Adenoviruses have been extensively studied in the field of gene regulation, since their genes are subjected to a tightly controlled temporal expression during the virus lifetime. The early-to-late shift in adenoviral gene expression distinguishes two completely different programs in gene expression. The adenoviral L4-33K protein, which is the subject of this thesis, was previously implicated to be a key player in the transition from the early to the late phase of infection. Here we show that L4-33K activates late gene expression by functioning as a virus-encoded alternative RNA splicing factor activating splicing of transcripts containing weak 3’ splice sites; a feature common to the viral genes expressed at late times of infection. The splicing enhancer activity of L4-33K was mapped to a tiny arginine/serine (RS) repeat in the carboxyl-terminal domain of the protein. Also, the subcellular distribution to the nucleus with enrichment in the nuclear membrane and subnuclear redistribution to viral replication centers during a lytic infection was observed to depend on this motif. RS repeats are common features for the cellular splicing factors serine/arginine-rich (SR) proteins, which in turn are regulated by reversible phosphorylation. We further show that L4-33K is phosphorylated by two cellular protein kinases, the double-stranded DNA-dependent protein kinase (DNA-PK) and protein kinase A (PKA) in vitro. Interestingly, DNA-PK and PKA have opposite effects on the control of the temporally regulated L1 alternative RNA splicing. DNA-PK functions as an inhibitor of the late specific L1-IIIa pre-mRNA splicing whereas PKA functions as an activator of L1-IIIa pre-mRNA splicing. In summary, this thesis describes L4-33K as an SR protein related viral alternative splicing factor. A tiny RS repeat conveys splicing enhancer activity as well as redistribution of L4-33K to replication centers. Finally, DNA-PK and PKA that phosphorylates L4-33K are suggested to be novel regulatory factors controlling adenovirus alternative splicing.

Une hyperactivation de la prolifération des cellules musculaires lisses vasculaires (CMLV) contribue à la pathogenèse des maladies des vaisseaux. Des travaux antérieurs suggèrent que l’augmentation de l'adénosine monophosphate cyclique (AMPc) inhibe la prolifération des CMLV. Provoquer une augmentation d’AMPc préviendrait aussi certaines maladies vasculaires qui sont associées à des altérations dans sa signalisation impliquant l'activité de la protéine kinase A (PKA). Des études ont démontré la contribution du facteur de transcription « early growth response protein-1» (Egr-1) dans la pathogenèse des maladies vasculaires et une surexpression d’Egr-1 a été rapportée dans des modèles d'athérosclérose et d'hyperplasie intimale. Divers agents vasoactifs contrôlent l'expression d’Egr-1 suivant des mécanismes qui ont fait l’objet de plusieurs études mais demeurent incomplètement élucidés. L'angiotensine-II (Ang-II) est l'un des principaux peptides vasoactifs impliqués dans la pathogenèse des maladies vasculaires. Une des voies de signalisation induite par l’Ang-II implique l’augmentation du calcium (Ca2+) intracellulaire. Celle-ci se produit par l’activation de l'entrée de calcium opérée par la relâche des réserves (SOCE) de Ca2+ réticulaire suite à l’activation du récepteur à l’inositol-3-phosphate (IP3R) et le recrutement ultérieur du complexe conducteur formé par la molécule d'interaction stromale 1 (STIM-1) et le canal Orai-1. Bien qu’il ait déjà été démontré que l’expression de l'Egr-1 est régulée par la signalisation calcique en réponse à plusieurs stimuli, l'implication du complexe STIM-1/Orai-1 dans l'expression d'Egr-1 dans la CMLV n’a jamais été étudiée. De même, la question de savoir si la signalisation induite par l'Ang-II conduisant à l'expression d'Egr-1 est modulée par l'AMPc n’a jamais été explorée. Par conséquent, les travaux menés dans cette thèse ont consisté à examiner le rôle de la signalisation du Ca2+ dans l'expression d'Egr-1 induite par l’Ang-II dans la CMLV avec une attention particulière portée sur le rôle joué par STIM-1 et Orai-1. En outre, nous avons examiné l'effet de l’augmentation de l’AMPc sur l'expression d'Egr-1 induite par l’Ang-II et étudié les voies de signalisation associées. Nos données montrent que l’inhibition du récepteur IP3R et du SOCE par le 2-aminoéthoxydiphénylborate atténue la libération de Ca2+ induite par l’Ang-II et ceci s’accompagne d’une baisse des niveaux d’expression de protéine et d’ARN messager de l’Egr-1. La stimulation de l’expression de l'Egr-1 a également été supprimée à la suite du blocage de la calmoduline et de la protéine kinase CaMKII. De plus, le blocage par interférence d’ARN de l’expression de STIM-1 et Orai-1 a atténué l'expression d'Egr-1 induite par l’Ang-II ainsi que la phosphorylation des protéines ERK et CREB. Par ailleurs, l'isoproterenol (ISO) et la forskoline (FSK), deux activateurs de l'adénylate cyclase ont atténué de manière dose-dépendante l'expression d'Egr-1 induite par l’Ang-II. Des réponses similaires ont été observées en utilisant des analogues non spécifique (dibutyryl-cAMP) et PKA-spécifique (Benzoyl-cAMP) de l’AMPc, ainsi qu'un inhibiteur à large spectre de l'activité phosphodiesterase intracellualaire (isobutylméthylxanthine). L'inhibition de l'expression d'Egr-1 induite par l’Ang-II s’accompagne d'une augmentation de l’activité de la PKA mesurée par la phosphorylation de la « phosphoprotéine activée par les vasodilatateurs (VASP) », et d’une diminution concomitante de la phosphorylation de la protéine ERK. Le blocage pharmacologique de la PKA a réduit la phosphorylation de VASP et restauré la phosphorylation de la protéine ERK ainsi que l'expression d'Egr-1 en présence de l’Ang-II. En résumé, nos données démontrent que la voie STIM-1/Orai-1 /Ca2+ médie l'expression de l'Egr-1 induite par l'Ang-II dans la CMLV et suggèrent que la suppression de la réponse à l’Ang-II menant à l’expression de l'Egr-1 peut expliquer les effets vasoprotecteurs de l’AMPc. En outre, ces travaux montrent que les mécanismes moléculaires de régulation de l’expression d’Egr-1 en réponse aux signaux externes culminent vers la modulation des cascades de signalisation en aval de la protéine ERK dans les CMLV. / Aberrant vascular smooth muscle cell (VSMC) proliferative responses contribute to the development of neointimal lesions. Cyclic adenosine monophosphate (cAMP) is believed to inhibit VSMC proliferation, and vascular diseases are associated with impairments in cAMP-induced signalling responses involving protein kinase A (PKA) signaling. An enhanced expression of the early growth response protein-1 (Egr-1), a zinc finger transcription factor, has been reported in models of vascular diseases and, a crucial role of Egr-1 in regulating the expression of genes implicated in neointimal formation leading to atherogenesis has been suggested. Various vasoactive factors have been shown to modulate Egr-1 expression in VSMC via mechanisms which remain to be completely understood. Angiotensin-II (Ang-II) is one of the key vasoactive peptides implicated in the pathogenesis of vascular diseases. Ang-II elevates intracellular calcium (Ca2+) through activation of voltage-gated calcium channels as well as store-operated calcium channels. The store-operated calcium entry (SOCE) involves an inositol-3-phosphate receptor (IP3R)-coupled depletion of endoplasmic reticular Ca2+ and a subsequent activation of the stromal interaction molecule 1 (STIM-1) /Orai-1 complex. Although Egr-1 has been demonstrated to be upregulated in a Ca2+-dependent fashion in response to several stimuli, the involvement of STIM-1/Orai-1-dependent signaling in Egr-1 expression in VSMC has never been addressed. Besides, whether Ang-II-induced signaling leading to Egr-1 expression is modulated by cAMP-dependent signaling pathway remains unexplored. Therefore, in the present studies, we have examined the role of Ca2+ signaling in Ang-II-induced Egr-1 expression in VSMC and investigated the contribution of STIM-1 or Orai-1. Additionnaly, we have examined the effect of cAMP on Ang-II-induced expression of Egr-1 and have investigated the associated signalling pathways. Pharmacological blockade of IP3R and SOCE by 2-aminoethoxydiphenylborate (2-APB) decreased Ang-II-induced Ca2+ release and attenuated Ang-II-induced enhanced expression of Egr-1 protein and mRNA levels. Egr-1 upregulation was also suppressed following blockade of calmodulin and CaMKII. Furthermore, RNA interference-mediated depletion of STIM-1 or Orai-1 attenuated Ang-II-induced Egr-1 expression, as well as Ang-II-induced phosphorylation of ERK1/2 and CREB. Moreover, isoproterenol (ISO) and forskolin (FSK), two respective receptor and non-receptor activators of adenylate cyclase, attenuated Ang-II-induced Egr-1 expression in a dose-dependent fashion. Similar responses were observed using non-specific (dibutyryl-cAMP) and PKA-specific (Benzoyl-cAMP) analogs of cAMP, as well as a broad spectrum inhibitor of intracellular phosphodiesterase activity (isobutylmethylxanthine). The inhibition of Ang-II-induced Egr-1 expression was accompanied by an increase in serine 157 phosphorylation of the vasodilator-activated phosphoprotein (VASP), a marker of PKA activity, and this was associated with a concomitant decrease in ERK phosphorylation. Pharmacological blockade of PKA using H89 decreased VASP phosphorylation, restored Ang-II-induced ERK phosphorylation and abolished ISO- and FSK-mediated inhibition of Ang-II-induced Egr-1 expression. In summary, our data demonstrate that STIM-1/Orai-1/Ca2+-dependent signaling pathways mediate Ang-II-induced Egr-1 expression in A-10 VSMC and suggest that PKA-mediated suppression of Ang-II-induced Egr-1 expression and phosphorylation of ERK may be among the mechanisms by which cAMP exerts its vasoprotective effects. In addition, our data supports the notion that stimuli-induced regulation of Egr-1 expression involves the participation of signaling cascades downstream of ERK in VSMC.

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No description available.

<p>Understanding and predicting chemical phenomena is the main goal of computational chemistry. In this thesis I present my work on applying computational approaches to study chemical processes in biological and supramolecular systems.</p> <p>pH-responsive molecular tweezers have been proposed as an approach for targeting drug-delivery to tumors, which tend to have a lower pH than normal cells. In chapter 2 I present a computational study I performed on a pH-responsive molecular tweezer using <em>ab initio</em> quantum chemistry in the gas phase and molecular dynamics simulations in solution. The binding free energy in solution was calculated using Steered Molecular Dynamics. We observe, in atomistic detail, the pH-induced conformational switch of the tweezer and the resulting release of the drug molecule. Even when the tweezer opens, the drug molecule remains near a hydrophobic arm of the molecular tweezer. Drug release cannot occur, it seems, unless the tweezer is a hydrophobic environment with low pH.</p> <p>The protonation state of amino acid residues in proteins depends on their respective pK_a values. Computational methods are particularly important for estimating the pK_a values of buried and active site residues, where experimental data is scarce. In chapter 3 I used the cluster model approach to predict the pK_a of some challenging protein residues and for which methods based on the numerical solution of the Poisson-Boltzmann equation and empirical approaches fail. The ionizable residue and its close environment were treated quantum mechanically, while the rest of the protein was replaced by a uniform dielectric continuum. The approach was found to overestimate the electrostatic interaction leading to predicting lower pK_a values.</p> / Master of Science (MSc)

Ao ser excitado do estado fundamental para o estado singlete excitado, o Pka do equilíbrio ácido-base das antocianinas apresenta um deslocamento de ca. 4 para ca. -1. Esta característica de superfotoácido, aliado à técnica de fotólise por pulso de laser, permite a perturbação da posição do equilíbrio ácido-base no estado fundamental. O acompanhamento da cinética de reestabelecimento do equilíbrio inicial, existente antes do pulso do laser, permite a determinação das constantes de desprotonação kd e protonação kp no estado fundamental. Este método foi aplicado para a determinação das constantes de protonação e desprotonação das seguintes antocianinas sintéticas e naturais: 4-metil-7-hidroxiflavílio (HMF), 4\', 7-dihidroxiflavílio (DHF), malvidina-3-glucosídeo (Oenina), malvidina-3,5-diglucosídeo (Malvina), pelargonidina-3,5-diglucosídeo (Pelargonina) e cianidina-3 ,5-diglucosídeo (Cianina). Assim, as constantes de desprotonação do estado fundamental são: kd = 1,4 x 106 s-1 (HMF), 3,1 x 106 s-1 (DHF), 5, 1 x 106 s-1 (Oenina), 3,8 x 106 s-1 (Malvina), 1,3 x 106 s-1 (Pelargonina) e 1,8 x 106 s-1 (Cianina). As constantes de protonação do estado fundamental são:kp = 3,56 x 1010 L mol-1 s-1 (HMF), 3,06 x 1010 L mol-1s-1 (DHF), 2,51 x 1010 L mol-1s-1 (Oenina), 2,9 x 1010 L mol-1s-1 (Malvina), 3,6 x 1010 L mol-1s-1 (Pelargonina) e 2,2 x 1010 L mol-1s-1 (Cianina). Nosso método é a única técnica que permite a determinação direta destas constantes. Este método também permite a determinação de kd e kp em meios micro-heterogêneos. Através de medidas de kd , foi possível provar que ocorre a estabilização do cátion flavílio de HMF em micelas aniônicas de SOS (dodecilsulfato de sódio). Em micelas não iônicas e catiônicas, por outro lado, ocorre a desestabilização do cátion flavílio, que se manifesta através do aparecimento de reatividade ausente em solução aquosa. Os resultados da aplicação deste método revelam ainda que o principal processo de dissipação da energia da luz, absorvida pelas antocianinas, é a transferência de próton no estado excitado e não a fluorescência. / Upon excitation from the ground state to the first excited singlet state, the pKa of the acid-base equilibrium of anthocyanins exhibits a large shift, from ca. 4 to ca. -1. This characteristic of super-photoacidity, coupled with the technique of laser flash photolysis, can be employed to perturb the position of the acid-base equilibrium in the ground state. Monitoring the kinetics of relaxation back to the initial equilibrium position that existed prior to the laser pulse permits determination of the rate constants for protonation (kp) and deprotonation (kd) in the ground state. This method was applied to the following natural and synthetic anthocyanins: 4-methyl-7-hydroxyflavylium (HMF); 4\', 7-dihydroxyflavylium (DHF); malvidine-3-glucoside (Oenin); malvidine-3,5-diglucoside (Malvin); Pelargonidine-3,5-diglucoside (Pelargonin); and. cyanidine-3,5-diglucoside (Cyanin). Thus, the ground state deprotonation rate constants (kd) for the acid form were found to be: 1.4 x 106 s-1 (HMF); 3.1 x 106 s-1 (DHF); 5.1 x 106 s-1 (Oenin); 3.8 x 106 s-1 (Malvin); 1.3 x 106 s-1 (Pelargonin); 1.8 x 106 s-1 (Cyanin). The corresponding rate constants for protonation of the ground state of the base (kp) were: 3.6 x 1010 M-1s-1 (HMF); 3.1 x 1010 M-1s-1 (DHF); 2.5 x 1010 M-1s-1 (Oenin); 2.9 x 1010M-1s-1 (Malvin); 3.6 x 1010 M-1s-1 (Pelargonin); 2.2 x 1010 s-1 (Cyanin). Our method is currently the only one that permits direct determination of these rate constants. This method also allows the determination of kpand kd in microheterogeneous media. Thus, employing measurements of kd, it was possible to demonstrate that anionic SDS (sodium dodecyl sulfate) micelles stabilize the flavylium cation form of HMF. In cationic and nonionic micelles, however, the flavylium cation is destabilized, which manifests itself via the appearance of reactivity that is absent in aqueous solution in the absence of these surfactants. Finally, the results of the application of this method show that the primary energy wasting process for radiant energy absorbed by anthocyanins is excited state proton transfer rather than fluorescence.

Ao ser excitado do estado fundamental para o estado singlete excitado, o Pka do equilíbrio ácido-base das antocianinas apresenta um deslocamento de ca. 4 para ca. -1. Esta característica de superfotoácido, aliado à técnica de fotólise por pulso de laser, permite a perturbação da posição do equilíbrio ácido-base no estado fundamental. O acompanhamento da cinética de reestabelecimento do equilíbrio inicial, existente antes do pulso do laser, permite a determinação das constantes de desprotonação kd e protonação kp no estado fundamental. Este método foi aplicado para a determinação das constantes de protonação e desprotonação das seguintes antocianinas sintéticas e naturais: 4-metil-7-hidroxiflavílio (HMF), 4\', 7-dihidroxiflavílio (DHF), malvidina-3-glucosídeo (Oenina), malvidina-3,5-diglucosídeo (Malvina), pelargonidina-3,5-diglucosídeo (Pelargonina) e cianidina-3 ,5-diglucosídeo (Cianina). Assim, as constantes de desprotonação do estado fundamental são: kd = 1,4 x 106 s-1 (HMF), 3,1 x 106 s-1 (DHF), 5, 1 x 106 s-1 (Oenina), 3,8 x 106 s-1 (Malvina), 1,3 x 106 s-1 (Pelargonina) e 1,8 x 106 s-1 (Cianina). As constantes de protonação do estado fundamental são:kp = 3,56 x 1010 L mol-1 s-1 (HMF), 3,06 x 1010 L mol-1s-1 (DHF), 2,51 x 1010 L mol-1s-1 (Oenina), 2,9 x 1010 L mol-1s-1 (Malvina), 3,6 x 1010 L mol-1s-1 (Pelargonina) e 2,2 x 1010 L mol-1s-1 (Cianina). Nosso método é a única técnica que permite a determinação direta destas constantes. Este método também permite a determinação de kd e kp em meios micro-heterogêneos. Através de medidas de kd , foi possível provar que ocorre a estabilização do cátion flavílio de HMF em micelas aniônicas de SOS (dodecilsulfato de sódio). Em micelas não iônicas e catiônicas, por outro lado, ocorre a desestabilização do cátion flavílio, que se manifesta através do aparecimento de reatividade ausente em solução aquosa. Os resultados da aplicação deste método revelam ainda que o principal processo de dissipação da energia da luz, absorvida pelas antocianinas, é a transferência de próton no estado excitado e não a fluorescência. / Upon excitation from the ground state to the first excited singlet state, the pKa of the acid-base equilibrium of anthocyanins exhibits a large shift, from ca. 4 to ca. -1. This characteristic of super-photoacidity, coupled with the technique of laser flash photolysis, can be employed to perturb the position of the acid-base equilibrium in the ground state. Monitoring the kinetics of relaxation back to the initial equilibrium position that existed prior to the laser pulse permits determination of the rate constants for protonation (kp) and deprotonation (kd) in the ground state. This method was applied to the following natural and synthetic anthocyanins: 4-methyl-7-hydroxyflavylium (HMF); 4\', 7-dihydroxyflavylium (DHF); malvidine-3-glucoside (Oenin); malvidine-3,5-diglucoside (Malvin); Pelargonidine-3,5-diglucoside (Pelargonin); and. cyanidine-3,5-diglucoside (Cyanin). Thus, the ground state deprotonation rate constants (kd) for the acid form were found to be: 1.4 x 106 s-1 (HMF); 3.1 x 106 s-1 (DHF); 5.1 x 106 s-1 (Oenin); 3.8 x 106 s-1 (Malvin); 1.3 x 106 s-1 (Pelargonin); 1.8 x 106 s-1 (Cyanin). The corresponding rate constants for protonation of the ground state of the base (kp) were: 3.6 x 1010 M-1s-1 (HMF); 3.1 x 1010 M-1s-1 (DHF); 2.5 x 1010 M-1s-1 (Oenin); 2.9 x 1010M-1s-1 (Malvin); 3.6 x 1010 M-1s-1 (Pelargonin); 2.2 x 1010 s-1 (Cyanin). Our method is currently the only one that permits direct determination of these rate constants. This method also allows the determination of kpand kd in microheterogeneous media. Thus, employing measurements of kd, it was possible to demonstrate that anionic SDS (sodium dodecyl sulfate) micelles stabilize the flavylium cation form of HMF. In cationic and nonionic micelles, however, the flavylium cation is destabilized, which manifests itself via the appearance of reactivity that is absent in aqueous solution in the absence of these surfactants. Finally, the results of the application of this method show that the primary energy wasting process for radiant energy absorbed by anthocyanins is excited state proton transfer rather than fluorescence.

No description available.

<p>Capillary Electrophoresis (CE) was explored as a means for physicochemical and biopharmaceutical characterisation of small drug molecules. Special attention was paid to the characterisation of acid-base and lipophilic properties of drug compounds by analysing their migration behaviour in different CE systems. The thesis comprises an overview of the field together with separate studies on the different topics.</p><p>The utility of CE for the determination of pK_a of labile drug compounds was investigated. A general methodology was developed comprising key steps such as the use of a stabilising sample diluent, electromigration injection, and analyte characterisation by UV-Vis spectroscopy. The methodology was successfully applied for two sets of drug compounds, labile at low and high pH, respectively.</p><p>CE was also evaluated for experimental modelling of passive intestinal membrane permeability by studying analyte migration in liposomal, microemulsion and micellar electrolytes. Good correlation is reported between CE migration and Caco-2 cell absorption estimates and for in vitro inhibition of thrombin. Interestingly, a slightly better correlation was obtained for liposomal electrolytes.</p><p>The utility of liposomes in CE was further extended by developing a novel procedure for immobilising liposomes inside fused silica capillaries. This approach enabled direct on-line coupling of liposome CE to high sensitivity mass spectrometry. The utility of liposome-coated capillaries is demonstrated for estimating drug passive intestinal membrane permeability. Its use in biopharmaceutical drug profiling is discussed.</p><p>Utilising advanced molecular descriptors, commonly applied to in silico prediction of passive intestinal membrane permeability, migration of analytes in micellar CE systems could be well predicted. The novel approach was based on hierarchical multivariate analytics and use of molecular descriptors for both analytes and micellar media surfactants. Demonstrated results propose that the CE format could be useful to validate how representative molecular descriptors are for describing molecular behaviour in complex liquid media, e.g. physiological systems.</p>