Anthracnose, caused by the fungal pathogen Colletotrichum trifolii, is one of the most destructive diseases of alfalfa worldwide. Cloning and characterization of the host resistance (R) genes against the pathogen will improve our knowledge of molecular mechanisms underlying host resistance and facilitate the development of resistant alfalfa cultivars. However, the intractable genetic system of cultivated alfalfa, owing to its tetrasomic inheritance and outcrossing nature, limits the ability to carry out genetic analysis in alfalfa. Nonetheless, the model legume Medicago truncatula, a close relative of alfalfa, provides a surrogate for cloning the counterparts of many agronomically important genes in alfalfa. In this study, we used genetic map-based approach to clone RCT1, a host resistance gene against C. trifolii race 1, in M. truncatula. The RCT1 locus was delimited within a physical interval spanning ~200 kilo-bases located on the top of M. truncatula linkage group 4. Complementation tests of three candidate genes on the susceptible alfalfa clones revealed that RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers broad spectrum anthracnose resistance. Thus, RCT1 offers a novel resource to develop anthracnose-resistant alfalfa cultivars. Furthermore, the cloning of RCT1 also makes a significant contribution to our understanding of host resistance against the fungal genus Colletotrichum.

Dagens dagvattenhantering i urbana områden är inte hållbar vilket resulterar i översvämningar, förfallav naturen och hälsoproblem. Fortsatt urbanisering och klimatförändringar påverkar systemet negativtoch kräver en brådskande förändring. Ett alternativ till traditionell dagvattenhantering är naturligalösningar, NBS, definierat som inspiration och användandet av naturen för att hanterasamhällsproblem. Det kan ge resilienta och adaptiva lösningar som främjar biologisk mångfald,människors välbefinnande, ett lovande alternativ för att tackla de här problemen. NBS är dock förenatmed osäkerheter såsom kunskapsbrister kring prestanda, underhåll och möjliga negativakonsekvenser. Även om NBS kan anses vara en hållbar lösning är den inte frekvent implementerad iurbana områden. Den här uppsatsen använder fallstudien Årstafältet för att samla och analysera föroch nackdelar av NBS och traditionella dagvattenlösningar. Med hjälp av intervjuer med inblandadeaktörer, plandokument, regleringar och en litteraturstudie visar resultatet att NBS kan ge ett resilient,flexibelt och kostnads-effektivt system med multipla fördelar vilket kan uppfylla t.ex. vattendirektivet(WFD), hållbarhetsmålen (SDGs) och miljömålen. Det traditionella systemet är en oelastisk lösninginkapabel att förebygga miljöförändringarna och med frekvent breddning av smutsigt dag- ochavloppsvatten. Systemet är sett som oförmögen att uppfylla mål kring biologisk mångfald, miljömåloch reningskrav och därav en ohållbar lösning. Existerande infrastruktur och ett välarbetat arbetssättgör att det traditionella systemet dock fortfarande förlitas på. Problem som identifierades med NBSvar kunskapsbrister, en ny process för att hantera dagvatten som inkluderar en omfattande inblandningav aktörer, övergångsbarriärer, oklarheter gällande ansvarsfördelning, brist på deltagande frånallmänheten, avsaknad av tekniskt vägledning, bristen och behovet av att kontrollera och mätalösningar samt beroendet av entusiastiska aktörer. / The current stormwater management in urban areas is not sustainable, resulting in frequent floodingevents, degeneration of the environment and human health issues. Increased urbanization and climatechange negatively impact the outcome and calls for an urgent change. An alternative to traditionalstormwater management is nature-based solution, NBS, broadly defined as the usage or inspiration ofnature to address societal challenges. It can provide resilient, adaptive solutions which promotebiodiversity and human well-being, a solution to address these challenges. NBS is however unitedwith uncertainties such as knowledge gaps of performance, maintenance, efficiency and potentialtradeoffs. Although NBS can be considered a sustainable solution, it is not widely adopted andimplemented in urban areas. This thesis uses the case study of Årstafältet project to identify andanalyze opportunities and challenges of NBS and conventional drainage system. Based on interviewswith involved actors, plans, regulation and a conceptual framework, the result shows that NBS canprovide a resilient, flexible and cost-effective system with multiple benefits which addresses allaspects of sustainability. It is widely supported by laws and policies, addressing the EU WaterFramework Directive (WFD), the Sustainable Development Goals (SDGs) and the EnvironmentalObjectives for example. The conventional drainage system is an inflexible system unable to mitigateclimate change, with frequent overflows (CSOs) of dirty storm and sewage water. It is unable toachieve biodiversity goals, environmental objectives and water quality targets and thus recognized asan unsustainable solution. With an existing infrastructure and an established way of managing theconventional drainage system, it is however still relied on. Identified challenges of NBS areknowledge gaps, a new management process with extensive stakeholder involvement, transitionbarriers, unclear division of responsibilities, lack of public participation and technical guidance, lackof and the need to monitor solutions and the dependence on enthusiastic actors.

Soybean mosaic virus (SMV), has been the cause of numerous and often devastating disease epidemics, causing reduction in both the quality and quantity of soybeans worldwide. Two important genes for resistance to SMV are Rsv1 and Rsv4. Alleles at the Rsv1 locus have been shown to control resistance to all but the most virulent strain of SMV. This locus has been mapped previously to the soybean F linkage group. Rsv4 is an SMV resistance locus independent of Rsv1 and confers resistance to all strains of SMV. This locus has not been mapped previously. The purpose of this study is to investigate the two genomic regions that contain these vitally important resistance genes. A population of 281 F2 individuals that had previously been genotyped for reaction to SMV was evaluated in a mapping study which combined bulk segregant analysis with Amplified Fragment Length Polymorphism (AFLP). A Rsv4-linked marker, R4-1, was identified that mapped to soybean linkage group D1b using a reference mapping population. More than 40 markers were mapped in the Rsv4 segregating population including eleven markers surrounding Rsv4. This will provide the necessary framework for the fine mapping of this important genetic locus. Previous work has located Rsv1 to a genomic region containing several important resistance genes including Rps3, Rpg1, and Rpv. An RFLP probe, NBS5, whose sequence closely resembles that of several cloned plant disease resistance genes has been mapped to this chromosomal region. The efficacy of using this sequence to identify potential disease resistance genes was assessed by screening a cDNA library to uncover a candidate disease resistance gene which corresponds to this NBS5 sequence. Two related sequence classes were identified that correspond to NBS5. Interestingly, one class corresponds to a full length gene closely resembling other previously cloned disease resistance genes offering evidence that this NBS5-derived clone is a candidate disease resistance gene. A new marker technique was developed by combining the speed and efficiency of AFLP with DNA sequence information from cloned disease resistance genes. Using this strategy, three new markers tightly linked to Rsv1 were identified. One of these markers, which maps 0.6 cM away from Rsv1, has motifs consistent with other cloned disease resistance genes, providing evidence that this approach is an efficient method for targeting genomic regions where disease resistance genes are located. / Ph. D.

The thesis discusses some issues on the topic of money velocity. Its goal is to summarise theroretical contributions in this area, which are followed by a practical application through the monetary analysis of the Slovak Republic from 1993 to 2000. The theoretical part involves the first approaches to money velocity, explanation of the quantity theory of money and monetarism and their critique. It also describes the monerary transmission mechanism. The practical application part evaluates the impact of the stability of money velocity on the decision of the National Bank of Slovakia to abandon monetary targeting. It also focuses on the other variables that influenced monetary policy execution. The last chapter is devoted to the importance of money velocity if the central bank uses inflation targeting as its policy.

La sénescence cellulaire est une réponse aux stresses selon laquelle des cellules pouvant proliférer optent pour entrer dans un arrêt du cycle cellulaire en réponse à une variété de stimulations intrinsèques et extrinsèques telle que, par exemple, le raccourcissement des télomères, un stress oxydatif, des dommages à l’ADN ou l’activation constitutive d’oncogènes. Toutes ces stimulations ont en commun le potentiel d’initier ou de promouvoir une transformation néoplasique qui peut dégénérer en cancer. En fait, la sénescence est maintenant acceptée comme un mécanisme cellulaire autonome pour empêcher le développement du cancer et elle est reconnue, particulièrement dans les cellules humaines, pour s’établir et se maintenir à l’aide d’au moins deux voies de suppression tumorale majeures : les voies de p53/p21CIP et de p16INK4A/pRB. Ces deux voies sont capables d’activer et d’augmenter l’expression d’un autre suppresseur tumoral : la protéine PML. En tant que suppresseur de tumeur, PML est suffisant pour induire la sénescence dans des cellules normales, mais il n’arrive généralement pas à activer une réponse complète de sénescence dans les cellules cancéreuses. Considérant ces faits, le premier objectif de cette thèse était d’étudier le mécanisme de résistance des cellules cancéreuses contre la sénescence induite par PML. Nous avons trouvé que, dans des cellules normales, la surexpression de CDK4 ou de CDK6 (CDK4/6) est suffisante pour contourner la sénescence induite par PML. De même dans les cellules cancéreuses, l’expression de ces kinases, souvent retrouvées augmentées dans de nombreux cancers, prévient probablement l’induction d’une sénescence par PML. Effectivement, grâce à l’inhibition de l’expression et/ou de la fonction kinase des CDK4/6, nous avons réussi à restaurer un programme de sénescence dans des cellules cancéreuses. En fait, l’utilisation de palbociclib, un inhibiteur spécifique de CDK4/6 maintenant en essais cliniques, permet d’augmenter l’habileté de PML à induire un arrêt de croissance plus fort et plus durable dans des cellules en cultures ainsi qu’une meilleure réduction de la progression de tumeurs dans des souris. Cette sénescence plus complète corrèle avec une augmentation de la présence de marqueurs d’autophagie, une meilleure répression des gènes cibles des E2F et une signature d’expression de gènes correspondant à l’inhibition de la méthylation de l’ADN. Ce dernier point découle du fait que l’inhibition de CDK4/6 par le palbociclib promeut une dégradation par autophagie de la DNA méthyltransférase DNMT1. Nous avons aussi démontré que CDK4 est capable d’interagir avec DNMT1 et de le phosphoryler in vitro. Ces résultats soulignent la valeur potentielle des inhibiteurs de CDK4/6 en tant que modulateurs épigénétiques pour faciliter l’activation de la sénescence dans des cellules cancéreuses. La sénescence induite par PML est fortement liée aux modifications post-traductionnelles. Parmi ces dernières, la SUMOylation joue un rôle important dans la fonction d’échafaudeur de PML et dans la formation des corps de PML. Les corps de PML sont des structures nucléaires dynamiques stimulées par des stresses, comme l’activation d’oncogènes menant à la sénescence, et dont la formation permet la séquestration de protéines spécifiques pour leur régulation et/ou pour leur modification post-traductionnelle. À travers le recrutement de protéines, les corps de PML régulent de nombreuses fonctions cellulaires telles que la sénescence, l’apoptose, la réponse antivirale, la réponse aux dommages à l’ADN et la régulation de l’expression de gènes. Compte tenu de cela, le deuxième objectif de cette thèse était de caractériser le rôle de la SUMOylation dans la sénescence induite par un oncogène, soit par l’expression de l’oncogène RAS. À l’aide d’une analyse du protéome de SUMO3 dans les cellules sénescentes versus des cellules en croissances, nous avons pu identifier 25 sites de SUMOylation dans 23 protéines dont l’incidence était significativement régulée par la sénescence. Il est à noter que la plupart de ces protéines (un tiers) sont connues pour être associées au corps de PML. Curieusement, UBC9 (la seule enzyme E2 pour la SUMOylation) a été retrouvée plus SUMOylée dans la sénescence sur sa Lys-49. Des études fonctionnelles d’un mutant d’UBC9 pour la Lys-49 ont démontré une diminution de son association aux corps de PML et la perte de la capacité d’UBC9 surexprimé à retarder la sénescence. De plus, la localisation forcée d’UBC9 dans les corps de PML gêne la sénescence induite par PML ou RAS. Ces résultats nous permettent de proposer des fonctions pro- et anti-sénescence de la SUMOylation des protéines, particulièrement pour UBC9. Mots-clés : Sénescence, PML, CDK4 et CDK6 (CDK4/6), méthylation de l’ADN, palbociclib, corps de PML, SUMOylation, UBC9 / Cellular senescence is a stress response wherein proliferating competent cells undergo a stable cell cycle arrest in response to a variety of intrinsic and extrinsic stimuli, including telomere shortening, oxidative stress, DNA damage or the constitutive activation of oncogenes among others. All these stimuli have in common the potential to initiate or promote neoplastic transformation that can degenerate in cancer. Senescence, particularly in human cells, is established and maintained by at least two major tumor suppressor pathways: the p53/p21CIP and p16INK4A/pRB pathways and is now accepted as a potent cell-autonomous mechanism for suppressing the development of cancer. Both pathways are able to activate and increase the expression of the tumor suppressor protein PML. As a tumor suppressor, PML is sufficient to induce senescence in normal cells; however, upon the same stimuli, cancer cells fail to engage a complete senescence response. Given this, the first aim of this thesis is to investigate the resistance mechanisms of cancer cells to PML-induced senescence. We found that overexpression of the CDK4 and CDK6 (which are often up-regulated in cancer) are sufficient to bypass PML-induced senescence in normal cells. In cancer cells the expression of these kinases impairs the PML-induced senescence. By inhibiting the expression and/or function of CDK4/6 we were able to restore the senescence program in cancer cells. Also, the specific CDK4/6 inhibitor palbociclib (currently used in clinical trials) increased the ability of PML to regulate a stronger and more permanent growth inhibition in cell culture and decreased tumor progression in mice. This complete senescence response correlated with an increase in autophagy markers, repression of E2F target genes and a gene expression signature of blocked DNA methylation. Furthermore, CDK4/6 inhibition by palbociclib promotes autophagy-dependant degradation of the DNA methyltransferase DNMT1. More important, we were able to demonstrate that CDK4 directly interacts and phosphorylates DNMT1 in vitro. These results highlight the potential value of CDK4/6 inhibitors as epigenetic modulators to facilitate activation of cellular senescence in cancer cells. PML-induced senescence is tightly regulated by post-translational modifications (PTMs). Among these PTMs, SUMOylation plays an important role in the scaffold function of PML and the formation of the PML-NBs (PML-Nuclear Bodies). PML-NBs are dynamic structures triggered by stress such as oncogene-induced senescence, and its formation allows the sequestration of target proteins for their regulation and/or its post-translational modification. By protein recruitment, PML-NBs regulate several cellular functions such as senescence, apoptosis, antiviral response, DNA repair and gene regulation. Given this; the second aim of this thesis is to characterize the role of SUMOylation in oncogene mediated cellular senescence, specifically by the expression of the oncogene RAS. By a SUMO3 proteome analysis of senescent cells we were able to identify 25 SUMO sites in 23 proteins that were significantly regulated during senescence. Importantly, most of these proteins were PML-NB associated. Interestingly, UBC9 (the only SUMO E2 enzyme), was found more SUMOylated in senescence on its Lys-49. Functional studies of a UBC9 mutant in Lys-49 showed a decreased association to PML-NBs and the loss of UBC9’s ability to delay senescence. Moreover, forced localization of UBC9 into PML-NBs counteracted RAS and PML-induced senescence. These results allowed us to propose a pro- and an anti-senescence function of protein SUMOylation, specifically for UBC9. Keywords: Senescence, PML, CDK4/6, DNA methylation, palbociclib, PML-NBs, SUMOylation, UBC9

http://www.pharmacol.usyd.edu.au/thesis This thesis is primarily concerned with a class of chemical compounds known as pyridazinediones, being 6-membered aromatic rings containing two adjacent nitrogen atoms (pyridazine), doubly substituted with oxygen. In particular, the work focuses on pyridazine-3,6-diones, derivatives of maleic hydrazide (1). Understanding of the chemistry of these compounds is extended, using theoretical and synthetic techniques. This thesis is also concerned with two very important classes of receptors which bind amino acids in the brain: firstly, the inhibitory GABA receptor, which binds g-aminobutyric acid (GABA) (2) in vivo, and for which muscimol (3) is an agonist of the GABAA subclass; secondly, Excitatory Amino Acid (EAA) receptors, which bind glutamate (4) in vivo, and in particular the AMPA subclass, for which (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) (5) is an agonist. The connection between pyridazinediones and amino acid receptors is the design, synthesis, and evaluation of structures based on pyridazinediones as potential GABA and EAA receptor ligands. Techniques of theoretical chemistry, molecular modelling, synthetic chemistry, and in vitro pharmacology are used to explore pyridazine-3,6-dione derivatives as ligands.

http://www.pharmacol.usyd.edu.au/thesis This thesis is primarily concerned with a class of chemical compounds known as pyridazinediones, being 6-membered aromatic rings containing two adjacent nitrogen atoms (pyridazine), doubly substituted with oxygen. In particular, the work focuses on pyridazine-3,6-diones, derivatives of maleic hydrazide (1). Understanding of the chemistry of these compounds is extended, using theoretical and synthetic techniques. This thesis is also concerned with two very important classes of receptors which bind amino acids in the brain: firstly, the inhibitory GABA receptor, which binds g-aminobutyric acid (GABA) (2) in vivo, and for which muscimol (3) is an agonist of the GABAA subclass; secondly, Excitatory Amino Acid (EAA) receptors, which bind glutamate (4) in vivo, and in particular the AMPA subclass, for which (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) (5) is an agonist. The connection between pyridazinediones and amino acid receptors is the design, synthesis, and evaluation of structures based on pyridazinediones as potential GABA and EAA receptor ligands. Techniques of theoretical chemistry, molecular modelling, synthetic chemistry, and in vitro pharmacology are used to explore pyridazine-3,6-dione derivatives as ligands.

Increased urbanisation and climate change are negatively affecting the water cycle and are increasing floods and creating concerns for the built environment and human wellbeing. This has created a need to research sustainable water management in cities. Nature-based solutions (NBS) can offer more sustainable ways of water management, but conventional systems are still favoured in governance. Hence, the aim of this thesis is to identify opportunities and challenges of implementing NBS at Årstafältet in Stockholm and the related governance processes from a Socio-Ecological-Technical system perspective in order to bring a holistic view on sustainable urban stormwater management. The methods used in triangulation for this case study are interviews, a site visit, and desktop study of associated planning documents. The collected data is analysed with an analytical framework that investigates the Social-Ecological-Technical System (SETS) dimensions, in relation to governance. The results show that the implementation of NBS at Årstafältet has been largely successful, due to contextual factors, as well as an adaptive and reflexive governance approach. However, identified system dynamics, interrelations and tensions have shown that there is room for improvements. By increasing transdisciplinarity in early stages of the process to overcome rigid governance structures and techno-centricity. The application of the SETS framework has proved to be successful in identifying dynamics, interrelations, and tensions but there are issues related to uncertainties in how to categorise system components. Nevertheless, the SETS perspective has been useful for identifying challenges and opportunities related to governance and planning processes of implementing NBS.

As rain events are likely to intensify and the proportion of impervious areas in cities increases, greater demands are put on stormwater management. Nature-Based Solutions (NBS) have become more popular over the past decades to be integrated into the stormwater strategy in order to reduce the volume of runoff in cities. For NBS to be effective over long term, maintenance must be prioritized. This study addressed two questions, what are the difficulties in modeling (lack of) maintenance over time and what are the consequences in terms of water quantity with inadequate maintenance on the long- term efficiency of NBS?. This was found by developing a hydrodynamic model in SWMM, where three NBS types were modeled, rain garden, permeable pavement, and infiltration trench. The efficiency of maintenance was investigated by creating several scenarios where different parameters were identified to represent maintenance. The parameters were varied for different precipitation events, as the effect of maintenance depends on the type of precipitation used. In general, inadequate maintenance has been proven to have little impact on rain events with a return period of 2-years, while a large difference could be seen for intense rains. Two consequences as a result of an absence of maintenance are overgrown vegetation and clogging of the media. Clogging was investigated for all three NBS types, and was found to have a large impact on its efficiency over time. The result also shows that despite the inadequate management, NBS can still contribute to manage stormwater, but with lower efficiency. / I takt med att regnen kommer att intensifieras och andelen hårdgjorda ytor i städer ökar, så ställs allt högre krav på dagvattenhantering. Naturbaserade lösningar (NBS) har under senaste decenniet blivit alltmer populära att integrera i dagvattenstrategin för att minska mängden dagvatten i städer. För att NBS ska vara effektiv över tid behöver skötseln prioriteras. Den här studien adresserar två frågor, vilka är svårigheterna med att modellera (bristfällig) underhåll över tid och vad är konsekvenserna av bristfälligt underhåll på NBS effektivitet över tid. Detta besvarades genom att skapa en modell i SWMM där tre NBS typer modellerades, regnbädd, genomsläpplig yta och infiltrationsdike. Effekten av underhåll undersöktes genom att skapa flera scenarion där olika parametrar som identifieras för att representera underhåll. Parametrarna förändrades sedan för olika nederbördstyper, då effekten av underhåll beror på vilken nederbördstyp som studeras. Bristfälligt underhåll sågs generellt inte ha någon större påverkan på 2-års regn, medan en stor differens kunde ses för de intensiva regnen. Igenvuxen vegetation på ytan och igentäppning av media, som konsekvens av undermåttligt underhåll, undersöktes bland annat för de tre NBS typerna, och resultatet visade att det har stor påverkar på dess effektivitet över tid. Resultatet visar också, trots otillräckligt underhåll, att kan NBS ändå bidra till att hantera dagvatten, men med betydligt lägre effektivitet.

[pt] A crescente ocorrência de eventos extremos, impulsionada pelas mudanças climáticas, tem um impacto direto nas cidades, onde aproximadamente 60 por cento da população mundial reside. As Soluções baseadas na Natureza (SbN) têm se tornado importantes estratégias de adaptação e mitigação climática nas políticas públicas. Esse estudo teve como objetivo orientar a Prefeitura da Cidade do Rio de Janeiro na implantação de SbN no Distrito de Baixa Emissão (DBE). Assim, foi elaborado um Caderno de Orientação para implantação de SbN que busca dar suporte à Prefeitura na ação estruturante de requalificação urbana do DBE. Uma ferramenta de Seleção de SbN foi utilizada e adaptada para o contexto do DBE e foram identificados os fatores de sucesso da capacidade institucional da Prefeitura na implementação das SbN, além da apresentação das 15 SbN recomendadas. Também foram gerados mapas de áreas prioritárias de infraestrutura verde para o distrito, baseados no Guia Metodológico para Implantação de Infraestrutura Verde. Por fim, foram elaborados um projeto conceitual de SbN para duas avenidas localizadas no DBE, a fim de ilustrar a aplicação prática dessas ações em escala local, e um estudo hidrológico do volume de águas pluviais amortecido nas sub-bacias dessas avenidas, para avaliar o desempenho das soluções propostas. Este estudo busca auxiliar a tomada de decisão da Prefeitura na implementação das SbN no DBE. A integração de SbN em programas e a realização de parcerias-público privadas são algumas estratégias importantes a serem consideradas pela Prefeitura, bem como a realização de outras pesquisas de SbN direcionadas para o DBE. / [en] The increasing occurrence of extreme events driven by climate change hasa direct impact on cities, where almost 60 percent of the world s population resides.Nature-based solutions (NbS) have become essential climate adaptation and mitigation strategies on public policies. This study aimed to guide the Rio de Janeiro City Hall in development of NbS in the Low Emission Zone (LEZ). Thus, a Guideline for the Deployment of NbS was formulated to support the City Hall instructuring urban renewal actions in the LEZ. An NbS Selection tool was used and adapted to the context of the LEZ, and the success factors of Rio s institutional capacity in implementing NbS were identified, in addition to the presentation of the 15 recommended NbS. Maps depicting priority areas for green infrastructure within the LEZ were also generated based on the Methodological Guide for Green Infrastructure Implementation. Finally, a conceptual project of NbS was created for two avenues located in the LEZ, to illustrate the practical application of the seactions on a local scale, and a hydrological study of the attenuated storm water volume in the sub-basins of these avenues, to evaluate the performance of the proposed solutions. This study seeks to assist the City Hall s decision-making in the implementation of NbS in the LEZ. The integration of NbS in programs and the realization of public-private partnerships are some relevant strategies to be considered by the City, as well as the realization of further research on NbS directed to the LEZ.