The p53 tumour suppressor protein has a highly complex pattern of regulation at transcriptional and posttranslationallevels. The discovery of p53 isoforms has added another layer of complexity to the mechanisms thatregulate p53 functions. Indeed, p53 is expressed as 12 isoforms that differ in their N- and C-terminus due toalternative splicing, promoter or codon initiation usage. So far, there is limited understanding of the patterns ofexpression and of the functions of each of these isoforms.In this Thesis, we have focused on the two major p53 N-terminal isoforms, Δ40p53 and Δ133p53. We haveanalysed their patterns of interactions with the full-length p53 and we have investigated whether their expressioncould be deregulated in melanoma, a cancer type in which TP53 mutations are rare. Our results show that Δ40p53 can modulate p53 function with a bi-phasic effect, acting as a repressor or activator of p53 to control itslevels and activity. Moreover, we demonstrate that the internal P2 promoter produces Δ133p53 and is regulatedby p53 in response to genotoxic stress, identifying a novel auto-regulatory loop by which p53 may control theexpression of an isoform acting as an inhibitor of p53 activities. Finally, we show that mRNAs encoding Nterminalisoforms are often over-expressed in highly metastatic melanoma when compared to non-invasiveforms, suggesting that N-terminal isoforms contribute to functionally inactivate p53. Thus, we propose that Δ40p53 and Δ133p53 modulate p53 functions within dynamic fluctuations of aprotein network. Hence, p53 isoforms may have a major role in basal p53 activities as well as in the functionalinactivation of p53 in cancer cells.

O presente trabalho visa contribuir para o esclarecimento da seqüência de eventos intracelulares produzidos pelas PKCs a e e, na modulação do pHi, via NHE1. Os estudos foram realizados em células MDCK e as medidas de pHi efetuadas por microscopia de fluorescência. A expressão das PKCs a e e, bem como do NHE1 foi investigada por western blot, utilizando anticorpos específicos para cada proteína. Os estudos foram realizados na situação controle ou na vigência de PMA ou ANG II, AVP e /ou inibidores específicos para cada receptor hormonal ou isoforma de PKC. Nossos resultados indicam que PMA (10-7 M) estimula a recuperação do pHi, por modular a atividade das PKCs a e e. ANG II e AVP em concentrações fisiológicas estimulam a recuperação do pHi após sobrecarga ácida, concomitante com o aumento da fosforilação da PKC a. Em concentração elevada, ambos hormônios não alteram estes parâmetros. O efeito de ANG II ou de AVP depende da interação de cada hormônio com receptores específicos para modular as vias de sinalização celular envolvidas com o aumento dos níveis de diacilglicerol, cálcio citosólico e AMPc. / The purpose of this work was to investigate the signaling events of PKCs a e e on the NHE1 activity. The effect of phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II) or arginine vasopressin (AVP) on the intracellular pH (pHi) was investigated in MDCK cells by using the fluorescence microscopy and fluorescent probe BCECF/AM. The NHE1 or PKCs a e e expression was examined by western blot and specific antibodies. Our results indicate that PMA (10-7 M) or low concentration of ANG II and AVP induced a significant increase of pH recovery rate and PKC a expression, after intracellular acidification with NH4Cl pulse. ANG II or AVP did not change the PKC a expression. However, in right concentration, both hormones did not change these parameter. In conclusion, the effect of ANG II and AVP on NHE1 activity, depend of specifics membrane receptors and cellular signaling of intracellular calcium, DAG and PKCs a and e.

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres, the individual contractile units. Mutations in many of the genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue, and in humans, often lead to myopathic conditions like cardiomyopathies and muscular dystrophies, which affect a considerable number of people the world over. As more information regarding causative mutations becomes available, it becomes imperative to explore mechanisms of muscle development, maintenance and pathology. In striated muscles, contraction is regulated by the thin filament-specific tropomyosin (Tm) – troponin (Tn) complex (Ca2+-binding troponin-C, inhibitory troponin-I and tropomyosin-binding troponin-T). These troponin subunits are present in 1:1:1 ratio on thin filaments, with 1 Tm-Tn complex present on every 7th actin molecule. This stoichiometry is tightly regulated, and disturbances have been associated with functional defects. Each of these proteins has multiple isoforms, whose expression is controlled both spatially and temporally. The expression of specific combination of isoforms confers specific contractile properties to each muscle subtype. Drosophila melanogaster has been a preferred model of choice to study various aspects of muscle development for decades. In this study, the Indirect Flight Muscles (IFMs) of Drosophila have been used to investigate developmental and functional roles of two temporally regulated isoforms of a vital structural and regulatory component of the sarcomere – Troponin T (TnT). On a larger scale, whole genome expression profiles of mutants that are null for major myofbrillar proteins have also been discussed. IFMs serve as an excellent model system to address these questions, owing to the extreme ease of genetic manipulability in this system, and high degree of homology between mammalian and Dipteran cytoskeletal proteins. Chapter 1 covers basics of muscle biology, and the role of TnT in muscle contraction. Phenomena responsible for generating diversity in genes encoding muscle proteins – alternative splicing and isoform switching – have also been discussed. These mechanisms are highly conserved, as are patterns of TnT splicing and isoform expression across phyla. Mutations leading to altered splicing patterns lead to myopathic conditions, and the importance of model systems to study muscle biology has been emphasized. The advantages of studying Drosophila IFMs and a comprehensive overview of IFM development has been covered. The resources and experimental tools used have been described in Chapter 2. Two isoforms of TnT are alternatively spliced in the Drosophila thorax – one containing alternative exon 10a (expressed in adult IFMs and jump muscle); and one containing alternative exon 10b (expressed in pupae and newly eclosed flies). These exons are spliced in a mutually exclusive manner, and defects in splicing have been reported to cause uncontrolled, auto-destructive contractions. In Chapter 3, a splice mutant of TnT, up1, has been discussed, with respect to its developmental profile. Transgenic rescue experiments with two separate isoforms demonstrate rescue at the structural as well as functional level. Transgenic over-expression, however, leads to functional abnormalities, highlighting the importance of stoichiometry in multi-protein complexes. In Chapter 4, molecular signals that bring about the developmentally regulated TnT isoform switch are discussed. A splicing factor, Muscleblind, has been transgenically knocked down in normal and mutant IFMs to study effects on muscle function. Chapter 5 looks at whole genome transcriptional alterations in muscles null for either actin or myosin. All significant expression changes have been classified into categories based on different biological processes, and an attempt to differentiate generic muscle responses from filament-specific responses has been made. In conclusion, the studies have highlighted the importance of TnT isoform switching, and that extended expression of a pupal stage-specific isoform can partially compensate for loss of the adult isoform. Also, in the absence of major myofibrillar proteins, stress response pathways like heat shock response and protein degradation pathways are activated, along with a subset of metabolic responses that are unique to the thin or thick filament systems.

A desnutrição acomete 925 milhões de pessoas em todo o mundo, independentemente da idade e classe social, os mais acometidos são indivíduos hospitalizados, crianças e idosos. A desnutrição causa alterações fisiológicas em diversos tecidos. O tecido hematopoiético é afetado na desnutrição protéica, por ser um tecido de elevada e constante necessidade de proteínas, levando a alterações hematológicas como anemia e leucopenia. Estudos do nosso laboratório têm demonstrado in vivo, alterações do microambiente hematopoiético, em camundongos submetidos à desnutrição protéica, bem como hipoplasia medular, mudanças quantitativas da matriz extracelular (MEC) como o aumento do deposito de fibronectina na região subendosteal (local da fixação das células tronco/ progenitoras hematopoiéticas), alterações do ciclo celular das células tronco/ progenitoras hematopoiéticas e alteração na expressão de VLA5; principal integrina na interação das células a fibronectina. Sendo assim propõe-se neste projeto avaliar possíveis isoformas da molécula de fibronectina, para melhor compreender o seu papel biológico no ciclo celular as células tronco/ progenitoras hematopoiéticas em um modelo de desnutrição protéica. Para isso foram utilizados camundongos C57BLI/6J machos, adultos, mantidos em gaioleiros metabólicos, separados em dois grupos. O grupo controle recebeu uma ração normoproteíca com 12% de proteína e o grupo desnutrido, uma ração hipoprotéica com 2% de proteína, num período de 5 semanas. Após este período os animais foram sacrificados para avaliação hematológica, celularidade da medula óssea, quantificação e pesquisa de isoformas da fibronectina, análise do perfil protéico no estroma medular em culturas de longa duração bem como a análise do estabelecimento do estroma medular em culturas de longa duração. Os animais desnutridos apresentaram uma menor celularidade e uma diminuição significativa de células jovens na medula óssea. O estroma medular estabelecido em culturas de longa duração dos animais do grupo desnutrido apresentaram uma menor confluência em relação ao grupo controle. Também foi observado nas culturas de longa duração um aumento significativo da fibronectina no 28° dia de cultura, mas com uma diminuição da fibronectina no 35° dia, porém com uma menor quantidade de região EDA nos dois períodos analisados (sítio de ligação das integrinas a fibronectina) quando comparados aos animais do grupo controle. A pesquisa de isoformas de fibronectina no estroma medular, por meio de RT-PCR, revelou que tanto os animais do grupo controle quanto os animais do grupo desnutrido apresentaram diferentes isoformas de fibronectina, porém não foi possível fazer uma análise quantitativa das regiões de splicing alternativo. O perfil protéico das culturas de longa duração analisado por meio de eletroforese bidimensional demonstrou que os animais do grupo desnutrido possuem um perfil protéico diferente dos animais do grupo controle, também foi observado uma diferença do perfil protéico entre os 28° e 35° dias de cultura. Portanto a alteração quantitativa da molécula de fibronectina e da região EDA, bem como a presença de diferentes isoformas de fibronectina, juntamente com as alterações do perfil protéico podem ser devido a um aumento e ou degradação das proteínas de matriz extracelular. E estas alterações podem ser responsáveis pela hipoplasia medular e alteração do ciclo celular das células tronco/ progenitoras hematopoiéticas e estromais, talvez pela menor interação com as integrinas, sendo esta interação fundamental para a modulação de diversas funções celulares tais como proliferação e diferenciação e para a regulação do remodelamento da matriz extracelular. Mas ainda se faz necessário a quantificação das regiões de splicing alternativo, seqüênciamento das proteínas da matriz extracelular e a identificação das possíveis metalaproteinases presentes no estroma medular para melhor elucidar as funções da fibronectina e outras proteínas da matriz extracelular na manutenção da hemopoese. / Malnutrition affects 925 million people worldwide, regardless of age and social class, the most affected individuals are hospitalized, children and elderly.Malnutrition causes physiological changes in various tissues.The hematopoietic tissue is affected in protein malnutrition, because it is a tissue of high and constant need of proteins, leading to hematological abnormalities such as anemia and leucopenia. Studies from our laboratory have demonstrated in vivo changes in the hematopoietic microenvironment in mice submitted to protein malnutrition, and bone marrow hypoplasia, quantitative changes of the extracellular matrix (ECM) as the increase in deposit fibronectin in the region subendosteal (site of attachment of the cells stem / progenitor), changes in cell cycle of stem cells / progenitor and changes in the expression of VLA5; main integrin interaction of cells to fibronectin. Therefore this project proposes to evaluate possible isoforms of fibronectin molecule, to better understand its biological role in cell cycle stem cells / progenitor in a model of protein malnutrition. Mice were used for this C57BLI/6J male adults were kept in metabolic gaioleiros, separated into two groups. The control group received a ration normoproteíca with 12% protein and the malnourished group, a low protein diet with 2% protein over a period of 5 weeks. After this period the animals were sacrificed for hematological evaluation, bone marrow cellularity, and quantification of fibronectin isoforms research, analysis of protein profiles in marrow stroma in long-term cultures and analysis of the establishment of bone marrow stroma in long-term cultures. The malnourished animals showed a lower cellularity and a significant decrease of young cells in the bone marrow.The bone marrow stromal cultures established in long-term animal malnourished group had a lower convergence in the control group. It was also observed in cultures long term a significant increase in fibronectin on the 28th day of culture, but with a decrease in fibronectin 35 th day, but with a smaller amount of EDA region in both periods analyzed (binding site of integrins to fibronectin) compared to the control group.The survey of isoforms of fibronectin in bone marrow stroma, by RT-PCR revealed that both control animals and animals in the malnourished group presented different isoforms of fibronectin, but it was not possible to make a quantitative analysis of the regions of alternative splicing.The protein profile of long-term cultures analyzed using two-dimensional electrophoresis showed that animals of the malnourished group have a protein profile different from the control group was also observed a difference in protein profile between 28° and 35° day of culture. Therefore, the quantitative change of the molecule fibronectin and the region of EDA, as well as the presence of different isoforms of fibronectin, together with changes in protein profile may be due to an increase, or degradation of extracellular matrix proteins. And these changes may be responsible for bone marrow hypoplasia and alteration of the cell cycle of stem cells / progenitor and stromal cells; perhaps because of less interaction with integrins, this interaction is essential for the modulation of various cellular functions such as proliferation and differentiation and to regulation of extracellular matrix remodeling. But it is still necessary to quantify the areas of alternative splicing, sequencing of the extracellular matrix proteins and identification of possible metalaproteinases present in bone marrow stroma to better elucidate the roles of fibronectin and other extracellular matrix proteins in maintaining hemopoese.

Les molécules HLA de class Ib ou nonclassiques, HLA-G, -E et -F, sont impliqués dans la régulation de la tolérance immunologique. En particulier, les polymorphismes génétiques ainsi que l’expression différentielle de HLA-G sont corrélés à l’évolution clinique des pathologies inflammatoires et des greffes d’organe. Notre premier travail a permis de montrer que la structure génétique de HLA-G était conservée dans des populations au patrimoine génétique différent (Mali et Sud de la France) et que, les haplotypes résultants (UTR), étaient corrélés à l’expression différentielle d’HLA-G soluble. Dans une deuxième étude on a pu montrer l’association différentielle des haplotypes HLA-G UTR avec certain allèles HLA-E, -A, -H, -G et -F suggérant une interaction privilégiée et/ou un effet tolérant synergique entre ces molécules. Une troisième étude a mis en évidence que deux haplotypes HLA-G UTR étaient corrélé à une évolution clinique péjorative dans la mucoviscidose et dans la transplantation pulmonaire. En fin, on a pu montrer une expression différentielle des isoformes de HLA-G dans les cellules épithéliales bronchiques (CEB), redifférencié in vitro, de témoins sains et patients atteints d’asthme avec une expression diminuée chez les asthmatiques. Finalement, l’ensemble de ces données suggèrent que la molécule HLA-G peut être un biomarqueur génétique et/ou sérologique attrayant et une cible thérapeutique potentielle via la modulation de son effet tolérogène dans un contexte de pathologies inflammatoires ou de conflit immunologique, tel que la transplantation d’organe et la transfusion. / HLA nonclassical class Ib genes HLA-G, -E and -F are involved in managing immune tolerance. Particularly for HLA-G, both its genetic polymorphism and its expression are correlated to clinical outcome in different pathologies, particularly in inflammatory disease and organ transplantation. In our first work we have shown that HLA-G genetic structure was conserved in distant healthy populations (Mali and South France) and that, the resulting haplotypes (UTR) were correlated to sHLA-G differential expression. In another study we observed a differential association between HLA-G UTR haplotypes and specific HLA-E, -A, -H, -F alleles that might reflect a privileged interaction and/or a synergistic tolerant effect between these molecules In a third study we showed that two different HLA UTR haplotypes were respectively associated with a worse evolution of cystic fibrosis and an impaired survival in lung transplant recipients. Afterwards, the analysis HLA-G isoforms expression in normal, mild and severe asthmatic human bronchial epithelial cells redifferentiated in vitro showed that the level of these transcripts was significantly reduced in asthmatics compared to controls. In conclusion, these results suggest that HLA-G molecule might be an attractive genetic and/or serologic biomarker and a potential therapeutic target, via the modulation of its tolerogenic effect, in specific allogenic and inflammatory contexts, such as solid organ transplantation or blood transfusion.

Tau ist ein Mikrotubuli-assoziiertes Protein, das die Polymerisation von Tubulin fördert und die Mikrotubuli stabilisiert. Folglich wird angenommen, dass Tau essentiell für die neuronale Morphogenese ist, vor allem für die Axonenausbildung und -aufrechterhaltung. Mittels tangentieller Nissl-gefärbter Schnitte von Mäusegehirnen konnte in der vorliegenden Arbeit gezeigt werden, dass Tau-knockout Mäuse die regelhafte thalamokortikale Barthaar-projektion („Barrel“ Konfiguration) entwickeln. Der Einfluss von Tau auf die Entstehung von Dendriten wurde anhand von Golgi-gefärbten Präparaten untersucht. Die Sholl-Analyse der gefärbten CA1-Pyramidenzellen zeigte, dass die Komplexität apikaler Dendriten durch das Fehlen von Tau reduziert wurde, während die Basaldendriten unbeeinflusst blieben. Das Tau-Protein scheint demzufolge unwesentlich für die Entstehung von axonalen Verbindungen im embryonalen Gehirn zu sein, ist aber beteiligt an der Steuerung des dendritischen Verzweigungsmusters. Ferner wurde beobachtet, dass sowohl die adulte Neurogenese, als auch die Mikrotubuli-Stabilität in den Apikal- und Basaldendriten und die Synapsen von dem Fehlen des Tau-Proteins unbeeinflusst blieben. In primären Zellkulturen aus dem Kleinhirn von Tau-knockout und Tau-wildtyp Mäusen konnten zwischen den zwei Genotypen keine signifikanten Unterschiede in der Länge oder im Verzweigungsmuster der Dendriten und der Axone von Körnerzellen beobachtet werden. Die Untersuchung der Effekte einzelner Tau-Isoformen auf die Morphologie von N2A-Zellen zeigte, dass es Unterschiede sowohl zwischen Tau-defizienten und Tau-positiven Zellen, als auch zwischen Zellen mit den verschiedenen Tau-Isoformen gibt. Das Tau-Protein übt demnach in vivo einen wichtigen Einfluss auf die Morphologie der Nervenzellen und besonders der Dendriten aus, welcher in vitro weiter analysiert wurde.:Abkürzungsverzeichnis 1 Einleitung 1 1.1 Das Mikrotubuli-assoziierte Protein Tau 1 1.2 Bedeutung des Tau-Proteins beim Menschen: Tauopathien 3 1.3 Bedeutung des Tau-Proteins beim Menschen: Mikrodeletion des MAPT-Lokus 4 1.4 Ergebnisse aus bisherigen Studien mit Tau-knockout Tieren 6 1.5 Aufgabenstellung 7 2 Material und Methoden 9 2.1 Material 9 2.1.1 Versuchstiere 9 2.1.2 Chemikalien 9 2.1.3 Häufig verwendete Lösungen 10 2.1.4 Geräte 10 2.2 Histologie 11 2.2.1 Fixierung 11 2.2.2 Golgi-Einzelschnittimprägnierung 11 2.2.3 Gefrierschnitte 11 2.2.4 Nissl-Färbung 12 2.2.5 Immunhistochemische Markierungen 13 2.3 Morphometrie 15 2.3.1 Sholl-Analyse 15 2.3.2 Volumenbestimmung 16 2.3.3 Zellzahl (Neurogenese) 17 2.3.4 Synapsenzahl 17 2.4 Proteinbiochemie 19 2.4.1 Proben 19 2.4.2 SDS-Polyacrylamid-Gelelektrophorese 19 2.4.3 Western Blot 20 2.4.4 Immundetektion am Western Blot 21 2.5 Transfektion von Nervenzellen in primärer Zellkultur 25 2.5.1 Primäre Zellkultur 25 2.5.2 Transfektion von primären Zellkulturen 25 2.5.3 Morphometrische Analyse von Körnerzellen des Kleinhirns 26 2.6 Klonierung von Tau-Protein-Isoformen 27 2.6.1 Klonierungsstrategie zur Herstellung eines pIRES-DsRed-Tau Vektors 27 2.6.2 Agarose-Gelelektrophorese 30 2.6.3 Gelextraktion der verschiedenen Tau-Isoform-Sequenzen 30 2.6.4 Herstellung chemisch kompetenter E.coli Zellen 31 2.6.5 Chemische Transformation kompetenter E. coli Zellen 32 2.6.6 Animpfen 32 2.6.7 Plasmid-DNA Purifikation aus 15ml Medium („Miniprep”) 32 2.6.8 Schneiden mit Restriktionsendonukleasen 33 2.6.9 Plasmidpräparation aus 100 ml Medium („Midiprep“) 33 2.6.10 Transfektion von N2A-Zellen mit pIRESRed-Tau 34 2.6.11 Rekonstruktion der transfizierten N2A-Zellen 35 2.7 Statistische Auswertung 36 3 Ergebnisse 37 3.1 Thalamokorticale Projektionen 37 3.2 Komplexität der Dendriten von CA1-Pyramidenzellen 37 3.3 Adulte Neurogenese im Hippocampus 41 3.4 Volumen des Hippocampus 43 3.5 Glia 45 3.6 Synapsen 46 3.7 Stabilität der Mikrotubuli 48 3.8 Mikrotubuli-assoziierte Proteine 50 3.9 Entwicklung in vitro 52 3.9.1 Primärkulturen 52 3.9.2 N2A-Zellkultur 54 4 Diskussion 58 4.1 Diskussion der Methoden 58 4.1.1 Herstellung von Tau-knockout Mäusen 58 4.1.2 Golgi-Einzelschnittimprägnierung und die dreidimensionale Zellrekonstruktion 59 4.1.3 Neurogenese 60 4.1.4 Volumenbestimmung von Hippocampus und Gyrus dentatus 61 4.1.5 Synaptische Marker 61 4.1.6 Western Blot 62 4.1.7 Transfektion von primären Zellkulturen 62 4.1.8 Transfektion von N2A-Zellen mit humanen Tau-Isoformen 63 4.2 Vergleich mit bekannten Daten aus der Forschungsliteratur 64 4.2.1 Axonogenese 64 4.2.2 Dendritogenese 64 4.2.3 Mikrotubuli-assoziierte Proteine und Mikrotubuli-Stabilität 67 4.2.4 Neurogenese 67 4.2.5 Synaptogenese 68 4.2.6 Rolle der einzelnen Isoformen 68 4.3 Bedeutung für die Medizin 71 4.4 Fazit 72 5 Zusammenfassung 73 6 Literaturverzeichnis 76 Posterpräsentationen 88 Danksagung 89 Erklärung über die eigenständige Abfassung der Arbeit 90 Lebenslauf 91

We have studied aspirin as a potential chemopreventive for colorectal cancer, completing Phase I studies on aspirin pharmacology and potential biomarker assays (prostaglandins, PGE2 and PGF(2α) and cyclooxygenase modulation) in normal human subjects. These studies have determined the optimal dose of aspirin for future Phase IIa and IIb chemopreventive trials in high-risk cohorts of patients for colon cancer. Aspirin's effects on rectal prostaglandins are prolonged, detectable even after aspirin and its metabolite are removed from the plasma. Aspirin-mediated inhibition of prostaglandin production in the human rectal epithelium may be related to direct suppression of cyclooxygenase transcription and not to enzyme inactivation by acetylation. A systematic method to monitor adherence (self- report, telephone contact, pill count, and microelectronic monitoring) has been established for future trials. Strategies to improve recruitment of high-risk cohorts have been developed. Phase IIa non-randomized studies with aspirin at 81 mg in high-risk cohorts (resected Duke's A colon cancer, Duke's C colon cancer treated with adjuvant therapy and disease-free at 5 years, history of colon adenomas > 1 cm, two or more first-degree relatives with colon cancer, and familial adenomatous polyposis and hereditary non-polyposis colorectal cancer syndromes) are currently being conducted for surrogate end- point biomarker (prostaglandins, cyclooxygenase, cellular mucins, and proliferation) modulation.

C-reactive protein (CRP) is synthesized in hepatocytes. The serum concentration of CRP increases dramatically during the acute phase response. In human hepatoma Hep3B cells, maximal CRP expression occurs in cells treated with the combination of IL-6 and IL-1β. IL-6 induces transcription of the CRP gene and IL-1β synergistically enhances the effects of IL-6. We investigated the role of IL-6-activated transcription factor STAT3, also known as STAT3α, in inducing CRP expression since we identified four consensus STAT3-binding sites centered at positions - 72, - 108, - 134 and - 164 on the CRP promoter. It has been shown previously that STAT3 binds to the site at - 108 and induces CRP expression. We found that STAT3 also bound to the other three sites, and several STAT3-containing complexes were formed at each site, suggesting the presence of STAT3 isoforms and additional transcription factors in the complexes. Mutation of the STAT3 sites at - 108, - 134 or - 164 resulted in decreased CRP expression in response to IL-6 and IL-1β treatment, although the synergy between IL-6 and IL-1β was not affected by the mutations. The STAT3 site at - 72 could not be investigated employing mutagenesis. We also found that IL-6 activated two isoforms of STAT3 in Hep3B cells: STAT3α which contains both a DNA-binding domain and a transactivation domain and STAT3β which contains only the DNA-binding domain. Taken together, these findings raise the possibility that IL-6 not only induces CRP expression but also regulates the induction of CRP expression by activating STAT3 isoforms and by utilizing all four STAT3 sites.

Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting proliferation and survival. While the survival and proliferative functions of IL-7 are well established, the identities of IL-7 signaling components in pathways other than JAK/STAT, that accomplish these tasks remain poorly defined. To this end, we used IL-7 dependent T-cells to examine those components necessary for cell growth and survival. Our studies revealed two novel signal transducers of the IL-7 growth signal: BimL and JunD. IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. After a bioinformatics survey to reveal possible JunD-regulated genes activated early in the IL-7 signaling cascade, our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism, including Pim-1. Pim-1, an IL-7 induced protein, was inhibited upon JNK or JunD inhibition. Our hypothesis that JunD positively regulated proliferation was confirmed when the proliferation of primary CD8+ T-cells cultured with IL-7 was impaired upon treatment with JunD siRNA. These results show that the IL-7 signal is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth through the expression of metabolic factors like HXKII and Pim-1. When metabolic activities are inhibited, cells undergo autophagy, or cell scavenging, to provide essential nutrients. Pro-apoptotic Bim was evaluated for its involvement in autophagy. Bim is a BH3-only member of the Bcl-2 family that contributes to T-cell death. Partial rescue of iv T-cells occurs when Bim and the interleukin-7 receptor are deleted, implicating Bim in IL-7- deprived T-cell apoptosis. Alternative splicing results in three different isoforms: BimEL, BimL, and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on IL-7 for growth, were used. Loss of Bim in IL-7-deprived T-cells delayed apoptosis, but blocked the degradative phase of autophagy. The conversion of LC3-I to LC3-II was observed in Bim-deficient T-cells, but p62, which is degraded in autolysosomes, accumulated. To explain this, BimL, was found to support acidification of lysosomes associated with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon IL-7 withdrawal only in Bimcontaining T-cells, indicating that in these cells autophagy was protective. IL-7 dependent Tcells lacking Bim were insensitive to inhibition of autophagy or lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosometracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, associated with intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of functional autolysosomes during autophagy

Reduced neo-adipogenesis and dysfunctional lipid-overloaded adipocytes are hallmarks of hypertrophic obesity linked to insulin resistance. Identifying molecular features of hypertrophic adipocytes requires appropriate in vitro models. We describe the generation of a model of human hypertrophic-like adipocytes directly comparable to normal adipose cells and the pathologic evolution toward hypertrophic state. We generate in vitro hypertrophic cells from mature adipocytes, differentiated from human mesenchymal stem cells. Combining optical, confocal, and transmission electron microscopy with mRNA/protein quantification, we characterize this cellular model, confirming specific alterations also in subcutaneous adipose tissue. Specifically, we report the generation and morphological/molecular characterization of human normal and hypertrophic-like adipocytes. The latter displays altered morphology and unbalance between canonical and dominant negative (PPARGΔ5) transcripts of PPARG, paralleled by reduced expression of PPARγ targets, including GLUT4. Furthermore, the unbalance of PPARγ isoforms associates with GLUT4 down-regulation in subcutaneous adipose tissue of individuals with overweight/obesity or impaired glucose tolerance/type 2 diabetes, but not with normal weight or glucose tolerance. In conclusion, the hypertrophic-like cells described herein are an innovative tool for studying molecular dysfunctions in hypertrophic obesity and the unbalance between PPARγ isoforms associates with down-regulation of GLUT4 and other PPARγ targets, representing a new hallmark of hypertrophic adipocytes.