Plasmodium falciparum, a parasite carried by mosquitoes as host organisms. As a potential way of treating malaria, research is being done on possible inhibitors of calmodulin (CaM) in the parasite. CaM is a highly conserved protein found in all eukaryotes, and is important in many essential biochemical reactions. The potential inhibitor analysed in this study is calmidazolium (CZM). This study aims to characterise structure and stability differences between the C-lobes of human and P. falciparum CaM, while analysing the effect of the presence of CZM.  Previous studies have proven that CZM acts as an inhibitor to human CaM by binding to the C-lobe, with a dissociation constant in the nano molar range. In other studies, thermal stability measurements have shown that the secondary structure of P. falciparum CaM is more stable than that of human CaM.  In this study, the stability measurements showed that for the ANS binding site and around tyrosines, the C-lobe of human CaM was more stable than the C-lobe of P. falciparum CaM, knowledge which was previously unknown. When studying the entire secondary structure, the C-lobe of P. falciparum CaM was found to be more stable, which is in agreement with previous studies for the secondary structure of the complete CaM variants. For binding, the dissociation constants for both the C-lobe of human CaM and for the C-lobe of P. falciparum CaM were proven to be at a lower range than micro molar, most likely in the nano molar range. This is in agreement with earlier findings regarding the entire human CaM. Furthermore, CaM and CZM were proven to have their absorbance at the same wavelengths. Finally, several amino acid differences between the C-lobes of human and P. falciparum CaM were found that could play a role in binding and stability. One specific amino acid that was suggested to contribute to the stabilisation of the C-lobe of P. falciparum CaM was isoleucine. In the C-lobe of human CaM, these isoleucines were exchanged to threonine and arginine. Another amino acid difference that could potentially play a key role was the valine versus isoleucine, where valine might contribute to the stabilisation of the ANS binding site of the C-lobe of human CaM. To perform this study, the methods fluorescence spectroscopy, UV spectroscopy and circular dichroism were used, as well as several bioinformatic tools.  Overall, both stability and structure analyses have helped determine several differences between the two CaM variants, opening up possibilities to find an inhibitor that targets only the CaM of P. falciparum. CZM still remains as an interesting potential inhibitor, and can hopefully be a part of future research in malaria treatment.

Puberty is a critical period for sexual maturation during which the sex-specific reorganization and remodelling of the pubertal brain facilitate sex biases in stress sensitivity. Pubertal (i.e., six-week-old) CD-1 mice treated with the bacterial endotoxin lipopolysaccharide (LPS; 1.5 mg/kg body weight, ip) show several sex-specific changes to the neuroendocrine and behavioural systems of several reproductive and non-reproductive functions. One promising explanation for the elusive mechanisms driving the sex-specific outcomes of pubertal immune challenge may lie in the cascade of neuroimmune events induced by this systemic immune stressor. This doctoral thesis tested the hypothesis that sex-specific responses of the pubertal neuroimmune network contribute to sex differences in the enduring outcomes of pubertal immune challenge on hippocampus-dependent cognitive processes. Male and female CD-1 mice are equally vulnerable to enduring impairments in spatial memory following pubertal LPS exposure. Across brain regions for cognition and stress regulation, pubertal LPS treatment alters baseline sex differences in microglial expression and morphology in a sex-dependent manner. The temporary female-specific increase in whole-brain blood-brain barrier permeability during LPS-induced sickness may have facilitated the apparent female bias in LPS-induced changes to pubertal microglia. In the context of sex- and region-specific residual effects of pubertal LPS-induced sickness on microglial expression and morphology, pubertal LPS treatment may accelerate certain neurodevelopmental processes in males but not females. The innate sex differences in the pubertal neuroimmune network highlighted by these studies underscore how a systemic immune challenge precipitates sex biases in immune-mediated disorders of brain and behaviour during adulthood.

The thesis simulates the problem of network connectivity that occurs due to the dynamic nature of a network during flight. Nine nodes are provided with initial positions and are flown based on the path provided by leader-follower control algorithm using the server-client model. The application layer provides a point to point connection between the server and client and by using socket programming in the transport layer, a server and clients are established. Each node performs a neighbor discovery to discover its neighbors in the data link layer and physical layer performs the CSMA/CA using RTS/CTS. Finally, multi hop routing is achieved in network layer. Each client connects with server at dedicated interval to share each other location and then moves to next location. This process is continued over a period of several iterations until the relative distance is achieved. The constraints and limitations of the technology are network connectivity is lack of flexibility for random location of nodes, links established with a distant node having single neighbor is unstable. Performance of a system decreases with increase in number of nodes.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a zinc-binding multi-functional protein comprising three disulfide bonds, that is involved in multiple disorders of worldwide concern. Recently LECT2 was found to be involved in amyloidosis (ALECT2) and is believed to be the third most common form of systemic amyloidosis. The disease progression of ALECT2 is relatively slow, and the aggregation assembly is foremostly associated with the kidneys and the liver, but also other organs in the later onset of the disease. This study involved developing a protocol for producing His6-TEV-LECT2 including expression in E.coli BL21(DE3), refolding, and purification. The protocol resulted in a sufficient yield for initial measurements for characterization and biophysical analysis with the following methods: mass spectrometry (MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorimetry. The produced protein was characterized as LECT2 predominantly in its oxidized form. A brief biophysical analysis was made where LECT2 started to unfold already at physiological temperature with a midpoint at 50°C. Additionally, under chemical denaturation LECT2 unfolded with a midpoint of 3 M urea in a cooperative transition without any intermediates. Further on, wavelengths for monitoring the unfolding and the aggregation simultaneously were identified. The unfolding process occurred under 20 sec in 6 M urea and correlates with a double-exponential model. The LECT2 aggregates resemble protofibril-like structures and aggregates species from monomer up to hexamer were found, suggesting simple monomeric addition towards a growing fibril as the aggregation mechanism. The content of aggregates in the sample was notably decreased upon disulfide bond reduction highlighting the importance of further investigating the role of the disulfide bonds in the destabilization and aggregate formation of LECT2.

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No description available.

Circulating lactate levels are a critical biomarker for sepsis and are positively correlated with sepsis-associated mortality. We investigated whether lactate plays a biological role in causing endothelial barrier dysfunction in sepsis. We showed that lactate causes vascular permeability and worsens organ dysfunction in CLP sepsis. Mechanistically, lactate induces ERK-dependent activation of calpain1/2 for VE-cadherin proteolytic cleavage, leading to the enhanced endocytosis of VE-cadherin in endothelial cells. In addition, we found that ERK2 interacts with VE-cadherin and stabilizes VE-cadherin complex in resting endothelial cells. Lactate-induced ERK2 phosphorylation promotes ERK2 disassociation from VE-cadherin. In vivo suppression of lactate production or genetic depletion of lactate receptor GPR81 mitigates vascular permeability and multiple organ injury and improves survival outcome in polymicrobial sepsis. Our study reveals that metabolic cross-talk between glycolysis-derived lactate and the endothelium plays a critical role in the pathophysiology of sepsis.

Yes / Purpose: Two binocular sources of information serve motion-in-depth (MID) perception: changes in disparity over time (CD), and interocular velocity differences (IOVD). While CD requires the computation of small spatial disparities, IOVD could be computed from a much lower-resolution signal. IOVD signals therefore might still be available under conditions of binocular vision impairment (BVI) with limited or no stereopsis, e.g. amblyopia. Methods: Sensitivity to CD and IOVD was measured in adults who had undergone therapy to correct optical misalignment or amblyopia in childhood (n=16), as well as normal vision controls with good stereoacuity (n=8). Observers discriminated the interval containing a smoothly-oscillating MID “test” stimulus from a “control” stimulus in a two-interval forced choice (2IFC) paradigm. Results: Of the BVI observers with no static stereoacuity (n=9), one displayed evidence for sensitivity to IOVD only, while there was otherwise no sensitivity for either CD or IOVD in the group. Generally, BVI observers with measurable stereoacuity (n=7) displayed a pattern resembling the control group: showing a similar sensitivity for both cues. A neutral-density (ND) filter placed in front of the fixing eye in a subset of BVI observers did not improve performance. Conclusions: In one BVI observer there was preserved sensitivity to IOVD but not CD, though overall only those BVI observers with at least gross stereopsis were able to detect disparity-based or velocity-based cues to MID. The results imply that these logically distinct information sources are somehow coupled, and in some cases BVI observers with no stereopsis may still retain sensitivity to IOVD. / UK Biotechnology and Biological 498 Sciences Research Council (BBSRC): BB/M002543/1 (Alex R. Wade) BB/M001660/1 (Julie 499 M. Harris) and BB/M001210/1 (Marina Bloj)

Ce travail montre l'intérêt, par deux aspects, des semiconducteurs semimagnétiques CdMnTe pour l'étude des propriétés structurales, magnétooptiques et électroniques des puits quantiques II-VI. La première partie est consacrée à la caractérisation des interfaces CdTe/CdMnTe dans des puits quantiques asymétriques. Le large effet Zeeman dû à l'interaction d'échange porteurs-Manganèse de la transition excitonique fondamentale, déterminé par des mesures de magnétoréflectivité et d'effet Kerr, permet une analyse précise du profil de concentration du Manganèse à proximité de l'interface. Nous mettons ainsi en évidence le mécanisme de ségrégation qui résulte l'échange d'atomes entre deux monocouches adjacentes en cours de croissance. Dans la seconde partie, l'effet Zeeman géant de la bande de conduction est utilisé pour polariser totalement le spin des électrons d'un gaz bidimensionnel de densité intermédiaire présent dans un puits quantique semimagnétique. Cette propriété permet de découpler les phénomènes dépendant et non-dépendant du spin des électrons et d'étudier ainsi les effets à N corps au sein d'un gaz d'électrons bidimensionnel. L'analyse des mesures de magnéto-transmission inter-niveaux de Landau met en évidence la création de paires électron-trou liés par l'interaction coulombienne. L'énergie de liaison de ce complexe est toutefois fortement réduite par deux mécanismes dus au gaz d'électrons : l'écrantage et le remplissage de l'espace des phases. Un autre effet à N corps est manifeste : l'interaction d'échange électron-électron qui permet d'expliquer, conjointement avec l'interaction d'échange porteurs-Manganèse, la polarisation complète du gaz à un faible champ magnétique. Un modèle au premier ordre, incluant ces trois effets, reproduit le comportement des énergies des transitions en champ.

Sur le site minier de Milluni (Bolivie, Altiplano, 4800 m asl), la contamination des eaux de surface et des sédiments par les métaux lourds a été étudiée en combinant des approches géochimiques, minéralogiques et hydrochimiques. Pour les différents métaux, les sources minérales de la contamination ont été établies, ainsi que les processus géochimiques d'altération des minéraux primaires en minéraux tertiaires. La compréhension des mécanismes géochimiques en jeu et les bilans réalisés ont permis de déterminer les origines et les modes de transport et de contrôle des métaux dans cet environnement. Enfin, les carottages des sédiments lacustres et des tourbières ont permis d'évaluer l'évolution historique de la contamination en métaux lourds, notamment au cours du siècle dernier.