La fibrose pulmonaire idiopathique (FPI) et le syndrome de détresse respiratoire aiguë (SDRA) de l’adulte constituent des affections sévères et létales du poumon profond associées à une hypoxie alvéolaire, pour lesquelles les ressources thérapeutiques sont limitées. La thérapie cellulaire utilisant des cellules souches mésenchymateuses humaines (CSMh) est actuellement envisagée dans ces pathologies, mais les mécanismes d’action des CSMh restent à élucider. Nous avons testé in vitro l’hypothèse selon laquelle les CSMh pourraient exercer un effet cytoprotecteur paracrine sur les cellules épithéliales alvéolaires (CEA) soumises à l’hypoxie.Dans une première étude, nous avons montré que l’exposition aiguë de CSMh dérivées de moelle osseuse à un environnement inflammatoire (traitement par cytomix constitué de TNFα, d’Interféron Ƴ et d’Interleukine 1β) et hypoxique (3% O₂) mimant le microenvironnement alvéolaire du SDRA ne modifiait pas le phénotype ou la viabilité des cellules mais induisait des modifications de leur secretome, notamment de facteurs connus pour réguler le transport trans-épithélial alvéolaire de sodium (Na) et la réabsorption de l’oedème alvéolaire : IL1-ra, PGE2 et KGF. Le milieu de culture des CSMh (MC-CSMh), qu’il soit obtenu en normoxie ou en hypoxie-cytomix, prévenait l’augmentation de la perméabilité épithéliale des CEA primaires de rat cultivées sur support semi-perméable et exposées à l’hypoxie et au cytomix. Le MC-CSMh provenant de CSMh cultivées en normoxie prévenait l’inhibition du transport transépithélial alvéolaire de Na en restaurant l’expression à la surface de la sous-unité α du canal sodique épithélial ENaC, tandis que le MC-CSMh obtenu sous hypoxie-cytomix n’avait pas d’effet protecteur. La sécrétion de Keratinocyte Growth Factor (KGF) par les CSMh était indispensable à leur effet protecteur.Dans une seconde étude, nous avons montré qu’une exposition prolongée à l’hypoxie telle que rencontrée au cours de la FPI induisait des modifications phénotypiques des CEA de rat évocatrices de transition épithélio-mésenchymateuse (TEM) avec perte progressive d’expression des marqueurs épithéliaux (TTF1, AQP5, ZO-1 et E-Cadhérine) couplée à l’apparition tardive de marqueurs mésenchymateux (α -SMA et Vimentine). Ces modifications phénotypiques s’accompagnaient de l’expression à la phase initiale de l’hypoxie de facteurs de transcription impliqués dans la TEM (SNAI1, TWIST1 et ZEB1) ou induits par l’hypoxie (HIF-1α et HIF-2α et de protéines induisant la TEM (TGFβ1 et CTGF). La co-culture des CEA avec des CSMh en fond de puits prévenait les modifications phénotypiques induites par l’hypoxie ainsi que l’expression des facteurs pro-TEM TWIST1, ZEB1, TGFβ1 et CTGF. Là encore, le KGF était au moins en partie responsable des effets protecteurs des CSMh.Ces deux études indiquent que les CSMh sont susceptibles d’exercer des effets cytoprotecteurs paracrines vis-à-vis des CEA soumises à l’hypoxie aiguë ou prolongée, en limitant d’une part les effets de délétères de l’hypoxie sur les propriétés de transport vectoriel de Na et en prévenant d’autre part les modifications phénotypiques évocatrices de TEM. La sécrétion par les CSMh de KGF, facteur de croissance épithélial bien connu pour ses effets bénéfiques sur les CEA, explique en partie les effets protecteurs paracrines des CSMh. Nos résultats suggèrent que les effets cytoprotecteurs des CSMh vis-à-vis des CEA pourraient contribuer aux effets bénéfiques des CSMh observés in vivo dans différents modèles animaux d’agressions alvéolaires aiguës ou à tendance fibrosante. / Non communiqué

Background: Liver injury is the most common severe adverse effect of first-line anti-tuberculosis therapy (ATT). Nacetylcysteine (NAC) has efficacy in patients with paracetamol toxicity, and may be of benefit in liver injury due to other causes, such as ATT-induced liver injury (AT-DILI). Rechallenge of first line ATT after liver injury is usually attempted and may result in recurrence of liver injury. Alanine transaminase (ALT) is the biomarker currently used in AT-DILI diagnosis. MicroRNA-122 (miR-122) is a sensitive biomarker for liver injury due to paracetamol, but data on utility as a biomarker for ATDILI are limited. Methods: We conducted a randomized double-blind placebo-controlled trial of intravenous NAC in adult hospitalized participants with AT-DILI. Primary endpoint was time to ALT < 100 U/L; secondary endpoints included length of hospital stay and 8-week mortality. We described outcomes of ATT rechallenge following AT-DILI. We quantified miR-122 and ALT concentrations before and after infusion of NAC/placebo, and explored the effect of NAC on miR-122. Results We enrolled 102 participants with AT-DILI, 53 randomized to NAC and 49 to placebo. Mean age was 38 (SD±10) years, 58 (57%) were female and 89 (87%) were HIV positive. Median time to ALT

There are a vast number of transcription factors that regulate plant growth and development. The NAC gene superfamily is one of the largest families of transcription factors in the plant kingdom. NAC gene expression profiles using Affymetrix ATH1 gene chips were obtained for different plant organs: heart embryo, mature embryo, leaf, root and flower. NAC gene expression profiles proved to be very complex, except for one NAC gene detected only in floral tissue, At1g61110. At1g61110 was shown to be specifically expressed in the anther tapetum of Arabidospis; therefore, its name was changed to TAPNAC. TAPNAC became the focus of our studies. We identified a tapnac T-DNA knockout (KO) line, SALK_069450. A molecular phenotype was observed. Several oligopeptide, sugar and metal transporters were differentially expressed. Coincidentally, a wheat NAC gene, named TaNAM-B1 for its high sequence similarity to ATNAM, TAPNAC and At3g15510 was found to be involved in nutrient remobilization. PHOSPHOLIPASE Dα1 (PLDα1) was also found to be down-regulated in the tapnac KO. PLDα1 is an enzyme which hydrolyzes phospholipids that are part of tapetal cell membranes and tapetal lipid bodies. Once these tapetal cell structures are disrupted, the secretion of the compounds that form part of the pollen coat (i.e. proteins, flavonoids and lipids) into the anther locule is facilitated. Promoter deletion analysis using a GUS reporter and later GUS immuno-localization confirmed the findings of Wellmer and others. TAPNAC is a tapetal specific gene. The cis-regulatory sequence that enhances tapetal expression in the TAPNAC promoter was identified. The consensus motif TCGTGT increased tapetal expression of a GUS reporter gene, only when flanked by the TAPNAC minimal promoter region (-217 bp to +51 bp). In summary, TAPNAC transcription factor has been characterized and data indicates that it could play a role in nutrient remobilization from the tapetum to the pollen grains, particularly during late floral stages. Also, important information on tapetal specifcation cis-regulatory sequences was discovered. The consensus motif TCGTGT, present in TAPNAC promoter, was shown to enhance tapetal expression of a GUS reporter gene.

There are a vast number of transcription factors that regulate plant growth and development. The NAC gene superfamily is one of the largest families of transcription factors in the plant kingdom. NAC gene expression profiles using Affymetrix ATH1 gene chips were obtained for different plant organs: heart embryo, mature embryo, leaf, root and flower. NAC gene expression profiles proved to be very complex, except for one NAC gene detected only in floral tissue, At1g61110. At1g61110 was shown to be specifically expressed in the anther tapetum of Arabidospis; therefore, its name was changed to TAPNAC. TAPNAC became the focus of our studies. We identified a tapnac T-DNA knockout (KO) line, SALK_069450. A molecular phenotype was observed. Several oligopeptide, sugar and metal transporters were differentially expressed. Coincidentally, a wheat NAC gene, named TaNAM-B1 for its high sequence similarity to ATNAM, TAPNAC and At3g15510 was found to be involved in nutrient remobilization. PHOSPHOLIPASE Dα1 (PLDα1) was also found to be down-regulated in the tapnac KO. PLDα1 is an enzyme which hydrolyzes phospholipids that are part of tapetal cell membranes and tapetal lipid bodies. Once these tapetal cell structures are disrupted, the secretion of the compounds that form part of the pollen coat (i.e. proteins, flavonoids and lipids) into the anther locule is facilitated. Promoter deletion analysis using a GUS reporter and later GUS immuno-localization confirmed the findings of Wellmer and others. TAPNAC is a tapetal specific gene. The cis-regulatory sequence that enhances tapetal expression in the TAPNAC promoter was identified. The consensus motif TCGTGT increased tapetal expression of a GUS reporter gene, only when flanked by the TAPNAC minimal promoter region (-217 bp to +51 bp). In summary, TAPNAC transcription factor has been characterized and data indicates that it could play a role in nutrient remobilization from the tapetum to the pollen grains, particularly during late floral stages. Also, important information on tapetal specifcation cis-regulatory sequences was discovered. The consensus motif TCGTGT, present in TAPNAC promoter, was shown to enhance tapetal expression of a GUS reporter gene.

Myogenesis is a complex and tightly regulated process, the end result of which is the formation of multinucleated myofibers. Muscle formation requires the precise expression of multiple myogenic regulatory factors (MRFs), whose expression regulates transcription of muscle specific proteins. Alteration in the expression of a muscle specific gene or protein ultimately results in muscle dysfunction. The inappropriate expression of factors that control muscle development may also be a contributing factor in Rhabdomyosarcoma, a pediatric cancer that accounts for most soft tissue sarcomas that arise in children. Previous studies suggest that the regulation of αNAC, BTF3, and skNAC are vital for normal myogenesis. Alterations in these factors results in retarded development and severe disorganization of muscle tissue. We hypothesized that αNAC, BTF3, and skNAC are imperative transcription factors whose dysregulation significantly alters the kinetics of C2C12 cell (murine skeletal muscle cells) myogenesis. We have shown that erroneous expression of these transcription factors is detrimental to myogenesis. In addition, we have shown that these transcription factors are recruited to muscle specific gene promoters during the myogenic differentiation program and the expression of αNAC, BTF3, and skNAC may potentiate the expression of the MRFs. Together, our experiments suggest that the expression of αNAC, BTF3, and skNAC are essential for the normal progression of myogenesis.

Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the molecular defects present in these cells that block myogenesis. We have found MEF2D is absent in RMS cell lines representing both major subtypes of RMS and primary cells derived from an embryonal RMS mice model. We have shown that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find MEF2D cannot bind to muscle specific gene promoters. Exogenous expression of MEF2D activates muscle specific luciferase constructs, upregulates p21 expression and increases muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility, anchorage independent growth in vitro, and tumor growth in vivo by xenograft assay. We also have found MEF2C is deregulated in rhabdomyosarcoma with the aberrant alternative splicing. We have shown that exon α in MEF2C is aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 being highly downregulated in RMS cells compared with normal myoblasts. We find that MEF2Cα1 is the ubiquitously expressed isoform which exhibits no myogenic activity and that MEF2Cα2, the muscle specific MEF2C isoform, is required for efficient differentiation. Compared with MEF2Cα2, MEF2Cα1 more strongly interacts with and recruits HDAC5 to myogenic gene promoters to repress muscle specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increases myogenic activity and promotes differentiation in RMS cells. We have also identified a serine protein kinase, SRPK3, which is downregulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage independent growth of RMS cells. The NAC complex performs many diverse biological functions, and the deregulation of its subunits has been correlated with many cancers. We sought to understand the function of the NAC complex in normal myogenesis and tumor progression in rhabdomyosarcoma cells. We found that the muscle specific subunit of the NAC complex, skNAC, which is the alternatively spliced isoform of NACα, was induced in normal cells and downregulated in RMS cells, while BTF3, also known as NACβ, was induced in normal cells and severely downregulated in RMS cells. We also showed that skNAC associated with muscle specific promoters together with BTF3 in differentiated normal cells, and this association was dependent on the expression of BTF3. We further investigated the involvement of skNAC in RMS progression. We found that the muscle specific expressed methyltransferase Smyd1 was nuclear localized in RMS cells and its interaction partner skNAC was switched with corepressors (HDAC1 and TBX2). We also confirmed the expression of skNAC was regulated by the splicing factor kinase SRPK3 and overexpression of SPRK3 induced skNAC expression and muscle differentiation in RMS cells. We also confirmed the overexpression of BTF3 in patient RMS tumors and depletion of BTF3 induced apoptosis in RMS cells and decrease RMS cell survival. BTF3 depletion also sensitized TRAIL induced cell apoptosis in RMS cells. However, BTF3 played a different role in normal cells. Deletion of BTF3 in C2C12 cells does not induce cell apoptosis, which suggests BTF3 functions as an anti-apoptosis factor in RMS cells and could be used as a cancer specific therapeutic target in RMS cells.

Syftet med detta arbete var att jämföra de två mjukvarorna Packetfence och Cisco Identity Services Engine. Målet är att bilda en förståelse kring hur den här typen av mjukvaror kan implementeras i ett nätverk av olika storlekar och även en insikt över hur detta kan minska den administrativa belastningen. Den här typen av system kan även användas för att centralt styra åtkomst till ett företags resurser och kan säkra upp olika typ av enheter och program. Det genomfördes en installation för att upptäcka skillnader mellan systemen, därefter konfigurerades de så likt varandra som möjligt för att upptäcka om det finns några olikheter i den här delen av mjukvaran. Rapporten tar upp grundläggande information kring de tjänster som systemen innehåller och beskriver även de steg som krävs för att utföra konfiguration. Rapporten redovisar den metod gruppen arbetat efter under dessa veckor för att uppnå ett tillfredsställande resultat för uppdragsgivaren. Resultatet presenterar arbetet gruppen utfört under arbetet och visar hur de olika systemen kan konfigureras för att uppnå ett säkert nätverk hos ett företag. Diskussionen tar upp att det är viktigt att vara flexibel i sin planering för att arbeta runt uppkomna problem. / The purpose of this study was to compare two security softwares, Packetfence and Cisco Identity Services Engin. The goal was to provide an understanding of how this kind of software can be implemented in a network of different sizes and also an insight into how this can reduce the administrative burden. This type of system can also be used to centrally control access to a company's resources and can secure different types of devices and applications. An installation was completed to detect differences between the systems, then they were configured as similarly as possible to detect if there were any differences in this part of the software. The report summarizes basic information about the services that the systems contain and also describes the steps required to perform configuration. The report presents the methodology the group worked for these weeks to achieve a satisfactory result for the client. The result presents the work the group completed during the study and shows how the various systems can be configured to achieve a secure network for a company. The discussion states that it is important to be flexible in its planning to work around problems that arise.

The leishmaniase is a serious public health problem in the world, demanding effective measures for its control and treatment. The patient's immune response in visceral leishmaniasis has a fundamental role in the prognosis of this infection inflammation, which can be fatal if untreated. So that chemotherapy can result in healing, it is important that the immune system is able and necessary stimulus to the development of appropriate cytokine profile and disease resistance to fighting agent.Thus, the purpose of this study was to evaluate the levels serum of patients on the adjuvant action of N-acetyl-L-cysteine (NAC) in the chemotherapy of human visceral leishmaniasis, made with pentavalent antimony. From an intervention study, clinical trial, blinded, randomized, 60 patients were investigated at the University Hospital with the diagnosis of visceral leishmaniasis. These patients were divided into two groups of 30: Study Group - which made use of pentavalent antimony standard dose supplemented with N-acetyl-L-cysteine (NAC) and the control group - which only used a standard dose of antimony. This study was approved by the ethics committee in search of UFS under CAAE 0151.0.107.000-07 number. The patients selected randomly and were assessed for clinical and laboratory parameters. To evaluate the rate of the patients were performed serum levels of cytokines IFN-γ, TNF-α, IL-10, IL-12p40 and of molecule sCD40L before, during and after treatment using the Luminex 100 analyzer. The results suggest that the addition of NAC to conventional therapy improves the immune response of patients, especially in lowering serum levels of IL-10, IL-12p40 and elevated sDC40L. This study showed that although both groups had healed up on the last day of treatment, the study group developed a response indicative of early healing, sugesting that the NAC has acted in a manner adjunct to pentavalent antimony, being also proposed the use of the sCD40L molecule as a prognostic marker for visceral leishmaniasis. / Para que a quimioterapia possa resultar em cura, é importante que o sistema imune tenha condições e estímulos necessários ao desenvolvimento do perfil de citocinas adequado à resistência à doença e ao combate do agente etiológico. Estudos demonstram que o N-acetil-L-cisteína (NAC), uma substância antioxidante, contriubi para a imunoregulação de determinadas doenças. Assim, o objetivo deste trabalho foi avaliar os níveis séricos dos pacientes quanto à ação adjuvante do N-acetil-L-cisteína (NAC) na quimioterapia da leishmaniose visceral humana, feita com antimônio pentavalente. A partir de um estudo de intervenção, tipo ensaio clínico, cego, randomizado, foram investigados 60 pacientes do Hospital Universitário com diagnóstico positivo para leishmaniose visceral aguda. Esses pacientes foram distribuídos em dois grupos de 30: Grupo Estudo que fez uso do antimônio pentavalente dose padrão complementado com o N-acetil-L-cisteína (NAC) e o Grupo Controle - que fez uso apenas do antimônio dose padrão. Este trabalho foi aprovado pelo comitê de ética em pesquisa da UFS sob número CAAE 0151.0.107.000-07. Os pacientes selecionados e randomizados foram avaliados quanto aos parâmetros laboratoriais. Para avaliar as taxas de citocinas dos pacientes, foram realizadas dosagens dos níveis séricos de IFN-γ, TNF-α, IL-10, IL-12p40 e IL-12p70, sendo também dosada a molécula sCD40L antes, durante e após o tratamento, com uso do analisador LUMINEX 100. Os resultados sugerem que a adição do NAC ao tratamento convencional melhora as taxas das citocinas dos pacientes, sobretudo a diminuição dos níveis séricos de IL-10, IL-12p40 e na elevação de sDC40L. Este estudo mostrou que, apesar de ambos os grupos apresentarem-se curados no último dia do tratamento, o grupo estudo desenvolveu uma resposta indicativa de cura mais precocemente, sugerindo que o NAC tenha atuado de forma adjuvante ao antimônio pentavalente, sendo proposto ainda o uso da molecula sCD40L como um marcador prognóstico para a leishmaniose visceral.

La sénescence constitue la dernière étape du cycle de vie de certains organes des plantes. Elle permet leur dégradation tout en réallouant les constituants des tissus sénescents vers d’autres organes. Dans le contexte de la nodulation symbiotique fixatrice d’azote entre certaines plantes légumineuses et des bactéries rhizobia, un processus de sénescence a été décrit. Cependant, les connaissances sur les mécanismes de régulation de la sénescence des nodosités symbiotiques sont limitées. Au sein du laboratoire, le facteur de transcription MtNAC969 a été identifié comme un régulateur de la sénescence des nodosités. L'objectif de cette thèse est d'identifier et de caractériser de nouveaux régulateurs de la sénescence de l'organe symbiotique. Nous avons développé : (i) une approche visant à identifier des facteurs de transcription corégulés avec MtNAC969 ou avec une cystéine protéase MtCP6 utilisée comme marqueur de la sénescence des nodosités ; et (ii), une approche avec "à priori" se focalisant sur la fonction des différentes voies de signalisation des cytokinines. Cette thèse a permis d'identifier deux facteurs de transcription, MtbHLH107 et MtNAC009 et de décrypter le rôle des cytokinines dans la sénescence des nodosités. Cette thèse a permis d'identifier d'une part, deux nouveaux gènes potentiellement régulateurs de la sénescence nodositaire, MtbHLH107 et MtNAC009; et d’autre partde décrypter le rôle des cytokinines dans la sénescence de cet organe symbiotique. / Senescence is the last step of plant organ lifespan and allows their degradation in order to remobilize components from senescent tissues toward others organs. In the nitrogen fixing symbiosis nodulation occurring between legume plants and rhizobia bacteria, a senescence process has been described. However, limited knowledge about regulatory systems controlling senescence in the symbiotic nodule is available. In the laboratory, the MtNAC969 transcription factor was identified as a regulator of nodule senescence. The aim of this PhD project is to identify and characterize new regulatory factors involved in nodule senescence. We developed two independent approaches : (i) the identification of genes coregulated with MtNAC969 or a cystein protease MtCP6 used as nodule senescence marker ; and (ii), targeted approach focused on the role of cytokinin signaling pathways in nodule senescence. This project allowed us to identify two regulator transcription factors, MtbHLH107 and MtNAC009 ; and to decipher the cytokinin role in the senescence of the symbiotic organ. This PhD thesis allowed us to identify two new potential regulators of nodule senescence, MtbHLH107 and MtNAC009; and to decipher the role of cytokinins in the senescence of this symbiotic organ.

Hunger is a problem faced by many people all over the world, and as the population grows, so does the need for food such as cereals. Because of this, the need for food with higher protein and nutrient content will be increasingly important. NAM-B1, a NAC-protein gene in wheat, has been shown to control the grain protein content and nutrient values, as well as senescence. In barley, two orthologous genes have been found, HvNAM1 and HvNAM2. This study focuses on Nordic barley accessions and how haplotypes of HvNAM1 and HvNAM2 correlate to the grain protein content (GPC) and nutrient content. No correlations between the different haplotypes of the HvNAM genes and the nutrient content and GPC were found. No differences in nutrient content and GPC were found in Nordic accessions originating from Sweden, Norway, Finland, or Denmark, nor were differences found for improvements status groups or for six-row barley and two-row barley. The Nordic accessions were shown to generally have high GPC when compared to control groups Karl and Lewis. However, even if the results of this study indicate that the HvNAM genes do not have major effects on the nutrient contents or GPC, Nordic barley might still be good material for plant improvement. Other factors such as other genes, environmental effects, and gene expression should therefore be investigated.