O processo de fixação biológica de nitrogênio (FBN) é a principal forma de entrada de nitrogênio (N) em ecossistemas naturais e é intermediado por microorganismos diazotróficos simbióticos ou de vida livre. A produção de biofertilizantes com bactérias diazotróficas é a principal alternativa ao uso de fertilizantes nitrogenados solúveis. Apesar da simbiose Rhizobium-leguminosas ser eficiente em promover o crescimento das plantas, a inoculação de bactérias diazotróficas em gramíneas vem apresentando resultados questionáveis, principalmente devido à baixa eficiência e incompatibilidade entre os atuais inoculantes bacterianos e as plantas. Os biofertilizantes para gramíneas utilizam bactérias endofíticas as quais desenvolvem interações pouco conhecidas com as plantas. Uma possibilidade para melhorar a eficiência da FBN em gramíneas é a aplicação de bactérias epifíticas de menor seletividade em relação às bactérias endofíticas. Os objetivos deste trabalho foram isolar novos genótipos de bactérias diazotróficas da filosfera de bambu da Mata Atlântica, verificar seu potencial biotecnológico in vitro e avaliar sua eficiência agronômica como biofertilizante em cana-de-açúcar em casa-de-vegetação. Foram obtidos 120 isolados bacterianos os quais foram caracterizados morfológica e filogenéticamente. Com relação ao potencial biotecnológico, 48 isolados bacterianos apresentaram resposta positiva nos testes in vitro de redução de acetileno (ARA) e para produção in vitro de ácido indol-3-acético (AIA) na presença de L-triptofano. Desses 48 isolados, 50% apresentaram capacidade de solubilizar fosfato de cálcio, 8% de produzir quitinases, 16% de produzir ACC desaminase e 17% de produzir sideróforos. O sequenciamento do gene rRNA 16S indicou que 75% dos isolados da filosfera de bambu foram similares à classe Gammaproteobacteria (Proteobacteria) , e a família Enterobacteriaceae. Dentre eles, 32% dos isolados apresentaram sequências similares a Klebsiella sp. e 2% foram similares a Serratia e Enterobacter. Das 48 sequências analisadas, 37% delas não foram classificadas quanto ao gênero, podendo então representar novos gêneros ou espécies. Dez isolados contendo o gene nifH foram selecionados para experimentos de eficiência agronômica em casa-de-vegetação usando bactérias inoculadas via pulverização foliar ou encapsuladas em micro-esferas de alginato e associados a diferentes níveis de adubação nitrogenada. A inoculação por meio de pulverização nas folhas de cana-de-açúcar resultou em aumento significativo de massa seca de parte aérea e concentração de N na parte aérea das plantas na sua fase inicial de desenvolvimento. A maior taxa de fixação nas folhas inoculadas foi observada sete dias após a inoculação no tratamento sem adição de N mineral. A inoculação no solo com os mesmos 10 isolados encapsulados em matriz polimérica em conjunto com diferentes níveis de adubação nitrogenada mostrou aumentos significativos de massa seca da parte aérea, raízes e concentração de N na parte aérea da cana-de-açúcar em comparação aos controles, principalmente nas fases tardias do desenvolvimento das plantas. O solo que recebeu as bactérias encapsuladas apresentaram elevadas taxas de FBN, oscilando entre 0 e 4 g de N g-1 h-1, 7 dias após a inoculação. Os resultados sugerem que as bactérias selecionadas possuem alto potencial biotecnológico para promover o crescimento das plantas em momentos diferentes do seu ciclo de desenvolvimento, dependendo do tipo de abordagem para inoculação. / The process of biological nitrogen fixation (BNF) is the most important form of nitrogen (N) input in natural ecosystems and is mediated by symbiotic or free-living diazotrophic microorganisms. The production of biofertilizers containing diazotrophic bacteria is the main alternative to the use of soluble nitrogen fertilizers, improving plant growth by nitrogen fixation or other plant growth promoting mechanisms. Despite the efficiency of the symbioses Rhizobium-legumes in promoting plant growth, the inoculation of diazotrophic bacteria in grasses, such as sugarcane, has shown variable results, mainly due to the low efficiency and incompatibility between the current bacterial strains used in inoculants and plant genotypes. Most of the biofertilizers for grasses uses endophytic bacteria that develop complexes interactions with the host plant, which are not totally understood. A possibility to improve the efficiency of BNF in grasses is the application of epiphytic diazotrophic bacteria that are less selective as compared to endophytes. The aims of this work were to isolate new genotypes of diazotrophic bacteria from the phyllosphere of bamboo from Atlantic Forest, determine their biotechnological potential based on in vitro assays, and evaluate their agronomic efficiency as biofertilizer for sugarcane under greenhouse conditions. A total of 120 bacterial isolates were obtained and characterized morphologically and phylogenetically. Regarding the biotechnological potential, 48 isolates showed positive responses under in vitro the acetylene reduction assay (ARA) and indole-acetic-acid (IAA) production in vitro assay in the presence of L-tryptophan. Among the 48 isolates evaluated, 50% were able to solubilize calcium phosphate, 8% produced chitinases, 16% were able to produce ACC deaminase, and 17% produced siderophores. The sequencing of the rRNA 16S gene revealed that 75% of the isolates were phylogenetically related to the family Enterobacteriaceae (Gammaproteobacteria) . The genus Klebsiella accounted for 32% of the isolates, whereas Serratia and Enterobacter accounted for 2%. Aproximatelly 37% of the isolates were assembled unclassified Bacteria. Ten isolates containing the nifH gene were selected for agronomic efficiency test under greenhouse conditions, using bacteria inoculated via foliar spraying, or encapsulation in alginate beads and inoculation in the soil, associated with different doses of nitrogen fertilizer. The inoculation on the sugarcane leaf surfaces resulted in significant increases in root biomass and N concentration in the shoots at the early stage of plant development. The highest N fixation rates in inoculated leaves were observed 7 days after inoculation in the absence of mineral N. The soil inoculation with the same 10 isolates immobilized in polymeric matrix in addiction to different rates of nitrogen fertilization showed significant increases in shoot and root biomass and N concentration in the shoots of sugarcane, when compared to the controls, mostly at later stages of plant development. The soil inoculated with encapsulated bacteria showed high rates of BNF even when nitrogen fertilizer was applied, ranging between 0 and 4 g of N g-1 h-1 seven days after the inoculation. The results suggest that the selected bacteria have high biotechnological potential to promote sugarcane growth at different stages of development, depending on the inoculation approach.

Context : Researchers have increasingly recognised the benefit of mining software repositories to extract information. Thus, integrating a version control tool (VC tool) and bug tracking tool (BT tool) in mining software repositories as well as synchronising missing bug tracking data (BT data) and version control log (VC log) becomes of paramount importance, in order to improve the quality of bug data in software repositories. In this way, researchers can do good quality research for software project benefit especially in open source software projects where information is limited in distributed development. Thus, shared data to track the issues of the project are not common. BT data often appears not to be mirrored when considering what developers logged as their actions, resulting in reduced traceability of defects in the development logs (VC logs). VC system (Version control system) data can be enhanced with data from bug tracking system (BT system), because VC logs reports about past software development activities. When these VC logs and BT data are used together, researchers can have a more complete picture of a bug’s life cycle, evolution and maintenance. However, current BT system and VC systems provide insufficient support for cross-analysis of both V Clogs and BT data for researchers in empirical software engineering research: prediction of software faults, software reliability, traceability, software quality, effort and cost estimation, bug prediction, and bug fixing. Aims and objectives: The aim of the thesis is to design and implement a tool chain to support the integration of a VC tool and a BT tool, as well as to synchronise the missing VC logs and BT data of open-source software projects automatically. The syncing process, using Bicho (BT tool) and CVSAnalY (VC tool), will be demonstrated and evaluated on a sample of 344 open source software (OSS) projects. Method: The tool chain was implemented and its performance evaluated semi-automatically. The SZZ algorithm approach was used to detect and trace BT data and VC logs. In its formulation, the algorithm looks for the terms "Bugs," or "Fixed" (case-insensitive) along with the ’#’ sign, that shows the ID of a bug in the VC system and BT system respectively. In i addition, the SZZ algorithm was dissected in its formulation and precision and recall analysed for the use of “fix”, “bug” or “# + digit” (e.g., #1234), was detected was detected when tracking possible bug IDs from the VC logs of the sample OSS projects. Results: The results of this analysis indicate that use of “# + digit” (e.g., #1234) is more precise for bug traceability than the use of the “bug” and “fix” keywords. Such keywords are indeed present in the VC logs, but they are less useful when trying to connect the development actions with the bug traces – that is, their recall is high. Overall, the results indicate that VC log and BT data retrieved and stored by automatic tools can be tracked and recovered with better accuracy using only a part of the SZZ algorithm. In addition, the results indicate 80-95% of all the missing BT data and VC logs for the 344 OSS projects has been synchronised into Bicho and CVSAnalY database respectively. Conclusion: The presented tool chain will eliminate and avoid repetitive activities in traceability tasks, as well as software maintenance and evolution. This thesis provides a solution towards the automation and traceability of BT data of software projects (in particular, OSS projects) using VC logs to complement and track missing bug data. Synchronising involves completing the missing data of bug repositories with the logs de tailing the actions of developers. Synchronising benefit various branches of empirical software engineering research: prediction of software faults, software reliability, traceability, software quality, effort and cost estimation, bug prediction ,and bug fixing.

Tropical rainforests have an unparalleled capacity to sequester carbon, harbor biodiversity, and cycle water and nutrients due to their high rates of primary production. The large biomass stocks and rapid regeneration rates of these forests are often attributed to ample soil nitrogen and quick recovery of the nitrogen cycle in tropical soils following disturbance. Symbiotic nitrogen-fixing trees, which are relatively abundant at tropical latitudes, have the greatest capacity to provide tropical rainforests with new nitrogen, yet the ecological drivers of tropical symbiotic nitrogen fixers and their effects on the forests they inhabit are not well understood. This dissertation consists of four chapters that examine the patterns, environmental controls, and ecological consequences of symbiotic nitrogen-fixing trees in regenerating and intact rainforests in the Caribbean lowlands of Costa Rica. In chapter 1, I use field sampling in a chronosequence of rainforest plots to show that symbiotic nitrogen fixation declines through succession despite increases in the basal area of nitrogen-fixing trees. Chapters 2 and 3 describe results from a controlled shadehouse experiment assessing the effects of light, soil nitrogen, and plant competition on nitrogen fixation rates and the growth and biomass allocation of nitrogen fixers and non-fixers. In chapter 2, I demonstrate that light regulates nitrogen fixation more strongly than soil nitrogen availability. This is a departure from the historical focus on soil nitrogen as the primary regulator of nitrogen fixation and has the potential to resolve longstanding paradoxes of tropical nitrogen cycling. In chapter 3, I show that nitrogen fixation provides some resistance to competitive effects from neighboring plants in nitrogen-limited conditions, and that nitrogen fixers in these conditions downregulate their fixation rates in the presence of a competitor. This chapter also demonstrates that nitrogen fixation does not represent a significant structural cost to the plant, as reduced root biomass of nitrogen fixers more than compensates for allocation to nodule production. Finally, in Chapter 4, I demonstrate that nitrogen-fixing trees in our chronosequence plots do not promote forest growth, as expected given their capacity to fertilize their neighbors, but rather inhibit forest growth because they are strong competitors. These chapters describe several unexpected findings – i.e. that light primarily drives nitrogen fixation and that nitrogen fixers slow forest growth – which provide new and important insight into the role that nitrogen-fixing trees play in the growth of Costa Rican rainforests.

In today’s technology driven society, software is becoming increasingly important in more areas of our lives. The domain of software extends beyond the obvious domain of computers, tablets, and mobile phones. Smart devices and the internet-of-things have inspired the integra- tion of digital and computational technology into objects that some of us would never have guessed could be possible or even necessary. Fridges and freezers connected to social media sites, a toaster activated with a mobile phone, physical buttons for shopping, and verbally asking smart speakers to order a meal to be delivered. This is the world we live in and it is an exciting time for software engineers and computer scientists. The sheer volume of code that is currently in use has long since outgrown beyond the point of any hope for proper manual maintenance. The rate of which mobile application stores such as Google’s and Apple’s have expanded is astounding. The research presented here aims to shed a light on an emerging field of research, called Genetic Improvement ( GI ) of software. It is a methodology to change program code to improve existing software. This thesis details a framework for GI that is then applied to explore fitness landscape of bug fixing Python software, reduce execution time in a C ++ program, and integrated into a live system. We show that software is generally not fragile and although fitness landscapes for GI are flat they are not impossible to search in. This conclusion applies equally to bug fixing in small programs as well as execution time improvements. The framework’s application is shown to be transportable between programming languages with minimal effort. Additionally, it can be easily integrated into a system that runs a live web service.

As bromélias habitam os mais diferentes ambientes sendo que muitas são encontradas como epífitas. Essas últimas estão sujeitas a condições de disponibilidade de água e nutrientes com intermitência. Elas conseguem sobreviver a essas circunstâncias por serem dotadas de diversas adaptações morfológicas e fisiológicas. A bromélia tanque Guzmania monostachia tem sido bastante estudada por possuir uma grande plasticidade fotossintética, porém, pouco é conhecido a respeito de outras possíveis adaptações como a interação com micro-organismos. Sendo assim, o presente trabalho isolou bactérias fixadoras de nitrogênio (diazotróficas) tanto da parte externa (epifíticas) quanto da parte interna (endofíticas) de diferentes porções (ápice, mediana e base) de folhas coletadas tanto no ambiente natural quanto na casa de vegetação. As bactérias foram selecionadas por meio do ensaio de redução de acetileno (ARA) e também pelo uso de quatro diferentes meios de cultura que não contém fonte de nitrogênio reduzido. As linhagens isoladas foram identificadas por meio do gene 16sRNA. Dois isolados Pseudomonas sp. e Burkholderia sp. foram escolhidos para serem marcados com um gene de fluorescência verde (GFP) e foram então inoculados (separadamente) em plantas de G. monostachia cultivadas em casa de vegetação. A colonização dos tecidos foliares foi monitorada com auxílio de um microscópio confocal. Além disso, foram estimadas as densidades populacionais epifíticas e endofíticas em diferentes grupos foliares (jovens e intermediários) e as folhas do grupo intermediário por serem maiores e totalmente expandidas foram divididas em porções (apical, mediana e basal). Também foram pesquisadas as seguintes moléculas descritas como importantes na interação entre planta e micro-organismo: óxido nítrico (NO), ácido salicílico (SA), etileno (ET) e ácido-indol-3-acético (IAA). Como resultados, a maioria das linhagens bacterianas foram classificadas como pertencentes ao grupo Proteobacteria, mas também foram encontrados isolados gram positivos pertencentes aos grupos Actinobacteria e Firmicutes. As bactérias endofíticas foram isoladas somente da porção basal foliar (tanto das folhas originadas do meio ambiente quanto das folhas originadas da casa de vegetação). Cabe ressaltar que após a inoculação de ambas bactérias marcadas com GFP, foi observado elas no interior dos tricomas foliares (estruturas presentes principalmente na base foliar). Após 20 horas da inoculação, ambas bactérias já foram visualizadas no interior da epiderme das folhas. Após 5 dias as bactérias foram se espalhando para regiões mais distantes do tricoma e também foram observadas no parênquima. Após 10 dias a bactéria Pseudomonas sp. foi encontrada nas paredes dos vasos condutores. Foram re-isoladas bactérias epifíticas e endofíticas da mediana e da base foliar, mas não da porção apical. Após 10 dias as bactérias foram isoladas como endofíticas somente da base. Essa porção não apresentou diferenças nas populações epifíticas e endofíticas. O NO aumentou nas folhas jovens e na base das intermediárias em um curto período de tempo após a inoculação. Aparentemente, ambas as bactérias não dispararam a via do SA. De acordo com os resultados aqui presentes, ambas as bactérias não pareceram ser prejudiciais à G. monostachia. Além disso, o presente trabalho mostra fortes evidencias de que as bactérias entram nos tecidos foliares por meio dos tricomas na base foliar e permanecem nessa porção, que é precisamente a mais importante para absorção de nutrientes / Bromeliads inhabit different environments and many are found as epiphytes. These plants are often subjected to periods of water and nutrient shortage. For their survival, epiphytic bromeliads are endowed with different morphological and physiological adaptations. Guzmania monostachia is a tank-bromeliad that has been extensively studied because of its great photosynthetic plasticity. However, little is known about other possible survival adaptations, such as beneficial interactions with microorganisms. Here, we isolated nitrogen fixing (diazotrophc) bacteria from both the outside (epiphytic) and the inside (endophytic) of different leaf portions (apex, middle and base), collected in natural environment and in greenhouse-cultivated plants. The bacteria were selected using the acetylene reduction assay (ARA) and four different media that do not contain reduced nitrogen source. The strains were identified by 16S rRNA. Two isolates, Pseudomonas sp. and Burkholderia sp. has been chosen to be tagged with green fluorescent protein (GFP) and then inoculated in G. monostachia plants cultivated in greenhouse. The colonization of the leaf tissues was monitored with the aid of confocal microscopy and also we estimate the external and internal bacterial population densities in different leaf groups (younger and expanded) and portions (apex, middle and base). In addition, we studied some important molecules in plant-microbe interactions: nitric oxide (NO), salicylic acid (SA), ethylene (ET) and indol-3-acetic acid (IAA). As a result, most of the isolated strains belong to the Proteobacteria group, but gram positive strains were also found belonging to the Firmicutes and Actinobacteria group. The endophytic bacteria were isolated only from the basal portion (both from leaves of natural environment and from leaves of greenhouse cultivated plants). Interestingly, after the inoculation of both bacteria tagged with GFP they were visualized entering by trichomes present mainly in the basal portion. Twenty hours after the inoculation, the bacteria were visualized inside the epidermis of the leaves. After five days, the bacteria were detected in the parenchyma and, ten days after the inoculation Pseudomonas sp. was found on the vessels walls. It was possible to re-isolate epiphytic and endophytic bacteria from the base and middle portions, but not from the apex. After 10 days the endophytic bacteria were found only in the base. The base did not show differences between epiphytic and endophytic populations. NO increased in a short time after the inoculation in the younger leaves and in the basal portion of intermediate leaves. Apparently, the SA pathway was not triggered by any of the bacteria used. According to these results, the bacteria tested do not seem to be harmful to the plant. Furthermore, we strongly suggest that they enter through the trichomes on the leaf base and remain in this portion, which is precisely the most important for the absorption of nutrients

O processo de fixação biológica de nitrogênio (FBN) é a principal forma de entrada de nitrogênio (N) em ecossistemas naturais e é intermediado por microorganismos diazotróficos simbióticos ou de vida livre. A produção de biofertilizantes com bactérias diazotróficas é a principal alternativa ao uso de fertilizantes nitrogenados solúveis. Apesar da simbiose Rhizobium-leguminosas ser eficiente em promover o crescimento das plantas, a inoculação de bactérias diazotróficas em gramíneas vem apresentando resultados questionáveis, principalmente devido à baixa eficiência e incompatibilidade entre os atuais inoculantes bacterianos e as plantas. Os biofertilizantes para gramíneas utilizam bactérias endofíticas as quais desenvolvem interações pouco conhecidas com as plantas. Uma possibilidade para melhorar a eficiência da FBN em gramíneas é a aplicação de bactérias epifíticas de menor seletividade em relação às bactérias endofíticas. Os objetivos deste trabalho foram isolar novos genótipos de bactérias diazotróficas da filosfera de bambu da Mata Atlântica, verificar seu potencial biotecnológico in vitro e avaliar sua eficiência agronômica como biofertilizante em cana-de-açúcar em casa-de-vegetação. Foram obtidos 120 isolados bacterianos os quais foram caracterizados morfológica e filogenéticamente. Com relação ao potencial biotecnológico, 48 isolados bacterianos apresentaram resposta positiva nos testes in vitro de redução de acetileno (ARA) e para produção in vitro de ácido indol-3-acético (AIA) na presença de L-triptofano. Desses 48 isolados, 50% apresentaram capacidade de solubilizar fosfato de cálcio, 8% de produzir quitinases, 16% de produzir ACC desaminase e 17% de produzir sideróforos. O sequenciamento do gene rRNA 16S indicou que 75% dos isolados da filosfera de bambu foram similares à classe Gammaproteobacteria (Proteobacteria) , e a família Enterobacteriaceae. Dentre eles, 32% dos isolados apresentaram sequências similares a Klebsiella sp. e 2% foram similares a Serratia e Enterobacter. Das 48 sequências analisadas, 37% delas não foram classificadas quanto ao gênero, podendo então representar novos gêneros ou espécies. Dez isolados contendo o gene nifH foram selecionados para experimentos de eficiência agronômica em casa-de-vegetação usando bactérias inoculadas via pulverização foliar ou encapsuladas em micro-esferas de alginato e associados a diferentes níveis de adubação nitrogenada. A inoculação por meio de pulverização nas folhas de cana-de-açúcar resultou em aumento significativo de massa seca de parte aérea e concentração de N na parte aérea das plantas na sua fase inicial de desenvolvimento. A maior taxa de fixação nas folhas inoculadas foi observada sete dias após a inoculação no tratamento sem adição de N mineral. A inoculação no solo com os mesmos 10 isolados encapsulados em matriz polimérica em conjunto com diferentes níveis de adubação nitrogenada mostrou aumentos significativos de massa seca da parte aérea, raízes e concentração de N na parte aérea da cana-de-açúcar em comparação aos controles, principalmente nas fases tardias do desenvolvimento das plantas. O solo que recebeu as bactérias encapsuladas apresentaram elevadas taxas de FBN, oscilando entre 0 e 4 g de N g-1 h-1, 7 dias após a inoculação. Os resultados sugerem que as bactérias selecionadas possuem alto potencial biotecnológico para promover o crescimento das plantas em momentos diferentes do seu ciclo de desenvolvimento, dependendo do tipo de abordagem para inoculação. / The process of biological nitrogen fixation (BNF) is the most important form of nitrogen (N) input in natural ecosystems and is mediated by symbiotic or free-living diazotrophic microorganisms. The production of biofertilizers containing diazotrophic bacteria is the main alternative to the use of soluble nitrogen fertilizers, improving plant growth by nitrogen fixation or other plant growth promoting mechanisms. Despite the efficiency of the symbioses Rhizobium-legumes in promoting plant growth, the inoculation of diazotrophic bacteria in grasses, such as sugarcane, has shown variable results, mainly due to the low efficiency and incompatibility between the current bacterial strains used in inoculants and plant genotypes. Most of the biofertilizers for grasses uses endophytic bacteria that develop complexes interactions with the host plant, which are not totally understood. A possibility to improve the efficiency of BNF in grasses is the application of epiphytic diazotrophic bacteria that are less selective as compared to endophytes. The aims of this work were to isolate new genotypes of diazotrophic bacteria from the phyllosphere of bamboo from Atlantic Forest, determine their biotechnological potential based on in vitro assays, and evaluate their agronomic efficiency as biofertilizer for sugarcane under greenhouse conditions. A total of 120 bacterial isolates were obtained and characterized morphologically and phylogenetically. Regarding the biotechnological potential, 48 isolates showed positive responses under in vitro the acetylene reduction assay (ARA) and indole-acetic-acid (IAA) production in vitro assay in the presence of L-tryptophan. Among the 48 isolates evaluated, 50% were able to solubilize calcium phosphate, 8% produced chitinases, 16% were able to produce ACC deaminase, and 17% produced siderophores. The sequencing of the rRNA 16S gene revealed that 75% of the isolates were phylogenetically related to the family Enterobacteriaceae (Gammaproteobacteria) . The genus Klebsiella accounted for 32% of the isolates, whereas Serratia and Enterobacter accounted for 2%. Aproximatelly 37% of the isolates were assembled unclassified Bacteria. Ten isolates containing the nifH gene were selected for agronomic efficiency test under greenhouse conditions, using bacteria inoculated via foliar spraying, or encapsulation in alginate beads and inoculation in the soil, associated with different doses of nitrogen fertilizer. The inoculation on the sugarcane leaf surfaces resulted in significant increases in root biomass and N concentration in the shoots at the early stage of plant development. The highest N fixation rates in inoculated leaves were observed 7 days after inoculation in the absence of mineral N. The soil inoculation with the same 10 isolates immobilized in polymeric matrix in addiction to different rates of nitrogen fertilization showed significant increases in shoot and root biomass and N concentration in the shoots of sugarcane, when compared to the controls, mostly at later stages of plant development. The soil inoculated with encapsulated bacteria showed high rates of BNF even when nitrogen fertilizer was applied, ranging between 0 and 4 g of N g-1 h-1 seven days after the inoculation. The results suggest that the selected bacteria have high biotechnological potential to promote sugarcane growth at different stages of development, depending on the inoculation approach.

ABSTRACT The success of land plants can be attributed to the evolution of beneficial associations between plant roots and soil microbes. Root-microbe mutualisms extend the range of plant nutrient acquisition delivered through the hyphal network of mycorrhiza, an ancient and widespread plant symbiosis, or by the more recent adaptive innovation of nitrogen-fixing nodule symbioses. A plant’s genetic toolkit governs its selection of beneficial symbionts and the developmental extent of these intimate interactions. However, the evolutionary origins and function for only a few symbiotic signaling components have been explored. The central aim of this dissertation is to resolve the evolutionary events that contributed two, novel genetic components for establishing root symbioses, NPF1B and NPF1C. The Medicago truncatula (Mt) LATD/NIP/NPF1.7C transporter functions in root and nodule meristems and is a member of the large NPF1 gene subfamily. Here, I propose that LATD/NIP’s role in establishing nitrogen-fixing symbioses is derived from the ancient mycorrhizal signaling pathway. I used a comparative phylogenomic approach to investigate the evolutionary origins of the NPF1 gene across flowering plants and then asked whether diversifying or purifying selection forces influenced NPF1 gene retention. I postulated that such gene retention correlates with the adaptive traits of mycorrhizal or nitrogen-fixing root nodule symbiosis; to test this I measured trait correlation within my dataset. I found that the NPF1 phylogeny is comprised of five well-supported angiosperm clades, A, B, C, D1 and D2, that arose by successive duplications and have unequal gene retention. NPF1B is present as a single copy gene or lost entirely, while the other major NPF1 clades expanded to multiple genes within angiosperms. The NPF1A, B and C genes are under strong purifying selection while the NPF1D genes display positive, diversifying selection. My data revealed a statistically significant correlation of NPF1A, B, C, and D2, but not NPF1D1, gene retention with the ability of a species to form mycorrhizal associations. Additionally, the retention of the NPF1B, C, D1, D2, but not NPF1A, genes within a species is statistically correlated with its ability to form nitrogen-fixing symbiosis. Supporting this correlation, NPF1B genes are expressed in plant root tissues with and without mycorrhizal fungi yet available datasets failed to detect NPF1B expression in nodule tissues whereas the NPF1C genes are expressed in both symbiotic and non-symbiotic plant root tissues. In support of functional conservation, expression of legume LATD/NIP cDNAs from Cicer arietinum (Ca) and Lotus japonicus (Lj) restored, in part, the root and nodule defects of the Mtlatd mutant and resulted in the formation of peculiar hybrid lateral root-nodule structures while, in wild-type M. truncatula, significantly augmented root development. In L. japonicus, the disruption of LATD/NIP alters the number of lateral roots and nodules My thesis data support the hypothesis for an ancestral NPF1 gene function in establishing mycorrhizal associations in angiosperms and, consequent to the monocot-eudicot divergence, co-opted this function for accommodating nitrogen-fixing symbioses in eudicots. Successive duplications then yielded the NPF1B and NPF1C genes that, by neofunctionalization and natural selection, further refined their roles in root organogenesis and symbiosis; a prerequisite for the evolution of nodule organs.

Australia is amongst the more efficient milk producers in the world.Milk production in the region of south-western Victoria relies mainly on rainfed white clover/perennial ryegrass pastures.As the demand for efficient and competitive milk production increases, the value of N2 fixation must be maximised. The objective of this thesis was to assess N2 fixation in grazed dairy pastures in south-western Victoria. Several tests and experiments were conducted and results noted. Studies revealed low white clover yields to be the major factor limiting N2 fixation in the region. For N2 fixation to have a significant impact on pasture quality and production, problems associated with legume persistence need to be addressed. Strategies may include the breeding of white clover cultivars with greater tolerance to water stress, improved winter production and increased competitiveness with companion species. Alternatively, the introduction of different legume species, better suited to the environment, may be appropriate. Where N2 fixation is unlikely to satisfy N demands, it may be necessary to introduce the strategic use of supplementary feeds or nitrogenous fertilisers. However, this would need to be carefully considered to ensure high input costs did not jeopardise the competitive advantage of low input pasture-based systems / Masters Thesis

Many bugs, even those that are known and documented in bug reports, remain in mature software for a long time due to the lack of the development resources to fix them. We propose a general approach, R2Fix, to automatically generate bug-fixing patches from free-form bug reports. R2Fix combines past fix patterns, machine learning techniques, and semantic patch generation techniques to fix bugs automatically. We evaluate R2Fix on three large and popular software projects, i.e., the Linux kernel, Mozilla, and Apache, for three important types of bugs: buffer overflows, null pointer bugs, and memory leaks. R2Fix generates 60 patches correctly, 5 of which are new patches for bugs that have not been fixed by developers yet. We reported all 5 new patches to the developers; 4 have already been accepted and committed to the code repositories. The 60 correct patches generated by R2Fix could have shortened and saved an average of 68 days of bug diagnosis and patch generation time.

To understand the soil microbial community structures of different locations and altitudes, as well as the fluctuation of microbial populations foward seasonal change, this study investigated the biodiversity of soil microbiota of eight sampling sites along the Lao-Nong River basin during 4/11/2003~3/21/2004. The results showed that the soil properties were different among these sampling sites. The soil temperatures were ranging in between 24~32¢J, except Tianchr, the highest altitude sampling site (7~19¢J) ; pH values were 4.00 (Tianchr) ~8.20 (Tauyuan) ; moisture contents were 2.31% (Liouguei) ~70.23% (Tianchr) ; total organic carbon contents were 0.38% (Linkou) ~36.21% (Tianchr) ; total organic nitrogen contents were 0.067% (Tauyuan) ~0.672% (Tianchr); the C/N ratio were 7.0~25.0, except Tianchr (42 in average) . In microbial community structures analysis, bacteria were the most abundant organisms among all soil samples (106~108 CFU per gram of soil) . The amount of other microbes were as follow: nitrogen-fixing microbes 105~107 CFU, actinomycetes 104~107 CFU, fungi 104~106 CFU, cellulolytic microbes 104~106 CFU, and the least one was phosphate- solubilizing microbes 103~105 CFU. For molecular biological microbiota analysis, the bead-beating DNA extraction method resulted the richest bacterial 16S rDNA DGGE profile. The16S rDNA DGGE profiles of various samples taken from different seasons showed somewhat seasonal variations that might relate to the specific climates of Taiwan, such as the plum rains and typhoons. Moreover, we also successfully cultured and preserved many microbes with specific biological functions. These microorganisms will provide a useful resource for further research and development in the biotechnology industries.