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Vaccinia virus (VACV) is the most intensively studied orthopoxvirus and acts as an excellent model to investigate host-pathogen interactions. VACV encodes about 200 proteins, many of which modulate the immune response. This study focusses on two of these: C16 and C4, that share 43.7 % amino acid identity. Given the sequence similarity, we explored whether C16 and C4 have any shared functions, whilst also searching for novel functions. To gain mechanistic insight, we sought to identify binding partners and determine the residues responsible. C16 has two reported functions. Firstly, it inhibits DNA-PK-mediated DNA sensing, and this study found that C4 can perform this function as well. Like C16, C4 associates with the Ku heterodimer to block its binding to DNA leading to reduced production of cytokines and chemokines. For both proteins, the function localised to the C termini and was abrogated by mutating three residues. Secondly, C16 induces a hypoxic response by binding to PHD2. This function was mapped to the N-terminal 156 residues and a full length C16 mutant (D70K,D82K) lost the ability to induce a hypoxic response. In contrast, C4 did not bind PHD2. C4 inhibits NF-κB signalling by an unknown mechanism. Reporter gene assays showed that C16 also suppresses NF-κB activity and, intriguingly, this was carried out by both the N and C termini. C16 acts at or downstream of p65 and the N terminus of C16 associated with p65 independently of PHD2-binding. Conversely, C4 acted upstream of p65, did not display an interaction with p65, and the function was restricted to its C-terminal region. Novel binding partners were identified by a screen utilising tandem mass tagging and mass spectrometry, and selected hits were validated. The C terminus of C16 associated with VACV protein K1, a known NF-κB inhibitor. Additionally, C16 bound to the transcriptional regulator ARID4B. C4 did not interact with these proteins, but the N-terminal region of C4 associated with filamins A and B. The functional consequences of these interactions remain to be determined. In vivo, C4 and C16 share some redundancy in that a double deletion virus exhibits an attenuated virulence phenotype that is not observed by single deletion viruses in the intradermal model of infection. However, non-redundant functions also contribute to virulence in that both single deletion viruses display attenuated virulence compared to a wild-type Western Reserve virus in the intranasal model of infection. Data presented also reveal that C4 inhibits the recruitment of immune cells to the site of infection, as was previously described for C16. Overall, this investigation highlights the complexity of host-pathogen interactions showing that VACV encodes two multifunctional proteins with both shared and unique functions. Moreover, their inhibition of DNA-PK emphasises the importance of this PRR as a DNA sensor in vivo.

Records of change of [delta]13C values in vertebrate teeth offer an opportunity to gain insight into changes in past vegetation. Increasingly, teeth from small mammals are used for such purposes, but because their teeth grow very rapidly, seasonal changes in vegetation potentially provide a large source of variability in carbon isotope composition, complicating interpretations of small mammal tooth isotope data. To investigate the controls of seasonality on the stable isotope composition of fossil teeth, we constructed a Monte-Carlo-based model to simulate the effects of changes in the seasonal pattern of diet in leporid lagomorphs (rabbits and hares) on the distribution of [delta]¹³C values in random populations of leporid teeth from the Edwards Plateau in central Texas. Changes in mean-state, seasonal vegetation range, and relative season length manifest themselves in predictable ways in the median, standard deviation, and skewness of simulated tooth [delta]¹³C populations, provided sufficient numbers of teeth are analyzed. This Monte Carlo model was applied to the interpretation of a 20,000 year record of leporid tooth [delta]¹³C values from Hall's Cave on the Edwards Plateau in central Texas. Variations in the [delta]¹³C values of teeth deposited at the same time (standard deviation = 1.69%) are larger than changes in the mean vegetation composition reconstructed from bulk organic carbon [delta]¹³C, indicating the influence of short-term variability, making it difficult to assess changes in mean C3/C4 vegetation from the tooth [delta]¹³C data. However, populations of teeth from different climate intervals (e.g., the late Glacial, Younger Dryas, and the Holocene) display changes in the shape of the tooth [delta]¹³C distributions. Interpretation of these changes as shifts in seasonal vegetation patterns that are based upon results from our model are consistent with hypothesized climatic changes. An increase in the standard deviation of the tooth population between the late Glacial and the Younger Dryas -- Holocene is consistent with an increase in seasonality. Furthermore, a shift to more C3-dominated vegetation in the tooth [delta]¹³C distribution during the Younger Dryas is accompanied by a more skewed population -- indicative of not only wetter conditions but an increase in the duration in the C3 growing season. However, late Holocene changes in vegetation are not clear in the tooth data, despite the evidence from bulk organic carbon [delta]¹³C values for an increase in % C3 vegetation of 57%. Small mammal teeth can potentially provide unique insights into climate and vegetation on seasonal and longer timescales that complement other data, but should be interpreted with a careful consideration of local conditions, taxon ecology and physiology, and the dominant timescales of isotope variability. / text

Approved for public release; distribution is unlimited. / MBA Professional Report / This project provides an analysis of the Army's acquisition of the Land Warrior (LW) Soldier System. Its objectives are to document the history of the LW and provide an overview of the program to establish the components of both it development and deployment and its associated business and management characteristics. The product is a document that provides an analysis of the actions taken and the obstacles encountered and how the materiel developers, warfighters, user representatives and lawmakers dealt with them. The LW was approved in 1993. The requirement was to provide improvements for dismounted soldiers in the five specific capabity caategories of lethality, command and control, mobility, survivability and sustainment. For a period lasting approximately 15 years, the LW has evolved. Despite this evolution, the Army in FY 2007 terminated it in FY 2007. Regardless, it has laid the foundation for follow-on soldier system initiatives. The LW was unsuccessfu initially due to the misalignment of three interrelated and supporting components; 1) technical immaturity, 2) poor user acceptance, and 3) lack of senior leadership support. Successes that are more recent can be attributed to: 1)soldier-driven design, 2) improved technical maturity, and 3) proven employment of the system in combat by warfighters.

Made available in DSpace on 2014-06-11T19:32:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-02-03Bitstream added on 2014-06-13T19:22:04Z : No. of bitstreams: 1 marchese_ja_dr_botfca.pdf: 1209124 bytes, checksum: 3852bccf50e18f3028e21b50674aba78 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Artemisia annua L. (Asteraceae) é uma planta herbácea altamente aromática, nativa da Ásia e aclimatada no Brasil. As folhas são fonte abundante de artemisinina, uma lactona sesquiterpênica que, conjuntamente aos seus derivados semi-sintéticos, apresentam ação efetiva contra as cepas resistentes das espécies de Plasmodium causadoras da malária. Os objetivos deste trabalho foram identificar o mecanismo fotossintético e avaliar o efeito de diferentes condições de temperatura e fotoperíodo no crescimento e desenvolvimento do acesso CPQBA 2/39x1V de A. annua. Para identificar o mecanismo fotossintético foram realizados experimentos para determinar composição dos isótopos do carbono (d 13C) e a anatomia foliar associada a testes histoquímicos. Para avaliar o efeito da temperatura e fotoperíodo no crescimento e desenvolvimento de A. annua foram realizados dois experimentos em câmaras fotoperiódicas (o primeiro com temperaturas médias máx. de 37ºC e mín. de 19ºC e o segundo com máx. de 29ºC e mín. de 13ºC) e um experimento com 6 épocas de plantio em campo no município de Pato Branco-PR (26º11' S, 52º36' W e 760 m de altitude), sul do Brasil. Um último experimento realizado, foi a aplicação exógena de artemisinina em plantas de A. annua para verificar o papel da molécula na indução do florescimento. Como resultados, A. annua apresentou uma d 13C - 31.76 l 0.07, valor típico de espécies com mecanismo fotossintético C3, que apresentam em média valores de d 13C - 28. Os estudos da anatomia foliar e testes histoquímicos confirmaram os resultados encontrados para a d 13C, onde, a despeito da existência de células parenquimáticas formando um anel ao redor do feixe vascular, estas não apresentaram cloroplastos e amido, confirmando ser A. annua uma espécie de mecanismo... / Leaves of Artemisia annua L. are a plentiful source of artemisinin, a drug with proven effectiveness against malaria. One of the objectives of this work was to identify the photosynthetic type of A. annua through studies of the carbon isotope composition (d 13C) and the leaf anatomy. We also verified the growth and development of the CPQBA 2/39x1V accession under moist subtropical climate. Two experiments were carried out in greenhouse using photoperiodic chambers. The plants were submitted to photoperiods of 7, 9, 11, 13, 15 and 17 h, in two natural conditions of temperature: spring/summer (maximum of 37ºC and minimum of 19ºC) and autumn/winter (maximum of 29ºC and minimum of 13ºC). Another experiment with different planting dates at field was carried out in Pato Branco, PR (26º11' S, 52º36' W and 760 m), Southern part of Brazil. The last experiment was the application of artemisinin (0, 500, 5000, and 10000 mg L-1) in A. annua plants to verify the role of the molecule on the flowering induction. A. annua presented a d 13C - 31.76 l 0.07, what characterizes it as typical species with a C3 photosynthetic mechanism, with an average of d 13C- 28, while C4 species possess an average of d 13C - 14. The study of A. annua leaf anatomy confirms the results of d 13C, where, in spite of the existence of parenchymatic cells forming a different sheath surrounding of the vascular tissue, these cells do not have chloroplast or starch. These features do not describe the Kranz anatomy typical of C4 species. The application of artemisinin did not induce the flowering of A. annua at any of the concentrations used. The results suggest that the accumulation of the artemisinin in the pre-flowering and flowering phases in A. annua is not the physiological signal of floral induction in this species...

The classical graph edge coloring problem deals in coloring the edges of a given graph with minimum number of colors such that no two adjacent edges in the graph, get the same color in the proposed coloring. In the following work, we look at the other end of the spectrum where in our goal is to maximize the number of colors used for coloring the edges of the graph under some vertex specific constraints. We deal with the MAXIMUM EDGE COLORING problem which is defined as the following –For an integer q ≥2 and a graph G, the goal is to find a coloring of the edges of G with the maximum number of colors such that every vertex of the graph sees at most q colors. The question is very well motivated by the problem of channel assignment in wireless networks. This problem is NP-hard for q ≥ 2, and has been well-studied from the point of view of approximation. This problem has not been studied in the parameterized context before. Hence as a next step, this thesis investigates the parameterized complexity of this problem where the standard parameter is the solution size. The main focus of the work is the special case of q=2 ,i.e. MAXIMUM EDGE 2-COLORING which is theoretically intricate and practically relevant in the wireless networks setting. We first show an exponential kernel for the MAXIMUM EDGE q-COLORING problem where q is a fixed constant and q ≥ 2.We do a more specific analysis for the kernel of the MAXIMUM EDGE 2-COLORING problem. The kernel obtained here is still exponential in size but is better than the kernel obtained for MAXIMUM EDGE q-COLORING problem in case of q=2. We then show a fixed parameter tractable algorithm for the MAXIMUM EDGE 2-COLORING problem with a running time of O*∗(kO(k)).We also show a fixed parameter tractable algorithm for the MAXIMUM EDGE q-COLORING problem with a running time of O∗(kO(qk) qO(k)). The fixed parameter tractability of the dual parametrization of the MAXIMUM EDGE 2-COLORING problem is established by arguing a linear vertex kernel for the problem. We also show that the MAXIMUM EDGE 2-COLORING problem remains hard on graphs where the maximum degree is a constant and also on graphs without cycles of length four. In both these cases, we obtain quadratic kernels. A closely related variant of the problem is the question of MAX EDGE{1,2-}COLORING. For this problem, the vertices in the input graph may have different qε,{1.2} values and the goal is to use at least k colors for the edge coloring of the graph such that every vertex sees at most q colors, where q is either one or two. We show that the MAX EDGE{1,2}-COLORING problem is W[1]-hard on graphs that have no cycles of length four.

The sterol biosynthetic pathway is essential for growth of all eukaryotic cells and the main target of antifungal agents. The emergence of resistance to these antifungals in an already ill patient population indicates a need to develop drugs that have a broad spectrum of activity among pathogenic fungi and have minimal patient toxicity. Squalene synthase is the first committed step in the sterol pathway and has been studied intensively for development of antifungal agents. While the overall architecture of this enzyme is identical throughout eukaryotes, it was shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implies that there is a component of the fungal carboxy-terminal domain that is responsible for the complementation phenotype and that is unique to the fungal kingdom of life. To determine the role of the carboxy-terminal domain of squalene synthase in the sterol pathway, we used the yeast Saccharomyces cerevisiae with a squalene synthase knockout mutation and expressed squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. When induced, non-fungal squalene synthases could not complement the knockout mutation and instead led to the accumulation of carboxysterol intermediates, suggesting an interaction between squalene synthase and the downstream sterol C4-decarboxylase. Overexpression of a sterol C4-decarboxylase from any kingdom of life both decreased the accumulation of carboxysterol intermediates and allowed non-fungal squalene synthases to complement the squalene synthase knockout mutation. Using chimeric squalene synthases from each kingdom of life, the motif in the C-terminal domain responsible for preventing this toxicity was mapped to a kingdom-specific 26-amino acid hinge motif adjacent to the catalytic domain. Furthermore, over-expression of the carboxy-terminal domain alone containing a hinge motif from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Since this hinge region is unique to and highly conserved within each kingdom of life, this data provides evidence for the development of an antifungal therapeutic as well as for tools to develop an understanding of triterpene catalytic activity and identify similar motifs in other biosynthetic pathways.

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第13107号 / 論生博第14号 / 新制||生||50(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 佐藤 文彦, 教授 河内 孝之, 教授 福澤 秀哉 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

L’auto-incompatibilité (AI) est une barrière reproductive prézygotique qui permet aux pistils d’une fleur de rejeter leur propre pollen. Les systèmes d’AI peuvent prévenir l’autofertilisation et ainsi limiter l’inbreeding. Dans l’AI gamétophytique, le génotype du pollen détermine son propre phénotype d’incompatibilité, et dans ce système, les déterminants mâles et femelles de l’AI sont codés par un locus multigénique et multi-allélique désigné le locus S. Chez les Solanaceae, le déterminant femelle de l’AI est une glycoprotéine stylaire extracellulaire fortement polymorphique possédant une activité ribonucléase et désignée S-RNase. Les S-RNases montrent un patron caractéristique de deux régions hypervariables (HVa et HVb), responsables de leur détermination allélique, et cinq régions hautement conservées (C1 à C5) impliquées dans l’activité catalytique ou la stabilisation structurelle de ces protéines. Dans ce travail, nous avons investigué plusieurs caractéristiques des S-RNases et identifié un nouveau ligand potentiel aux S-RNases chez Solanum chacoense. L’objectif de notre première étude était l’élucidation du rôle de la région C4 des S-RNases. Afin de tester l’hypothèse selon laquelle la région C4 serait impliquée dans le repliement ou la stabilité des S-RNases, nous avons généré un mutant dans lequel les quatre résidus chargés présents en région C4 furent remplacés par des résidus glycine. Cette protéine mutante ne s’accumulant pas à des niveaux détectables, la région C4 semble bien avoir un rôle structurel. Afin de vérifier si C4 est impliquée dans une liaison avec une autre protéine, nous avons généré le mutant R115G, dans lequel un acide aminé chargé fût éliminé afin de réduire les affinités de liaison dans cette région. Ce mutant n’affectant pas le phénotype de rejet pollinique, il est peu probable que la région C4 soit impliquée dans la liaison des S-RNases avec un ligand ou leur pénétration à l’intérieur des tubes polliniques. Enfin, le mutant K113R, dans lequel le seul résidu lysine conservé parmi toutes les S-RNases fût remplacé par un résidu arginine, fût généré afin de vérifier si cette lysine était un site potentiel d’ubiquitination des S-RNases. Toutefois, la dégradation des S-RNases ne fût pas inhibée. Ces résultats indiquent que C4 joue probablement un rôle structurel de stabilisation des S-RNases. Dans une seconde étude, nous avons analysé le rôle de la glycosylation des S-RNases, dont un site, en région C2, est conservé parmi toutes les S-RNases. Afin d’évaluer la possibilité que les sucres conjugués constituent une cible potentielle d’ubiquitination, nous avons généré une S11-RNase dont l‘unique site de glycosylation en C2 fût éliminé. Ce mutant se comporte de manière semblable à une S11-RNase de type sauvage, démontrant que l’absence de glycosylation ne confère pas un phénotype de rejet constitutif du pollen. Afin de déterminer si l’introduction d’un sucre dans la région HVa de la S11-RNase pourrait affecter le rejet pollinique, nous avons généré un second mutant comportant un site additionnel de glycosylation dans la région HVa et une troisième construction qui comporte elle aussi ce nouveau site mais dont le site en région C2 fût éliminé. Le mutant comportant deux sites de glycosylation se comporte de manière semblable à une S11-RNase de type sauvage mais, de manière surprenante, le mutant uniquement glycosylé en région HVa peut aussi rejeter le pollen d’haplotype S13. Nous proposons que la forme non glycosylée de ce mutant constitue un allèle à double spécificité, semblable à un autre allèle à double spécificité préalablement décrit. Il est intéressant de noter que puisque ce phénotype n’est pas observé dans le mutant comportant deux sites de glycosylation, cela suggère que les S-RNases ne sont pas déglycosylées à l’intérieur du pollen. Dans la dernière étude, nous avons réalisé plusieurs expériences d’interactions protéine-protéine afin d’identifier de potentiels interactants polliniques avec les S-RNases. Nous avons démontré que eEF1A, un composant de la machinerie de traduction chez les eucaryotes, peut lier une S11-RNase immobilisée sur résine concanavaline A. Des analyses de type pull-down utilisant la protéine eEF1A de S. chacoense étiquetée avec GST confirment cette interaction. Nous avons aussi montré que la liaison, préalablement constatée, entre eEF1A et l’actine est stimulée en présence de la S11-RNase, bien que cette dernière ne puisse directement lier l’actine. Enfin, nous avons constaté que dans les tubes polliniques incompatibles, l’actine adopte une structure agrégée qui co-localise avec les S-RNases. Ces résultats suggèrent que la liaison entre eEF1A et les S-RNases pourrait constituer un potentiel lien fonctionnel entre les S-RNases et l’altération du cytosquelette d’actine observée lors des réactions d’AI. Par ailleurs, si cette liaison est en mesure de titrer les S-RNases disponibles à l’intérieur du tube pollinique, ce mécanisme pourrait expliquer pourquoi des quantités minimales ou « seuils » de S-RNases sont nécessaires au déclenchement des réactions d’AI. / Self-incompatibility (SI) is a prezygotic reproductive barrier that allows the pistil of a flower to specifically reject their own (self-) pollen. SI systems can help prevent self-fertilization and avoid inbreeding. In gametophytic SI (GSI), the genotype of the pollen determines its breeding behaviour and in this system both female and male specificity determinants of SI are under the control of a multigenic and multiallelic locus called the S-locus. In Solanaceae, the female determinant of SI is a highly polymorphic stylar-expressed extracellular glycoprotein with RNase activity called the S-RNase. S-RNases show a distinct pattern of two hypervariable (HVa and HVb) regions, responsible for their allelic specificity, and five highly conserved regions (C1 to C5) thought to be involved in either the catalytic activity or the structural stabilization of the protein. In this work, we analyzed and characterized several conserved features of the S-RNases and also identified a potential novel S-RNase interactant in Solanum chacoense. The aim of our first study was to investigate the role of the C4 region of S-RNases. To test the hypothesis that the C4 region may be involved in S-RNase folding or stability, we examined a mutant in which the four charged residues in the C4 region were replaced with glycine. This mutant did not accumulate to detectable levels in styles, supporting a structural role for C4. To test the possibility that C4 might be involved in binding another protein, we prepared an R115G mutant, in which a charged amino acid was eliminated to reduce any potential binding to this region. This mutant had no effect on the pollen rejection phenotype of the protein, and thus C4 is likely not involved in either ligand binding or S-RNase entry inside pollen tubes. Finally, a K113R mutant, in which the only conserved lysine residue in all the S-RNases was replaced with arginine, was generated to test if this residue was an S-RNase ubiquitination site. However, S-RNase degradation was not disrupted in this mutant. Taken together, these results indicate that the C4 region likely plays a structural role. In a second study, we analyzed the role of S-RNase glycosylation. All S-RNases share a conserved glycosylation site in the C2 region. To test the possibility that the sugar residues might be a target for ubiquitination, a transgenic S11-RNase lacking its single glycosylation site was examined. This construct behaved similarly to a wild type S11-RNase, demonstrating that the lack of glycosylation does not confer constitutive pollen rejection. To determine if the introduction of an N-linked glycan in the HVa region would affect pollen rejection, a construct containing a second N-glycosylation site inside the HVa region of the S11-RNase and a construct containing only that N-glycosylation site inside the HVa region were prepared. The first construct rejected S11 pollen normally, but surprisingly, plants expressing the construct lacking the C2 glycosylation site rejected both S11 and S13 pollen. We propose that the non-glycosylated form is a dual specific allele, similar to a previously described dual-specific allele that also had amino acid replacements in the HV regions. Interestingly, this phenotype is not observed in the mutant containing two glycosylation sites, which suggests that the sugar residues are not removed during S-RNase entry into the pollen. In the final study, S-RNase-binding assays were performed with pollen extracts to detect potential interacting proteins. We found that concanavalin A-immobilized S11-RNase bound eEF1A, a component of the eukaryotic translational machinery. This interaction was validated by pull-down experiments using a GST-tagged S. chacoense eEF1A. We also found that a previously documented actin binding to eEF1A was markedly increased in the presence of S-RNases, although S-RNases alone do not bind actin. Lastly, we observed that actin in incompatible pollen tubes has an unusual aggregated form which also co-labels with S-RNases. This suggests that binding between S-RNases and eEF1A could provide a potential functional link between the S-RNase and the alteration of the actin cytoskeleton that occurs during the SI reaction. Furthermore, if eEF1A binding to S-RNases acted to titrate the amount of free S-RNase in the pollen tube, this binding may help explain the threshold phenomenon, where a minimum quantity of S-RNase in the style is required to trigger the SI reaction.