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Conselho Nacional de Desenvolvimento Científico e Tecnológico / Polyamines (putrescine, spermidine and spermine) are aliphatic amines produced by the action of ornithine descarboxylase (ODC), a rate-limiting and protein kinease C (PKC)-regulated step in polyamine synthesis. Since the levels of polyamines were found to be high in synovial fluid of arthritic patients, the aim of the present study was to identify the role of peripherally produced polyamines in the model of inflammatory pain induced by adjuvant arthritis. The subcutaneous injection of complete Freund s adjuvant (CFA, 50 μL/paw) caused the development of mechanical allodynia and edema. Moreover, it increased ODC expression and activity as well as PKC activation. The previous administration of the selective ODC inhibitor DFMO (10 μmol/paw) was capable of preventing the development of allodynia and edema as well as the increase in ODC activity produced by CFA injection. Furthermore, the previous administration of the PKC inhibitor GF109203X (1 nmol/paw) reduced allodynia and the increase of ODC activity in animals injected with CFA. Accordingly with the synthesis inhibition, we have observed that subcutaneous injection of putrescine (10 μmol/paw), spermidine (3-10 μmol/paw) or spermine (0.3-3 μmol/paw) into the rat paw also caused mechanical allodynia and edema. The present results suggest that endogenously synthesized polyamines are involved in the development of nociception and edema caused by the adjuvant. Moreover, the polyamine production in inflammatory site seems to be related with the increase in ODC activity stimulated by PKC activation. Thus, the control of polyamine synthesis and action could be an interesting target to control inflammatory pain. / Poliaminas (putrescina, espermidina e espermina) são aminas alifáticas produzidas pela ação da ornitina descarboxilase (ODC), enzima limitante e proteína quinase C (PKC), passo regulatório da síntese de poliaminas. Desde que níveis elevados de poliaminas foram encontrados no fluído sinovial em pacientes com artrite, o objetivo do presente estudo foi investigar a produção de poliaminas perifericamente em modelo de dor inflamatória induzido por CFA. A injeção subcutânea do adjuvante completo de Freund (CFA, 50 μL/pata) causou o desenvolvimento de alodínia mecânica e edema, bem como um aumento na expressão e atividade da ODC e na ativação da PKC. A administração prévia do inibidor seletivo da ODC, o DMFO (10 μmol/pata), foi capaz de prevenir o desenvolvimento da alodínia e edema, bem como o aumento da atividade produzida pela injeção de CFA. Além disso, a pré-administração do inibidor da PKC o GF109203 (1 nmol/pata), reduziu a alodínia e o aumento da atividade da ODC em animais injetados com CFA. De acordo com a inibição da síntese, também observamos que a injeção subcutânea de putrescina (10 μmol/pata), espermidina (3-10 μmol/pata) ou espermina (0,3-3 μmol/pata) na pata de ratos desenvolveu alodínia mecânica e edema. O presente estudo sugere que as poliaminas sintetizadas endogenamente estão envolvidas no desenvolvimento da nocicepção e edema causado pelo CFA. Além disso, as poliaminas produzidas nos sítios de inflamação estão relacionadas com o aumento na atividade da ODC estimulada pela ativação da PKC. Assim, o controle da síntese das poliaminas e função poderia ser um interessante alvo para o controle da dor inflamatória.

Cette thèse entre dans le contexte des études portant sur l’endommagement laser des optiques en silice utilisées sur le Laser MégaJoule (LMJ) au CEA/CESTA. L’étude par spectroscopie de luminescence des défauts présents dans les micro-fractures sous surfaciques des pièces de silice polie, supposés être à l’origine de l’amorçage de l’endommagement laser, a été choisie pour cette recherche. Les micros fractures étant difficiles à détecter, nous avons créé des fractures modèles en faisant des indentations dans la silice. Une comparaison des spectres de luminescence sous excitation à 325 nm, proche de la longueur d’onde utilisée sur le LMJ, entre les indentations et les dommages laser dont l’analyse a donné lieu à des interprétations indiscutables dans la bibliographie, est proposée. Des expériences de luminescence à basse température ou sur des pièces acidées sont présentées pour compléter les informations obtenues sur les différents défauts observés. / Defects present in subsurface damage, supposed to be possible damage precursors, have been studied by luminescence spectroscopy. Because of the difficulty to detect micro cracks, we have selected a model cracks based on indentations. Luminescence spectra performed under a 325 nm excitation wavelength (experimental condition close to that used on the LMJ) are be compared on indentation as well as laser damages. Luminescence experiments at low temperature and on etched samples are reported in order to complete data obtained for the different observed defects.

Malaria is one of the most life-threatening diseases affecting mankind, with over 3 billion people being at risk of infection, with most of these people living in Africa, South America and Asia. As the malaria parasite is rapidly becoming resistant to many of the possible treatments on the market, it is of upmost importance to identify new possible drug targets and describe drugs against these that are inexpensive, easy to manufacture and have a long shelf-life in order to combat malaria. One such target is the polyamine pathway. The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation. Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections. Usually, both enzymes are individually transcribed and highly regulated as monofunctional proteins. However, ODC and AdoMetDC from P. falciparum (PfODC and PfAdoMetDC, respectively) are found as a unique bifunctional protein (PfAdoMetDC/ODC) in the malaria parasite, making it an enticing target for new, selective antimalarial chemotherapies. In order to apply structure-based drug discovery strategies to design inhibitors for PfAdoMetDC/ODC, the atomic resolution structures of these proteins are needed. Each individual domain has had its structure proposed through homology modelling; however atomic resolution structures of these domains are not yet available. The homology model of PfAdoMetDC/ODC has not yet been elucidated due to the interactions between the domains of the bifunctional protein not being fully understood. High levels of recombinant expression of the bifunctional protein have been either unsuccessful or resulted in the formation of insoluble proteins being produced. The purpose of this project is to optimise the recombinant expression of PfAdoMetDC/ODC, and the PfODC domain, to produce high yields of pure, soluble protein for subsequent atomic resolution structure determination. Ultimately, this will enable the utilisation of PfAdoMetDC/ODC in structure-based drug discovery strategies. Overexpression of P. falciparum proteins in E. coli is notoriously difficult, mainly due to the codon bias between the two species. Comparative studies were performed on four constructs of the PfAdoMetDC/ODC gene, containing either the wild-type, fully codon harmonised, or partially codon harmonised gene sequences to analyse the effect codon harmonisation had on protein expression and activity of both domains of PfAdoMetDC/ODC as well as on the monofunctional PfODC domain. Codon harmonisation did not improve the expression levels or the purity of recombinantly expressed PfAdoMetDC/ODC or the monofunctional PfODC domain. Truncated versions of both proteins, and contamination by the E. coli chaperone proteins DnaK and GroEL, were present in the protein samples even after purification by affinity chromatography. However, codon harmonisation improved the activity levels of the PfAdoMetDC domain, while decreasing the activity of the PfODC domain of PfAdoMetDC/ODC. Harmonisation of the monofunctional PfODC domain resulted in a decrease in the activity of the protein. In order to identify possible inhibitors of the PfODC domain of the bifunctional protein, a structure-based drug discovery study was initiated based on a homology model for PfODC. Four hundred compounds with known antimalarial activity were virtually screened against the PfODC homology model and the top two scoring compounds were selected for enzyme inhibition assays based on their predictive binding affinity against the enzyme, and two medium scoring compounds were selected as controls. Enzyme inhibition studies were performed on the bifunctional PfAdoMetDC/ODC to determine the effect the compounds had on both domains of the protein. Of the compounds assayed one of the compounds significantly reduced the activity levels of both domains of PfAdoMetDC/ODC. Additionally, one compound significantly reduced the activity level of the PfAdoMetDC domain of PfAdoMetDC/ODC. This work therefore contributes towards characterisation of the unique PfAdoMetDC/ODC in malaria parasites as a novel drug target. / Dissertation (MSc)--University of Pretoria, 2012. / Biochemistry / unrestricted

Cette thèse entre dans le contexte des études portant sur l'endommagement laser des optiques en silice utilisées sur le Laser MégaJoule (LMJ) au CEA/CESTA. L'étude par spectroscopie de luminescence des défauts présents dans les micro-fractures sous surfaciques des pièces de silice polie, supposés être à l'origine de l'amorçage de l'endommagement laser, a été choisie pour cette recherche. Les micros fractures étant difficiles à détecter, nous avons créé des fractures modèles en faisant des indentations dans la silice. Une comparaison des spectres de luminescence sous excitation à 325 nm, proche de la longueur d'onde utilisée sur le LMJ, entre les indentations et les dommages laser dont l'analyse a donné lieu à des interprétations indiscutables dans la bibliographie, est proposée. Des expériences de luminescence à basse température ou sur des pièces acidées sont présentées pour compléter les informations obtenues sur les différents défauts observés.

In all mammalian species examined thus far, the ovaries produce a burst of ornithine decarboxylase (ODC) and putrescine during ovulation or after application of a bolus of human chorionic gonadotropin (hCG). Aged mice are deficient in this peri-ovulatory ODC and putrescine burst. Moreover, peri-ovulatory putrescine supplementation in aged mice increases egg quality and reduces miscarriage rates. These studies suggest that peri-ovulatory putrescine supplementation may be a simple and effective therapy for reproductive aging for women. However, putrescine has never been used in humans and, currently no pure source of putrescine is suitable for human trials. Given that ODC is highly expressed in the ovaries during ovulation but otherwise exhibits low activity in most tissues, we hypothesized that L-ornithine, the substrate of ODC, might be a better alternative. In this study, we have demonstrated that systemic application of L-ornithine increased ovarian putrescine levels; the increase was restricted to animals that had been injected with hCG. Furthermore, L-ornithine specifically increased ovarian putrescine levels without affecting putrescine levels in most other tissues. Unfortunately, thus far peri-ovulatory L-ornithine supplementation in mouse drinking water produced mixed effects on reproductive outcome in aged mice. Therefore, our studies demonstrated the potential of L-ornithine supplementation as a possible therapy for aging-related infertility, but further work is required to produce an effective application method.

This report discusses the importance of test process evaluation in order to improve a test model and to provide developer- and management feedback. The report results in a test evaluation framework, developed in cooperation with a department at Ericsson Software Technology in Karlsrona. The framework is a result of discussions with the developers regarding performed testing, studying defect types from past projects and by analyzing the result from a small survey answered by some of the developers at Ericsson. The overall project aim was to evaluate performed testing in order to improve the test model. This requires a good insight of the test process, which is provided by the developed test evaluation framework. The test process is visualized by extracting test process data, making it possible to achieve the project aim. The project aim can be divided into the three following areas: Firstly to evaluate if the current test model is followed as expected, for example are all test techniques used according to the test model? Secondly to evaluate how well the test model fulfills predefined expectations, i.e. is a defect detected with the expected test technique and in the expected test phase? Finally to evaluate if there are any problematic defects that should receive extra attention during a project such as if one or several defect types occurs more frequently than others? The framework is based on another framework, Orthogonal Defect Classification [Chillarege92], combined with the research area Test Technique Effectiveness. The aim of this combination was to support the developed framework. Further a specific part of the framework is focusing on developer- and management feedback. / Dan Bengtsson Västra Stationstorget 7 222 37 Lund

Malaria is one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapy and -prophylaxis drugs. The need for novel therapeutic targets and drugs is therefore enormous and the understanding of the biochemistry of the parasite is imperative. The aim of this study was the identification and molecular characterisation of the eDNA of one such metabolic target protein, ornithine decarboxylase (ODC), in the human malaria parasite P. falciparum. The P. falciparum ODC eDNA was isolated by means of a modified RT-PCR technique, RACE. No sequence data were available and the primers used were based on consensus areas identified in the protein sequences from other related organisms. The isolation and identification of the eDNA with degenerate primers was successful in 3' -RACE, but necessitated the optimisation of the eDNA synthesis protocol and the use of total RNA as starting material. The sequence obtained facilitated the application of 5' -RACE with ODC-specific primers based on the 3' -RACE sequence data. The full-length ODC eDNA sequence was obtained by overlap-alignment of various segments. A novel suppression PCR technology was applied during the 5' -RACE in order to create an uncloned eDNA library of amplified cDNAs representing only the mRNA population. The P. falciparum ODC eDNA contains an open reading frame of ---2847 bp and translates to a large 939 amino acid protein. The protein contained large internal insertions and was extended by '""273 N-terminal residues compared to ODCs from other organisms. Several possible signature motifs were identified for phosphorylation, glycosylation and transamidation. The P. falciparum ODC protein seems to contain more hydrophilic and a-helix forming residues. These characteristics should be further investigated after expression of the recombinant protein. The isolation of the P. falciparum ODC eDNA facilitates the validation of this protein as an antimalarial target. / Dissertation (MSc)--University of Pretoria, 1998. / gm2014 / Biochemistry / unrestricted

Probiotic food products - food additives contain different species of probiotic bacteria. Accurate species identification with their characteristics is very important from the view of products quality. Methods of DNA diagnostics are used for these purposes. In this thesis DNA was isolated from 4 probiotic products. The presence of bacterial of genus Lactobacillus and species L. acidophilus, L. casei, L. plantarum, L. rhamnosus were detected in three products by PCR. This information was in accordance with the data provided by the manufacturer. Two sets of primers were used for identification of species. Using other primers sequences of genes such as bsh, lai and odc were detected in DNA isolated from the products. Differences were estimated among products concerning the detection of lai gene Lactobacillus acidophilus.

Ce travail s'inscrit dans le cadre de l'étude des systèmes physiques de basse dimension. Nous présentons une étude des propriétés électroniques locales de deux composés quasi-unidimensionnels (Q1D) à onde de densité de charge (ODC), le bronze bleu Rb0.3MoO3 et NbSe3, par microscopie à effet tunnel (STM), sous ultra-haut vide (UHV) et à basse température. Ces matériaux ont été clivés in-situ suivant leur plan naturel, (-201) pour Rb0.3MoO3 et (100) pour NbSe3. Notre étude montre que la distribution spatiale de surface des ODC peut être qualitativement différente de celle du volume et qu'une préparation in-situ des surfaces de ces composés est nécessaire pour avoir accès à leurs propriétés électroniques véritables. En surface de Rb0.3MoO3, l'ODC a été visualisée par STM. Un phénomène nouveau a été observé: la composante qb=2kf du vecteur d'onde de l'ODC n'est pas homogène en surface. Nous proposons que ces déviations résultent d'inhomogénéités de surface du vecteur de Fermi, dues à la distribution inhomogène des atomes alcalins sur la surface, après clivage du matériau. Les implications de ces résultats sont discutées. Nos mesures STM sur NbSe3 offrent une première étude cohérente des états ODC q1 et q2, et de leur distribution spatiale sur les différentes chaînes de la structure. A basse température, l'existence d'une interaction entre les ODC q1 et q2 est révélée par l'apparition d'une nouvelle superstructure. Enfin, tandis que l'ODC q1 possède en surface une température de transition (Tc) comparable à celle du volume, l'ODC q2 possède une Tc de 10 à 15K plus élevée en surface. Différents mécanismes reposant sur des effets spécifiques de surface sont discutés.