The present study examined adverse effects of sunlight exposure (mimicked by intradermal cis-urocanic acid, cUCA) on local and systemic immune responses, with or without co-exposure to the immunotoxic insecticide permethrin. A single exposure to cUCA caused diminished splenic macrophage phagocytosis that was persistent up to 30 days post-exposure. Five-day exposure to cUCA subtly increased splenocyte proliferation in response to the T cell mitogen Concanavalin A. Four-week exposure to cUCA caused increased splenic lymphocyte cellularity, thymic hypocellularity, and enhanced hydrogen peroxide production by splenic leukocytes. Single exposure to topical permethrin resulted in decreased thymic and splenic weight and cellularity, and inhibited antibody production by splenic B cells. cUCA worsened the negative effect of permethrin on both thymic weight and cellularity, and depressed splenocyte blastogenesis, hydrogen peroxide production, and antibody production. Five-day exposure to either cUCA or permethrin also caused persistent decreased contact hypersensitivity responses, an effect that became more than additive when the chemicals were administered concurrently. Defects in antigen processing and presentation by cutaneous Langerhans cells were evaluated as possible contributing mechanisms to the cutaneous immunosuppression, using mice with deleted genes. Vehicle-exposed IFNg knockout mice displayed approximately a 22.1% depression in the ear swelling response as compared to control C57BL/6N mice, suggesting that this cytokine may be required for mounting a control-level hypersensitivity response. Ear swelling in cUCA-exposed IFNg knockout mice displayed a 21.4% depressed response as compared to cUCA-exposed wild-type C57BL/6N mice, again suggesting that IFNg is an important cytokine in the contact hypersensitivity (CH) response. TNFaR knockout mice exposed to cUCA displayed 33.9% greater ear swelling than cUCA-exposed wild-type C57BL/6N mice, suggesting that increased TNFa may be involved in inhibited CH by cUCA. TNFaR knockout mice exposed to permethrin displayed 33.9% greater ear swelling than permethrin-exposed C57BL/6N mice, suggesting that increased TNFa may also be involved in inhibited CH by permethrin. C57BL/6N mice exposed to cUCA + permethrin displayed severe reduction of the CH response to 8.7% of the control level. IFNg knockout mice exposed to permethrin + cUCA showed essentially identical depression of the CH response as IFNg knockout mice exposed to either permethrin or cUCA alone. These results suggest that IFNg is required for the greater than additive immunotoxic effect that occurred when these two agents were co-administered. TNFaR knockout mice exposed to cUCA + permethrin displayed 8.7 fold greater ear swelling than similarly exposed C57BL/6N mice, again suggesting that increased TNFa is involved in inhibited CH by both cUCA and permethrin. / Ph. D.

This thesis entitled “Design and Access to Disallowed and Unusual Conformers of Peptides in Crystals and in Solution: Structural Consequences of the Imidate and Thioimidate Isosteres for the Peptide bond” is divided into eight chapters. Imidate Modification The range of disallowed dihedral angles for residues in peptides is governed by their local steric and electrostatic clashes. Rare tolerances of violations in these angles are attributed to distortions in both local and global bond characteristics of the peptides. Discerning the origins of such disallowed angles and the consequent distortions in body of the peptides is essential, for a complete understanding of the protein fold, to improve the crystal database for validation of rare but acceptable residue conformations and for validation and improvement of theoretical models that evaluate the interactions that define the Ramachandran space. Unlike for the ordered secondary structures such as β-sheets α-helices and β-turns currently there are no models for residues constrained in disallowed folds. We reasoned that residues may be stabilized in disallowed folds in peptides if a neighbouring group and The range of disallowed dihedral angles ( , ψ) for residues in peptides is governed by their hence its local unfavorable clashes can be selectively modified to a motif that favors such space Steric clashes of the type H•••Xi±n involving the backbone amide hydrogen (H) contribute to ~60% of disallowed ,ψspace. Conversion of an amide to an imidate (A→I) will remove the corresponding H and hence the steric clashes related to it in peptides. Importantly, this will introduce an H-bond acceptor N (of imidate) in place of an H-bond donor NH (of amide), which will allow formation of unusual H-bonding interactions between the imidate N and the neighbouring Hs and hence constrain residues in otherwise inaccessible dihedral angles. The conversion of A→I is challenging owing to difficulties in selective synthesis, stability and purification of the imidate motif. We address all these concerns by the selective conversion of a backbone amide in peptides to the relatively stable cyclic 5,6-dihydro-4H-1,3-oxazine imidate isostere, by an intra¬molecular nucleophilic cyclo-O-alkylation reaction. Chapter 1:SectionB: Autocyclo-O-Alkylation of N-(3-Bromopropyl)amides into 2-Alkyl-5,6-Dihydro-4H-1,3-Oxazinehydrobromides We are describing the reactivity of N-(3-bromopropyl)amides that are precursors for 2-peptide-5,6-dihydro-4H-1,3-oxazine. The starting materials, 3-bromopropylamides, were synthesized in good yields by coupling the corresponding carboxylic acids and anhydrides with 3-bromopropylaminehydrobromide using standard mixed anhydride peptide coupling protocol. N-(3-bromopropyl)-acylamides are unstable during the isolation. Time-dependent 1H NMR of all the acetamides revealed that they underwent clean auto-cyclization to form the corresponding 2-alkyl-5,6-dihydro-4H-1,3-oxazine hydrobromides following first order rate. The salts were easily isolated in high purity by trituration of the mixtures with ether. The t1/2 of autocycliation of decreased upon increase in electron density on the R-carbon. Notably, the tert-butyl substituent cyclized significantly faster than acetamide which have enolizable hydrogens at the R-carbon. Thus, the cyclization rate is affected predominantly by the inductive effect of the R-carbon substituents. The formamide remained stable and unchanged due to the poor electron-donating ability of hydrogen. Chapter 1: Section C: Intramolecular Hydrogen Bond Assistance Improves Autocyclization in N-(3-Bromopropyl)amides The autocyclisation do not go to 100% completion. This is because the released hydrobromic acid quenches the nucleophilicity of amide carbonyl oxygen. In order to scavenge hydro bromic acid, we used 1 equivalent of DIEA base is acting only acid scavenger which conformed by unaffecting the reaction rate upon increasing equivalents of DIEA. We found that autocyclisation of N-(3-bromopropyl)amides rates in peptides involved in intramolecular backbone H-bonding interactions improve the autocyclization rates significantly than unstructured (random coil) peptides. Even with in the ordered structures the rate depends on the proximity of H-bonding distances as well as the H-bond acceptor strength. Half-life of autocyclisation in various peptide secondary structures are determined from time variant 1H NMR studies performed at 60 mM peptide concentration in CDCl3 at 32 oC. Chapter 2: Section A: Synthesis and Isolation of 5,6 Dihydro-4H-1,3-Oxazine Containing Peptidomimetics We have introduced 5,6-Dihydro-4H-1,3-oxazine as the imidate isostere at C-terminus of a number of peptides through NaH (base) mediated intramolecular cyclo-O-alkylation of N-(3-bromopropyl)amides. The amide to imidate (A→I) modification reaction is faster (1-5.5 h), Exhibiting no electronic and structural effects under these conditions. The side product NaBr can be easily separated by filtration through celite. No side products were observed and there is no need of further purification to get pure 1,3-oxazines in quantitative yields in all the peptidomimetics. Using this synthetic protocol we have synthesised a variety of 1,3-oxazine containing peptide analogues including aliphatic, branched aliphatic, polar side chains and larger peptides. We show that the retention of configuration at Cαof peptides during the base mediated cyclo-O-alkylation reaction. that the C5i.structures are more populated at Aib due to operation of The Thorpe-Ingold effect. The strength of hydrogen bonding interaction in C5i structure is similar to those of the highly buried backbone hydrogen bonding interaction found in the middle of a model 310-helical peptide as indicated by DMSO titration experiments. Chapter 3: Section A: Consequences of "Disallowed" Conformations on Constrained β-Turn Peptides Here we are describe the consequence of disallowed conformations the on a C-terminus of a type-II β-turn. We choose stereochemically constrained Type-II β-turn Pro-Aib dipeptide analogue which is the ideal model to mirror the structural effects of introducing the A→I modification at the C-terminus. The imidate containing peptidomimetic crystallised in dichloromethane and hexane mixture. Analysis of crystal structure revealed that Aib NH is involved in 3-centered H-bonding interactions with the N of oxazine and N of proline. This constrains Aib in a conformation that is natively disallowed to it. The (, ) angles of Aib residue fall in the (180,0) region which is strictly disallowed for natural peptides due to steric clashes involving the back bone amide N-H. More importantly there are two C•••O interactions which are accomidated in the crystal structures. Both oxygen‟s were place in staggered orientation of the Pro oxygen (OPro) between the two β-CH3 groups of Aib, which is again strictly disallowed in natural peptides due to strong C•••Oi-1 hard sphere clashes. However no vdW space violations are observed between these atoms. Chapter 3: Section B: Conformational Effects of “Disallowed Aib on a 310-Helical peptide In order to investigate the origins and consequences of “disallowed” conformations on a folded helical peptide body, the conformationally stable peptide sequence Boc-Leu1-Aib2-Ala3-Leu4-Aib5-Ala6-Phe7-Aib8-OMe (310-helix-OMe)was chosen which is known to adopt 310-helix in crystal structure. Analyses of the ROESY spectra, DMSO titration experiments, and CD spectra of 310-helix-OMe and its Oxa analogue reveal that their solution conformations are identical to those of the crystal structure of 310-helix-OMeSix sequential i+3→i intramolecular backbone H-bonds stabilize the 310-helical peptide fold in both peptides in solvents of varying polarity. The N-terminal and central segments of the helical molecules are quite structurally rigid and are not deformed. The presence of the disallowed Aib*8 residue in Oxa analogue has a clear conformational effect mainly on the residue Phe7. It looks like the Phe7 amide H is involved in shielding, the Aib*8 amide H through a bifurcated hydrogen bonding interactions with the nitrogen of oxazine and carbonyl oxygen of Ala6 residue. Maximum structural distortion effect on the registers closest to the putative imidate bond. Our results show that “disallowed folds need not denature order in the peptide fold”. Chapter 4: Synthetic Methods for Introducing the A → I Modification anywhere along the Peptide Chain Here we describe the incorporation of imidate isostere in the middle of any peptide sequence. In Oxa selectivity is towards 5-exo-cyclo-O-alkylation in 1 : 4. In Thi it is towards 6-exo-cyclo-S-alkylation in 3 : 1 ratio. This is because of better nucleophile of sulphur (S). We saw that Thi is stable to peptide coupling, N-and C-terminus protection, deprotection conditions and can be easily incorporated in middle of peptide. Chapter 5: Section A: Cis-trans Isomerism in the X-Pro Peptide Bond In tertiary amides like X-Pro peptides having high propensity to access cis conformations due to similar environment in both cis and trans around the Cof X. X-Pro peptide bonds, constrained in s-cis conformations are prevalently found in the turn regions of peptides with the residue „X‟ in the i+1position and Pro at the i+2position of the β¬turn. These types of turns are termed as the type VI β-turns. For better understanding of the molecular recognition at specific cis X-Pro peptide bonds in biological events, the structure and dynamics of various constrained cis X-Pro peptide bond analogues with varying steric and electronic perturbations have been studied. Many models have been developed for stabilizing cis conformer by perturbation of molecular recognition surface of proline by employing steric and electronic interaction. In biological functions proline molecular recognition surface and cis X-Pro peptide bond more important. There is need of novel method for stabilizing X-Pro peptide bond in cis conformer without modifying the pyrolidine ring in proline. Chapter 5: Section B: Biasing the cis/trans Equilibrium in X Pro Peptides using Reverse ni → ni-1 * Interactions Here we present our findings that peptidomimetics containing the 5,6-dihydro-4H-1,3¬oxazine (Oxa) and 5,6-dihydro-4H-1,3-thiazine (Thi) functional groups at the C-terminus of Pro selectively and remotely stabilize the s-cis rotamers of the preceding pyrrolyl (Xaa-Pro) 3° amide bonds, while conserving these recognition elements. The cis/trans equilibrium of Xaa-Pro peptide bonds is shifted significantly in favor of the satirically disfavored cis isomers in these peptidomimetics (upto ~90%). We also provide evidence for the influence of an unusual n→ πi-1 * interaction in the cis, and the n)(n repulsion in the trans, conformers of these molecules to beat the origin of such the origin of such cis stabilization. Chapter 6: Steric Interactions in the cis Piv-Pro Peptide Bond The inaccessibility of cis Piv-Pro rotamer in any peptide is believed to be because the steric clashes between substituents on CX and CPro are unavoidable in this conformer. Here we access the cisPiv-Pro conformer in crystal structure of Piv-Pro-Aib-OMe and that it is sufficiently flexible to undergo bond distortions and avoid all steric clashes between substituents on CPiv and CPro . It is however the unavoidable distortions in the dihedral angle of the Prothe cisPiv-Proconformer. The cisPiv -Pro conformer is indeed accessible, if such distortions are accommodated in the peptide. Chapter 7: Steric and Electronic Interactions in the cis Isomer of Piv-Pro Peptide Bond in Solution We have studied the electronic and steric interactions and the conformational equilibrium in two sets of homologous peptides, X-Pro-Aib-OMe (which contain Aib) and X-Pro-NH-Me, where X is acetyl, propionyl, isobutyryl and pivaloyl, in solvents of varying polarities consisting of carbontetrachloride, chloroform or dimethylsulfoxide, by means of their 1H and 13C-NMR, and FT-IR spectra. Formation of n * interactions between the carbonyls that flank the Aib residue, influences the alleviation of steric interactions that are believed to preclude access to the cis conformer of the Piv-Pro peptide bond. The cis Piv-Pro conformer is observable in the Aib containing peptides, at ambient conditions by FT-IR and at temperatures as low as 273 K by NMR. We estimate that the steric interactions contribute < 0.5 kcal/mol to the conformational free energy of X-Pro peptide bond isomerism, irrespective of the steric bulk on the acyl (X) group. The relative strengths of intramolecular hydrogen bonding interactions involving the X-Pro peptide motif in different conformers of these peptides influence their relative conformational stabilities. Chapter 8: Remote Effect of Oxa and Thi Functional Groups on cis-trans Isomerism at X-Pro Peptide Bonds The C5a interaction at Pro residue occurs in the transition states for the intramolecular acid catalysis of cis → trans isomerization in peptidyl prolyl isomerases (PPIs) and enables the decrease in transition energy barrier for the isomerization process. We show that the NPro….HAib interactions in C5a structures can be remotely effected in order to control in equilibrium constant values of the cis/trans isomerism (Kc/t) in X-Pro¬Aib-Oxa and Thi containing peptides. By this method we observed improvement in Kc/t values from 0.18 in esters to 0.56 in Thi and 0.66 in Oxa containing peptides. Analyses of the ROESY spectra, DMSO titration experiments, variable temperature experiments and FT-IR spectra of R-CO-Pro-Aib-Oxa (R = Me, Et, iPr) and its Thi analogues reveals that both interactions (C5a and C5i) are persistent in cis and trans conformers of this peptidomimetics. (for structural formula pl. see the abstract.)

Utifrån dagens litteratur kring projektbaserade organisationer (PBO) kan tre problem identifieras: 1) motsägande resultat gällande om organisationsformen PBO främjar innovation, 2) överdrivet fokus på teknisk innovation i dessa sammanhang samt 3) bristfällig systematik i dessa typer av studier. Av denna anledning har vi genomfört en kvantitativ studie för att bidra med mer systematik i debatten kring PBO och innovation. Vi valde också att studera en annan typ av innovation i PBO-sammanhang, nämligen managementinnovation (MI). Studien syftar till att identifiera om det föreligger ett samband mellan MI och organisationsfaktorerna centralisering, samarbete och kommunikation, överkapacitet samt formalisering. Utöver detta skall studien identifiera om det föreligger något samband mellan den formella organisationsstrukturen PBO och MI, samt om sambanden mellan organisationsfaktorerna och MI är starkare eller svagare i en PBO-kontext. Studien genomfördes i Sverige med hjälp av en enkätundersökning. Urvalet uppgick till 500 företag vilka blev identifierade genom ett slumpmässigt stratifierat urval. Svarsfrekvensen uppgick till 192 företag vars svar sedan analyserades i statistikprogrammet SPSS genom regressionsanalyser. Slutsatsen av studien blev att vi på en femprocentig signifikansnivå bekräftade en av femton hypoteser, nämligen H8c. Hypotesen beskriver att sambandet mellan personalöverkapacitet och MI är starkare i en PBO än i andra organisationstyper. Utöver detta kunde vi bekräfta hypotes 9b på en tioprocentig signifikansnivå. Denna hypotes behandlade formalisering 2 och innebar att sambandet mellan formalisering 2 och MI är starkare i andra organisationstyper än i en PBO. Med detta sagt positionerar sig denna studie bland författarna som hävdar att det inte föreligger något samband mellan PBO och innovation. / We were able to identify three problems from today’s literature about project-based organizations: 1) conflicting results about whether PBOs further innovation or not, 2) excessive focus on technical innovation in this context, and 3) insufficient systematics in these kinds of studies. For that reason, we have carried through a quantitative study to contribute with more systematics in the debate about PBO and innovation. We also chose to study a different kind of innovation in the PBO context, namely Management Innovation (MI) This study aims to identify if there exist a correlation between MI and the organizational factors centralization, teamwork and communication, slack of resources and formalization. Beyond this the study aims to identify if there exist a correlation between the formal organizational structure PBO and MI, and if the connection between the organizational factors and MI is stronger or weaker in a PBO context.  A survey was carried out in Sweden. 500 companies were asked to participate and they were all selected through a random stratified sampling. The response rate was 192 and the information from the questionnaires was then analysed in the statistical software SPSS through regression analysis. With a significance level of five percent this study confirms one out of fifteen hypotheses. The confirmed hypothesis was H8c which states that the connection between personnel slack and MI is stronger in a PBO than other kinds of organizational structures. We also confirmed hypothesis 9b on a significance level of ten percent. H9b states that the connection between formalization 2 and MI is weaker in PBO than other kinds of organizations. Consequently, this study position itself alongside authors and studies that claim that it doesn’t exist a correlation between PBO and innovation.

Les membres de la famille Homeobox jouent un rôle critique dans le développement hématopoïétique normal. L'expression ectopique des gènes Homeobox provoque des désordres dans l'hématopoïèse et le développement de leucémies. L'oncogène TLX3 s'exprime de manière ectopique exclusivement dans les Leucémies Aiguës Lymphoblastiques T (LAL-T), avec un blocage des thymocytes à un stade de différentiation précoce cortical CD4+CD8+ DP. De nombreuses études ont investigué les mécanismes d'action des oncogènes TLX1/3, mais plusieurs questions restent en suspens. Durant ma thèse, j'ai étudié l'impact de l'expression ectopique de l'oncogène TLX3 sur le développement lymphocytaire T et les mécanismes de transformation leucémique associés. L'expression de TLX3 a provoqué le blocage des thymocytes à un stade DN2 avec une immortalisation des clones preleucémiques. Les souris transplantées avec les cellules TLX3 ont développé des tumeurs similaires aux LAL-T. Les analyses de ChIP-Seq et d'expression génique ont identifié un recrutement de TLX3 sur les enhancers spécifiques aux cellules T par le motif de fixation Ets/Runx1. Nos résultats suggèrent que la fixation de TLX3 sur les éléments cis-régulateurs peut contribuer à la transformation maligne des thymocytes en perturbant les réseaux transcriptionnels responsables de l'oncogenèse LAL-T. / It is now well established that members of the homeobox gene family play a critical role in normal hematopoietic cell development and that their unbalanced or ectopic expression can lead to characteristic perturbations in haemopoiesis and the onset of leukaemia. TLX3 expression in human haematologic malignancies is exclusive to T-ALL, where it is almost universally associated with transformation of early cortical CD4+CD8+ DP thymocytes. Multiple studies intensively investigated the mechanisms by which TLX1/3 oncogenes could promote complex tumor development, but many questions remain still unclear. During my thesis I investigated the impact of ectopic TLX3 expression on T cell development, and the initiating mechanisms of T-cell transformation leading to leukemia onset. Forced expression of TLX3 disrupted the thymic develoment at DN2-like stage giving rise to immortalized preleukemic clones. Following the transfer into immunodeficient mice TLX3 preleukemic cells initiated malignant cell transformation resulting into leukemia-like disease. Applying a combination of ChIP sequencing and gene expression profiling, we identified TLX3 recruitment onto T-cell specific enhancers via interaction with Ets1/Runx1 composite motif sites as preferential molecular events in the initial steps of TLX3-induced transformation. Thus our findings suggest that the genome-wide binding properties of TLX3 on cis-regulatory elements may contribute to its ability to promote thymocyte preleukaemic state via perturbation of transcriptional regulatory networks responsible for T-ALL oncogenesis.

Cette thèse présente trois chapitres qui mobilisent un cadre d’analyse évolutionniste et qui étudient empiriquement la relation « innovation-performance » à partir de données CIS. Nous souhaitons montrer que les sources de l’innovation et les méthodes d’appropriation varient en fonction des secteurs d’activité et des stratégies d’innovation des firmes. Dans un premier temps, nous décrivons les tendances de l’innovation à partir de quatre vagues d’enquêtes CIS (1994-2006) et nous analysons la persistance de l’innovation sur un échantillon de 431 firmes avec une régression logistique binaire. Nous montrons que la persistance de l’innovation est plus élevée pour les firmes qui innovent en produits car ces firmes sont contraintes d’investir de manière continue dans des projets d’innovation pour rester compétitives. Les firmes qui innovent en procédés sont moins persistantes car leur stratégie est plus orientée vers des ajustements de la qualité des produits ainsi que vers l’amélioration des processus de production. Les deux derniers essais explorent avec la méthode des doubles moindres carrés le lien innovation-performance économique sur un échantillon de 7 742 firmes portant sur la période 2002-2005. Nous expliquons que la source principale de l’innovation des firmes à « forte intensité scientifique » est la R&D, d’une part, et que les méthodes d’appropriation des rentes de l’innovation passent par l’acquisition d’actifs complémentaires (tels que l’utilisation combinée de titres de propriété intellectuelle et de secrets de fabrication), d’autre part. En revanche, les firmes dans les autres catégories (notamment celles à fortes économies d’échelle) fondent leurs avancées technologiques sur des sources externes de l’innovation telles que les concurrents, les fournisseurs et les utilisateurs avancés. De plus, ces firmes utilisent de manière plus importante des méthodes d’appropriation commerciale telles que les marques ou les stratégies marketing, car leurs produits sont moins exposés au risque d’imitation certes, mais aussi parce qu’elles sont sensibles aux changements de coûts. / This thesis is structured in three essays based on evolutionary theoretical grounds and provides empirical evidence from CIS. It aims at showing that the sources of innovation and the appropriation of innovation rents vary in function of firms’ activities and innovation strategies.In essay 1, we describe four waves of CIS, covering the period 1994-2006 and we study persistent innovation behavior with a discrete choice model on a data set of 431 firms. We find that innovation persistence is more important for product innovators because they need novel products to be more competitive and therefore enrich their base of knowledge continuously. By contrast, process innovators are less persistent because innovation strategy is less “market” oriented and intends to meet quality or production adjustments. The two last essays explore with the two stage least squares method how firms benefit economically from their innovations on a sample of 7 742 firms, on the period 2002-2005. We show that science-based firms rely more on R&D investments to develop their products and maintain their leads by acquiring complementary assets, i.e. they use mixed methods to appropriate the rents of innovation (the combined use IPRs and strategic methods for instance secrecy). By contrast, firms in other categories (for instance firms using cost-cutting strategies) draw more on external sources of knowledge coming either from suppliers or advanced users. Additionally, these firms use more extensively trademarks or non technological methods of appropriation (as marketing devices), because they are less exposed to potential imitation and because they are price sensitive.

Die Identifizierung von Apoptose-modifizierten Proteinen erfolgte durch Proteomanalyse per 2D-Gelelektrophorese und Massenspektrometrie. Zunächst wurde eine 2DE-Datenbank errichtet, die im Internet zugänglich ist. Die Identifikation von Proteinen durch Peptidmassenfingerabdruck konnte durch Verwendung einer zweiten Matrix verbessert werden. Die Analyse ausgelassener tryptischer Spaltstellen, N-terminaler Pyroglutamatbildung und Oxidation von Tryptophan konnte die Identifikation von Proteinen mittels Peptidmassenfingerabdruck verbessern. Die Apoptose wurde über den Fas-Rezeptor-Signalweg oder durch DNA-Schädigung mittels Cis-Platin eingeleitet und das Totallysat oder die Kompartimente von Jurkat T-Zellen der Proteomanalyse unterworfen. Große Übereinstimmungen der beiden Prozesse wurden festgestellt. 95 Apoptose-modifizierte Proteine wurden identifiziert, wovon 78 Proteine bisher unbekannt für den Apoptoseprozess waren. Auffällig war, dass 40 % der Proteine RNA-Bindungsmotive enthielten und das 21 Onkoprotein oder Onkoprotein-interagierende Proteine identifiziert wurden. Für 39 Proteine konnten bisher proteolytische Spaltungen vorausgesagt werden. Eine Fülle von Informationen wurde über putative Translokationen der Proteine erhalten. Für das Protein p54nrb wurden drei Caspase-3 Spaltstellen durch die Einführung von Mutationen und die Abhängigkeit der Caspase-3 Spaltung von RNA bewiesen. Mitochondriale ribosomale Proteine von Mensch, Maus und Ratte wurden durch Abgleichung von EST-Datenbanken mit partiellen Aminosäuresequenzen aus dem Rind bestimmt. Die Konservierung der Sequenzen der Säugetierproteine der mitochondrialen ribosomalen Proteinen war geringer als von den bekannten cytosolischen ribosomalen Proteinen. Weiterhin wurden unterschiedliche Ergebnisse bzgl. der mitochondrialen Signalsequenzen der Proteine gefunden. RNA-Protein-Wechselwirkungen im Ribosom wurden nach Quervernetzung auf einzelne Aminosäuen bzw. Nukleotide bestimmt. Die Daten wurden zur Verbesserung von ribosomalen Modellen verwendet. Die mittlerweile erhaltenen Kristallstrukturen des Ribosoms zeigten, dass die Ergebnisse der Quervernetzungsexperimente mit den tatsächlichen RNA-Protein-Wechselwirkungen weitgehend übereinstimmen. Die Affinität von verschiedenen Komponenten zu einem Zielmolekül zur Bildung von nicht-kovalenten RNA-Peptid-Wechselwirkungen wurde mit Hilfe von MALDI-MS ermittelt. Die Interaktionen sind stark abhängig von der Anzahl der Arginine. / Apoptosis-modified proteins were identified by proteome analysis via 2D gel electrophoresis and mass spectrometry. First, a internet-accessible 2DE database was rendered. The identification of the proteins by peptide mass fingerprinting was improved using a second matrix. The analysis of missed tryptic cleavage sites, the formation of N-terminal pyroglutamine and oxidation of tryptophan could improve the identification of proteins by peptide mass fingerprinting. Apoptosis was induced via the Fas-receptor signaling pathway or by means of DNA damage by cis-platin. The total lysate and the compartments of Jurkat T cells were analyzed by proteome analysis. High similarities between both processes were observed. 95 apoptosis-modified proteins were identified, 78 of these were until now unknown to be involved in apoptosis. Noticeable, 40% of the proteins include a RNA-binding motif and 21 oncogene or oncogene-interacting proteins were identified. A proteolytic cleavage could be predicted for 39 proteins. Some information was received about the putative translocation of the proteins. Three caspase-3 cleavage sites were shown for the protein p54nrb with the incorporation of mutations. Furthermore, the caspase-3 cleavage was dependent on the occurrence of RNA. Mitochondrial ribosomal proteins of human, mouse and rat were determined by screening of EST-databases with partial amino acid sequences from bovine. The conservation of sequences of mammalian proteins of the mitochondrial ribosomal proteins was less than for known cytosolic ribosomal proteins. Furthermore, different results were obtained considering mitochondrial signal sequences. RNA-protein interaction within the ribosome were determined on single amino acids and nucleotides, respectively, after cross-linking. These data were used to improve models of the ribosome. The in the meantime obtained 3D-structures of the ribosome showed high consistency with the revealed RNA-protein interaction sites after cross-linking. The affinity of different components to a target molecule to form RNA-peptide interactions was determined by MALDI-MS. The interactions were strongly dependent on the number of arginines.

In dieser Dissertation wurde eine isolierte Brachydaktylie vom Typ E (BDE) untersucht. Grundlage war eine Familie mit autosomal-dominanten Erbgang BDE. Der genetische Hintergrund ist eine balancierte Translokation t(8;12)(q13;p11.2). Der Bruchpunkt auf derivativem Chromosom der(8) liegt 86 kb strangaufwärts des chondrogenetisch essentiellen Kandidatengens PTHLH (Parathyroid hormone like hormone). PTHLH ist für die Differenzierungsrate von proliferativen Chondrozyten verantwortlich. Positiv oder negativ reguliertes Pthlh führen zu einer Dysbalance mit Brachydaktylie-ähnlichen Phänotypen in murinen Tiermodellen. Der Leserahmen des Kaliumkanals KCNB2 auf Chromosom 8 wurde durch die Translokation in Intron 2 getrennt. Chrondrogenetische KCNB2 Funktionen konnten durch in situ Hybridisierungen ausgeschlossen werden. Der Translokationsbruchpunkt auf der(8) liegt in einer in Mammalia hochkonservierten Region und beeinhaltet ein Bindungsmotiv für AP1 Transkriptionsfaktoren. Durch die Translokation befindet sich in unmittelbarer Nähe eine Kernkonsensussequenz für ETS Transkriptionsfaktoren. AP1 und ETS Transkriptionsfaktoren interagieren und wurden auf eine potentielle PTHLH Regulation untersucht. Epigenetische Histonmodifizierungen, charakteristisch für cis-regulatorische Elemente, sowie Reportergenassays mit AP1 und ETS1 Bindungsmotiven zeigten einen Bezug zur PTHLH Regulation. Bindungsassays mit AP1 und ETS1 Transkriptionsfaktoren an den Bruchpunktsequenzen, sowie funktionelle in vitro Experimente mit Chondrozyten verifizierten die Hypothese, dass der Translokationsbruchpunkt strangaufwärts von PTHLH regulatorische Eigenschaften besitzt. Die AP1 und ETS1 Transkriptionsfaktoren regulierten PTHLH positiv in ATDC5 und C28/I2 Chondrozyten. In chondrogeninduzierten Patientenfibroblasten war die PTHLH Expression inhibiert. Die molekulare Pathogenese der BDE wurde durch die bisher unbekannte chondrogene PTHLH Fehlregulation dargestellt. / We studied a 3-generation family with Brachydactyly Type E (BDE) and identified a t(8;12)(q13;p11.2) translocation. We identified PTHLH (Parathyroid hormone like hormone) on chromosome 12p11.2 and the ionchannel KCNB2 on chromosome 8q13 as candidate genes. KCNB2 was disrupted in intron 2, while the chromosome 12 breakpoint is localized 86 kb upstream of PTHLH; only the latter gene is involved in chondrogenesis. The 12p11.2 breakpoint is conserved and features an AP1 binding site 86 kb upstream of PTHLH. Due to the translocation, an ETS binding site from 8q13 resided near the AP1 site. Since both transcription factors interact, we tested if AP1 and ETS1 can activate PTHLH in ATDC5 and C28/I2 chondrocytes. We used the breakpoint sequences of the derivative chromosomes 8 and 12 and the nonaffected chromosome 8 and 12 allele sequences in reporter-gene assays. Reporter-gene constructs containing the der(8) breakpoint revealed activation in murine and human chondrocytes. The enrichment of histone modifications, implicating cis-regulatory effects were investigated in the breakpoint area. We found the enriched histone H3K4me1 modification at the chromosome 12 breakpoint position in murine and human chondrocytes, while affected fibroblasts showed higher H3K4me1 enrichment at the der(8) breakpoint compared to wt(12) allele. Furthermore, the breakpoint sequence bound to AP1 and C-ets-1 in EMSA. Western blotting after PMA-stimulated AP1 and ETS1 activation and overexpression of different AP1 and ETS1 combinations showed activated PTHrP expression in chondrocytes. In chondrogenic induced BDE fibroblasts PTHLH was inhibited, while IHH was upregulated. We suggest that PTHLH was dysregulated by the translocation in BDE chondrocytes. This could lead to BDE. We highlight the impact to characterize genomic breakpoints in detail and demonstrate a novel AP1- and ETS1-directed chondrogenic PTHLH regulation in wild-type chondrocytes and dysregulation in the pathogenesis of BDE.

Made available in DSpace on 2015-12-04T11:45:12Z (GMT). No. of bitstreams: 1 Mauro_Fernando_Gallo.pdf: 1955869 bytes, checksum: ae2de252f340b3da6adc7d24bdc3d4c3 (MD5) Previous issue date: 2002-10-24 / The participation and importance of the business combinations through the consolidation, merger and spin-off operations have been increasing in the present context of the world economy. This fact is occurring also in Brazil, mainly due to the world changes that have been causing the competition increasing. This importance is due to not only the transaction quantities but mostly because many times the values involved are extremely high. It is not possible to say it is only a business concentration phenomenon: there are several reasons for the realization of these transactions, with a crescent utilization to reduce effectively the tributary charge assessed to the companies. In Brazil, the utilization of these operations started due to some tax advantages offered by the Federal Government. In 1971, it was created the Comiss?o de Fus?o e Incorporac?o de Empresas - COFIE, a commission connected to Ministry of Finance, in order to coordinate and analyze these tax advantages. The merger transactions were regulated for a long time past, but the spin-off transaction was inserted in the Brazilian legislation only in 1976, through the federal Law nr. 6404/76, known as Lei das S/A - the corporation acts. With this legal regulation, the utilization of the business company combinations has increased in spite of the Brazilian concept differences in relation to the United States of America and Europe ones. It is the case, for instance, of the merger, that they consider as a special type of consolidation. The professionals that work in tributary planning, that is, attorneys, accountants, managers and economists, amplified the business company combination use in Brazil, mainly merger and spin-off, due to the tax advantages reached through the Income Tax reduction, elimination or postponement, for several reasons. The main point of this study is the accounting and tributary ones to show the employment of these operations to substitute the acquisitions, aiming mainly the elimination of the capital gain of the stockholders, being an individual person or a company. The objective of this study was confirmed by the searches: this way of using the business company combination causes problems in the areas of law and accountancy, because in Brazil people are more connected to the juridical way of the fact than the economic one. So it may be concluded that the accountancy and the law professionals have many things to search and develop to improve and differentiate the types of transactions - those involving companies that have the same corporate control and those involving companies pertaining to different economic groups, explaining also the method differences used in the accountancy registers. / As reorganiza????es societ??rias atrav??s dos institutos da fus??o, incorpora????o e cis??o v??m tendo sua participa????o e import??ncia ampliadas no cen??rio atual da economia mundial, inclusive no Brasil, principalmente devido ?? globaliza????o e ao crescimento da concorr??ncia. Essa import??ncia deve-se n??o apenas ao quantitativo de opera????es, mas tamb??m ao fato de que os valores envolvidos muitas vezes s??o bem elevados. Tamb??m n??o se pode afirmar que ?? apenas um fen??meno de concentra????o empresarial: h?? in??meras e variadas raz??es para a efetiva????o dessas opera????es, com sua crescente utiliza????o como forma de redu????o do ??nus tribut??rio aplicado ??s empresas. No Brasil, o emprego dessas opera????es foi implementado utilizando-se alguns benef??cios fiscais concedidos pelo Governo Federal; criou-se em 1971 a Comiss??o de Fus??o e Incorpora????o de Empresas - COFIE, ligada ao Minist??rio da Fazenda, com a finalidade de coordenar e analisar a concess??o desses benef??cios fiscais. A fus??o e a incorpora????o j?? eram devidamente regulamentadas h?? um bom tempo, mas a cis??o somente foi introduzida na legisla????o brasileira atrav??s da Lei das Sociedades An??nimas, em 1976. Com isso, ampliou-se a utiliza????o de tais institutos, apesar de em nossa legisla????o societ??ria e fiscal haver algumas diferen??as conceituais em rela????o aos Estados Unidos e Europa: ?? o caso da incorpora????o, que l?? ?? considerada como um tipo especial de fus??o. Os profissionais que operam com planejamento tribut??rio, sejam advogados, contadores, administradores e economistas, ampliaram o emprego principalmente da incorpora????o e cis??o no Brasil devido ??s vantagens fiscais alcan??adas atrav??s da redu????o, elimina????o ou posterga????o de Imposto de Renda, por diversas raz??es. O foco principal deste estudo compreende as ??reas cont??bil e tribut??ria, com o fim de demonstrar o emprego dessas opera????es em substitui????o ??s de aquisi????o, visando principalmente a elimina????o do ganho de capital dos acionistas ou quotistas, sejam pessoas f??sicas ou jur??dicas. Conforme era o objetivo do estudo, pelas pesquisas desenvolvidas confirmou-se tal utiliza????o, provocando essa forma de emprego distor????es tanto no campo jur??dico como nos procedimentos cont??beis adotados, vez que no Brasil preocupa-se mais com a forma jur??dica do que com a econ??mica dos fatos. Isso leva a concluir que tanto os profissionais da contabilidade como os do direito t??m muito a pesquisar e desenvolver para aperfei??oar e distinguir essas opera????es, tanto as ocorridas entre empresas sob o mesmo controle societ??rio quanto entre as pertencentes a grupos econ??micos distintos, clarificando inclusive as diferencia????es dos m??todos adotados nos registros cont??beis.

Fatores de transcrição (FT) são proteínas que se ligam em sequências específicas e bem conservadas de nucleotídeos no DNA, denominadas sítios de ligação dos fatores de transcrição (SLFT), localizadas em regiões de regulação gênica conhecidas como módulos cis-reguladores (CRM). Ao reconhecer o SLFT, o fator de transcrição se liga naquele sítio e influencia a transcrição gênica positiva ou negativamente. Existem técnicas experimentais para a identificação dos locais dos SLFTs em um genoma, como footprinting, ChIP-chip ou ChIP-seq. Entretanto, a execução de tais técnicas implica em custos e tempo elevados. Alternativamente, pode-se utilizar as sequências de SLFTs já conhecidas para um determinado fator de transcrição e aplicar técnicas de aprendizado computacional supervisionado para criar um modelo computacional para tal sítio e então realizar a predição computacional no genoma. Entretanto, a maioria das ferramentas computacionais existentes para esse fim considera independência entre as posições entre os nucleotídeos de um sítio - como as baseadas em PWMs (position weight matrix) - o que não é necessariamente verdade. Este projeto teve como objetivo avaliar a utilização de gramáticas regulares estocásticas (GRE) como técnica alternativa às PWMs neste problema, uma vez que GREs são capazes de caracterizar dependências entre posições consecutivas dos sítios. Embora as diferenças de desempenho tenham sido sutis, GREs parecem mesmo ser mais adequadas do que PWMs na presença de valores mais altos de dependência de bases, e PWMs nos demais casos. Por fim, uma ferramenta de predição computacional de SLFTs foi criada baseada tanto em GREs quanto em PWMs. / Transcription factors (FT) are proteins that bind to specific and well-conserved sequences of nucleotides in the DNA, called transcription factor binding sites (TFBS), contained in regions of gene regulation known as cis-regulatory modules (CRM). By recognizing TFBA, the transcription factor binds to that site and positively or negatively influence the gene transcription. There are experimental procedures for the identification of TFBS in a genome such as footprinting, ChIP-chip or ChIP-Seq. However, the implementation of these techniques involves high costs and time. Alternatively, one may utilize the TFBS sequences already known for a particular transcription factor and applying computational supervised learning techniques to create a computational model for that site and then perform the computational prediction in the genome. However, most existing software tools for this purpose considers independence between nucleotide positions in the site - such as those based on PWMs (position weight matrix) - which is not necessarily true. This project aimed to evaluate the use of stochastic regular grammars (SRG) as an alternative technique to PWMs in this problem, since SRGs are able to characterize dependencies between consecutive positions in the sites. Although differences in performance have been subtle, SRGs appear to be more suitable than PWMs in the presence of higher base dependency values, and PWMs in other cases. Finally, a computational TFBS prediction tool was created based on both SRGs and PWMs.

O cloridrato de tramadol, analgésico sintético de ação central, possui dois centros quirais: o isômero cis é ativo e o isômero trans é uma impureza de processo. Ambos os enantiômeros do cloridrato de cis-tramadol contribuem para o efeito analgésico, mas através de mecanismos diferentes, complementares e interativos farmacologicamente. Os dois isômeros do cis-tramadol apresentam efeitos terapêuticos, e a presença de impurezas, incluindo os isômeros trans - decorrentes do processo de síntese ou devido à decomposição - podem comprometer a qualidade do produto comercializado. Assim, este trabalho teve como objetivo desenvolver e validar metodologia estereosseletiva para análise do cloridrato de cis-tramadol e das possíveis impurezas quirais ou não na matéria-prima e formulações farmacêuticas. Para a separação e quantificação dos enantiômeros do cloridrato de cis-tramadol e das impurezas trans-tramadol, 1,2-olefina e 1,6-olefina, foi utilizada a coluna Chiralcel® OD-H, fase móvel constituída por hexano (60% e 100% de n-hexano, 1:1, v/v):isopropanol:dietilamina:ácido trifluoracético (99,5:0,5:0,3:0,1, v/v/v/v), na vazão de 0,7 mL min-1 e detecção em 274 nm. A coluna Chiralpak® AD fase móvel constituída por hexano (60% de n-hexano):etanol absoluto:dietilamina (95:5:0,1, v/v/v), na vazão de 1,0 mL min-1 e o comprimento de onda para detecção dos compostos foi de 228 nm foi utilizada para a separação e quantificação das impurezas O-desmetiltramadol, N-desmetiltramadol e tramadol N-óxido. Os métodos desenvolvidos foram devidamente validados através dos parâmetros seletividade, linearidade, precisão, exatidão, intervalo, limite de detecção e limite de quantificação. Os resultados obtidos na validação mostraram que os métodos são adequados para a determinação do cis-tramadol e de suas impurezas na matéria prima e na formulação farmacêutica. / Tramadol hydrochloride is a centrally acting analgesic with two chiral centers; the cis isomer is the active drug and the trans isomer is a process impurity. Both enantiomers of cis-tramadol hydrochloride contribute to the analgesic effect through different, but complementary and interactive pharmacological mechanisms. Although both isomers of cis-tramadol hydrochloride show therapeutic effects, the presence of impurities, originated from the synthesis process or due to degradation, can compromise the quality of the marketed product. The aim of this present work was the development and validation of a stereosselective methodology for the analysis of the drug cis-tramadol hydrochloride and the possible chiral or non-chiral impurities in raw materials and pharmaceutical formulations. The separation and quantitation of cis-tramadol enantiomers and the impurities trans-tramadol, 1,2-olefin and 1,6-olefin were carried out using a Chiralcel® OD-H column, mobile phase of hexane (60% and 100% of n-hexane, 1:1, v/v):2-propanol:diethylamine:trifluoroacetic acid (99,5:0,5:0,3:0,1, v/v/v/v) at a flow rate of 0,7 mL min-1 and detection at 274 nm. For the separation and quantitation of the impurities O-desmethyltramadol, N-desmethyltramadol and tramadol N-oxide, a Chiralpak® AD column was used with a mobile phase of hexane (60% of n-hexane):ethanol absolute: diethylamine (95:5:0,1, v/v/v) at a flow rate of 1,0 mL min-1 and detection at 228 nm. The methods were validated using the parameters selectivity, linearity, precision, accuracy, range, detection limit and quantitation limit. The results obtained show that the methods are suitable for the analysis of cis-tramadol and its impurities in raw material and pharmaceutical formulation.