Thesis advisor: Daniel A. Kirschner / In the present study, the ability of small compounds to inhibit the fibrillogenesis of beta-amyloid 12-28 was explored. Beta-amyloid 12-28 is a synthetic fragment of Alzheimer's beta-amyloid, which contains the core hydrophobic residues thought to be significant for fiber formation. Using x-ray diffraction, preliminary screening of over sixteen compounds was performed. Cotinine, PTI-00703®, and tetracycline were chosen because of their ease of solubility, the effect on the coherent domain size of the beta-crystallite subunit in the presence of chosen small molecules as shown by x-ray diffraction, as well as their presence in previously published literature. This conformational-driven inhibition of fibrillogenesis was explored in the current research using circular dichroism spectroscopy and x-ray diffraction. Circular dichroism spectroscopy revealed the nascent beta-sheet structure of beta-amyloid12-28 when first dissolved and only cotinine, out of all three inhibitors, was able to shift the equilibrium away from the fibrillogenic beta-sheet structure toward a random coil secondary structure after 36 hours of incubation. X-ray diffraction in this study demonstrated no change in hydrogen bond spacing at ~4.7Å and intersheet spacing at ~10-12Å both alone and in the presence of all small molecules. With increasing concentration of inhibitor, however, the widths of these reflections increased, indicating a decrease in the coherent domain size. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

La presente memoria tiene como objetivo generar un Plan de Acción que sería utilizado el próximo año 2008 en el inicio de las actividades de diseño y planificación del Programa Territorial Integrado Salmón Aysén (PTISA), instrumento de fomento de la Corporación de Fomento de la Producción (CORFO) Cabe destacar que esta memoria se realizó en el marco del "Estudio de caracterización y determinación del potencial de atracción de inversiones del sector salmón en la XI Región" y del "Estudio para la determinación de la Línea Base de la Industria del Salmón en la XI Región de Aysén", trabajo que la empresa Knowledge Management Technology (KMT Consultores) realizó por encargo de CORFO, y en los cuales el autor de esta memoria fue miembro del equipo de trabajo.

Plants rely on innate immunity to perceive and respond to pathogenic microbes. Pathogenic microbes suppress and evade plant immune responses to obtain nutrients and multiply resulting in plant diseases and death. One battleground for the arms race between plants and microbial invaders is located in the leaf intercellular space, specifically between Pseudomonas bacteria and Arabidopsis. This thesis seeks to understand the virulence mechanisms that allow Pseudomonas bacteria to grow within the leaves of Arabidopsis and how the plant immune response reduces pathogen growth and reproduction. Some plant pathogens produce specific extracellular polysaccharides to potentially enhance pathogenicity during infection of plants. The objective of this thesis is to understand the importance of biofilms for Pseudomonas success and determine if Arabidopsis suppresses biofilm formation as part of the plant immune response. It was hypothesized that biofilm formation contributes to Pseudomonas success in planta and Arabidopsis suppresses biofilm formation during PAMP-Triggered Immunity (PTI) to reduce bacterial growth. Wild-type plants and defense mutants were infiltrated with flg22 or mock (water) treatments to induce or mock-induce PTI in plants, followed by observing GFP-expressing Pseudomonas via florescence microscopy to determine if biofilm-like aggregate formation was occurring. In vivo studies in this thesis indicate that biofilm-like aggregate formation contributes to bacterial success during Arabidopsis infection. Additionally, the phytohormone, salicylic acid (SA), accumulates in leaf intercellular spaces of resistant plants during PTI that suppresses biofilm formation, suggesting that SA acts as an anti-microbial and anti-biofilm agent that contributes to the suppression of pathogen growth during plant defense. / Thesis / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24675号 / 農博第2558号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5456(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 寺内 良平, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Sjukdom på grödor orsakad av bakterier kan bidra till ekonomiska förluster för bönder samt brist på mat, därför är det viktigt att utveckla nya hållbara sätt att motverka och behandla grödor mot bakterier. Idag är det mest vanliga tillvägagångssättet antibiotika men detta är inte hållbart p.g.a uppkomst av antibiotikaresistens. Antibiotika är inte heller tillgängligt för alla bönder och grödor då kostnaden blir för hög. Världsbefolkningen växer och om 80 år beräknas det bo mellan 9.9 och 12.7 biljoner (95% konfidens) människor på jorden. Växande befolkning samt ökande klimatförändringar, som torka och höjda temperaturer kräver nya bekämpningsmetoder mot bakterier för att tillgodose behoven i framtiden. Det saknas information om hur växter hanterar och reglerar bakteriella hot, därför är målet med denna studie att bidra med kunskap kring den transkriptionella regleringen av växters immunsystem mot bakterier. För att göra detta har promotorsekvenser hos gener som är förknippade med immunförsvaret i växter undersökts efter konserverade regulatoriska element. En känd receptor, FLS2 har en stor roll i växters försvar mot bakterier och känner igen en peptid från bakteriers flagell. Denna studie har undersökt FLS2 och den sammankopplade receptorn SERK1. Hos FLS2 kunde ingen konserverad modul hittas i uppströmsekvensen, däremot observerades ett 8 bp långt motiv, CAACTTG, i alla undersökta arter. I SERK1 hittades en lång konserverad modul bestående av flera motiv. Både FLS2-motifet och två motiv i SERK1-modulen binds av transkriptionsfaktorn MYC2. För att testa hypotesen att MYC2 bidrar till den transkriptionella regleringen av FLS2 och SERK1 har en experimentell plan utformats, där Nicotiana benthamiana transfekteras av Agrobacterium tumefaciens innehållandes promotorsekvenserna samt generna till transkriptionsfaktorn MYC2. En ökad förståelse kring de olika delarna och mekanismerna som medverkar inom växters immunförsvar kan bidra till den fortsatta forskningen mot hållbara lösningar till att säkra mat i framtiden. / Several factors contribute to the demand of new, sustainable solutions to bring food security to the world population. The United Nations predicts, with a confidence of 95%, that the world population will be between 9.9 and 12.7 billion by year 2100. At the same time plant agriculture as seen today is threatened by climate changes e.g., rising temperatures and more extreme weather conditions. In addition, plant bacterial pathogens reduce yields, and cause losses of over $1 billion dollars worldwide every year to the food production chain. The currently most used and effective treatment against bacterial infections on crops is antibiotics, but this is not a viable alternative for most growers due to increasing antibiotic resistance and the high development, production, and distribution cost. During the upcoming years development of new approaches against bacterial infections on crops is of high importance but currently there are information gaps in the field of plant defense regulation systems. This study was aimed to provide knowledge about the transcriptional regulation of genes that are included in plant immune system towards bacteria. To investigate this, conserved regulatory elements of the upstream sequences of two defense-related plant receptor kinases, FLS2 and SERK1, was searched for in different species. FLS2 is a surface receptor that recognizes a peptide derived from the bacterial flagellin protein, and is part of the pathogen-triggered immunity response of most of higher plants. In FLS2 no conserved module was found but a single motif, CAACTTG, is conserved in all chosen species. In SERK1 a strikingly long and conserved module was found. Both the FLS2 motif and two motifs in the SERK1 module are recognition motifs with MYC2, a transcription factor involved in different plant mechanisms and the regulation of phytohormones like abscisic acid and auxin. To address whether MYC2 is involved in the transcriptional regulation of FLS2, an experimental approach is described, involving transactivation by MYC2 of FLS2 reporter constructs, studies using agroinfiltration in Nicotiana benthamiana. An increased knowledge about the different components and mechanisms of plant defense regulation will help the research towards new bactericides, transgenic plants, and other ways to secure food for upcoming generations.

No description available.

This work investigated the subcellular dynamics of the plant elicitor peptide AtPep1 and its interplay with plant defense responses. First, an introduction of the plant innate immunity system is provided with emphasis on pattern trigger immunity (PTI), which is based on the recognition of \"non-self\" and \"self\" elicitor molecules by surface-localized patternrecognition receptors (PRRs). Then, the Arabidopsis endogenous peptides that act as selfelicitor molecules are presented, with details on AtPep1 and its PEPR receptors. Plant endomembrane trafficking is described, encompassing endocytic pathways, clathrin mediated endocytosis (CME) and receptor-mediated endocytosis (RME). In the next chapter, we explored strategies for the in vivo study of the subcellular behavior of AtPep1; to this end, we fused the precursor protein of AtPep1 (PROPEP1) to GFP and assessed its localization. We found that PROPEP1 was associated with the tonoplast and accumulated in the vacuole, suggesting that this organelle could work as the station where PROPEP1 is stored and later released, only in a danger situation, hence initiating AtPep1. Moreover, we generated AtPep1 versions labeled with fluorescent dyes and demonstrated that this peptide could be fluorescently tagged without loss of its biological activity. In chapter 3, we combined classical and chemical genetics with life imaging to study the behavior of a bioactive fluorescently labeled AtPep1 in the Arabidopsis root meristem. We discovered that the labeled AtPep1 was able to bind the plasma membrane very quickly in a receptor-dependent manner. Subsequently, the PEPR-AtPep1 complex was internalized via CME and transported to the lytic vacuole, passing through early and late endosomal compartments. Impairment of CME compromised the AtPep1 responses. Our findings provide for the first time an in vivo visualization of a signaling peptide in plant cells, thus giving insights into its intracellular fate and dynamics. The role of the coregulatory receptor BRI1-associated kinase 1 (BAK1) in AtPep1-responses was also investigated (chapter 4). Our results confirmed that BAK1 interacts with PEPRs in a ligand-dependent manner and indicate that BAK1 modulates AtPep1 signaling and endocytosis, but that, when absent, it might be replaced by homologous SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) proteins that could have additional functions during the AtPep1 signaling. Furthermore, phosphorylation events after the formation of PEPR-BAK1 complexes seem to dictate the molecular bases of AtPep1 internalization and signaling. Finally, we discussed our findings in a more general perspective, highlighting the important findings for the plant endomembrane trafficking field, the potential use of fluorescently labeled ligands as a tool to study ligand-receptors pairs, the availability of AtPep1-PEPRs as an excellent model to study endocytosis and its interplay with signaling, and the future challenges in the field. / Neste trabalho, foi investigada a dinâmica subcelular do peptídeo elicitor de planta AtPep1 e suas implicações nas respostas de defesa. Primeiramente, é fornecida uma introdução do sistema imune inato de plantas com ênfase na imunidade ativada por moléculas elicitoras derivadas de organismos invasores ou da mesma planta, após seu reconhecimento por receptores localizados na membrana plasmática (PTI responses). Peptídeos endógenos que têm sido reportados em Arabidopsis como ativadores de PTI são descritos, dando especial destaque para o peptídeo AtPep1 e seus receptores PEPRs. O tráfego de endomembranas em plantas é introduzido, abrangendo as vias de internalização, endocitose mediada por proteínas clathrinas (CME) e endocitose mediada por receptor (RME). No capítulo seguinte, foram avaliadas estratégias para o estudo in vivo da dinâmica subcelular do AtPep1. Para isso a proteína precursora do AtPep1 (PROPEP1) foi fusionada a GFP e sua localização visualizada, encontrando que PROPEP1 é associado com o tonoplasto e acumula dentro do vacúolo, fato que sugere uma função de armazenamento do PROPEP1 para esta organela, desde onde é liberado em caso de uma situação de perigo dando origem ao AtPep1. Adicionalmente, foram produzidas versões biologicamente ativas do AtPep1 marcado com fluróforos. No capítulo três foram combinados genética clássica e genética química com visualizações in vivo para estudar o comportamento de um AtPep1 bioativo e marcado fluorescentemente na células meristemática da ponta da raiz de Arabidopsis, sendo encontrado que AtPep1 se liga rapidamente na membrana plasmática numa forma dependente de receptor. Em seguida, o complexo AtPep1-PEPR foi internalizado via CME e transportado para o vacúolo, passando através do endossomo primário e secundário. Quando o funcionamento da CME foi comprometido, as respostas ao AtPep1 também foram afetadas. Estes resultados fornecem a primeira visualização in vivo de um peptídeo de sinalização em plantas, mostrando sua dinâmica e destino intracelular. O papel regulatório durante as respostas induzidas pelo AtPep1 do co-receptor BRI1-associated kinase 1 (BAK1) foram investigadas (Capítulo quatro). Nossos resultados confirmaram que BAK1 interage com PEPRs numa forma dependente do ligante e indicam que BAK1 modula sinalização e endocitose do AtPep1, no entanto quando ausente, BAK1 pode ser substituído por seus homólogos SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE os quais poderiam ter funções adicionais durante as repostas induzidas pelo AtPep1. Eventos de fosforilação após a formação do complexo PEPR-BAK1 parecem ditar as bases moleculares da internalização e sinalização do AtPep1. Finalmente, são discutidos os resultados encontrados nesta pesquisa numa perspectiva geral, destacando a relevância destas descobertas na área de pesquisa em que estão inseridos, o potencial que representa o uso de ligantes marcados fluorescentemente como ferramenta para o estudo de complexos entre ligante-receptor, a disponibilidade do sistema AtPep1-PEPRs como modelo de estudo da endocitose em plantas e sua relação com sinalização, e os futuros desafios na área.

L'expression des gènes peut être contrôlée à différents niveaux : transcriptionnel post-transcriptionnel, traductionnel et post­-traductionnel. A ce jour la majorité des études se sont concentrées au niveau transcriptionnel, néanmoins l'importance des mécanismes de régulation post-transcriptionnels se fait de plus en plus évidente. Chez les procaryotes cette régulation post-transcriptionnelle est assurée par les ARN régulateurs dont le mécanisme d'action passe par l'interaction directe avec les ARN messagers ou les protéines. Grâce à l'essor des analyses transcriptomiques haut-débit (RNA-seq), l'identification de ces ARN est devenue accessible, en revanche leur caractérisation fonctionnelle demeure toujours un défi. Nous avons identifié de nombreux ARN régulateurs candidats chez Agrobacterium fabrum C58 (anciennement Agrobacterium tumefaciens C58). Cette bactérie commune du sol devient phytopathogène lorsqu'elle porte le plasmide Ti (pour Tumor inducing). Elle est alors responsable de la maladie dite de la galle du collet qui se traduit par la formation de tumeurs chez les plantes. Ces travaux de thèse ont eu pour objectif de caractériser fonctionnellement des ARN régulateurs présent sur le plasmide Ti. Deux candidats ont été étudiés en combinant prédiction de cibles et analyses phénotypiques. Le premier, nommé RNA1111, a été caractérisé tant que régulateur de la virulence. Quant au deuxième, nommé QfsR, nous avons démontré qu'il régulait des gènes responsables du transfert conjugatif du plasmide Ti et de la production du signal de quorum sensing associé, mais également des gènes chromosomiques responsables de la motilité et de la production de succinoglycane. En utilisant un système rapporteur, nous avons également démontré que QfsR agissait via une interaction directe avec les ARN messagers des gènes cibles. QfsR représente le premier exemple d'ARN régulateur plasmidique régulant des cibles chromosomiques. L'existence d'un tel régulateur chez un plasmide présent transitoirement au sein des populations d'Agrobacterium illustre le dialogue entre plasmide et chromosome / Gene expression can be controlled at different levels: transcriptional, post-transcriptional, translational and post-translational. To date, the majority of studies have been concentrated on the transcriptional level, but the importance of post-transcriptional regulation mechanisms is becoming more and more evident. In prokaryotes this post-transcriptional regulation is ensured by regulatory RNAs whose mechanism of action passes through direct interaction with messenger RNAs or proteins. With development of high-throughput transcriptomic analyzes (RNA-seq), the identification of these RNAs has become accessible, but their functional characterization remains challenging. We have identified many candidate regulatory RNAs in Agrobacterium fabrum C58 (formerly Agrobacterium tumefaciens C58). This common bacterium of the soil becomes phytopathogenic when carrying the plasmid Ti (for Tumor inducing). It is then responsible for the so-called grown gall disease which results in the formation of tumors in plants. The objective of this thesis was to characterize functionally regulatory RNAs present on the Ti plasmid. Two candidates were studied by combining target prediction and phenotypic analysis. The first, named RNA1111, was characterized as a regulator of virulence. As for the second, named QfsR, we demonstrated that it regulates genes responsible for the conjugative transfer of the Ti plasmid and the production of the associated quorum sensing signal, as well as chromosomal genes responsible for the motility and succinoglycan production. Using a reporter system, we also demonstrated that QfsR was acting via direct interaction with the messenger RNAs of the target genes. QfsR represents the first example of plasmid regulatory RNA regulating chromosomal targets. The existence of such a regulator in a plasmid transiently present in the populations of Agrobacterium illustrates the dialogue between the plasmid and the chromosome

This work presents a spectroscopic and theoretical study of several energy materials using synchrotron-based techniques. Two classes of materials are studied: solids that have reported photocatalytic properties, and lithium compounds that are thought to form during the cycling of modern battery electrodes. An overview of synchrotron soft X-ray spectroscopic techniques is presented, along with the theory and procedures associated with performing such measurements. These measurements are compared to density functional theory (DFT) calculations, as implemented by the WIEN2k package, along with a description of the DFT method. Calculated electronic structure is shown to be a useful aid in interpreting the results of X-ray emission and X-ray near-edge absorption measurements (XES and XANES), allowing conclusions about the physical structure and properties of the materials to be reached. Two photocatalytic systems are outlined, the first of which is a solid solution of GaN and ZnO (GaN:ZnO) that exhibits an unexpected reduction in band gap. By carefully comparing common hybridized features from O, N and Zn core emission lines, a binding energy picture of the valence and conduction bands of GaN:ZnO is constructed, allowing its band gap reduction to be described as a consequence of heterojunctions between predominantly GaN and ZnO regions within the solid solution. This description attempts to resolve controversy in the literature regarding the origin of the band gap reduction, as well as to rule out a hypothesized oxynitride superlattice structure as the explanation. The second photocatalytic system studied is a carbon nitride derivative, poly(triazine imide) (PTI) that displays high crystallinity and that could be very inexpensive to produce due to its elemental abundance. Through resonant excitation, two inequivalent N sites in PTI can be probed by X-ray emission spectroscopy, indicating the material is not a conjugated polymer like other reported carbon nitrides. The band gap of the system is observed to decrease in response to disordered Li loading, an e ect that is con rmed by DFT calculation. Several potential disorder models of the Li loading of PTI are investigated with DFT force minimization in order to choose a structural candidate capable of producing calculated X-ray spectra that agree with our measurements. The presented lithium study attempts to use a modern soft X-ray absorption facility to characterize the Li surface by-products inherent to the charge-discharge cycling of a battery electrode. A survey of potential Li compounds was performed using Li K-edge XANES will be compared to DFT calculations and X-ray Raman Scattering measurements performed by collaborators in the future. Correlating measurements of the survey compounds with charge-cycled electrode measurements will be an area for future work.

abstract: Design and mitigation of infrastructure on expansive soils requires an understanding of unsaturated soil mechanics and consideration of two stress variables (net normal stress and matric suction). Although numerous breakthroughs have allowed geotechnical engineers to study expansive soil response to varying suction-based stress scenarios (i.e. partial wetting), such studies are not practical on typical projects due to the difficulties and duration needed for equilibration associated with the necessary laboratory testing. The current practice encompasses saturated “conventional” soil mechanics testing, with the implementation of numerous empirical correlations and approximations to obtain an estimate of true field response. However, it has been observed that full wetting rarely occurs in the field, leading to an over-conservatism within a given design when partial wetting conditions are ignored. Many researchers have sought to improve ways of estimation of soil heave/shrinkage through intense studies of the suction-based response of reconstituted clay soils. However, the natural behavior of an undisturbed clay soil sample tends to differ significantly from a remolded sample of the same material. In this study, laboratory techniques for the determination of soil suction were evaluated, a methodology for determination of the in-situ matric suction of a soil specimen was explored, and the mechanical response to changes in matric suction of natural clay specimens were measured. Suction-controlled laboratory oedometer devices were used to impose partial wetting conditions, similar to those experienced in a natural setting. The undisturbed natural soils tested in the study were obtained from Denver, CO and San Antonio, TX. Key differences between the soil water characteristic curves of the undisturbed specimen test compared to the conventional reconstituted specimen test are highlighted. The Perko et al. (2000) and the PTI (2008) methods for estimating the relationship between volume and changes in matric suction (i.e. suction compression index) were evaluated by comparison to the directly measured values. Lastly, the directly measured partial wetting swell strain was compared to the fully saturated, one-dimensional, oedometer test (ASTM D4546) and the Surrogate Path Method (Singhal, 2010) to evaluate the estimation of partial wetting heave. / Dissertation/Thesis / Masters Thesis Engineering 2017