Interleukin-18 (IL-18) is a pro-inflammatory cytokine known to be involved in maintaining metabolic homeostasis; however, also capable of inducing cardiac dysfunction. Additionally, IL-18, has been shown to bind to a novel receptor, the Na-Cl Co-transporter (NCC). We hypothesized that NCC mediates IL-18 metabolic and cardiac signaling in mice. Using male C57BL/6J mice, we compared the metabolic and cardiac function changes after at least 8 weeks of high-saturated fat high sugar diet (Western Diet) in NCC knockout (NCCKO), IL-18 knockout (IL-18KO), and wild-type mice. We show that NCCKO mice have significantly increased body weight gain from baseline, no difference in fasting blood glucose, and attenuated cardiac diastolic dysfunction after WD compared to wild-type mice. Collectively, the metabolic and cardiac phenotypes of NCCKO mice resembled that of the IL-18KO mice, indicating that NCC may mediate IL-18 signaling in a mouse model of diet-induced obesity and cardiac dysfunction.

Le nitrate, source majeur d’azote pour la plupart des plantes,n’est pas seulement un élément nutritif mais est aussi une molécule signale. Il existe cependant des réponses au nitrate contrastées entre les différentes plantes supérieures. Chez Medicago truncatula, espèce modèle de la famille des légumineuses, le nitrate a un effet inhibiteur sur la croissance de la racine primaire en phase post-germinative. Une étude de génétique quantitative a montré qu’un transporteur de nitrate se situe au pic d’un QTL impliqué dans la croissance de la racine primaire. La caractérisation fonctionnelle de ce transporteur, nommé MtNRT1.3 et renommé MtNPF6.8, a montré que celui-ci est à double affinité pour le nitrate. Ce transporteur est alors susceptible de participer à l’influx de nitrate dans la plante. Après l’obtention de trois génotypes mutants RNAi stables, les expérimentations utilisant duK15NO3 ont montré que ce transporteur participe effectivement à l’influx de nitrate lié au système de transport à faible affinité inductible dans la plante (iLATS). En revanche,la mutation de MtNPF6.8 ne semble pas avoir de conséquence sur le métabolisme azoté. Par ailleurs, les études sur la croissance de la racine primaire ont permis de confirmer l’implication du transporteur sur ce caractère phénotypique. L’inhibition de croissance de la racine primaire observée sur nitrate chez le génotype sauvage est alors imputée, à l’échelle cellulaire, à une modulation de l'élongation cellulaire. La possibilité que l’ABA, hormone végétale, joue un rôle dans la médiation de cette réponse dépendant du nitrate, est fortement favorisée. L’ensemble de résultats, conforté par une étude de mutants exprimant ce transporteur chez A. thaliana, indique donc que MtNPF6.8 est un senseur de nitrate pour la plante en phase post germinative,ceci indépendamment de sa fonction de transport de nitrate. / Nitrate, a major nitrogen source for most plants, is not only anutrient but also a signaling molecule. However, there arecontrasting responses to nitrate between different higherplants. In the model legume Medicago truncatula, nitrate hasan inhibitory effect on the primary root growth in postgerminationphase. A quantitative genetic study has shownthat a nitrate transporter is localized at the peak of a QTLinvolved in the primary root growth. Functionalcharacterization of the transporter, named MtNRT1.3 andrenamed MtNPF6.8, showed that it encodes a dual affinitynitrate transporter. MtNPF6.8 is likely to participate in thenitrate influx in the plant. After obtaining three knockdownlines by RNA interference, experiments using K15NO3 showedthat this transporter is effect involved in nitrate influx relatedto the inducible low affinity transport system (iLATS).However, mutation in MtNPF6.8 does not any effect onnitrogen metabolism. In addition, studies on the primary rootgrowth have confirmed the involvement of the transporteron phenotypic trait. In wild-type plants, cortical cell sizedecreased after nitrate treatment, showing that primary rootgrowth was due to this reduced cell elongation. Thepossibility that ABA also plays a role in mediating this nitratedependent response is heavily favored. All these results,reinforced by a study of mutants expressing this transporterin A. thaliana, indicate that MtNPF6.8 is a nitrate sensor forMedicago in the post-germination phase, independently ofits nitrate transport activity.

Eisenmangel ist ein weltweites Ernährungsproblem für Pflanzen und allen von Ihnen abhängigen Sekundärkonsumenten. Er reduziert den Ertrag, die Qualität und Produktivität von z.B. Kulturpflanzen, was wiederum zu Mangelerscheinungen beim Menschen führen kann. Die Nahrungsmittelforschung hat ein großes ökonomisches und wirtschaftliches Interesse daran, die Eisenverfügbarkeit und -Aufnahme der Pflanzen zu erhöhen. In der vorliegenden Arbeit wurde eine kleine Familie von fünf (VTL1-5) neuen potentiellen Eisentransportern in Arabidopsis thaliana identifiziert und charakterisiert und somit ein weiterer Baustein zu der Aufklärung der Eisenhomöostase hinzugefügt. Bei den fünf Transportern handelt es sich um CCC1-like Proteine, von denen vier (VTL1-3 und 5) eine eisenabhängig regulierte Expression zeigen. Für VTL1 kann mittels Protein-tag Markierung sehr überzeugend eine Lokalisation in der Vakuolenmembran und ein damit verbundener Eisentransport in die Vakuole, analog zur Funktion von VIT1, gezeigt werden. Da vermutlich ein knockout von VTL1 zur Embryo Lethalität führt und auch die vit1-1 Mutante einen sehr verkümmerten Phänotyp unter Eisenmangel zeigt, scheinen beide Proteine getrennt voneinander an verschiedenen Schlüsselpositionen im Eisenhaushalt zu wirken. Auch für VTL2 und VTL5 kann eine Lokalisation an der Vakuolenmembran und der damit verbundene Eisenimport postuliert werden. Die heterologe Expression aller drei Gene in ∆ccc1 Zellen führt zur Erhöhung der vakuolären Eisenkonzentration. Für VTL4 wird mittels Protein-tag Markierung eine Lokalisation an der Plasmamembran mit einer Exportfunktion vorgeschlagen. VTL3 und VTL4 heterolog exprimierende ∆ccc1 Hefe-Zellen weisen einen geringeren Eisengehalt als Kontrollzellen auf. Alle fünf Proteine sind in der Lage sowohl den Mutanten-Phänotyp der ∆ccc1 Hefe-Mutante, wie auch der Arabidopsis vit1-1 und nramp3/nramp4 Doppelmutante zu komplementieren. Anhand dieser Tatsachen konnte die Eisentransportfähigkeit bewiesen werden. / Iron deficiency is a worldwide nutritional problem for plants and in general all heterotrophic organisms. Iron deficiency reduces crop productivity, quality and yield, which in consequence lead to iron deficiency symptoms in humans. Because approximately 50 % of the global human caloric intake is derived from a cereal grain diet, it is not surprising that the emphasis of nutrition research is on uptake, transport and storage of iron in plants, specifically in seeds. In this doctoral thesis a small family of five potential iron transporters (VTL1-5) in Arabidopsis thaliana has been identified and characterized. These five transporters are CCC1-like proteins, four of which (VTL1-3, 5) show a pattern of iron-dependent expression. Through the use of a protein tag, VTL1 is shown to be localized on the vacuolar membrane and associated with import of iron into the vacuole. In this respect VTL1 displays an analogous function to VIT1. Since the knockout of VTL1 likely leads to embryo lethality and seedlings of the vit1-1 mutant display an ephemeral phenotype caused by iron deficiency, both proteins seem to uniquely influence the iron homeostasis. As for VTL1 both VTL2 and VTL5 are localized on the vacuolar membrane and catalyze iron import. The heterologous expression of all three genes in ∆ccc1 cells leads to an increased vacuolar iron concentration. In contrast, VTL3 and VTL4 may function as iron exporters and play possibly roles in xylem loading. This conclusion is supported by the facts that a mCherry-VTL4 signal is localized to the plasma membrane of tobacco leaf cells and that ∆ccc1 cells in which VTL3 and VTL4 are heterologous expressed, show a decreased iron content compared to control cells. All five proteins are able to complement the ∆ccc1 yeast mutant and the two Arabidopsis vit1-1 and nramp3/nramp4 mutant phenotypes, thereby demonstrating a function in iron transport.

Orientador: Doris Hissako Matsushita / Banca: Maria Aparecida Visconti / Banca: Flávia Lombardi Lopes / Banca: Joel Claudio Heimann / Banca: Fernando Yamamoto Chiba / Resumo: Atualmente, está bem estabelecido que o ambiente fetal está ligado à saúde materna, e estímulos ou agressões anormais durante a vida intra-uterina podem resultar em mudanças na fisiologia e metabolismo da prole, aumentando o risco de doenças na vida adulta, este fenômeno é conhecido como programação fetal. Alterações na metilação do DNA e expressão gênica são consideradas mecanismos moleculares responsáveis por esta programação. Estudos anteriores demonstraram que a doença periodontal (DP) materna promove resistência insulínica, aumento nas concentrações plasmáticas de citocinas, redução do conteúdo de GLUT4 e do seu índice de translocação para membrana plasmática em sua prole adulta. E citocinas, como por exemplo, o TNF-α, têm sido relacionadas com a redução da expressão de GLUT4 por meio da ativação do fator de transcrição nuclear κappa B (NF-κB). Além disso, esta citocina pode estimular algumas serinas quinases, incluindo IκB quinase (IKK), c-Jun amino-terminal kinase (JNK) e quinases reguladas por sinais extracelulares (ERKs) que estão envolvidas na resistência insulínica. Tais achados evidenciam a necessidade de realizar mais estudos para verificar os mecanismos envolvidos nestas alterações. Portanto, os objetivos do presente estudo foram avaliar em ratos adultos, proles de ratas com DP: 1) glicemia e insulinemia; 2) expressão do RNAm da proteína transportadora de glicose GLUT4 e do IRS1 em muscular esquelético gastrocnêmio (MG); 3) o grau de metilação do DNA na região p... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: It is well establishedthat the fetal environment is linked to maternal health, and abnormal stimuli or aggressions during intrauterine life can result in changes in the physiology and metabolism of offspring, increasing the risk of disease in adult life, this phenomenon is known as fetal programming. Changes in DNA methylation and gene expression are considered molecular mechanisms responsible for this programming. Previous studies have demonstrated that maternal periodontaldisease (PD) promotes insulin resistance, increased plasma concentrations of cytokines, reduced GLUT4 content and its plasma membrane translocation index in its adult offspring. And cytokines, such as TNF-α, have been linked to reduced GLUT4 expressionthrough the activation of nuclear transcription factor kappa B (NF-κB). In addition, this cytokine can stimulate some serine kinases including IκB kinase (IKK), c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinases (ERKs)that are involved in insulin resistance. These findings evidenced the need for further studies to verify the mechanisms involved in these changes. Therefore, the objectives of the present study were to evaluate in adult rats, offspring of rats with PD: 1) birth weight and during the75 days of age;2) glycemia and insulinemia; 3) GLUT4 and IRS1mRNA expression in skeletal muscle gastrocnemius (MG); 4) the degree of DNA methylation in the promoter region of the GLUT4 gene in MG; 5) phosphorylation of JNK, IKKα/β, ERK 1/2, N... (Complete abstract click electronic access below) / Doutor

<p>This licentiate thesis consists of two free-standing papers and a linking essay that links the papers by articulating an overall research aim and common theoretical background. The research aim is kept general and focuses on the role public transport can play in sustainable development. The work aims to contribute to a better understanding of why people do or do not choose to use public transport, to grasp the ideas/attitudes underlying the decisions made regarding the future of public transport, and to make suggestions for solving problems that may arise in public transport.</p><p>The thesis has three theoretical bases: transport research, cultural theory, and sustainable development. Transport research is the starting point of the work, and I outline a gap in it that needs to be filled. This gap is filled by drawing on cultural theory connected to geographic terms. Sustainability discourses, on one hand, legitimate the work’s importance but also further stimulate the linking essay. Methodologically, the thesis can be placed in the hermeneutic tradition. Due to the work’s particular alignment with cultural theory, cultural relativism is supported. In addition, the interdisciplinary and multi-methodical approach can be explained by the work’s cultural focus.</p><p>Against this background, the research concluded that, as a business, public transport has difficulties achieving the vaguely formulated ideological goals set for it at the national political level. As well, citizens have been marginalised in the goal-setting process. Citizen empowerment and a clearer formulation of the ideological goals are needed. Furthermore, public transport seems to be poorly rooted in society, and the present research found subregional differences that might be culturally based or explainable. The fact that public transport is poorly rooted in society can partially be explained by the marginalisation of citizens from decision making. Cultural differences in and between subregions should thus be taken into account by a farther-reaching customer perspective that acknowledges the importance of subregions. In conclusion, one should be sceptical as to public transport’s role in sustainable development. This pessimism arises from the technological step backward people would have to take in changing their main mode of transportation. Therefore, more all-embracing technical innovations are needed in the transport sector.</p><p> </p>

This licentiate thesis consists of two free-standing papers and a linking essay that links the papers by articulating an overall research aim and common theoretical background. The research aim is kept general and focuses on the role public transport can play in sustainable development. The work aims to contribute to a better understanding of why people do or do not choose to use public transport, to grasp the ideas/attitudes underlying the decisions made regarding the future of public transport, and to make suggestions for solving problems that may arise in public transport. The thesis has three theoretical bases: transport research, cultural theory, and sustainable development. Transport research is the starting point of the work, and I outline a gap in it that needs to be filled. This gap is filled by drawing on cultural theory connected to geographic terms. Sustainability discourses, on one hand, legitimate the work’s importance but also further stimulate the linking essay. Methodologically, the thesis can be placed in the hermeneutic tradition. Due to the work’s particular alignment with cultural theory, cultural relativism is supported. In addition, the interdisciplinary and multi-methodical approach can be explained by the work’s cultural focus. Against this background, the research concluded that, as a business, public transport has difficulties achieving the vaguely formulated ideological goals set for it at the national political level. As well, citizens have been marginalised in the goal-setting process. Citizen empowerment and a clearer formulation of the ideological goals are needed. Furthermore, public transport seems to be poorly rooted in society, and the present research found subregional differences that might be culturally based or explainable. The fact that public transport is poorly rooted in society can partially be explained by the marginalisation of citizens from decision making. Cultural differences in and between subregions should thus be taken into account by a farther-reaching customer perspective that acknowledges the importance of subregions. In conclusion, one should be sceptical as to public transport’s role in sustainable development. This pessimism arises from the technological step backward people would have to take in changing their main mode of transportation. Therefore, more all-embracing technical innovations are needed in the transport sector.

Malaria is a life threatening parasite disease caused and transmitted by infected female anopheles mosquito. However, the parasite, Plasmodium falciparum, has become resistant to most anti malarial drugs, such as chloroquine, which contributes to fever and anaemia because of its ability to digest the haemoglobin in the red blood cells. The aims of this project were to establish whether “Bac to Bac” Baculoviral Expression System is suitable for expression of pfmdr 1 gene and for purification of the pgh 1 protein. The pfmdr 1 gene encodes an ABC transporter protein, pgh 1, fixed in the cell membrane of the Plasmodium falciparuum gut, which assist in elimination of drug compounds. Furthermore, “Bac to Bac” Baculoviral Expression System uses vectors with histidine tags to clone the pfmdr 1 gene and subsequently transform these into DH10Bac cells to produce the recombinant bacmid DNA. Since pfmdr 1 gene is an AT-rich sequence, PCR was optimized, by lowering the annealing and extension temperature to 47Co and 66Co respectively. The results show that “Bac to Bac” Baculoviral Expression System can be used to express the pfmdr 1 gene, though further experiments has to be performed.

Cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC form the bacteriocin colicin V (ColV) secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter with nucleotide-binding motifs in the C-terminal domain (CTD). To study the role of CvaB-CTD in the ColV secretion, a truncated construct of this domain was made and over-expressed. Different forms of CvaB-CTD were obtained during purification, and were identified as monomer, dimer, and oligomer on gel filtration. Nucleotide binding was shown critical for the CvaB-CTD dimerization: oligomers could be converted into dimers by nucleotide bindings; the removal of nucleotide from dimers resulted in transient monomers followed by CTD oligomerization and aggregation; no dimer form could be cross-linked from the nucleotide-binding deficient mutant D654H. The spatial proximity of the Walker A site and ABC signature motif in CTD dimer was identified through disulfide cross-linking of mixed CvaB-CTD with mutants A530C and L630C, while mutations did not dimerize individually. Those results indicated that the CvaB-CTD formed a nucleotide-dependent head-to-tail dimer. Molecular basis of differential nucleotide bindings was also studied through bioinformatics prediction and biochemical verification. Through sequence alignment and homology modeling with bound ATP or GTP, it was found that the Ser503 and Gln504 on aromatic stacking region (Y501DSQ-loop) of CvaB-CTD provided two additional hydrogen-bonds to GTP, but not to ATP. Site-directed mutations of the S503A and/or Q504L were designed based on the model. While site-directed mutagenesis studies of Walker A&B sites or the ABC signature motif affected little on the GTP-binding preference, the double mutation (S503A/Q504L) on the Y501DSQ-loop increased both ATP-binding and ATPase activity at low temperatures. The double mutant showed slight decrease of GTP-binding and about 10-fold increase of the ATP/GTP-binding ratio. Similar temperature sensitivity in nucleotide-binding and activity assays were identified in the double mutant at the same time. Mutations on the Y501DSQ-loop did not affect the ColV secretion level in vivo. Together, the Y501DSQ-loop is structurally involved in the differential binding of GTP over ATP.

<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

Denna rapport utreder översiktligt regional och kommunal infrastruktur ochmiljöpåverkan från transporter av bergmassor, bentonit och lera vid ett slutförvar avkärnavfall eller om det inte blir ett slutförvar i Oskarshamns kommun. Strategin harvarit att utefter de regionala miljökvalitetsmålen begränsad klimatpåverkan, frisk luftsamt det länsegna målet fossilbränslefri zon år 2030 undersöka hur olika lastbärare(lastbil, tåg och fartyg) bidrar till måluppfyllelse. Slutsatsen är att det mest troligascenariot med transporterna är olika transportalternativ beroende på vart materialetskall någonstans och vad det skall användas till. Oavsett om det blir ett slutförvar ellerej i Oskarshamns kommun, kommer infrastrukturen att bli förändrad, speciellt för deboende i Misterhults socken. Fossilbränslefrizon år 2030 är det målet som inte skullenå måluppfyllelse ifall enbart lastbärare med fossilt bränsle skulle användas vidtransporterna. 2008:Nr 3 Teknik