HPr est une protéine du système phosphoénolpyruvate:sucre phosphotransférase impliquée dans le transport, la régulation d'enzymes et de transporteurs et la régulation de la transcription de certains gènes. La protéine HPr peut être retrouvée sous quatre formes différentes. Elle peut être phosphorylée sur un résidu histidyl par l'enzyme 1 pour former HPr(His"-'P). Sous cette forme, HPr peut, entre autres, contrôler par phosphorylation certains régulateurs transcriptionnels contenant des domaines PRD. Chez les bactéries à Gram positif, HPr peut être phosphorylée sur un résidu séryl par la HPr kinase/phosphorylase. Le produit formé, HPr(Ser-P), est impliqué dans la répression catabolique selon un mécanisme impliquant la formation d'un complexe entre HPr(Ser-P), la protéine CcpA et une séquence spécifique d'ADN située dans la région promotrice des opérons cibles, la séquence cre. HPr(Ser-P) est également impliquée dans le phénomène d'exclusion d'inducteur. Enfin, des taux importants de HPr(Ser-P)(His-P) ont été détectés chez Streptococcus salivarius, Streptococcus mutans et Streptococcus thermophilus. Afin de caractériser HPr(Ser-P)(His-P), nous avons vérifié si certaines bactéries de la classe des Bacilli pouvaient produire et maintenir des taux importants de HPr(Ser-P(His-P) et vérifié si HPr(Ser-P)(His-P) pouvait participer au transport des sucres. Les quantifications de HPr ont été effectuées par immunoélectrophorèse croisée et la capacité de HPr(Ser-P)(His-P) à phosphoryler les enzymes IIABMan et la glycérol kinase ont été étudiées. Des taux importants de HPr(Ser-P)(His-P) ont été détectés chez toutes les souches de streptocoquès et lactocoques testées mais pas chez Bacillus subtilis, Enterococcus faecalis et Staphylococcus aureus. HPr(Ser-P)(His-P) était en mesure de transférer son groupement phosphoryle aux enzymes IIABMan mais' pas à la glycérol kinase, suggérant que HPr(Ser-P)(His-P) participe au transport des sucres PTS mais ne serait pas impliquée dans le contrôle de l'activité de la glycérol kinase. Nous avons finalement étudié le rôle de HPr(His"-'P) dans la régulation de la transcription chez Streptococcus salivarius via une étude comparative des protéomes de la souche parentale ATCC 25975 et du mutant G71 , une souche Er, incapable de produire HPr(His-P). Les analyses protéomiques différentielles ont été réalisées à partir d' extraits provenant de cellules récoltées en phase exponentielle et stationnaire de croissance. Chez les cellules récoltées en phase stationnaire de croissance, l'analyse des profils protéiques a révélé que 1 % à 2% des protéines étaient exprimées différentiellement chez S. salivarius G71 suggérant que HPr(His-P) serait impliquée dans le contrôle de l'expression de gènes chez les streptocoques.

Neisseria meningitidis possède un PTS incomplet. Constitué des composants générales EI et HPr et de deux EIIAs (EIIANtr et EIIAMan), ce système ne permet pas le transport des sucres chez cette bactérie. Cependant, nous avons confirmé que la cascade de phosphorylation (EI HPr EIIANtr) est fonctionnelle et que HPr est aussi phosphorylée sur sa Ser-46 par une HprK/P.Dans l’objectif d’étudier l’effet de HPr sur la virulence de N. meningitidis, nous avons construit un mutant ΔptsH chez N. meningitidis 2C4-3. Le mutant ΔptsH a montré une faible survie chez la souris, une faible production de la capsule, une meilleure adhérence aux cellules épithéliales et un niveau élevé de cellules apoptotiques, par rapport à la souche sauvage. HPr semble intervenir dans la virulence de N. meningitidis en interagissant avec la protéine CrgA. L’interaction HPr/CrgA est plus forte quand HPr est phosphorylée sur sa Ser-46 par HprK/P.N. meningitidis utilise le glucose et le maltose comme seuls sucres. Nous avons identifié une perméase à glucose (GlcP) et une perméase à maltose (MalY), responsables du transport de ces sucres. Une perméase putative à gluconate (GntP) a été également identifiée chez N. meningitidis 2C4-3. Cette perméase n’assure pas le transport du gluconate, dans les conditions testées. La délétion de gntP chez N. meningitidis 2C4-3 induit une meilleure croissance sur glucose et une bonne survie du mutant ΔgntP chez la souris, par rapport à la souche sauvage. La fonction réelle de la perméase GntP chez N. meningitidis reste inconnue et suscite des études ultérieures. / Neisseria meningitidis has an incomplete PTS composed of general proteins EI and HPr and two EIIAs (EIIANtr and EIIAMan). This system does not allow the transport of sugars in this bacterium. However, we confirmed that the phosphorylation cascade (EI HPr EIIANtr) is functional and HPr is also phosphorylated at its Ser-46 by an HprK/P.In order to study the effect of HPr on meningococcal virulence, we constructed a ΔptsH mutant in N. meningitidis 2C4-3. Compared to the wild-type strain, the ΔptsH mutant showed poor survival in mice, low production of capsule, better adherence to epithelial cells and high levels of apoptotic cells. HPr appears to be involved in the virulence of N. meningitidis by interacting with CrgA protein. The HPr/CrgA interaction is stronger when HPr is phosphorylated at its Ser-46 by HprK/P.N. meningitidis uses glucose and maltose as the only sugars. We identified permeases for glucose (GlcP) and maltose (MalY), which catalyze the uptake of these sugars.A putative gluconate permease (GntP) has also been identified in N. meningitidis 2C4-3. Under the conditions tested, this permease did not catalyze the transport of gluconate. Compared to the wild-type strain, deletion of gntP in N. meningitidis 2C4-3 induced faster growth on glucose and a better survival of the mutant in mice. To underst and the function of the GntP permease in N. meningitidis further studies will have to be carried out.

Die Infektionskrankheit Malaria wird von einzelligen Parasiten der Gattung Plasmodium verursacht und stellt vor allem im südlichen Afrika eine große Herausforderung dar. Die Zellen der Parasiten enthalten zwei endosymbiontische Organellen, den Apicoplast und das Mitochondrium. Beide Organellen besitzen ein reduziertes Genom. Die Struktur des mitochondrialen Genoms ist ungewöhnlich. Mit nur 6 kb gehört es zu den kleinsten beschriebenen Genomen und enthält neben drei proteinkodierende Gene auch 34 kleine rRNA-Gene. Um das Genom zu exprimieren wird eine Vielzahl von kernkodierten Faktoren benötigt. Die Regulation der Expression, die Prozessierung der polycistronischen Primärtranskripte und die Regulation des RNA-Metabolismus des Mitochondriums ist jedoch weitestgehend unbekannt. In dieser Arbeit konnten kurze RNAs in den Mitochondrien von P. falciparum mittels Hochdurchsatzsequenzierung identifiziert werden. Solche RNA-Akkumulationen an Transkriptenden werden in den Organellen höherer Pflanzen durch PPR-Proteine (Pentatricopeptide repeat) verursacht. Um zu untersuchen, ob in P. falciparum PPR-ähnliche, helikale Proteine vorhanden sind, wurde genomweit nach Proteinen mit repetitiven, helikalen Elementen gesucht. Dabei konnte eine vorher unbekannte Proteinfamilie identifiziert werden, die aufgrund ihrer 37 Aminosäure langen Motive Heptatricopeptide repeat Proteine (HPR) genannt wurde. In P. berghei konnte für 7 HPR-Proteine eine mitochondriale Lokalisation betätigt werden. Außerdem zeigten Deletionsversuche, dass die meisten HPR-Proteine in den Blutstadien essentiell sind. In vitro RNA-Bindestudien konnte für ein rekombinantes HPR-Protein eine unspezifische Interaktion mit mitochondrialen Transkripten nachgewiesen werden, während keine Bindung an DNA erfolgt. Eine breite Suche in verschiedenen phylogenetischen Gruppen zeigte, dass HPR-Proteine in verschiedensten eurkaryotischen Taxa vorhanden sind, mithin früh in der Evolution der eukaryotischen Zelle entstanden sind. / Malaria is caused by a single celled parasite of the genus Plasmodium. Especially in Sub-Saharan Africa, -this disease is a huge challenge for the health system. The cells of the parasites contain two organelles of endosymbiotic origin, the apicoplast and the mitochondrion. Both organelles still contain a reduced genome. For the expression of the genome, the organelles depends on a large set of nuclear encoded proteins. The mitochondrial genome has a unique structure. With only 6 kb it is one of the smallest genomes discovered to date and it contains only three protein coding genes along with 34 small ribosomal genes. The regulation of expression, the processing of the polycistronic primary transcript and the regulation of the RNA metabolism in the mitochondria of Plasmodium remains largely unknown. Through high-throughput sequencing of cellular RNA, we discovered a population of small RNAs originating in the mitochondria of P. falciparum. Similar RNA accumulations can be detected in the organelles of higher plants and are caused by helical-hairpin repeat proteins like PPR proteins (pentatricopeptide repeat). To search for plant-like RNA binding proteins similar to PPR proteins we scanned the nuclear genome of P. falciparum for helical-hairpin repeat proteins. We found a novel protein family with repetitive helical elements of 37 amino acid length we termed heptatricopeptide repeat (HPR) proteins. In the rodent Malaria parasite P. berghei, the mitochondrial localization for 7 HPR-Proteins was verified. In knockout studies, we also showed that almost all HPR proteins are essential for blood stages of P. berghei. In RNA-binding assays, one recombinant HPR protein showed unspecific interaction with mitochondrial transcripts but not with DNA. By broadening the search, we discovered that HPR proteins are found in multiple eukaryotic taxa.

Title from PDF of title page. / Document formatted into pages; contains 48 pages. / Thesis (M.S.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: C-Terminal glycine-extended prohormones are enzymatically converted to á-amidated peptides, by peptidylglycine á-amidating monooxygenase (PAM). PAM is a bifunctional enzyme with two catalytic domains: peptidylglycine á -hydroxylating monooxygenase (PHM) and peptidylglycine peptidylglycineaminoglycolate lyase (PAL). PAM has a significant role in the proliferation of androgen-independent prostate cancer. Thus, the inhibition of PAM could halt cancer growth. Hippurate and hippurate analogs were used as lead compounds for developing inhibitors for PAM. The hippurate analogs exhibiting the highest affinity to PAM (lowest inhibition constant) did inhibit the growth of human androgen-independent prostate cancer DU 145 cells. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

Clostridium perfringens is a versatile pathogen capable of causing a wide array of diseases, ranging from clostridial food poisoning to tissue infections such as gas gangrene. An important factor in virulence as well as in the distribution of C. perfringens is its ability to form an endospore. The symptoms of C. perfringens food poisoning are directly correlated to the release of an enterotoxin at the end of the sporulation process. The sporulation process in C. perfringens is subject to carbon catabolite repression (CCR) by sugars, especially glucose. CCR is a regulatory pathway that alters transcription based on carbon source availability. In Gram-positive bacteria, the HPr kinase/phosphatase is responsible for this nutritional sensing by phosphorylating or dephosphorylating the serine-46 residue of HPr. HPr-Ser-P then forms a complex with the transcriptional regulator CcpA to regulate transcription. We were able to show here that purified recombinant C. perfringens HPr kinase/phosphatase was able to phosphorylate the serine-46 residue of HPr. When the codon for this serine residue is mutated through PCR mutagenesis to encode alanine, phosphorylation could not take place. We have also shown that in gel retardation assays, CcpA and HPr-Ser-P were able to bind to two DNA fragments containing putative C. perfringens CRE-sites, sequences where CcpA binds to regulate transcription. The genome sequence of a food poisoning strain of C. perfringens was searched for potential CRE-sites using degenerate sequences designed to match those CRE-sites CcpA was shown to bind. DNA fragments containing these newly identified CRE-sites were then used in gel retardation assays to determine whether CcpA binds to these CRE-sites, making them candidates for CCR regulation. These results, combined with comparisons of metabolic characteristics of a ccpA- strain versus wild-type C. perfringens, provide evidence that CcpA participates in the regulation of carbon catabolite repression in the pathogenic bacterium C. perfringens / Master of Science

In den Firmicutes wird der Induktorausschluss (Katabolitrepression) durch das am Serin46 phosphorylierte HPr (PTS) vermittelt. Der genaue Mechanismus war jedoch unklar. Um diese Frage auf der Grundlage von isolierten Proteinen zu klären, wurde ein zum Escherichia coli Maltose-/Maltodextrin-ABC-Transporter homologes System aus Lactobacillus casei BL23 (MalE1-MalF1G1K12) als Modellsystem genutzt. Im Rahmen der Promotion wurde über isothermale Titrationskalorimetrie und Fluoreszenzspektroskopie gezeigt, dass das Bindeprotein MalE1 lineare und zyklische Maltodextrine, aber keine Maltose bindet. Experimentell ermittelte dreidimensionale Strukturen von MalE1 im Komplex mit diesen Zuckern belegten eine vergleichbar geschlossene Konformation und dienten zusätzlich als Grundlage, um die fehlende Maltosebindung zu erklären. Die Stimulierung der ATPaseaktivität des in Liposomen und Nanodiscs eingebauten Komplexes wurde jedoch hauptsächlich durch eine MalE1-Beladung mit linearen Maltodextrinen bewirkt. Eine bis zu 85 %ige Inhibierung der ATPaseaktivität durch P-Ser46-HPr belegte erstmals in vitro eine Interaktion von mehr als einem phosphorylierten Protein mit dem Transporter. Analog zum EIIAGlc-Inhibitor des homologen Systems aus E. coli wurden über Quervernetzungsexperimente und massenspektrometrische Analysen Interaktionen mit dem MalK1-Dimer als interagierende Komplexeinheit in der Nähe des Walker A-Motivs nachgewiesen. Über Fluoreszenzmessungen in Anwesenheit des ATP-Analogons TNP-ATP wurde eine unbeeinflusste ATP-Bindung und damit eine fehlende Blockade der γ-Phosphatbindestelle des Walker-A Motivs durch die Phosphorylgruppe von P-Ser46-HPr bestimmt. Die folgende Substitution verschiedener positiv geladener MalK1-Reste, die als potenzielle Interaktionsstellen für die Phosphorylgruppe fungieren könnten, identifizierte K63 in der Nähe des Walker A-Motivs als ersten möglichen Partner. Der genaue Mechanismus der Inhibierung bleibt jedoch unklar. / Catabolite repression is a global mechanism which controls the utilization of carbohydrates in bacteria. In Firmicutes HPr, a component of the phosphoenolpyruvate carbohydrate phosphotransferase system, prevents the uptake of less preferred sugars but only when it is phosphorylated at serine46. However the exact mechanism was unclear. To address this question the purified ATP-binding cassette transporter from Lactobacillus casei BL23 (MalE1-MalF1G1K12) was used as a model system, which is homologous to the Escherichia coli maltose/maltodextrin ABC importer. Isothermal titration calorimetry and fluorescence spectroscopy revealed that the binding protein MalE1 binds linear and cyclic maltodextrins but not maltose. Experimentally determined three-dimensional structures from MalE1 in complex with these sugars show a comparably closed conformation and served as a basis to explain the lack of maltose binding. The stimulation of the ATPase activity of the transporter incorporated in liposomes and nanodiscs however, was mainly caused by MalE1 loaded with linear maltodextrins. For the first time an inhibition of ATPase activity by P-Ser46-HPr up to 85 % and an interaction of more than one phosphorylated protein with the transporter was demonstrated. Analogous to the EIIAGlc inhibitor of the homologous system from E. coli, cross-linking experiments and mass spectrometric analyzes revealed interactions with the MalK1 dimer near the Walker A motif. Fluorescence measurements in the presence of the ATP analogue TNP-ATP, however, revealed an unaffected ATP binding and thus a lack of blockade of the γ-phosphate binding site (Walker A motif) by the phosphoryl group from P-Ser46-HPr. The following substitution of several positively charged MalK1 residues that could act as potential sites of interaction for the phosphoryl group, identified K63 near the Walker A motif as the first potential partner. The exact mechanism of inhibition, however, remains unclear.

No description available.

Datavisualisering är en vanlig metod för att öka förståelsen av data genom att omvandla den från text till bild. Genom datavisualisering kan en gigantisk mängd data omvandlas till diagram och bilder som bidrar till att ge en användare en tydlig förståelse över vad denna data representerar. Det medlemsägda företaget Biometria verkar inom skogsnäring och har som uppgift att mäta skogsprodukter, så som trädstammar och stockar. Mätdatat lagras i skördspecifika XML-filer och görs tillgängligt för de som är involverade i skörden. Problemet är att dessa filer tenderar att bli extremt stora och svårtydda för någon som söker en överblick av en specifik skörd. Detta projekt har gått ut på att undersöka huruvida Biometrias data kan visualiseras och på så sätt förtydligas. Undersökningen resulterade i utvecklingen av en prototyp av en webbapplikation som tar emot en skördefil och visualiserar vissa mätdata från den. Den slutgiltiga prototypen bestod av en punktkarta som visar var stammarna fälldes, två cirkeldiagram som visar proportioner av trädarter samt sortiment av stockar, och ett stapeldiagram som visar max, minimi och medelvärden av stammarna och stockarnas diametrar. Resultatet var lyckat, eftersom de anställda på Biometria som resultatet presenterades för var överens om att visualiseringen bidrog till en mycket högre förståelse av den data som visualiserades. / Data visualization is a common method to increase the understanding of data by converting it from text to image. Through data visualization, a large amount of data can be transformed into diagrams and images that helps giving a user a clear understanding of what the data represents. The member-owned company Biometria operates in the forest industry and is tasked with measuring forest products, such as tree trunks and logs. The measurement data is stored in harvest-specific XML-files and are made available for those involved in the harvest. The problem is that these files tend to get extremely large and difficult to read for someone who wants an overview of a specific harvest. This project has aimed to investigate whether Biometria's data can be visualized and thus clarified. The study resulted in the development of a prototype of a web application that receives a harvest file and visualizes some measurement data from it. The final prototype consisted of a dot map showing where the trees were felled, two pie charts showing proportions of tree species and assortment of logs, and a bar chart showing the maximum, minimum and mean values of the trees and log diameters. The result was successful, since the employees at Biometria for whom the result was presented all agreed that the visualization contributed to a much higher understanding of the data that was visualized.

No description available.

The aim of this study was to investigate if SI from HPR-data from harvesters and soil moisture classes from digital depth to water maps could be used to support site adaption of regenerations within stands. The study was made on pine and spruce stands in central Sweden. The number of plants/ha, plant height, growth and damage were measured at plot level and for these plots also soil moisture classes and SI were derived from digital maps and HPR. The study shows a potential using SI from HPR and depth to water maps for site adaption of regenerations and to vary tree species within stands. Variations of SI and soil moisture are important within stands regarding different tree species establishment, growth and damage. But also, for sites with medium SI were the choice between pine and spruce is not obvious and in stands with a high spread in SI.