Caprine Arthirtis Encephalitis Virus (CAEV) and Visna Virus are two viruses of the lentivirus family. They encode three structural genes (gag, pol, and env) and two regulatory genes (rev and tat). The Rev protein regulates Gag, Pol and Env expression by transporting their mRNAs to the cytoplasm by binding to the RRE (Rev Response Element) found on their mRNAs. Previous studies have indicated that Rev may be toxic to transfected cells, overexpression of exogenous RREs or a better binding RRE can inhibit Rev activity and Rev-C (CAEV Rev) can trans-activate RRE-V (Visna Virus RRE). To test these possibilities FACS analysis, RNA binding assays, cotransfections, and SELEX were done. The results indicated that Rev is not acutely toxic to cells, inhibition of Rev activity could not be achieved by making a better binder or through expression of exogenous RREs, and Rev-C can trans-activate RRE-V implicating conservation of Rev/RRE interactions in lentiviruses.

Redan i förskolan läggs grunden för barns utveckling av matematikkunskaper. Därför är det oerhört viktigt att arbeta med matematik i förskolan. Syftet för detta arbete handlar om att förstå hur förskollärare och förskolechefer synliggör och förhåller sig till den grundläggande matematiken samt uppmärksamma om fortbildning har påverkat deras syn på arbete med matematik. Vår undersökning baseras på intervjuer av sex förskollärare och två förskolechefer från sex olika förskolor. Resultatet av intervjuerna visade att alla förskollärare och förskolechefer var överens om att matematiken finns överallt i barns vardag. Matematik handlar inte enbart om siffror och att räkna, utan matematik är också språk och begrepp. Både litteraturen och respondenterna uttrycker att förskollärare behöver sätta på sig sina ”matematikglasögon” för att medvetandegöra barnen om matematik vilket innebär att i vardagliga situationer och på ett lustfyllt sätt uppmärksamma allt som har med matematik att göra. Resultatet visade att några förskollärare och förskolechefer har fördjupade kunskaper utifrån hur de beskriver sitt sätt att synliggöra matematiken. För dem har fortbildningen haft stor betydelse för hur de kan uppmärksamma matematiken i förskolan. Matematiken har förtydligats i förskolans läroplan Lpfö98/10 vilket inneburit att förskollärare upplever att det är enklare att ta vara på barnens utforskande och undersökande för att på detta viset konkretisera och synliggöra matematiken.

SAMMANFATTNING Syftet med detta arbete är att beskriva och analysera på vilket sätt Freinetpedagogikens syn på barn och dess lärande är förenligt med Läroplanen för förskolan, Lpfö 98, (Skolverket 2010). För att få svar på detta har jag intervjuat fyra pedagoger på två olika Freinetförskolor, som beskriver sina erfarenheter och upplevelser av att arbeta med den franske pedagogen Freinets tankar och idéer i en svensk förskola. Jag har i detta arbete utgått från en kvalitativ forskningsansats där jag via litteraturstudier och kvalitativa djupintervjuer söker besvara studiens frågeställningar och syfte. Vad gäller resultatet så ser samtliga pedagoger Freinetpedagogiken mer som ett medvetet förhållningssätt än en metod, där det utmärkande är synen på barnen. Mycket av respekten för barnen och deras kompetens befästs i det trevande försöket, handen och hjärnans arbete värderas lika. Att barnen ges stort utrymme till inflytande och medbestämmande, samt att de ges ansvar utifrån sin förmåga, är något som samtliga respondenter menar som karakteristiskt för Freinet. Detta ansvar anser de genererar i en självkänsla hos barnen som yttrar sig i ”jag kan själv” och ”allt är möjligt”, och som i sin tur ger barnen styrka i att växa som egna individer i demokratisk anda. ”Läroplanen är vår bibel” och genomsyrar allt i verksamheten menar en av respondenterna. Utifrån min analys av resultatet är det i mångt och mycket sant, Lpfö 98 (Skolverket 2010) används som ett levande dokument och används i såväl dokumentation som i temaplanering och som samtalsämne på föräldramöten. Samtliga respondenter verkar bekväma i att omvandla teorier i praktiken, teorier från såväl Lpfö 98, (Skolverket 2010) som Freinets teorier kring barn och dess lärande.

Nuclear hormone receptors (NRs) are ligand-dependent DNA binding proteins that translate nutritional and physiological signals into gene regulation. The significance of NRs in human health and disease is underscored by the availability of drugs that targets NRs for treating several diseases. In this context, a subgroup of NR family has been proposed to regulate metabolism in a cell/tissue specific manner. The Rev-erb subgroup of NRs consists of two isoforms Rev-erb and Rev-erb. These two receptors have been shown to regulate different aspects of human physiology such as metabolism, inflammation, and circadian rhythm. Many NRs are expressed in skeletal muscle, a major mass peripheral tissue that accounts for ~40% of the total body weight and energy expenditure. This lean tissue is a major site for lipid and glucose homeostasis. Skeletal muscle express and secrete cytokines which perform autocrine and paracrine function with other tissues such as adipose. Accordingly, skeletal muscle plays important role in blood lipid profile, insulin sensitivity and progression of diseases such as type 2 diabetes and obesity. In addition, many studies have shown that NRs in skeletal muscle regulate glucose, lipid and energy homeostasis. Therefore, understanding the function of NRs in skeletal muscle provides a platform for potential new therapeutic treatments for metabolic disease. Rev-erb is expressed in skeletal muscle; however the function of this receptor in skeletal muscle metabolism has not been examined. Nevertheless, considering the importance of Rev-erb subfamily in metabolism, circadian control and the role of skeletal muscle in lipid homeostasis, the function of Rev-erb in skeletal muscle metabolism needs to be further investigated. We tested the hypothesis that Rev-erb (directly and/or indirectly) regulated the genetic programs that control lipid homeostasis in skeletal muscle. Initially, we exogenously expressed a truncated version of Rev-erb without the ligand-binding domain (Rev-erb) in vitro (in the C2C12 skeletal muscle cell culture system); and in vivo (in mouse tibialis anterior muscle. Moreover, we also attenuated Rev-erb expression in skeletal muscle cells using siRNAs targeting N-terminus and hinge regions of Rev-erb. We performed candidate based expression profiling utilizing quantitative RT-PCR analysis on the Taqman Low Density Array (TLDA) platform to identify putative downstream primary and/or secondary targets of Rev-erb action in skeletal muscle cells in the context of metabolism and muscle growth. Exogenous expression of Rev-erb in skeletal muscle cells in vitro decreased the expression of several genes involved in fatty acid/lipid absorption (including Cd36, and Fabp3 and 4). Interestingly, the mRNA encoding the master lipogenic regulator, SREBP-1c was also increased after ectopic Rev-erb expression. Moreover, we observed significant induction in the mRNAs encoding interleukin-6 and IKB that are involved in the regulation of the inflammatory cascade. Finally, we also observed the marked repression of myostatin mRNA, an important protein implicated in negative regulation of muscle hypertrophy/hyperplasia and a positive regulator of body fat accumulation. In summary, our in vitro study suggested that Rev-erb regulates genes involved in metabolism, inflammation and muscle growth. Quantitative PCR analysis that utilised the Taqman Low Density Array (TLDA) platform revealed Rev-erb siRNA expression down-regulated (in a subtle but significant manner) several genes involved in lipid/glucose homeostasis and the TGF- signalling pathway. Interestingly, genes that are involved in the myostatin and TGF- signalling pathway such as Activin A receptor type 2a (ACVR2A), Smad specific E3 ubiquitin protein ligase 1 (Smurf1), and TGF- receptor 2 (TGFBR2) were identified potential (direct and/or indirect) target of Rev-erb action in skeletal muscle cells. Moreover, genes such as Citrate Synthase (CS), V-akt murine thymoma viral oncogene homolog 2 (Akt2), Peroxisome proliferator- activated receptor- coactivator (PGC)-1 (PGC1) were also significantly modulated by Rev-erb in these analyses. The expression of two mRNAs encoding a) SREBP1c and b) IKB increased by ectopic Rev-erb expression was examined in more detail. These were selected because Rev-erb has been presumed to function as a transcriptional silencer. Secondly, we had demonstrated that in vivo expression of Rev-erb (after injection and electroporation of mouse tibialis anterior muscle) increased SREBP-1c expression, and Rev-erb siRNA studies suggested that this orphan NR was necessary for optimal SREBP-1c mRNA expression. Consequently, we tested the hypothesis that Rev-erb encodes the potential to function as a transcriptional activator in skeletal muscle. To test this hypothesis, we examined whether the SREBP1c and IKB promoters were trans-activated by co-transfected Rev-erb in skeletal muscle cells. We initially tested whether Rev-erb regulates the SREBP1c promoter. Transfection experiments showed Rev-erb expression trans-activated this promoter. This observation was in contrast to previous promoter studies showing that Rev-erb is a potent repressor of gene transcription. Therefore, we subsequently performed an experiment in which we simultaneously used the Rev-erb promoter (previously characterized to be repressed by Rev-erb) and SREBP1c promoter to examine the effect of Rev-erb expression. This experiment showed that Rev-erb repressed the activity of Rev-erb promoter, and in parallel trans-activated the SREBP1c promoter. Bioinformatics analysis identified two regions covering putative Rev-erb response elements RERE1 (-1342 to -1158) and RERE2 (-525 to -401) in the SREBP1c promoter. Chromatin immuno-precipitation assays demonstrated that Rev-erb is selectively recruited to RERE2 between nucleotide positions –525 to –401 in the promoter. Unidirectional deletion analysis of the SREBP1c promoter coupled with the analysis of mutants in the LXR response elements (of the SREBP-1c promoter) confirmed that Rev-erb mediated trans-activation of SREBP1c promoter does not function through LXR response elements. Interestingly, treatment of skeletal muscle cells with Hemin, a molecule recently proposed to function as a ligand for Rev-erbs, increased SREBP1c mRNA expression. In summary these data show that Rev-erb is a novel positive regulator of SREBP1c mRNA expression in skeletal muscle. We subsequently cloned the previously characterised human IKB promoter region spanning the potential ROR and Rev-erb binding site. Transfection experiments showed that in accordance to previously published observation, ROR trans-activated the IKB promoter. However, both Rev-erb and Rev-erb when co-transfected with the IKB promoter had minimal effects on the activity of this promoter. Studies have shown that Rev-erb functions as a competitor for ROR and block ROR mediated trans-activation of its target gene expression. Interestingly, our co-transfection experiments showed that both Rev-erb and Rev-erb blocks ROR-mediated trans-activation of IKB promoter. Together, this data suggests that Rev-erb-mediated regulation of IKB transcription in skeletal muscle cells could occur through indirect mechanisms. In conclusion, our studies have shown Rev-erb directly and indirectly regulates the expression of genes involved in metabolism, inflammation and muscle growth suggesting that Rev-erb in skeletal muscle has the potential to be exploited in a therapeutic manner.

Glucocorticoids (Gcs) are a commonly used drug to target the glucocorticoid receptor (GR). The GR has a myriad of cellular and physiological effects, however, Gcs are clinically used for the treatment of inflammatory conditions due to the potent anti-inflammatory actions of GR. The anti-inflammatory effects come with serious side effects e.g. metabolic disease. I examine the role of lipid rafts in modulating the anti-inflammatory actions of Gcs, and the role of circadian rhythms in the control of Gc side effects. I tested the role of caveolin-1 (Cav1), a constituent of membrane lipid rafts, and its role in Gc suppression of inflammation. Gene expression analysis of mouse lung tissue showed that genetic depletion of Cav1 (CAV1KO) results in increased transactivation of Gc target genes. The increased Gc action, however, does not result in an increased effect on suppression of inflammation in a model of innate immunity: aerosolised lipopolysaccharide (LPS) induced lung inflammation or in a model of adaptive immunity: Ovalbumin. CAV1KO mice were protected from LPS induced inflammation, despite increased cytokine production. This suggests a differential response to LPS in lung parenchyma and alveolar macrophages dependent on Cav1. CAV1KO results in a pro-inflammatory phenotype in macrophages, and the opposite in parenchymal tissue. These data suggest that while Cav1 is an upstream regulator of Gc response, it does not have a strong enough effect to alter the ability of GR to repress inflammation in vivo. Gc treatment results in a strong metabolic phenotype, with aberrant energy metabolism, insulin resistance and hepatosteaotosis, I investigated how this side effect interacts with circadian rhythms, another key determinant of energy metabolism. Using transcriptomics of whole lung and liver taken during the day or the night, I demonstrate that the metabolic actions of Gc in the liver can be temporally separated, whilst maintaining consistent anti-inflammatory actions in both liver and lung. This temporal gene regulation by Gc is controlled by REV-ERB, a rhythmically expressed, orphan nuclear receptor, part of the core clock machinery, via a direct interaction with GR at key regulatory DNA loci. Genetic deletion of REV-ERB protects mice from the hepatosteotosis associated with Gc treatment. Taken together, these data suggest that Gcs are regulated upstream of the receptor by the core consitutent of membrane lipid rafts; Cav1, which modulates the Gc response in vivo. Also, that the GR action can be controlled by dosing at different times of day, separating the detrimental metabolic effects of Gcs from the beneficial anti-inflammatory effects. This is enabled through a direct interaction between GR and REV-ERB at key gene regulatory sites.

MEDEIROS, Melissa Soares. Polimorfismo do RRE e resistência aos antirretrovirais do vírus da imunodeficiência humana e efeito citopático e replicativo in vitro da enfuvirtida no códon 36 do vírus modificado pNL4-3. 2011. 255 f. Tese (Doutorado em Farmacologia) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2011. / Submitted by denise santos (denise.santos@ufc.br) on 2016-03-28T11:42:34Z No. of bitstreams: 1 2011_tese_msmedeiros.pdf: 18233541 bytes, checksum: 79da766383cb7344f8326013c6aa304c (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2016-03-28T11:45:13Z (GMT) No. of bitstreams: 1 2011_tese_msmedeiros.pdf: 18233541 bytes, checksum: 79da766383cb7344f8326013c6aa304c (MD5) / Made available in DSpace on 2016-03-28T11:45:13Z (GMT). No. of bitstreams: 1 2011_tese_msmedeiros.pdf: 18233541 bytes, checksum: 79da766383cb7344f8326013c6aa304c (MD5) Previous issue date: 2011 / Introduction: Rev Responsive Element (RRE) is a RNA molecule responsible to mRNA from HIV-1 virus nuclear transportation to cytoplasm through RRE-Rev pathway, essential to virus replication. Enfuvirtide resistance mutations are primary located in a perimeter of 10 amino acids of HR1, a corresponded region of RRE. Characterize RRE should provide a new approach for HIV therapy. Objectives: Sequence and characterize RRE from gp41 to evaluate variability and correlate with laboratory parameters in sequences from HIV-1-infected patients, which were receiving regimens including Enfuvirtide, naïve or rescue therapy. Also evaluated mutation G to D at codon 36 in the presence of fusion inhibitor (enfuvirtide). Methods: Sixty-two samples from HIV patients in Ceara/Brazil were collected and Thirty-five RRE sequences and clinical follow-up were analyzed, distributed into three groups: N (naïve therapy), T (treated patients with rescue regimens) and F (rescue regimens containing Enfuvirtide). Sequences obtained were aligned with Los Alamos HIV sequence database by using the HIV BLAST Search. Culture Study was performed using two different pNLA-3 (36D and 36G) with increasing amounts of enfuvirtide. Results: A phylogenetic analyses demonstrated higher prevalence of HIV-1 subtypes B (97.2%). An increased immunology response was observed in CD4 count higher on group T (71.5%) compared with F (2.98%). Group N most common mutations and polymorphisms were Q32L (41.6%), N42S (8.3%), R46K (33.3%), L54M (41.6%); group T: Q32R (8.3%), R46K (25%), L54M (33.3%); and group F: Q32L (18.2%), G36D (9.1%), V38A (9.1%), N42S (27.3%), N42T (9.1%), R46K (27.3%), L54M (45.4%), K77R (54.5%). Three samples demonstrated significant resistance mutations to fusion inhibitors. Analysis of RRE nucleotide primary sites observed mutation 28A in 27.2% and 8.3% on groups F and N respectively, and 27S in 8.3% on group T. There was selective pressure on HR1 region from HIV-1 patients using antiretroviral, independent of enfuvirtide exposure. There was no statistical difference between p24 curves of virus 36D compared with 36G, independent of T20 concentrations (p>0.05). It was observed less syncytial formation in 36D virus, with diminished fusogenic activity besides keeping infectivity. Conclusions: This study defined most prevalent RRE polymorphisms in Ceara/Brazil and suggests highly preserved regions primary sites to Rev connection. Observed a low resistance profile to enfuvirtide in failing regimens with this drug. Selective pressure on HR1 region in failed regimens with out fusion inhibitors was detected. A less syncytial formation in 36D virus with diminished fusogenic activity was detected. / Introdução: Rev Responsive Element (RRE) é uma molécula RNA responsável pelo transporte do mRNA viral do HIV-1 do núcleo para o citoplasma da célula CD4, através da via RRE-Rev, essencial para a replicação viral. As mutações de resistência a Enfuvirtida são primariamente localizadas no perímetro de 10 aminoácidos do HR1, região correspondente no RRE. Caracterizar o RRE poderá fornecer uma nova abordagem terapêutica para a terapia do HIV. Objetivos: Sequenciar e caracterizar o RRE da gp41 para avaliar sua variabilidade e correlação com parâmetros laboratoriais em sequências de pacientes infectados pelo HIV-1 que receberam terapia antirretroviral ou virgens. Em estudo in vitro avaliar a mutação 36D na presença de Enfuvirtida. Metodologia: 62 amostras de pacientes com HIV-1 do Ceará foram coletadas e 35 sequências de RRE foram obtidas e distribuídas em três grupos para fins de análise comparativa: N (virgens de terapia), T (uso de antirretroviral sem inibidor de fusão) e F (uso de antirretroviral associados a Enfuvirtida). Sequências obtidas foram alinhadas com o banco de dados de Los Alamos para HIV usando HIV BLAST Search. Estudo in vitro utilizou dois vírus de laboratório pNLA-3 (36D e 36G) observando citopatogenicidade e proliferação na presença de doses crescentes de Enfuvirtida. Resultados: A análise filogenética demonstrou alta prevalência do HIV-1 subtipo B (97,2%). Observou-se aumento da resposta imunológica no grupo T (71,5%) comparado ao F (2,98%). Mutações mais comuns e polimorfismos do Grupo N foram Q32L (41,6%), N42S (8,3%), R46K (33,3%), L54M (41,6%); no grupo T: Q32R (8,3%), R46K (25%), L54M (33,3%); e no grupo F: Q32L (18,2%), G36D (9,1%), V38A (9,1%), N42S (27,3%), N42T (9,1%), R46K (27,3%), L54M (45,4%), K77R (54,5%). Três amostras demonstraram mutações de resistência significativas para os inibidores de fusão. Análise dos sítios primários de ligação do RRE observou presença de mutação 28A em 27,2% e 8,3% nos grupos F e N respectivamente, e 27S em 8,3% no grupo T. Houve pressão seletiva da região HR1 do HIV-1 de pacientes usando antirretroviral, independente da exposição à Enfuvirtida. Não houve diferença estatística significativa nas curvas de p24 do vírus 36D comparado com 36G, independente de concentrações de T20 (p>0.05). Observou-se menor formação de sincício, com diminuição da capacidade fusogênica, sem impacto na infectividade. Conclusão: O estudo definiu as mutações e polimorfismos mais prevalentes no Ceará, sugerindo alta preservação nas regiões de sítio primário de ligação do Rev-RRE. Evidenciou baixo perfil de resistência a Enfuvirtida em regimes com falha utilizando esta medicação. Detectou-se pressão seletiva no HR1 do HIV-1 de pacientes em uso de Antirretroviral, independente de exposição à Enfuvirtida. Evidenciado in vitro menor formação sincicial no vírus 36D, com diminuição na atividade fusogênica, mantendo infectividade.

The thesis focus on the availibility of the time series of producer price index (PPI) in the selected countries from Eastern Europe. The PPI time series are necessary for assets price estimation. The aim of the thesis is to proove if there could be a "converter" between the PPI total and PPI for individual activity which could be used for creating time series in countries where the data are not available.

No description available.

L’intestin joue un rôle clé dans le contrôle de l’homéostasie énergétique. Les entérocytes sont des cellules polarisées qui permettent les échanges entre la lumière intestinale (membrane apicale) et le compartiment lymphatique et sanguin (membrane baso-latérale). Dans cette thèse, nous nous sommes particulièrement intéressés au contrôle par les entérocytes de deux processus liés au métabolisme des lipides et du cholestérol : l’excrétion trans-intestinale de cholestérol (TICE) et l’absorption des lipides alimentaires.Très récemment, il a été montré que l’intestin contribue à 20-30% de l’excrétion fécale du cholestérol chez la souris. Ce mécanisme, appelé TICE, implique le passage direct du cholestérol provenant de la circulation sanguine à travers les entérocytes vers les fèces. De par son caractère modulable par des substances pharmacologiques comme l’ézétimibe et les statines, le TICE représente une cible thérapeutique potentielle pour corriger les dyslipidémies athérogènes du diabétique. Cependant, les mécanismes moléculaires gouvernant le transport rétrograde du cholestérol (du pôle baso-latéral au pôle apical) dans l’entérocyte lors du TICE, sont complètement inconnus. Dans une première étude, nous avons mis en évidence la lignée entérocytaire humaine Caco-2/TC7 comme un modèle d’étude des processus trans-entérocytaires liés au TICE. Nous avons d’abord montré que suite à l’incubation avec du plasma humain dans le compartiment baso-latéral et des micelles lipidiques dans le compartiment apical, les cellules Caco-2/TC7 miment des caractéristiques du TICE in vivo. De plus, grâce à ce modèle in vitro, nous avons pu identifier les microtubules comme acteurs nécessaires au transport rétrograde du cholestérol dans l’entérocyte. Dans une seconde étude, nous nous sommes intéressés au contrôle par le récepteur nucléaire Rev-erbα de la production des chylomicrons (CM) par les entérocytes. En effet, bien qu’essentiellement vue comme la conséquence d’une clairance retardée, des données émergentes présentent la surproduction de CM par l’intestin comme un contributeur majeur de la dyslipidémie chez l’insulino-résistant. Il existe une balance, au sein de l’entérocyte, entre l’utilisation des lipides absorbés pour un stockage transitoire sous forme de gouttelettes lipidiques (GL) cytosoliques ou pour l’assemblage de lipoprotéines riches en triglycérides (LRT). Le récepteur nucléaire Rev-erbα est un répresseur transcriptionnel impliqué dans le métabolisme énergétique et le rythme circadien. Rev-erbα contrôle particulièrement le métabolisme lipidique au niveau du foie et le catabolisme des LRT. Pour cette seconde étude, une lignée Caco-2/TC7 invalidée pour Rev-erbα (sh Rev-erbα) a donc été développée par infection lentivirale et différenciée sur insert. Les résultats indiquent que suite à l’incubation avec des micelles lipidiques dans le compartiment apical, les cellules Caco-2/TC7 sh Rev-erbα sécrètent plus de LRT dans le milieu baso-latéral et stockent moins de lipides sous la forme de GL cytosoliques. De plus, la lignée Caco-2/TC7 sh Rev-erbα présente une activité lipophagique plus importante et l’inhibition de l’autophagie par la bafilomycine dans cette lignée restaure la sécrétion baso-latérale de LRT et le stockage intracellulaire de GL aux mêmes niveaux que ceux de la lignée sh control. Cette seconde étude montre donc que l’invalidation de Rev-erbα dans l’entérocyte entraîne une augmentation de la mobilisation des lipides des GL via le processus de la lipophagie résultant en une augmentation de la sécrétion de LRT. Notre hypothèse est que Rev-erbα joue un rôle clé dans le contrôle de la balance GL/LRT et donc de la triglycéridémie post-prandiale.Les deux études présentées dans cette thèse permettent une meilleure compréhension des mécanismes liés au contrôle du métabolisme lipidique par l’intestin et mettent ainsi en avant l’intestin comme une cible thérapeutique potentielle pour corriger les dyslipidémies du diabétique. / The intestine plays a key role in the control of energy homeostasis. Enterocytes, which constitute the main cellular type of intestinal epithelium (> 90%), are polarized cells allowing exchanges between intestinal lumen (apical membrane) and lymph/blood compartment (basolateral membrane). In this thesis, cholesterol and lipid metabolism control by enterocytes was studied and particularly, trans intestinal cholesterol excretion (TICE) and dietary lipid absorption.Recently, it has been estimated that intestine contributes 20-30% of fecal neutral sterol excretion in chow-fed mice. This pathway called TICE involves the direct luminal secretion of plasma-derived cholesterol by enterocytes. Moreover, TICE can be pharmacologically modulated, for instance by ezetimibe and statins and so, represents a new therapeutic target in order to prevent atherosclerosis in type 2 diabetic patients. However, at present, the molecular mechanisms behind the trans-enterocytic process of TICE are still unknown, especially the steps sustaining cholesterol entry, trafficking and efflux in enterocytes. In the first study of this thesis, we highlighted the human enterocytic Caco-2/TC7 cell line as a suitable model to study the enterocyte-related processes of TICE. We have first shown that upon basolateral incubation with human plasma and apical incubation with lipid micelles, differentiated Caco-2/TC7 cells mimic some of the in vivo TICE features. Moreover, using this model, we have identified a key role of the microtubule network in the process.In the second study of this thesis, chylomicron secretion by enterocytes and its control by the nuclear receptor Rev-erbα were investigated. Indeed, although chylomicron remnant accumulation has been associated to a delayed clearance by the liver, some recent studies show that chylomicron overproduction by the intestine is a major contributor to dyslipidemia in insulin resistant patients. Dietary lipid absorption results from a balance between transient storage in enterocytes as cytosolic lipid droplets (LD) and secretion as triglyceride-rich lipoproteins (TRL). The nuclear receptor Rev-erbα is a transcriptional repressor involved in the energy metabolism and the circadian rhythm. Particularly, Rev-erbα controls lipid metabolism in the liver and thus the catabolism of TRL. The aim of this second study was to investigate the role of Rev-erbα in intestinal lipid metabolism and particularly in TRL secretion. To study that, Caco-2/TC7 cells infected with lentivirus encoding or not a shRNA targeting Rev-erbα (sh Rev-erbα) were grown on transwells. Compared to sh control, sh Rev-erbα Caco-2/TC7 cells secrete higher amounts of micelle-derived LRT in the basolateral medium and exhibit lower quantity of neutral lipids stored as cytosolic LD, whereas the apical uptake is not different. Activation of lipophagy in sh Rev-erbα compared to sh control cells was evidenced by a higher autophagic flux and an increased colocalization of the autophagy marker LC3 with LD. Finally, autophagy inhibition with bafilomycin in sh Rev-erbα cells restores lipid secretion to the same level as in sh control cells. This second study show that Rev-erbα knock-down in enterocytes leads to a higher lipophagy-mediated remobilization of intracellular lipids and an increased TRL secretion. Our hypothesis is that Rev-erbα may be a molecular gear in the control of chylomicron secretion and a major regulator of post-prandial triglyceridemia.In conclusion, these two studies allow to better understand lipid metabolism control by the intestine: the first one by identifying the contribution of the microtubule network in enterocytes for trans-enterocytic retrograde cholesterol transport; the second one by highlighting the nuclear receptor Rev-erbα as a regulator of TRL secretion by enterocytes. These two studies point out the intestine as a potential therapeutic target to treat dyslipidemia in type 2 diabetic patients.

This study has attempted to show the overarching significance of the conquering motif in relation to discourse dynamics of the entire book of Revelation and the significance of salvific history for its syntagmatic understanding. Based on language-in-use as a whole between the model author and the model audience, syntagmatic analysis (i.e., SVU analysis) and associative analysis (i.e., sign-intertextual reading) are eclectically and concertedly utilized by means of sampling analysis. Utilizing this integrative method, the findings are as follows: (1) the interwoven network of the prologue (Rev 1:1-8) programmatically provides the paradigmatic reading strategy for understanding the key paraenetic motif in the rest of the book against the background of salvific history; (2) by summarizing the churches’ earthly prophetic roles – withdrawal and witness through martyrdom – in terms of conquering, the model author alerts his audience to the military significance of their daily actions or choices in their ordinary earthly lives through visionary communication; (3) just as the prologue preliminarily guides, the ever-forward-moving historical framework serves as an incentive device for the paraenetic-imperative in Rev 2-3 and 4-22.