Le polyomavirus BK (ou BKPyV) est un virus ubiquitaire qui infecte plus de 80% de la population adulte. Asymptomatique chez le sujet immunocompétent, il peut être la cause de cystites hémorragiques chez les greffés de cellules souches hématopoïétiques ou de néphropathies interstitielles après transplantation rénale. Parmi les facteurs de risques impliqués dans le développement des néphropathies à BKPyV, nous nous sommes intéressés à l’étude de la variabilité de la région régulatrice non codante (NCCR) du génome viral et à la réponse immunitaire cellulaire spécifique anti- BKPyV en post-greffe. La région NCCR du BKPyV est caractérisée par l’apparition de réarrangements (rrNCCR) chez certains patients présentant une virémie intense et prolongée. Ces réarrangements sont également observés in vitro au cours de la multiplication virale sur cellules permissives. Dans le but de caractériser les rrNCCR et d’étudier leur impact sur la réplication virale, nous avons étudié l’émergence de ces réarrangements in vitro et in vivo sur des échantillons cliniques d’une cohorte de transplantés du rein du CHU de Nantes. Par ailleurs, nous avons étudié la réponse spécifique anti-BKPyV dans les premiers mois postgreffe dans une cohorte prospective de patients adultes transplantés de rein. Nos résultats montrent une augmentation du taux des anticorps au cours de l’infection, et l’absence de valeur prédictive de la réponse médiée par les lymphocytes T sur la survenue d’une infection active à BKPyV. L’ensemble de ces résultats contribue à une meilleure compréhension des mécanismes en jeu au cours des infections à BKPyV en transplantation rénale. / The BK polyomavirus is ubiquitous and infects the majority of the adult population. It is not associated with any specific disease in immunocompetent individuals, but can be responsible for hemorrhagic cystitis after stem cell transplantation or interstitial nephropathy after kidney transplantation. Among the different risk factors involved in the development of such opportunistic diseases, we focused on the genetic polymorphism of the non coding control region of the viral genome (NCCR) and on the specific immune responses directed against BKPyV after kidney transplantation. The NCCR region is characterized by the emergence of rearrangements in vitro on permissive cells, and in vivo in case of prolonged infection and high viral loads. We described the emergence of such rearranged strains in vitro, correlated them with increased viral replication and transcription, and compared these sequences with clinical strains obtained from kidney transplanted patients. Our second objective was to study the specific immune responses in the first months following kidney transplantation. We showed that the active infection was associated with an increase in the anti-BKPyV IgG levels, and that the detection of a CD4+ or CD8+ mediated response was not predictive of a protection toward viral reactivation. Our results contribute to a better understanding of the different factors involved in the pathogenesis of BKPyV infections.

University, Diss.--Jena, 2006.

Bausmės skyrimas – tai toks procesas, kai teismas parenka konkrečią baudžiamajame įstatyme numatytą prievartos priemonę asmeniui, pripažintam kaltu dėl jo padarytos nusikalstamos veikos. Kartu teismas siekdamas bausmės tikslų privalo paskirti teisingą bausmę t.y. ją individualizuoti. Skiriant asmeniui konkrečią bausmę, atsižvelgiama į bausmės skyrimo pagrindus, numatytus BK 54 straipsnyje. Prie tokių pagrindų, turinčių įtakos bausmės skyrimui, yra priskiriamos ir atsakomybę lengvinančios bei sunkinančios aplinkybės. Atsakomybę sunkinančių aplinkybių sąrašas yra įtvirtintas Lietuvos Respublikos Baudžiamojo kodekso 60 straipsnyje. Atsakomybę sunkinančios aplinkybės yra tokios aplinkybės, kurios apibūdindamos kaltąjį asmenį ir jo padarytą veiką, teismui parodo tas bylos aplinkybes, kurios didina nusikalstamos veikos bei kaltojo asmens pavojingumą ir leidžia griežtinti baudžiamąją atsakomybę. Teismui nustačius tokias aplinkybes, kaltininko teisinė padėtis yra griežtinama. Teismas kaltinamajam skiria griežtesnės bausmės rūšį ar dydį. Be to, atsakomybę sunkinančios aplinkybės turi įtakos atleidimo nuo baudžiamosios atsakomybės instituto taikymui. Darbe aiškinama atsakomybę sunkinančių aplinkybių samprata, jas reglamentuojančių baudžiamosios teisės normų istorinė raida. Taip pat atskleidžiamas kiekvienos atsakomybę sunkinančios aplinkybės turinys, taikymas praktikoje. Be to, palyginamas mūsų šalies baudžiamajame įstatyme įtvirtintas atsakomybę sunkinančių aplinkybių sąrašas su kitų... [toliau žr. visą tekstą] / The imposition of punishment is a process when the court selects a specific coercive measure provided by the law for a person, who was pleaded guilty. Because of the purposes of the punishment, the court must impose a fair punishment i.e. individualize it. Also the court must consider the grounds of imposition of punishment defined in the article 54 of the Criminal Code. Circumstances aggravating responsibility and circumstances extenuating responsibility are one of these grounds. The list of circumstances aggravating responsibility is established in the article 60 of the Criminal Code. Circumstances aggravating responsibility are those circumstances that aggrandize the danger of the criminal and his act committed and thus indicate a greater level of criminal responsibility. If the court determines such circumstances, legal status of criminal will be strengthened. The court will impose stricter type of penalty or size of penalty. Moreover, circumstances aggravating responsibility influence the application of the institution of exemption from criminal liability. This master paper clarifies the definition of circumstances aggravating responsibility and discusses the historical development of legal norms of criminal law. Also it explains content of the each one circumstance aggravating responsibility and application of those in practice. Finally, this master paper compares the list of circumstances aggravating responsibility applicable in Lithuania with the list of other... [to full text]

Palmitoylation is a post-translational modification that has been implicated in the control of multiple proteins, including ion channels. S-Palmitoylation is a lipophilic modification that involves the attachment of palmitate through a thioester linkage to a cysteine residue in a target protein. By increasing the hydrophobicity of the target region, palmitoylation can promote membrane targeting. Here, palmitoylation is shown to play an important role in regulating large conductance calcium- and voltage- activated (BK) potassium channels. The STREX splice variant of the BK channel contains a 58 amino acid insert at the splice site C2 within the intracellular C-terminal RCK1-RCK2 linker that confers increased calcium sensitivity to the channel and determines PKA inhibition of channel activity. The cysteine rich STREX domain was predicted to be palmitoylated, and using an imaging assay STREX was shown to act as a membrane targeting domain through palmitoylation of a di-cysteine motif (C645:C645). A membrane potential assay and electrophysiological analysis demonstrates that palmitoylation at the C645:C646 site in STREX is important in mediating the increased calcium sensitive properties inherent to the STREX channel. Palmitoylation is also shown to modulate PKA channel inhibition. The stability of palmitoylation can often be reliant on the local environment within the protein. Generally in most proteins; lipidated regions, basic domains or transmembrane domains are found adjacent to a palmitoylation site. In STREX, a polybasic domain composed of 11 basic residues just upstream from the C645:C646 palmitoylation site, functions to control the palmitoylation status of the STREX insert. A site directed mutagenesis approach to disrupt the polybasic domain revealed an important role in controlling membrane targeting of the STREX C-terminus, mediating the increased calcium sensitivity inherent to STREX channels and controlling the palmitoylation status of the C645:C646 palmitoylation site using multiple techniques involving electrophysiology, fluorescent imaging and biochemical assays. Further to this, using imaging to examine the membrane association of fluorescently tagged C-terminal proteins, phosphorylation is shown to function as a physiological electrostatic switch to regulate the polybasic region in controlling palmitoylation of the STREX insert. Finally, an additional palmitoylation site that is constitutively expressed in all BK channels was identified to be located in the S0-S1 linker (C53:C54:C56). Mutation of the C53:C54:C56 palmitoylation site in the S0-S1 linker was shown to abolish all palmitoylation in BK channels that did not contain the STREX insert. Palmitoylation allows the S0-S1 linker to associate with the plasma membrane however the mutated de-palmitoylated channels did not affect channel conductance or the calcium/voltage sensitivity of the channel. Palmitoylation of the S0-S1 linker was shown to be a critical determinant of cell surface expression of BK channels, as steady state surface expression levels were reduced by ~55% in the C53:C54:C56 mutant. STREX channels that could not be palmitoylated in the S0-S1 linker also showed decreased surface expression even through STREX insert palmitoylation was unaffected. Palmitoylation is rapidly emerging as an important post-translational mechanism to control ion channel behaviour. This work reveals that palmitoylation of the BK channel can control channel function of the STREX splice variant channel and can regulate cell surface expression in all other channel variants. Palmitoylation appears to be functionally independent at these two distinct sites expressed within the same channel protein.

S-palmitoylation is a reversible post-translational lipid modification of proteins by adding a 16-carbon palmitate onto a cysteine residue. Palmitoylation has been shown to control the trafficking and function of many signalling proteins including ion channels. In this Thesis, palmitoylation is shown to control both the plasma membrane expression and gating properties of large conductance calcium- and voltage- dependent potassium (BK) channels. The BK channel is assembled from four pore-forming α-subunits. Each α-subunit contains seven transmembrane domains (S0-S6), with an extracellular N-terminus and a large intracellular C-terminus. BK channel α-subunit is encoded by a single gene Kcnma1 that undergoes extensive pre mRNA splicing at various splice sites, thus there are a number of alternatively spliced variants of α-subunits. Using quantitative imaging assays, palmitoylation of the intracellular S0-S1 loop controlled trafficking of full length ZERO variant BK channels to the plasma membrane in HEK293 cells as well as neuronal N2a cells. Importantly, all four α-subunits need to be palmitoylated for robust surface expression. Thus, palmitoylation of the S0-S1 loop of the α-subunit is important for surface expression of BK channels. The BK channel may also assemble with auxiliary β-subunits (β1-4) that regulate surface expression and gating properties of BK channels. The N-terminus of the β1- subunit and the C-terminus of the β4-subunit were shown to be palmitoylated using [3H]-palmitate incorporation, respectively. However, mutation of the palmitoylated cysteine (C18 in β1 and C193 in β4) to alanine to generate depalmitoylated β- subunits had no significant effects on the electrophysiological properties resulting from co-expression with the ZERO variant of the BK channel. However, although palmitoylation of the S0-S1 loop does not affect the electrophysiological properties of the ZERO channels alone, it is important for the shift in the V0.5max of ZERO channel when co-expressed with the β1-subunit, but not β4-subunit. These data suggest that palmitoylation of the S0-S1 loop controls the functional coupling between the ZERO α-subunit and β1-subunit. Although palmitoylation of C18 in the N-terminus of the β1-subunit was not required for functional coupling to α-subunits, we identified other critical residues within the short intracellular N-terminus of the β1-subunit that are essential. The functional coupling between BK α- and β1-subunit was predicted to be controlled by the interaction between a non-classic amphipathic α-helix in the β1 subunit N-terminus and the plasma membrane. Deletion, or mutations predicted to disrupt the interaction significantly decreased the β1-subunit induced left shift in the BK channel V0.5max. This suggests that the amphipathic in-plane anchor is critical for functional coupling of β1-subunits with BK channel α-subunits. In this Thesis, we demonstrated: i) palmitoylation of the α-subunit S0-S1 loop controls surface membrane expression of BK channels, and also controls functional regulation by β1, but not β4-subunits; and ii) a potential non-classical amphipathic in-plane anchor in the β1 N-terminus is essential for functional coupling with α- subunits. These studies help us further understand the regulation of BK channels and suggest potential therapeutic targets for various diseases related to dysfunctional BK channels, such as hypertension.

The theory of quantum chromodynamics (QCD) predicts that at high energies, such as those investigated in deep inelastic scattering experiments, hadrons evolve into dense gluonic states described by the BFKL equation, and at very high densities, the more general BK equation. In certain approximations, the BK equation reduces to a well studied reaction-diffusion type nonlinear partial differential equation, the FKPP equation, for which analytical results are known. In this work, we model the BK equation using a classical branching process rooted in the dipole model of QCD evolution. Because the BK equation is inherently two dimensional, our model allows dipole impact parameters to occupy the full transverse space. A one dimensional limit of this model is studied as well. Results are compared with the predictions of the FKPP equation, and correlations between evolution at different impact parameters are presented. The general features of previously studied one dimensional impact parameter models are verified, but the details are refined in what we believe to be a more accurate model. / text

Tolerance to anesthetic drugs is mediated partially by homeostatic mechanisms that attempt to restore normal neural excitability. The BK-type Ca2+-activated K+ channel, encoded by the slo gene, plays an important role in this neural adaptation. In Drosophila, a single sedative dose of the organic solvent anesthetic benzyl alcohol induces dynamic spatiotemporal changes in histone H4 acetylation across the slo regulatory region and leads to slo induction and tolerance. Mutations ablating the expression of slo also block the acquisition of tolerance, whereas activating the expression of a slo transgene results in resistance to drug sedation. Moreover, artificially inducing histone acetylation with the histone deacetylase inhibitor causes similar acetylation changes, slo induction, and functional tolerance to the drug. Histone acetylation changes occur over two highly conserved non-coding DNA elements, 6b and 55b, of the slo control region. To investigate the function of these two elements, I generated individual knockout mutants by gene targeting. Both knockout alleles are backcrossed into the CS wild type background. The 6b element seems to repress slo induction after drug sedation, because the 6b knockout allele overreacts to the drug. Compared to the wild type, 6b knockout allele shows a much greater slo message induction after drug sedation, it also displays stronger enhancements in seizure susceptibility and following frequency. In addition, the 6b deletion causes a persistent tolerance for at least a month, while tolerance only lasts about 10 days in wild type flies. My investigation also indicates that the 55b element limits basal slo expression in muscle. Finally, to investigate if the particular histone acetylation spikes are required for drug-induced slo induction and tolerance, I tether the histone-modifying enzymes, HDAC or HAT, to the 6b and 55b DNA elements, respectively. I observe that the positioning of an HDAC on these two elements blocks drug-induced slo induction and the development of tolerance. Therefore, histone acetylation across slo control region is required for the activation of slo and the acquisition of tolerance. / text

Šiame darbe analizuojama karo nusikaltimų traktuotė tarptautinės teisės aktuose bei Lietuvos Respublikos Baudžiamajame kodekse. Pirmiausia yra tiriama šių nusikalstamų veikų samprata tarptautiniuose aktuose ir, siekiant pateikti išsamų ir aiškų karo nusikaltimų apibrėžimą, yra išnagrinėjama šių nusikaltimų sampratos raida, taip pat analizuojamas jų ryšys su tarptautine humanitarine teise bei, viso to pasekoje, išskiriami pagrindiniai karo nusikaltimų požymiai, išskiriantys juos iš visų kitų nusikalstamų veikų: nusikaltimų padarymo laikas; sunkūs ir rimti tarptautinės teisės normų pažeidimai; dalykas ir nukentėjusieji; subjektas; subjektyvioji pusė; ryšys su ginkluotu konfliktu. Minėti požymiai darbe išsamiai analizuojami, apžvelgiant bei lyginant Tarptautinio baudžiamojo tribunolo buv. Jugoslavijai praktiką, taip pat Tarptautinio baudžiamojo teismo, tarptautinių baudžiamųjų tribunolų statutus, užsienio bei Lietuvos teisės doktriną. Nagrinėjant šiuos požymius yra akcentuojami daugiausia diskusijų sukėlę klausimai bei problemos, su kuriomis buvo susidurta reglamentuojant karo nusikaltimus bei iki šiol susiduriama juos taikant. Darbe taip pat analizuojama Lietuvos Respublikos baudžiamajame kodekse įtvirtintų karo nusikaltimų samprata, jų rūšinė sudėtis, jos atitiktis tarptautinės teisės nuostatoms bei atskirų LR BK XV skyriuje nurodytų karo nusikaltimų atitiktis tarptautinės teisės normoms, taip pat akcentuojami kiti probleminiai LR BK XV skyriaus klausimai, pateikiami... [toliau žr. visą tekstą] / This thesis provides a detailed analysis of the approach to war crimes in the instruments of the International law and in the Criminal code of the Republic of Lithuania. First of all, the conception of war crimes in international documents is analized, with an objective to provide a clear and comprehensive definition of war crimes; also the link between war crimes and the humanitarian law is defined and the main elements that distinguishes war crimes from other crimes are provided. Those elements are: the circumstances in which war crimes are commited; grave breaches and other serious violations of the norms of the International law; the object and victims; subject; mens rea; nexus between war crime and armed conflict. The mentioned elements are analized in view and comparison of the case law of International Criminal Tribunal for former Yugoslavia (ICTY), the statutes of International Criminal Court and the statutes of the International criminal tribunals and the approach to war crimes in Lithuanian and in International law. While analysing, the most controversional issues are being held and problems that appeared in implementing war crimes and are still appearing in practice are accented. This thesis also provides analysis of the concept of war crimes in the Criminal Code of Lithuania, the elements of war crimes, implemented in the Criminal code of Lithuania and the coincidence with the documents of International law. Also the problematic issues are held and some brief... [to full text]

Large conductance Ca2+-activated K+ channels (BK channels) are expressed ubiquitously in both excitable and non-excitable cells and are important for a range of physiological functions. BK channels gate K+ efflux in response to concurrent depolarized membrane voltage and increased intracellular Ca2+ to modulate action potential shape and duration in neurons, regulate contractility in smooth muscle, and control fluid secretion by epithelial cells in the airway and gut. In addition, mutations in the human BK channel gene (KCNMA1) are linked to neurological disease, such as epilepsy and paroxysmal dyskinesia. Thus, BK channel modulators may provide treatment avenues for BK channelopathies. It will be important to expand our arsenal of BK channel-selective activators and inhibitors and to grow our understanding of their molecular mechanisms of action. Discovery of new channel modulators will further lead to a greater understanding of BK channel structure and function. To better understand the basic structure-function relationship of BK channel gating in response to increased intracellular Ca2+ concentration, in this work I initially investigate structural determinants of BK channel activation in response to conformational changes following Ca2+ binding. I analyze crystal structures of the BK channel cytosolic Ca2+-sensing domain (CSD), also known as the “gating ring”, formed by the C-terminal domains of each of the four identical pore-forming subunits. In the Ca2+-bound state, N449 from the adjacent subunit contacts the bound Ca2+ ion, forming a “Ca2+ bridge.” Mutating N449 to alanine eliminates this coordinate interaction, and using electrophysiology, I found that BK channels with the N449A mutation exhibit a shift in the voltage required for half maximal activation (V1/2) towards more positive voltages. Using size-exclusion chromatography, I observed that the purified BK channel CSD with the N449A mutation shows reduced gating ring oligomerization in response to Ca2+ compared to the wild-type CSD. To further probe molecular determinants of BK channel gating and increase our arsenal of BK channel gating modulators, I optimized a fluorescence-based high throughput screening approach to discover compounds with BK channel inhibitor activity with 99.73% confidence. Through this approach I discovered that the -opioid receptor agonist, loperamide, is a potent BK channel inhibitor. Loperamide (LOP) reduced the open probability of channels at depolarized voltages, but not at very negative voltages when the voltage-sensor is at rest. I observed a weak voltage dependence of loperamide inhibition, consistent with loperamide binding shallow within the inner cavity to block the channel pore. I quantified the inhibitory effect of LOP using an allosteric model in which LOP blocks conduction through open channels and binds with 45-fold higher affinity to the open state over the closed state. These data suggest that loperamide may represent a new class of “use-dependent,” open channel blockers. Together this work describes an approach to understanding BK channel structure and function with the goal of identifying and developing novel therapeutics for the treatment of BK-related diseases. / Biochemistry

Šiame magistriniame darbe atskleidžiamas Lietuvos Respublikos baudžiamajame kodekse numatytų baudžiamąją atsakomybę lengvinančių aplinkybių turinys, šio instituto aiškinimas baudžiamosios teisės teorijoje ir teismų praktikoje, identifikuojami teorijos ir praktikos neatitikimai ir pagrindinės kylančios problemos. Magistrinio darbo tikslas yra ir atsakomybę lengvinančių aplinkybių instituto reikšmės bausmės skyrimui išsamus ištyrimas ir aprašymas bei pagrindinių kylančių problemų analizė. Taip pat šiame darbe tiriami naujuoju LR BK įvesti atsakomybę lengvinančių aplinkybių reglamentavimo pakeitimai, jų priežastys, atliekant lyginamąją analizę su senuoju (1961 m.) baudžiamuoju kodeksu, nagrinėjama kaip šis institutas aprašomas kitų valstybių baudžiamuosiuose kodeksuose bei kitose LR teisės šakose. / Criminal code of Lithuanian Republic, article number 59 regulates mitigating circumstances. In this yearly paper, author tries to find out the main specific features of this article, the evolution of it, comparing to old criminal code, and the main principles, which courts have created, when trying of this kind of cases.