Black band disease (BBD) of corals is a temperature dependent, highly virulent, polymicrobial disease affecting reef-building corals globally. The microbial consortium of BBD is primarily comprised of functional physiological groups that include photosynthetic cyanobacteria, sulfate reducers, sulfide oxidizers and a vast repertoire of heterotrophic bacteria. Quorum sensing (QS), the cell-density dependent communication phenomenon in bacteria, is known to induce expression of genes for a variety of virulence factors in diseases worldwide. Microbes capable of QS release signals such as acyl homoserine lactones (AHLs) and autoinducer-2 (AI-2), which coordinate microbial interaction. The focus of the present study was to investigate the presence and potential role of QS in BBD pathogenicity, utilizing culture dependent and independent methodologies. Isolates across coral health states including BBD, were screened for production of QS signals, and AHL and AI-2 production capabilities were analyzed via LC-MS/MS. The effect of temperature on AHLs was also examined. Additionally, antimicrobial production capabilities of isolates were tested. BBD metagenomes were utilized to screen for sequences related to QS, antimicrobial synthesis, and antimicrobial resistance genes. BBD isolates represented a significantly higher proportion of isolates capable of producing QS signals in comparison to healthy coral isolates. Several AHLs produced by coral derived bacterial cultures were identified, and three AHLs, specifically 3OHC4, 3OHC5 and 3OHC6, showed a significant increase in production at an elevated temperature of 30 °C, which correlates with increased BBD incidence on reefs with increasing water temperature. Most of the BBD cultured isolates were identified as vibrios. Several sequences related to QS, antimicrobial synthesis and resistance genes were detected in the BBD metagenomes. Based on the findings of this study, a model for potential microbial interactions amongst BBD heterotrophs, centered around QS, is proposed. Taken together, the findings from this study provide a clearer understanding of the potential role of QS in BBD, and serve as the basis for further studies aimed at elucidating the pathogenesis of an intricate coral disease.

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Följande uppsats kommer att utreda samt komparera den juridiska institutionen för grundlagsändring mellan länderna Sverige och Spanien. Detta kommer att genomföras i en komparativrättslig metod för att kunna analysera och diskutera vardera lands reglering kring detta ämne. Uppsatsen redovisar hur en grundlagsändringsprocedur går till i praktiken, men även vilka som kan initiera ändringen samt vad för regleringar som finns gällande grundlagsändring. Materialet som kommer att användas är väsentligen de traditionella juridiska källorna samt somliga publikationer från media. Det kommer att dras slutsatser gällande skillnaderna respektive likheterna i slutet av uppsatsen för att summera all information samt analysen. / This following essay will investigate and compare the legal institution for constitutional amendment between the countries of Sweden and Spain. This will be carried out in a comparative legal method in order to be able to analyze and discuss each country’s regulation around this subject. The essay describes how a constitutional amendment procedure works in practice, but also who can initiate the amendment and what regulations exist regarding the constitutional amendment. The material that will be used in this essay is essentially the traditional legal sources as well as some publications from the media. Conclusions will be drawn regarding the differences and similarities at the end of the essay to summarize all the information and the analysis.

公司法上股東會與董事會之定足數門檻（Quorum），規制股份有限公司作成決策時，應有一定比例之股份數及董事人數出席會議。然而隨著現代公司經濟規模擴張，面對眾多的股東人數，及瞬息萬變的資訊，既有規制方式是否應一併調整值得研議。為深入研究此議題，本文將先介紹定足數門檻所涉之上位概念，說明多數決原則下，為保障公司決議之作成具有一定民主基礎，應設定法定出席門檻規制，確保公司治理運行。 就股東會決議之定足數門檻，目前我國法院見解認為，此係股東會之法定成立要件，如未達定足數，股東會決議當然不成立，顯示公司法對於資本多數決原則「程序正義」與「實質正義」面向之重視。然而，我國法律實踐上，為使一般股東亦有意願出席股東會，以達定足數要求，部分公司股東會開會成本偏高。是以，本研究擬參酌外國法制及分析我國規範後，重新思考就目前規範放寬之可能性及其限度，以合理減輕實務負擔。 至於董事會決議部分，我國法亦定有定足數門檻要求，彰顯立法者認為在於追求議事效率同時，應同樣重視董事之出席義務及受任人義務。惟我國法上董事會決議區分普通與特別決議，而有不同之定足數門檻，係比較法上較為少見之立法模式。因此，本文將分析現行規範，對公司董事會運作產生何種影響、實務上發生之爭議，及是否有調整規範之必要性，期望能使公司運作更為流暢。

The Quorum of the Twelve Apostles for the Church of Jesus Christ of Latter-day Saints is an administrative and ecclesiastical quorum. The Church, first organized in 1830, did not organize the Quorum of Twelve Apostles until 1835. When it was organized, Joseph Smith outlined the quorum's responsibilities through revelation. The Twelve were assigned two unique and specific responsibilities: to take the gospel to the nations of the earth and to form a traveling high council for the regulating of the Church outside of its stakes. The first opportunity for the Twelve to fulfill their responsibilities was in May 1835 when they were assigned to travel to the eastern United States and southern Canada. There they both preached the gospel and regulated the branches of the Church. This mission represents not only the first time the Apostles fulfilled their assigned responsibilities but the only time that they filled their responsibilities as an entire quorum. It is surprising that more secondary literature on this mission is not available. This thesis seeks to commence an academic conversation regarding this mission and its impact both on the quorum's development and on the Church in its outlying areas. Chapter 1 details the preparation of the individual members of the Twelve to fulfill this mission. It discusses the preparation of the Twelve prior to their call to the apostleship. It also discusses the training that took place between their call and the commencement of this mission. As an administrative body for the membership of the Church, the Twelve spent the majority of their time on this mission with the members of the Church. Chapter 2 identifies the unique purpose of the Twelve on this mission and how that purpose was fulfilled. Joseph Smith originally laid out the geographic framework for this mission, which sent the Twelve into Canada and throughout much of the northeastern United States. Chapter 3 identifies the locations of the Twelve based on available records and seeks to provide an answer to how the Twelve decided which areas to preach in. Many individuals were baptized during this five-month mission. Chapter 4 identifies what the Twelve taught and the sources that they used. It also discusses the reaction of the people they taught. The concluding chapter summarizes the thesis and identifies areas for further research.

The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.

Plants are continuously confronted with a multitude attack either abiotic but also biotic in nature. Interestingly, despite the abundance of bacteria that plant has to face, only few are able to induce death or disease in the host plant. It is therefore likely that, in addition to secondary metabolites with antimicrobial properties, plants also synthesize secondary metabolites which are able to inhibit the expression of virulence genes in bacteria without affecting either growth or viability, which allows plants to host willingly or not bacterial populations. This work focuses on the identification of such metabolites in Malagasy plants (genera Dalbergia and Combretum) and the demonstration of their inhibitory effect on the expression of virulence genes in two different pathosystems: Rhodococcus fascians (a phytopathogen) and Pseudomonas aeruginosa (an opportunistic pathogen). Thus, two metabolites were isolated using a combination of chromatographic techniques coupled with tests that evaluate the expression of certain genes involved in the virulence mechanisms of these bacteria. The first is a new prenylated isoflavanone, named perbergin, isolated from the bark extract of D. pervillei. It was shown that the perbergin target attR gene expression, encoding a LysR-type transcriptional regulator that plays a key role in regulating the expression of virulence genes of R. fascians and the transition from an epiphytic to a pathogenic lifestyle. Therefore, we have also shown that the expression of all virulence genes known to date in R. fascians is also affected while the expression of genes involved in epiphytic fitness of the bacteria is not altered. In addition, the application of perbergin at the time of infection of plants susceptible to R. fascians shows that this molecule reduces in vivo the virulence of R. fascians, highlighting the potential of perbergin as an anti-infective agent. The second is a flavonoid known as catechin, isolated from the bark extract of C. albiflorum. Catechin significantly inhibits the expression of genes that regulate the mechanism of quorum sensing in P. aeruginosa such as lasI, LasR, rhlI and rhlR but also lasB and rhlA which expression depends on quorum sensing. Therefore, the production of virulence factors such as pyocyanin and elastase is significantly affected. Because of the limited number of our arsenal of antibiotics and their increasing ineffectiveness, the identification of these compounds create a path to an alternative in the fight against pathogenic bacteria and multidrug resistance of pathogenic bacteria to antibiotics. Our results also demonstrate the richness of Malagasy plants as (re)sources of new therapeutic molecules and the importance of widening the range of bacterial targets to be investigated to develop new strategies to fight within the endless war that we are waging against bacteria pathogens.<p><p>Les plantes sont continuellement confrontées à une multitude d’attaques qu’elles soient de nature abiotique ou surtout biotique. Il est intéressant de noter que malgré la multitude de bactéries auxquelles les plantes doivent faire face, seules quelques unes sont capables d’induire la mort ou une maladie chez la plante hôte. Il est dès lors fort probable que, outre les métabolites secondaires ayant des propriétés antimicrobiennes, les plantes synthétisent également des métabolites secondaires capables d’inhiber l’expression des gènes de virulence chez les bactéries sans toutefois affecter ni leur croissance ni leur viabilité, ce qui permet aux plantes de contenir les populations bactériennes qu’elles hébergent de gré ou de force. Ce travail porte sur l’identification de ce type de métabolites dans des plantes malgaches (genres Dalbergia et Combretum) et la démonstration de leurs effets inhibiteurs sur l’expression de gènes de virulence chez deux pathosystèmes différents: Rhodococcus fascians (un phytopathogène) et Pseudomonas aeruginosa (un pathogène opportuniste). Ainsi, deux métabolites ont été isolés en utilisant une combinaison de techniques chromatographiques couplées avec des tests qui évaluent l’expression de certains gènes impliqués dans les mécanismes de virulence de ces bactéries. Le premier est un nouvel isoflavanone prénylé, nommé perbergine, isolé à partir de l’extrait d’écorces de D. pervillei. Il a été montré que la perbergine cible l’expression du gène attR, codant un régulateur transcriptionnel de type LysR qui joue un rôle clé dans la régulation de l’expression des gènes de virulence de R. fascians et qui assure la transition entre un mode de vie épiphyte et le mode pathogène. En conséquence, nous avons également montré que l’expression de l’ensemble des gènes de virulence connu à ce jour chez R. fascians est également affectée alors que l’expression de gènes impliqués dans l’aptitude épiphyte de la bactérie n’est pas altérée. Par ailleurs, l’application de perbergine au moment de l’infection de plantes sensibles à R. fascians montre que cette molécule atténue la virulence de R. fascians in vivo, mettant en exergue le potentiel de la perbergine comme agent anti-infectieux. Le deuxième est un flavonoïde, connu sous le nom de catéchine, isolé de l’extrait d’écorces de C. albiflorum. La catéchine inhibe significativement l’expression des gènes régulateurs du mécanisme du quorum sensing chez P. aeruginosa tels que lasI, lasR, rhlI et rhlR et également lasB et rhlA dont l’expression dépend du quorum sensing. En conséquence, la production des facteurs de virulence tels que la pyocyanine et l’élastase est significativement affectée. Compte tenu de l’appauvrissement de notre arsenal d’antibiotiques et de leur inefficacité croissante, l’identification de ces composés ouvre une voie alternative de lutte contre les bactéries pathogènes et la multirésistance des bactéries pathogènes aux antibiotiques. Nos résultats démontrent également la richesse des plantes malgaches comme (res)sources de nouvelles molécules thérapeutiques et l’importance d’élargir le champ des cibles bactériennes à investiguer pour développer de nouvelles stratégies de lutte dans la guerre sans fin que nous menons contre les bactéries pathogènes. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished