5 Abstract Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) belongs to the serine/ threonine protein kinase family, which is involved in the calcium signaling pathway. The increase of intracellular calcium concentration induces the activation of calmodulin (CaM), which then activates its binding partners including CaMKII, CaMKIII, CaMKK1 and CaMKK2. CaMKK2 activates CaMKI, CaMKIV and AMP-dependent kinase, AMPK, by phosphorylation. CaMKK2 is naturally present in cells in an autoinhibited state, which is caused by the steric hindrance of the active site by the autoinhibitory domain. When calmodulin binds to the calmodulin-binding domain, the autoinhibitory domain is removed and the active site becomes accessible. Upon activation, CaMKK2 undergoes autophosphorylation, which increases its enzyme activity. Negative regulation of CaMKK2 is mediated by cAMP-dependent protein kinase A (PKA)- and GSK3-dependent phosphorylation. Sites phosphorylated by PKA have been identified for both CaMKK1 and CaMKK2. Two of them are also motifs recognized by scaffolding 14-3-3 proteins. Previous studies have shown that the 14-3-3 protein binding maintains phosphorylated CaMKK2 in an inhibited state by blocking the dephosphorylation of S495, which prevents the binding to calmodulin. However, it is unclear if it is the...

This diploma thesis focuses on the MANET Network (Mobile Ad-Hoc Network). There are theoretically elaborated basic information, the characteristics of the MANET network and great emphasis is placed on routing in these networks. In the next chapter the diploma thesis focuses on the Ad Hoc Demand Distance Vector (AODV) routing protocol. It features the characteristics of this protocol and the principle of communication and routing in MANET networks. Hereinafter, the individual reports of the AODV protocol are described in detail. In the third chapter, the thesis introduces the NS-3 simulation environment (network Simulator - 3) and then deals with the design of the MANET network in NS-3 using the AODV routing protocol. The practical part of the diploma thesis is the MANET network simulation and testing with the AODV routing protocol. Laboratory task is built from these simulations and the results of the testing.

An ad hoc mobile network is a collection of mobile nodes that are dynamically and arbitrarily located in such a manner that the interconnections between nodes are capable of changing on a continual basis. In order to simplify communication within the network, a routing protocol is used to discover routes between nodes. The primary goal of such an ad hoc network routing protocol is correct and efficient route establishment between a pair of nodes so that messages may be delivered on time. Route construction should be done with a minimum of overhead and bandwidth consumption. This document describes the Optimized Link State Routing (OLSR) protocol for mobile ad hoc networks. The key concept used in the protocol is that of multipoint relays (MPRs). MPRs provide an efficient mechanism for flooding control traffic by reducing the number of transmissions required.

La maturation 3’ des pré-ARNm constitue une étape majeure dans la régulation post-transcriptionnelle de l’expression des gènes, indispensable à la stabilité, l’export vers le cytoplasme et la traduction des ARNm. Elle est composée de deux réactions : un clivage à l’extrémité 3’ suivie de l’addition d’une queue poly(A). Des études ont montré que la maturation en 3’ est inhibée en réponse aux dommages de l’ADN. Cependant, la cellule a mis en place des mécanismes compensatoires qui permettent à certains pré-ARNm d’être correctement maturés assurant ainsi le maintien de son intégrité. Les travaux que nous avons menés ont mis en évidence un mécanisme de résistance à l’inhibition de maturation en 3’ du pré-ARNm codant pour le suppresseur de tumeur p53. Ce mécanisme fait intervenir l’hélicase DHX36 qui déplie une structure secondaire appelée G-quadruplexe située en aval du site de clivage. Par ailleurs dans une deuxième étude, nous avons montré que la maturation en 3’ maintenue du pré-ARNm p53 en réponse aux dommages de l’ADN, est découplée du processus de transcription, contrairement au pré-ARNm TBP dont la maturation 3’ est inhibée en réponse aux dommage de l’ADN. Ce découplage a lieu grâce à un clivage co-transcriptionnelle du pré-ARNm p53 au niveau de la chromatine qui entraîne sa libération dans le nucléoplasme où il subit sa maturation en 3’. Une étude à grande échelle nous a permis de montrer que ce mécanisme de maturation en 3’ survenant dans le nucléoplasme est associé au maintien d'une maturation en 3’ efficace en réponse aux dommages de l’ADN. / The 3’-end processing of pre-mRNA, a key step in the post-transcriptional gene expression regulation, is essential for mRNA stability, export and translation. This process is a two-step reaction composed of a cleavage at the 3’-end followed by the addition of a poly(A) tail. Studies have shown that pre-mRNA 3’-end processing is inhibited in response to DNA damage. However, compensatory mechanisms exist to allow some pre-mRNA to be properly processed at their 3’-end in order to maintain cell integrity. For instance, in response to DNA damage, the 3’-end processing of the pre-mRNA coding for the tumor suppressor p53 is able to escape from its inhibition. In the present work, we have shown that the underlying mechanism involves the DHX36 helicase that unwinds a secondary structure called G-quadruplex located downstream of the cleavage site of the p53 pre-mRNA. Moreover, in a second study, we have shown that the maintained p53 pre-mRNA 3’-end processing in response to DNA damage is uncoupled from the transcription process, unlike the inhibited TBP pre-mRNA 3’-end processing. This uncoupling takes place through a co-transcriptional cleavage of p53 pre-mRNA from the chromatin and its release in the nucleoplasm where it undergoes its 3’-end processing. A genome-wide study allowed us to show that the pre-mRNA 3’-end processing occurring in the nucleoplasm is associated with a maintained 3’end processing in response to DNA damage

Orientador: Cleslei Fernando Zanelli / Resumo: eIF5A é um fator de elongação da tradução conservado em arqueias e eucariotos, que interage com a subunidade maior do ribossomo 80S. É a única proteína que possui o aminoácido hipusina, formado pós-traducionalmente, essencial para sua função. eIF5A auxilia na tradução de proteínas que contêm motifs ricos em prolina, pois sequência repetida deste aminoácido pode causar parada do ribossomo em tradução devido à rigidez estrutural que forma. Na ausência de eIF5A funcional ocorre parada do ciclo celular em G1, na transição de G1 para S, tanto em Saccharomyces cerevisiae quanto em mamíferos, sugerindo papel essencial desta na tradução de proteínas importantes para a progressão do ciclo celular. Neste trabalho foi proposta a identificação das proteínas relevantes para a fase G1 que se encontram diferencialmente presentes durante desativação de eIF5A em S. cerevisiae. Foram utilizados dados de larga escala de perfil proteômico diferencial entre linhagens selvagens e mutantes de eIF5A, previamente obtido por nosso grupo, como ponto de partida para identificação das proteínas relevantes. A confirmação de níveis diferenciais destas proteínas foi realizada utilizando metodologia de Western blot após indução da desativação de eIF5A, utilizando o mutante sensível a temperatura hyp2-3. A desativação foi feita através do cultivo da linhagem mutante em temperatura semi-permissiva, que induz perda da função de eIF5A, porém mantendo viabilidade celular. Foi observado que a proteína Cka2 está di... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: eIF5A is a translation elongation factor conserved in archaea and eukaryotes, that interacts with the major subunit of the 80S ribosome. It is the only protein to contain the amino acid hypusine, formed post-translationally, essential to its function. eIF5A helps the translation of proteins that contain proline enriched motifs, as repeated sequence of this amino acid can cause ribosome stall during translation due to the rigid structure that it forms. In the absence of functional eIF5A occurs cell cycle arrest in G1, on the transition of G1 to S, both in Saccharomyces cerevisiae and mammals, suggesting an essential role of it on the translation of important proteins to the progression of the cell cycle. In this work, it was proposed the identification of relevant proteins to the G1 phase that are present differentially during deactivation of eIF5A in S. cerevisiae. It was used data of high-throughput differential proteomic profile between wild type strains and eIF5A mutant strains, previously obtained by our group, as a starting point to the identification of the relevant proteins. The confirmation of the differential levels of these proteins was made using Western blot methodology after induction of eIF5A deactivation, using the temperature-sensitive mutant hyp2-3. The deactivation was performed through cultivation of the mutant strain in semipermissive temperature, that induces loss of function of eIF5A but maintain cell viability. It was observed that the protein Cka2 is d... (Complete abstract click electronic access below) / Mestre

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Martin Svoreň Supervisor: PharmDr. Martina Čečková, Ph.D. Specialized supervisor: PD Dr. Bernd Evert Title of diploma thesis: Analysis of genomic regions bound and regulated by Ataxin-3 Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a dominantly inherited neurodegenerative disease. In SCA3, the disease protein ataxin-3 (ATXN3) contains an abnormally long polyglutamine (polyQ) tract encoded by CAG repeat expansion. ATXN3 binds DNA and interacts with transcriptional regulators pointing toward a direct role of ATXN3 in transcription. It is conceivable that mutant ATXN3 triggers multiple, interconnected pathogenic cascades leading to neurotoxicity, however, the principal molecular pathomechanism remains elusive. Here, PCR analyses of 16 ATXN3-bound genomic regions recently identified by next generation sequencing of immunoprecipitated ATXN3-bound chromatin fragments confirmed enriched binding of ATXN3 to 5 genomic regions next to genes encoding CCAAT/enhancer binding protein delta (CEBPD), period circadian clock-2 (PER2), phosphatase and tensin homolog (PTEN), serine protease inhibitor family F2 (SERPINF2) and thrombospondin-1 (THBS1). To investigate putative...

Was führt kriminaltechnische Sachverständige zur Konferenz Entwerfen Entwickeln Erleben Es mag ungewöhnlich sein, dass sich hier Kriminaltechniker bzw. kriminaltechnische Sachverständige zu Wort melden. Bedenkt man, dass sich die Kriminaltechnik ja nahezu ausschließlich naturwissenschaftlicher Methoden bedient, so sind allein hierin schon Berührungspunkte vorgegeben. Für uns ist es die Erprobung einer neuen Methode, sich ganz konkret an den Lehrstuhl Konstruktionstechnik/CAD zu wenden. Wir glauben im Übrigen nicht, dass es ungewöhnlich ist, dass die Kriminaltechnik mit universitären Einrichtungen zusammenarbeitet. Immerhin, wenn man in ihre Geschichte schaut, hat sich die Kriminaltechnik aus Zweigen einzelner Naturwissenschaften entwickelt.

The synthesis of 3- and 6- aminosugars from the old route requires many synthetic steps and is challenging. An alternative approach is to utilize acid-catalyzed hydrolysis of kanamycin derivatives. The 3-and 6 aminosugar donor was synthesized in just two steps with excellent yield and cost-effective. The acidic hydrolysis of these aminoglycosides provided not only the 3-and 6-aminosugars, but the direct chemical glycosylation of these aminosugars was proven feasible using isopropanol and octanol as the acceptor.

No description available.

Shape synthesis (also called topological synthesis or inverse design in other disciplines) has the potential to provide antenna engineers with a useful addition to their design tools. Transmitarray antennas, which consist of a feed antenna plus a printed planar structure that emulates a lens, are able to provide high directivity antenna performance, and have been the subject of sustained research over the past ten years. The transmitarray lens consists of a lattice of cells, with each cell occupied by an element that includes conductors of specific shape. The feed field incident on each element on the input surface side of the transmitarray is transformed by each element into a field of different amplitude and phase on the output surface side of each element, providing some desired aperture distribution on the output surface. In this thesis we develop a technique, and the overall computational tool to implement it, that fundamentally allows the electromagnetics to dictate how the conducting portions of a 3-layer element must be shaped in order to obtain some specific transmission coefficient. Such shape synthesis of the elements offers the possibility of obtaining elements that have properties not obtainable using conventional elements. These techniques were applied to the shape synthesis of dual-band elements (18 GHz and 24 GHz). A transmitarray using these elements was designed and fabricated, its performance measured and compared to simulated results. An in-depth discussion of the outcome experimentally validates the shape synthesis procedure.