To analyze the role of protein kinase B(PKB)on developmental and functional aspects of T cells, we have generated transgenic mouse lines expressing a constitutively active form of PKB (myrPKB) in early stages of T cell development.Peripheral CD4+ T cells from PKB tg mice are hyperreactive, more efficient in producing th1 and th2 cytokines and show faster and CD28 co-stimulation independent cell cycle progression.Interestingly PKB tg T cells are resistant to CsA treatment in proliferation and cytokine production.Further analysis show PKB tg CD4+ T cells have a drastically reduced nuclear translocation of NFAT proteins and this is due to a direct interaction between PKB and NFAT. To study whether the negative regulatiopn of NFATs by PKB affects T cell development, we analyzed double tg mice expressing both, a constitutively active version of calcineurin (dCam) and myrPKB. dCam tg mice have a severe block in thymocyte development at the DN3 stage.But in the dCam/PKB double tg mice this developmental block is significantly rescued.This rescue of thymocyte development by PKB is due to the expression of RAG1 and subsequent TCRb chain expression. CsA treatment of neonatal thymic lobes from dCam mice restores normal thymocyte development, indicating involvement of NFATs in the severe block in dCam thymocyte development.Confocal studies clearly established that compared to dCam DN cells there is a significant reduction in the nuclear levels of NFATc1 and NFATc3 in dCam/PKB cells.Downregulation of nuclear NFAT levels by myrPKB thus seems to be an essential parameter in dCam cells to proceed with normal differentiation. In summary, the data from PKB tg peripheral CD4+ T cells and dCam/PKB double tg thymocytes clearly establish PKB as an important modulator of T cell development and function and PKB as a novel negative regulator of NFAT activation.

Die Lamine gehören zu einer Familie von Proteinen, die als strukturelle Hauptelemente die Kernlamina ausbilden, einen wesentlichen Bestandteil der Kernhülle eukaryontischer Zellen. In Säugern exprimieren differenzierte somatische Zellen die Lamine A, C, B1 und B2. Die Kernhülle in Keimzellen unterscheidet sich in Bezug auf Struktur und Proteinzusammensetzung deutlich von der einer somatischen Zelle. So exprimieren Keimzellen Lamin B1 als einziges der somatischen Lamine und zwei kurze keimbahnspezifische Spleißvarianten, die Lamine C2 und B3. Die vorliegende Arbeit enthält eine detaillierte Analyse des Expressionsmusters und der zellulären Verteilung von Lamin B3 im Verlauf der Spermatogenese der Maus. Die Daten aus RT-PCR, Western Blot und Immunfluoreszenz belegen eindeutig, dass Lamin B3 ausschließlich in postmeiotischen Stadien während der Spermiogenese exprimiert wird. In runden Spermatiden konnte das Protein an der Kernhülle und überraschenderweise auch im Nukleoplasma nachgewiesen werden. Im weiteren Verlauf der Spermiogenese kommt es zu einer Umverteilung des Proteins, es konzentriert sich zunehmend am posterioren Pol des Spermatidenkerns. Damit ist die Lamina während der Säuger-Spermiogenese nur aus B-Typ-Laminen aufgebaut und Lamin B3 ist in Säugern das erste Beispiel für ein Lamin, das selektiv nur in postmeiotischen Stadien der Spermatogenese exprimiert wird. Die ektopische Expression von Lamin B3 in Kulturzellen führt zu einer Deformation der Zellkerne, die eine hakenförmige Gestalt annehmen. Mit Hilfe von Transfektionsexperimenten in COS-7-Zellen konnte eindeutig gezeigt werden, dass die auftretenden morphologischen Veränderungen der Kerne transfizierter Zellen auf die trunkierte zentrale Stäbchendomäne in Lamin B3 zurückzuführen ist. Darüber hinaus zeigte das Protein eine stark erhöhte Löslichkeit im Vergleich zu Lamin B2 und die Analyse transfizierter Kulturzellen mit „fluorescence recovery after photobleaching“ (FRAP) und „fluorescence loss in photobleaching“ (FLIP) ergab, dass ein erheblicher Anteil der Lamin-B3-Moleküle eine hohe Mobilität aufweist, die ebenfalls ausschließlich durch die kurze Stäbchendomäne begründet ist. Die Ergebnisse führen zu dem Schluss, dass Lamin B3 die Kernhülle in Keimzellen flexibler macht, was eine Voraussetzung für einige Vorgänge in der Spermiogenese sein könnte. Mit einem Fusionsprotein aus GST und dem 84 Aminosäuren umfassenden N-Terminus von Lamin B3 wurde über einen „Pull-Down-Assay“ nach möglichen Interaktionspartnern in Keimzellen gesucht. Mit MSY2, MSY2a und MSY4 wurden drei hoch interessante Kandidaten identifiziert. Sie gehören zu den Y-Box-Proteinen, DNA- und RNA-bindende Proteine, die bei der Speicherung und späteren Translation von mRNAs beteiligt sind, u.a. die mRNA von Protamin 1 (diese Form der Regulation von Genexpression hat in der Spermatogenese große Bedeutung). Die Interaktion von Lamin B3 mit diesen Proteinen muss noch überprüft werden, würde aber einen weiteren Bezug zwischen Kernhülle und Chromatinreorganisation in der Spermiogenese herstellen, wie es für die Kernhüllenproteine GCL und LBR bereits gezeigt werden konnte. Außerdem wäre es ein erster Hinweis auf eine funktionelle Bedeutung der N-terminalen Domäne von Lamin B3. / Lamins are members of a protein family that are the main structural elements of the nuclear envelope in eukaryotic cells. Differentiated mammalian somatic cells express lamins A, C, B1 and B2. The composition and structural organisation of the nuclear lamina in spermatogenic cells differ significantly from that of somatic cells: among the somatic lamins they only express lamin B1 but, additionally, two germ line-specific isoforms could be found, namely lamins C2 and B3. This study contains a detailed investigation of the expression pattern and localisation of lamin B3 during mouse spermatogenesis. By combining RT-PCR, immunoblotting, and immunofluorescence microscopy, it turned out, that lamin B3 is selectively expressed in postmeiotic stages during spermiogenesis. In round spermatids, lamin B3 is distributed in the nuclear periphery and, notably, also in the nucleoplasm. In the course of spermiogenesis, lamin B3 becomes redistributed as it concentrates progressively to the posterior pole of spermatid nuclei. The results show that during mammalian spermiogenesis the nuclear lamina is composed of B-type isoforms only, namely lamin B1 and the germ line-specific lamin B3. Lamin B3 is the first example of a mammalian lamin that is selectively expressed during postmeiotic stages of spermatogenesis. When ectopically expressed in culture cells, lamin B3 causes severe deformation of nuclei which adopt a hook-like configuration. Transfection experiments in COS-7 cells could prove that the observed nuclear deformations are due to the shortened rod domain of lamin B3. Cell fractionation experiments revealed that lamin B3 can be solubilised more easily than lamin B2. In addition, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) analyses of transfected COS-7 cells showed that considerable amounts of lamin B3 molecules exhibit a significantly increased mobility compared to lamin B2. The increased solubility of lamin B3 compared to lamin B2 as well as the mobility of that protein is only determined by its shortened rod domain. Taken together, these data lead to the conclusion that lamin B3 reduces the stability of the nuclear periphery, what might be an important prerequisite for some reorganisation processes during spermiogenesis to occur. Via a pull-down assay using a fusion protein containing GST and the 84 amino acid long N-terminal domain of lamin B3 a screen for interaction partners of lamin B3 was performed. With MSY2, MSY2a and MSY4 three interesting candidates were found. These proteins belong to the large family of Y-box containing proteins, which are DNA and RNA binding proteins. They are involved in storage and subsequent translation of various mRNAs, e.g. the mRNA of protamine 1 (this mechanism for regulation of gene expression is a major principle in spermatogenesis). The interaction between lamin B3 and the Y-box proteins has to be verified but it would provide an additional link between reorganisation of chromatin and the nuclear envelope as it has already been reported for other proteins of the nuclear envelope like GCL or LBR. Besides that it could be a first evidence for a specific function of the lamin B3 N-terminal domain.

Thesis advisor: Rafael Luciani / Thesis advisor: Casey Beaumier / Thesis (STL) — Boston College, 2018. / Submitted to: Boston College. School of Theology and Ministry. / Discipline: Sacred Theology.

Ph.D. thesis, Faculty of Health Sciences, University of the Witwatersrand, 2009

Thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science (Biochemistry), University of the Witwatersrand, Johannesburg, 1992 / An investigation has been made into aspects of the expression of oncogenes in normally dividing cells and in hepatoceilular carcinoma (Hee). HOC occurs commonly in Southern Africs, and thf1aetiology ·ofthis tumour lsaseccieted with hepatitis a virus (HBV) infection. c·erbA, c..mva and e-tos but not c~Ha..res mANA were elevatad in tumours and adjacent hepatic tissue from the same petiEJ;htswhen compared to normal liver. Amounts of Fos and MYQ prot~in in the liver tumour specimens were else raised. The"e was some correlation between the patients' serum a..fetoproteirt concentretlons, histological features of tumour differentiatic)t"l, c..mvc and c40s r.ixpression. expression of e-tas and c..myc has been reportec to be elevated after stimulation of cells to alvlde, ,'1$ occurs during liver r19ganeration. This was corroborated by the findin~ that c-mvc, c·fo~· and c-jun mRNA concentratlona "Jere increased it"! cultured 3T6 mouse fibroblasts following treatment with alkaline medium aa a mitogenlo stimulus. The time course of the expression of these oncogenes was similar to that reported after gro\l'l/th factor sttmulation, The H[~V X..gene ma\' be responsible for increased oncogene expression it' YCC as a result of its documented trans activating properties. This vi!'a~ gene is unusual in that it has a codon preferanc";which is similar to that of eukarvotic ceU genes. Also HBV may ha'V& evolved from ti similar ancestral virus to that giving rise to retroviruses. These ideas suggest that the HBV X·gene is a viral oncogene derived from a host homologue. Low stringency Northern brot hybridisation using a X-gene probe denlonstrated a murine transcrlpt in heart and thymus. Attempts to isolate the sequence from mouse heart and thymus eDNA libraries ware unsuccessful despite ext,~n$jve screening with sensitive probes (SP6 palymerfjsa and peR fab(':.lUed X~gen~~fragments). Conserved X~gene \ . I sequences were also used fot the desigr:Jof primers in .~.peR bas£'d method " . II aimed at isolating a mammalian sequence. No sinnificant sequsnce \\ homology was found bet\lveen the HBVI\X..gene and Ol\A ampllfle'd from \1 l! gen(llmic and eDNA I1br'srytemplate sou~\pes.The peR preducts ttppeared to have been artef.,ots of arnplWaation. ~~n'IJreto detect the hQrtll.)logous gene may have resu~ted from poo' complS,JIlentarity between the VIral ant! \\ mammalian secuencec, 1\ \\ Non..~pecific amplification is commonly enct~unter&d when u$1110 PCli'. A qtJick asvmmatrlc re·ampW~catj(ii1 method I,?ssed on eXUOSilin of an " interm.uly' hybrfdising X·gelllapfimar we! davisQ\j to confirm FICRprOdu(,ts. The l"n1ithodwas specific irlthat "ver~ single bas~ mlsmatohe$ betwsen the internal primer and tem1>late re;.,ultad in fatJut~ of dete(;tabla \tUim$f extension. / GR 2016

Master of Science / The hepatitis B virus (HBV) vaccine has contributed greatly to decreasing the HBV epidemic. However, it remains unclear why 5-10% of individuals do not mount an adequate antibody response. Previous studies have shown that genetic variation influences HBV vaccine response. Since such studies are lacking in South African individuals, we examined the associations between HBV vaccine response and genetic variation in HLA-DPB1, additional candidate genes and HLA-DPB1 expression levels in a South African cohort. HLA-DPA1 and -DPB1 allele typing was performed using Luminex technology, twenty-four candidate SNPs were typed by MassArray Analysis and HLA-DPB1 mRNA expression levels were measured by qPCR. HLA-DPB1*01:01, *04:01:01G and *09:01 and SNPs and haplotypes in IL1B, IL4, IL12B, IFNG and the HLA region were significantly associated with HBV vaccine response. A trend of lower HLA-DPB1 expression associating with better anti-HBs response was observed, although this was not significant. Response to the HBV vaccine is multi-genic but HLA-DP plays an important role. / CR2017

Les deux problèmes majeurs considérés dans cette thèse : le b-coloration problème et le graphe emballage problème. 1. Le b-coloration problème : Une coloration des sommets de G s'appelle une b-coloration si chaque classe de couleur contient au moins un sommet qui a un voisin dans toutes les autres classes de couleur. Le nombre b-chromatique b(G) de G est le plus grand entier k pour lequel G a une b-coloration avec k couleurs. EL Sahili et Kouider demandent s'il est vrai que chaque graphe d-régulier G avec le périmètre au moins 5 satisfait b(G) = d + 1. Blidia, Maffray et Zemir ont montré que la conjecture d'El Sahili et de Kouider est vraie pour d ≤ 6. En outre, la question a été résolue pour les graphes d-réguliers dans des conditions supplémentaires. Nous étudions la conjecture d'El Sahili et de Kouider en déterminant quand elle est possible et dans quelles conditions supplémentaires elle est vrai. Nous montrons que b(G) = d + 1 si G est un graphe d-régulier qui ne contient pas un cycle d'ordre 4 ni d'ordre 6. En outre, nous fournissons des conditions sur les sommets d'un graphe d-régulier G sans le cycle d'ordre 4 de sorte que b(G) = d + 1. Cabello et Jakovac ont prouvé si v(G) ≥ 2d3 - d2 + d, puis b(G) = d + 1, où G est un graphe d-régulier. Nous améliorons ce résultat en montrant que si v(G) ≥ 2d3 - 2d2 + 2d alors b(G) = d + 1 pour un graphe d-régulier G. 2. Emballage de graphe problème : Soit G un graphe d'ordre n. Considérer une permutation σ : V (G) → V (Kn), la fonction σ* : E(G) → E(Kn) telle que σ *(xy) = σ *(x) σ *(y) est la fonction induite par σ. Nous disons qu'il y a un emballage de k copies de G (dans le graphe complet Kn) s'il existe k permutations σi : V (G) → V (Kn), où i = 1, …, k, telles que σi*(E(G)) ∩ σj (E(G)) = ɸ pour i ≠ j. Un emballage de k copies d'un graphe G est appelé un k-placement de G. La puissance k d'un graphe G, noté par Gk, est un graphe avec le même ensemble de sommets que G et une arête entre deux sommets si et seulement si le distance entre ces deux sommets est au plus k. Kheddouci et al. ont prouvé que pour un arbre non-étoile T, il existe un 2-placement σ sur V (T). Nous introduisons pour la première fois le problème emballage marqué de graphe dans son graphe puissance / Two problems are considered in this thesis: the b-coloring problem and the graph packing problem. 1. The b-Coloring Problem : A b-coloring of a graph G is a proper coloring of the vertices of G such that there exists a vertex in each color class joined to at least a vertex in each other color class. The b-chromatic number of a graph G, denoted by b(G), is the maximum number t such that G admits a b-coloring with t colors. El Sahili and Kouider asked whether it is true that every d-regular graph G with girth at least 5 satisfies b(G) = d + 1. Blidia, Maffray and Zemir proved that the conjecture is true for d ≤ 6. Also, the question was solved for d-regular graphs with supplementary conditions. We study El Sahili and Kouider conjecture by determining when it is possible and under what supplementary conditions it is true. We prove that b(G) = d+1 if G is a d-regular graph containing neither a cycle of order 4 nor of order 6. Then, we provide specific conditions on the vertices of a d-regular graph G with no cycle of order 4 so that b(G) = d + 1. Cabello and Jakovac proved that if v(G) ≥ 2d3 - d2 + d, then b(G) = d + 1, where G is a d-regular graph. We improve this bound by proving that if v(G) ≥ 2d3 - 2d2 + 2d, then b(G) = d+1 for a d-regular graph G. 2. Graph Packing Problem : Graph packing problem is a classical problem in graph theory and has been extensively studied since the early 70's. Consider a permutation σ : V (G) → V (Kn), the function σ* : E(G) → E(Kn) such that σ *(xy) = σ *(x) σ *(y) is the function induced by σ. We say that there is a packing of k copies of G into the complete graph Kn if there exist k permutations σ i : V (G) → V (Kn), where i = 1,…, k, such that σ*i (E(G)) ∩ σ*j (E(G)) = ɸ for I ≠ j. A packing of k copies of a graph G will be called a k-placement of G. The kth power Gk of a graph G is the supergraph of G formed by adding an edge between all pairs of vertices of G with distance at most k. Kheddouci et al. proved that for any non-star tree T there exists a 2-placement σ on V (T). We introduce a new variant of graph packing problem, called the labeled packing of a graph into its power graph

Les lymphomes B primitifs du médiastin (LBPMs) constituent une entité anatomo-clinique particulière au sein des lymphomes diffus à grandes cellules B. Les analyses du transcriptome des LBPMs ont montré une forte expression des gènes induits par l’IL-4 ou l’IL-13 et des effecteurs de cette voie de signalisation. L’objectif de ce travail a été d’étudier la voie IL-4/IL-13 dans les LBPMs. Dans une 1ere partie, nous avons montré que le facteur de signalisation et de transcription 6 (STAT6) est constitutivement phosphorylé et possède une activité de liaison à l’ADN dans les lignées dérivées de LBPM (MedB1, Karpas1106). Le STAT6 phosphorylé (-P) est présent dans les noyaux des cellules tumorales de LBPMs (73% des cas). Cette activation est due en partie à l’activité de la kinase JAK2 et aux altérations du gène régulant négativement cette voie de signalisation, SOCS1. Dans une 2eme partie, nous avons étudié le rôle de STAT6 dans la physiopathologie de ces lymphomes en inhibant son expression par un siRNA dans les lignées. Nous avons montré une diminution de la prolifération et une augmentation de la mort cellulaire, ainsi qu’une diminution du taux du mRNA Bcl-xL dans les cellules MedB1. Nous avons observé une corrélation entre l’accumulation nucléaire de STAT6-P et l’expression cytoplasmique de Bcl-xL dans les cellules tumorales. Enfin, nous avons mis récemment en évidence des mutations du domaine de liaison à l’ADN de STAT6, dans 35% des LBPMs. L’étude des mécanismes oncogéniques activés dans la voie IL-4/IL-13 devrait permettre de comprendre le dysfonctionnement cellulaire à l’origine des LBPMs et pourrait aussi donner de nouvelles cibles pour le diagnostic et la thérapie / Primary mediastinal large B-cell lymphomas (PMBCLs) are a particular anatomo-clinical entity among diffuse large B-cell lymphomas (DLBCLs). The transcriptome analyses of PMBCLs showed high expression of genes activated by IL-4 or IL-13 and effectors of this signaling pathway. The objective of this work was study the IL-4 / IL-13 signaling pathway in PMBCLs. In a 1st part, we demonstrated that signal transducer and activator of transcription 6 (STAT6) is constitutively phosphorylated and exhibits DNA binding activity in PMBCL derived cell lines (MedB1, Karpas1106). This phosphorylated STAT6 (P-) is present in nuclei of PMBCL neoplastic cells (73 % of cases). This activation is partially due to the activity of JAK2 kinase and to the alteration of a gene which regulates negatively this signalling pathway, SOCS1. In a 2nd part, we studied the STAT6 role in physiopathology of these lymphomas by inhibiting its expression with a siRNA in cell lines. We showed proliferation decrease and cell death increase, as well as diminution of Bcl-xL mRNA in MedB1 cells. We observed a correlation between P-STAT6 nuclear accumulation and Bcl-xL cytoplasmic expression in neoplastic cells. In a last part, we recently demonstrate mutations of STAT6 DNA binding domain, in 35 % of PMBCLs. The study of oncogenic mechanisms activated in IL-4/IL-13 signaling pathway could allow to understand the cellular dysfunction at the origin of the PMBCLs and could also identify new targets for the diagnostic and the therapy

Byskolor lever i ständig ovisshet gällande om de ska få fortsätta sin verksamhet eller behöva läggas ned. I en liten skola med ett lågt elevantal placeras eleverna oftast i åldersintegrerade klasser, vilket kan ge en större känsla av närhet och gemenskap. Men elever och lärare i den mindre skolan upplever också, likt vilken annan skola som helst, svårigheter och utmaningar inom verksamheten och undervisningen. Lärarna, som är en viktig del av byskolorna, kan ibland antas ha en lättare arbetsbörda än lärare som arbetar på större skolor med fler elever. Men faktum är att byskolornas lärare ständigt påverkas av flera utomstående faktorer som inverkar på deras undervisning. Denna studie syftar därmed till att ta del av sju byskolelärares upplevelser och tankar om hur deras undervisning och arbete påverkas av ett antal ramfaktorer, det vill säga faktorer utanför lärarens direkta kontroll som påverkar undervisningen. Därför har kvalitativa intervjuer genomförts och innehållet har analyserats utifrån Urban Dahllöfs och Ulf P. Lundgrens ramfaktorteori enligt ett särskilt analysupplägg som skapats i enlighet med studien. De ramfaktorer som undersöks är tid, personal, gruppstorlek, lokaler, ekonomiska resurser, skolledning och organisation, närsamhället samt kulturer och vårdnadshavare. Resultatet av studien visar att ramfaktorerna tid, personal, gruppstorlek, ekonomiska resurser samt kulturer och vårdnadshavare påverkar byskolelärarnas undervisning och arbetssituation mest, både positivt och negativt. Resultatet visar även att ramfaktorerna tid, ekonomi och skolledning och organisation samverkar på så sätt att de ekonomiska beslut som fattas av skolledningen påverkar hur mycket personal som kan organiseras inom skolans verksamhet, vilket i sin tur påverkar hur mycket tid läraren upplever sig kunna ägna åt undervisning och andra arbetsuppgifter. I jämförelsen mellan de två byskolorna framkom fler likheter än skillnader i hur lärarna uppfattar sin undervisning och arbetssituation. Två av de likheter som framkom var exempelvis upplevd tidsbrist, främst vid planering, och att den nationella lärarbristen inte upplevs påverka byskolorna som undersöks. En av de skillnader som framkommer är byskolornas skilda perspektiv på utmaningar som gruppstorleken kan medföra där den ena byskolan har ett elevperspektiv, medan den andra har ett perspektiv med fokus på åldersintegrering i klasserna. Avslutningsvis visar resultatet att de ramfaktorer som undersöks påverkar lärarnas undervisning och arbetssituation i stor utsträckning.

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