Jaburetox (Jbtx) é um peptídeo de 10 kDa derivado de uma das isoformas de urease de Canavalia ensiformis. Em um estudo anterior realizado com o triatomíneo vetor da doença de Chagas Triatoma infestans, esse peptídeo foi encontrado interagindo com a proteína UDP-N-acetilglicosamina pirofosforilase (UAP), alterando também sua atividade enzimática no sistema nervoso central, in vivo e in vitro. A UAP já foi encontrada em eucariotos, bactérias e vírus, estando relacionada com as rotas de produção de quitina, N-glicosilação e síntese de glicoinositolfosfolipídeos. Assim, o presente trabalho tem três objetivos: i) investigar o efeito de Jbtx sobre a atividade enzimática e a expressão gênica da UAP do inseto modelo Rhodnius prolixus, ii) clonar e expressar a UAP e iii) estudar a UAP filogeneticamente. Para a primeira parte, foram avaliados, no triatomíneo R. prolixus, a atividade enzimática da UAP e o perfil de expressão dessa enzima e da quitina sintase em insetos controles e alimentados com Jbtx. Para a segunda, o cDNA da enzima de R. prolixus foi clonado em vetor pET-15b e expressado em Escherichia coli Rosetta 2. A purificação da enzima recombinante foi feita por cromatografia de afinidade a níquel. Para a terceira parte, foram buscadas sequências de aminoácidos homólogas às da UAP de R. prolixus no servidor pHmmer e foi construída uma árvore filogenética com o método de Máxima Verossimilhança. Os resultados obtidos indicam que o Jbtx aumenta a atividade enzimática da UAP em glândulas salivares, corpo gorduroso e epiderme, enquanto diminui a expressão da UAP em intestino médio anterior, túbulos de Malpighi, glândulas salivares, corpo gorduroso, epiderme e sistema nervoso central, assim como a expressão da quitina sintase nos mesmos órgãos e no intestino médio posterior. Foi obtida uma UAP recombinante de 56 kDa, compatível com peso molecular previsto in silico. A árvore filogenética construída contém 40 sequências, sendo 38 de insetos e 2 sequências de grupo externo. A árvore segue o padrão de evolução dos insetos e foi identificado um novo organismo com potenciais dois genes codificantes de UAP. Esse trabalho apresenta a primeira evidência de que Jbtx altera a expressão gênica em R. prolixus. O resultado obtido pela análise filogenética indica que a UAP é uma enzima ancestral à diversificação em Insecta. / Jaburetox (Jbtx) is a 10 kDa peptide derived from a urease isoform of Canavalia ensiformis. In a previous work with the triatomine vector of Chagas’ disease Triatoma infestans, this peptide was found interacting with the protein UDP-N-acetylglucosamine pyrophosphorylase (UAP), also increasing the UAP enzymatic activity in the central nervous system in vivo and in vitro. UAP has been described in eukaryotes, bacteria and virus, and is involved in chitin production, N-linked glycosylation and glyco inositol phospholipids synthesis pathway. Thus, the present work has three main aims: i) to understand the effect of Jbtx on this enzyme on the model insect Rhodnius prolixus, ii) to clone and express UAP and iii) to study UAP from a phylogenetic point of view. Firstly, UAP enzymatic activity and its expression profile, as well as the chitin synthase expression, were analysed in the triatomine R. prolixus in saline- or Jbtx-fed insects. Secondly, the cDNA from R. prolixus’ UAP was cloned into the pET-15b vector and expressed in Escherichia coli Rosetta 2. The recombinant enzyme was purified through a nickel affinity chromatography. Thirdly, homolog sequences to R. prolixus’ UAP were searched in pHmmer database and a phylogenetic tree was built using the Maximmum Likelihood method. The results obtained indicate that Jbtx increases UAP enzymatic activity in salivary glands, fat body and epidermis, while decreasing UAP’s expression in the anterior and posterior midgut, Malpighian tubules, salivary glands, fat body, epidermis and central nervous system, as well as the chitin synthase expression in the same organs and the posterior midgut. A 56 kDa recombinant UAP was obtained, in agreement with the in silico estimated size. The phylogenetic tree built has 40 sequences, from which 38 are from insects and 2 are from mammals (external group). The tree follows the insect evolution patterns and a new organism containing two potential UAP coding genes was identified. This work presents the first evidence that Jbtx is able to interfere in the gene expression in R. prolixus. The results obtained through phylogenetic analysis shows that UAP is an enzyme ancestral to the diversification in Insecta.

The stability of the Internet is at risk if the amount of voice and video traffic continues to increase at the current pace. While current transport layer protocols do work well for most applications, they still present some problems. TCP is reliable, tracks the state of some network conditions and reacts drastically to an indication of congestion. TCP serves data-oriented applications very well but it can lead to unacceptably low quality for streaming applications by multiplicatively reducing the congestion window upon a sign of congestion. The other main transport layer protocol, UDP, provides good service for streaming applications but is not friendly to TCP and can cause the well-known existing congestion collapse problem in the Internet. This thesis proposes a new protocol to provide a good service for voice and video applications while being friendly to TCP and solving the congestion collapse problem. The protocol utilizes a fuzzy logic controller that considers network related information to govern the applications sending rate while satisfying the users needs. Using network information such as the available bandwidth, Packet Loss Rates (PLR), and Round Trip Times (RTT) a fuzzy inference system optimizes the applications send rate to meet the requested rate in a smooth manner without wasting network resources unnecessarily. The fuzzy logic controller is designed and its performance evaluated using MATLAB model simulations. The results indicate that the fuzzy controller solves the congestion collapse problem by reducing the number of undelivered packets into the network by nearly 100%. It provides smooth transition changes as demonstrated by the controlled UDP flow utilizing an estimated 44% more of the available bandwidth to smooth the send rate than the TCP flow in a highly varying bandwidth environment. The controller also remains friendly with TCP which was demonstrated to share the bandwidth at nearly 50% with one other competing controlled UDP flow.

<p>To detect network changes and network troubles, Transpondertech needs a tool that can make network measurements.</p><p>The purpose of this thesis has been to find measurable network properties that best reflect the status of a network, to find methods to measure these proerties and to implement these methods in one single tool. The resulting tool is called NetworkPerf and can measure the following network properties: availability, round-trip delay, delay variation, number of hops, intermediate hosts, available bandwidth, available ports, and maximum allowed packet size. Together, these properties give a good picture of the status of a network connection.</p><p>The thesis also presents the methods used for meassuring these properties in the tool.</p>

No description available.

<p>With the Internet expansion, real-time transmission over Internet is becoming a new promising application. Successful real-time communication over IP networks requires reasonably reliable, low delay, low loss date transport. Since Internet is a non-synchronous packet switching network, high load and lack of guarantees on data delivery make real-time communication such as Voice and Video over IP a challenging application to become realistic on the Internet. </p><p>This thesis work is composed of two parts within real-time voice and video communication: network simulation and measurement on the real Internet. In the network simulation, I investigate the requirement for the network"overprovisioning"in order to reach certain quality-of-service. In the experiments on the real Internet, I simulate real-time transmission with UDP packets along two different traffic routes and analyze the quality-of- service I get in each case. </p><p>The overall contribution of this work is: To create scenarios to understand the concept of overprovisioning and how it affects the quality-of-service. To develop a mechanism to measure the quality-of-service for real-time traffic provided by the current best-effort network.</p>

<p>Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures<i> </i>and growing<i> </i>plants<i>. </i>The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated.</p><p>A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb.</p><p>Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis.</p><p>Expression of the bacterial UGDH in <i>E. coli</i> resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. </p><p>Overexpression of UGD1, one of four <i>A. thaliana</i> UGDH genes, in <i>A. thaliana,</i> resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.</p>

<p>The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix in all higher organisms, affecting cellular processes such as migration, proliferation and differentiation. Hyaluronan is synthesized by a plasma membrane bound hyaluronan synthase (HAS) which exists in three genetic isoforms. This thesis focuses on the understanding of the hyaluronan biosynthesis by studies on the expression and regulation of the HAS proteins.</p><p>In order to characterize the structural and functional properties of the HAS isoforms we developed a method to solubilize HAS protein(s) while retaining enzymatic activity. The partially purified HAS protein is, most likely, not asscociated covalently with other components. Cells transfected with cDNAs for HAS1, HAS2 and HAS3 were studied and all three HAS isozymes were able to synthesize high molecular weight hyaluronan chains in intact cells. The regulation of the hyaluronan chain length involves cell specific elements as well as external stimulatory factors. HAS3 transfected cells with high hyaluronan production exhibit reduced migration capacity and reduced amounts of a cell surface hyaluronan receptor molecule (CD44) compared to wild-type cells.</p><p>The three HAS isoforms were studied and shown to be differentially expressed and regulated in response to external stimuli. Platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-<i>β</i>1) are important regulators of HAS at both the transcriptional and translational level. The HAS2 isoform is the isoform most susceptible to external regulation.</p><p>The role of the UDP-glucose dehydrogenase in mammalian glycosaminoglycan biosynthesis was assessed. The enzyme is essential for hyaluronan, heparan sulfate and chondroitin sulfate biosynthesis, but does not exert a rate-limiting effect.</p>

The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix in all higher organisms, affecting cellular processes such as migration, proliferation and differentiation. Hyaluronan is synthesized by a plasma membrane bound hyaluronan synthase (HAS) which exists in three genetic isoforms. This thesis focuses on the understanding of the hyaluronan biosynthesis by studies on the expression and regulation of the HAS proteins. In order to characterize the structural and functional properties of the HAS isoforms we developed a method to solubilize HAS protein(s) while retaining enzymatic activity. The partially purified HAS protein is, most likely, not asscociated covalently with other components. Cells transfected with cDNAs for HAS1, HAS2 and HAS3 were studied and all three HAS isozymes were able to synthesize high molecular weight hyaluronan chains in intact cells. The regulation of the hyaluronan chain length involves cell specific elements as well as external stimulatory factors. HAS3 transfected cells with high hyaluronan production exhibit reduced migration capacity and reduced amounts of a cell surface hyaluronan receptor molecule (CD44) compared to wild-type cells. The three HAS isoforms were studied and shown to be differentially expressed and regulated in response to external stimuli. Platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-β1) are important regulators of HAS at both the transcriptional and translational level. The HAS2 isoform is the isoform most susceptible to external regulation. The role of the UDP-glucose dehydrogenase in mammalian glycosaminoglycan biosynthesis was assessed. The enzyme is essential for hyaluronan, heparan sulfate and chondroitin sulfate biosynthesis, but does not exert a rate-limiting effect.

Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures and growing plants. The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated. A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb. Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis. Expression of the bacterial UGDH in E. coli resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. Overexpression of UGD1, one of four A. thaliana UGDH genes, in A. thaliana, resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.

UDP-galactopyranose mutase (EC 5.4.99.9; UGM), the product of the glf gene, is an enzyme that catalyzes the conversion of uridine diphosphate galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM activity is found in bacteria, parasites and fungi, however is absent in higher eukaryotes. This enzyme is essential for the viability of many pathogenic organisms, such as Mycobacterium tuberculosis and Escherichia coli, due to the broad distribution of Galf in crucial structures such as the cell wall or capsular polysaccharide. Not surprisingly, galactofuranose biosynthesis has become an attractive antimicrobial target due to the absence of these sugars in higher eukaryotes. The UGM homologue, UDP-Nacetylgalactopyranose mutase (UNGM), was identified in Campylobacter jejuni 11168, encoded for by the cj1439c gene. UNGM is known to function as a bifunctional mutase, which catalyzes the reversible ring contraction between the pyranose-furanose forms of UDPgalactose (UDP-Gal) and UDP-N-acetylgalactosamine (UDP-GalNAc). UNGM is essential for the virulence of C. jejuni, due to the incorporation of UDP-N-acetylgalactofuranose into the capsular polysaccharide. We report the first structure of UNGM determined by X-ray crystallography, to a resolution of 1.9 Å. Analysis of the dimeric, holoenzyme structure of UNGM has identified that the cofactor flavin adenine dinucleotide is bound within each monomer of the enzyme. Comparative analysis with UGM homologues has confirmed the conserved active site residues involved in the binding of various substrates. Docking studies suggest that UNGM binds its natural substrates in a productive binding mode for catalysis with the flavin cofactor, which is consistent with the proposed mechanism for UNGM. The mobile loops are essential for substrate binding, and we have identified that the conserved arginine residue, Arg169, and the neighboring Arg168, function to stabilize the diphosphate region of UDP, although not concurrently. The non-conserved arginine residue, Arg168, appeared to favor the stabilization of N-acetylated sugars, which is in agreement with the enzyme’s higher binding affinity for UDP-GalNAc over UDP-Gal by a factor of 0.9. We have also identified that the active site Arg59 exists in two conformations in the structure of UNGM, with one conformation directed toward the active site. Arg59 is 2.5 to 3.0 Å from the acetamido moiety of GalNAc, which is favorable for stabilization and is believed to confer specificity for this substrate.