Pinus radiata is the most planted tree in the southern hemisphere. The planted trees are especially susceptible to pathogens, but even the native population, nowadays limited tomerely five locations, are threatened by diseases caused by arthropods, fungi and dehydration. Endophytes are bacteria or fungi that reside inside healthy plant tissue, and often have a beneficial effect on their hosts. Endophytes can help plants adapt to abiotic stress such as drought and protect them against pathogens and insect pests. Given the roles that endophytes play in host stress responses, it is possible that without studying endophytes we may not fully understand a plant’s response to increased temperatures and climate-induced disease.Using Illumina-sequencing of the 16S rRNA-gene the bacterial endophyte community in 15 trees from three of the remaining native populations were studied. By investigating trees from several sites geographical community differences were discovered. The three overall most dominating bacterial taxa can all be connected with genera known to contain members withanti-fungal properties.

The ubiquitous nature of microorganisms and their specificity to certain locations make them potentially useful for forensic investigators. Advances in microbial profiling techniques have made it possible to compare microbial community profiles obtained from evidence or crime scenes to individuals and vice versa. Profiling microbial communities associated with cadaver decomposition may provide useful information concerning post-mortem intervals and aid in the identification of clandestine graves. Four experiments using pigs as human decomposition analogues were performed over the course of 2011 and 2012 in southern Ontario to document changes in soil microbiology following decomposition. Studies were conducted in the spring and summer to document the effect of environmental conditions on the decomposition process and subsequent changes in gravesoil microbiology. Microbial activity was measured using a fluorescein diacetate assay as a preliminary indicator of changes within the soil microbial population. Both decreases and increases in microbial activity were observed throughout each decomposition experiment indicating that the microbial response to decomposition is complex. It is believed that environmental conditions and decomposition rates play a role in determining how taphonomic events affect soil microbial activity. Fatty acid methyl esters (FAME) profiling was used document community level changes throughout decomposition. Shifts in FAMEs profiles were brought on by the onset of active decay and persisted through to the dry remains stage. The fatty acids 3OH 12:0, 12:0, 16:0 and 18:0 were frequently found in higher amounts in gravesoils and may prove useful as markers of cadaver decomposition. Metagenomic profiles of soil microbial communities were obtained using Illumina?? sequencing. Decomposition was associated with changes v in microbial community composition. This allowed gravesoil samples to be differentiated from control samples for an extended period of time. Bacteria responsible for the shift in microbial profiles are those commonly associated with cadaver decomposition. Both sets of soil profiles indicated that weather had an effect on microbial community composition. Results highlight the need to document natural changes in microbial communities over seasons and years to establish normal microbial patterns to effectively use soil microbial profiles as post-mortem interval or clandestine grave indicators.

Mean winter air temperatures have risen by 2.5˚C over the last 50 years in the northeastern U.S., reducing mean annual winter snowpack depth by 26 cm and the duration of winter snow cover by four days per decade. Because snow cover insulates soil from below-freezing air temperatures, continued declines in snowpack depth are projected to be accompanied by colder winter soil temperatures and more frequent soil freeze-thaw events. Soil bacteria and fungi will play a significant role in the forest ecosystem response to snowpack loss because they are the primary agents that carry out soil organic matter decomposition and soil nutrient cycling. Additionally, the effect of winter snowpack decline on soil bacterial and fungal communities may act indirectly via winter climate change effects on plant roots. The objectives of my dissertation research were to first determine the effect that reductions in winter snow cover has on microbial exoenzyme activity, microbial respiration, net nitrogen (N) mineralization, and net nitrification rates in two mixed-hardwood forests (Harvard Forest, MA and Hubbard Brook Experimental Forest, NH). Additionally, I sought to determine the relative role that abiotic factors (i.e., winter snow cover or soil frost) versus biotic factors (i.e., altered root-microbe interactions) contribute to overall changes in soil biogeochemical processes as winter snow cover declines. I found that winter snow depth and duration are related positively to microbial exoenzyme activity and microbial respiration following snowmelt in spring, but this relationship is transient and attenuates into the growing season. By contrast, soil freeze-thaw events during winter result in persistent declines in microbial oxidative enzyme activity that are not compensated for by warming soils during the growing season. Together, these results suggest that loss of winter snow cover will result in lower rates of nutrient cycling in northeastern U.S. hardwood forests. Tree roots interact with winter snow depth to affect net mineralization and nitrification rates, as well as bacterial and fungal community composition. Thus, winter climate change portends a reorganization of root-microbe interactions with important consequences for soil biogeochemical cycling in mixed hardwood forests of the northeastern U.S.

Los alimentos fermentados son una excelente fuente de microorganismos con una serie de beneficios para el consumidor. En las zonas altoandinas de Perú, uno de los fermentados más consumidos por los pobladores es el tocosh, al cual se le han atribuido efectos benéficos en la salud del consumidor. Los efectos benéficos podrían originarse en la microbiota asociada al tocosh y/o a sus procesos de elaboración. Por lo tanto, en el presente estudio, se buscó determinar la composición microbiana del tocosh y como esta cambia en tres etapas de elaboración. La tipificación de la microbiota del tocosh se analizó mediante secuenciación de próxima generación (NGS del inglés) y herramientas bioinformáticas. Se determinó que en las tres muestras predominan los filos Clostridium y Prevotella. Asimismo, se evidenció variaciones entre las muestras con relación a la microbiota del alimento, por ejemplo, en los filos bacterianos Firmicutes y Bacteroidetes. También se encontraron géneros y especies con capacidad productora de antibióticos, como es el caso de Actinomicetales. Por último, este estudio es el primero en utilizar secuenciamiento de próxima generación para obtener un mapeo general de las bacterias existentes al interno del tocosh con un total de 720 especies identificadas adicionales a más del 50% de especies aun no identificadas. Este estudio podría servir como guía para posibles investigaciones futuras que busquen cultivar las bacterias encontradas en el tocosh y posiblemente probar la actividad antimicrobiana atribuida durante muchos años por el poblador altoandino. / Fermented foods are an excellent source of microorganisms with a number of benefits for the consumer. In the high Andean areas of Peru, one of the most consumed fermented products by the inhabitants is the tocosh, which has been attributed beneficial effects on the health of the consumer. The beneficial effects could originate in the microbiota associated with tocosh and / or its manufacturing processes. Therefore, in the present study, we sought to determine the microbial composition of tocosh and how it changes in three stages of elaboration. The typification of the tocosh microbiota was analyzed by next generation sequencing (NGS) and bioinformatics tools. It was determined that the Clostridium and Prevotella phylum predominate in all three samples. Likewise, variations between the samples in relation to the microbiota of the food are evidenced, for example, in the bacterial phylum Firmicutes and Bacteroidetes. We also found genera and species with antibiotic-producing capacity, such as Actinomycetals. Finally, this study is the first to use next-generation sequencing to obtain a general mapping of the bacteria inside the tocosh with a total of 720 additional identified species and more than 50% of species not yet identified. This study could serve as a guide for future research that seeks to cultivate the bacteria found in tocosh and possibly test the antimicrobial activity attributed for many years by the high Andean population. / Tesis

It is well known that the elderly often manifest chronic low-grade inflammation. This phenomenon, called “inflamm-aging,” is postulated to contribute to increased susceptibility towards infectious diseases and an overall increase in frailty. We have proposed examining the gut microbiome as a potential mediator of these changes. Gut microbial communities influence the host immune system; often dictating an individual’s health status. Thus, harmful gut microbiome changes, termed dysbiosis, are associated with poor health in the elderly. We first sought to understand the key immunological, physiological and microbiome changes occurring with age (Chapter 3). Our data reveals immune impairments in aged mice, with increased intestinal permeability, systemic inflammation and alterations in the functions of myeloid cell populations. However, our aged germ-free (GF) mice are protected from these outcomes, indicating that the old microbiome may play a strong role in these age-associated impairments. To study this further, we have colonized young and old GF mice with the “young” or “old” microbiota in order to determine whether the relationship between microbial dysbiosis with age and health status is correlative or causative (Chapter 4). Interestingly, young GF mice colonized with old microbiota have significantly increased permeability, systemic inflammation and an influx of Ly6Chigh monocytes when compared to those colonized with the young microbiota. By using transgenic mice (TNF-/- mice), or by reducing systemic TNF levels via therapeutics, we were able to reduce some aspects of microbial dysbiosis and age-associated inflammation (Chapter 5). Our data suggests that harmful changes to the gut microbiome composition with age initiate a cycle of negative events that ultimately result in increased inflammatory myeloid cell recruitment, increased intestinal permeability and an overall increase in systemic inflammation in old mice. By identifying these key changes, we can work towards developing effective therapeutics that promotes healthy aging and protection against infectious diseases. / Thesis / Master of Science (MSc) / Élie Metchnikoff first coined the term “dysbiosis” when he described the imbalance in microbial populations that could result anywhere in the body. Since then, numerous studies have examined the role of the intestinal microbiota in defense against pathogens. Metchnikoff also suggested that the gut composition and function is altered with age and this can in turn; increase the host’s susceptibility towards infectious diseases. My research aims to characterize the role of microbial dysbiosis on the immune defects with age. To do so, I will be utilizing a unique set of mice, called gnotobiotic mice. These mice are housed under specific germ-free conditions and contain no microbiome. Thus, they provide us with the ideal model to study the effects of the microbiome on immune function. The findings from these studies will help in the development of preventative and therapeutic alternatives to provide the elderly with more years of healthy, independent living.

Drought is a major environmental stress factor that poses a serious threat to food security. The effects of drought on early reproductive tissue at 1-2 DAP (days after pollination) is irreversible in nature and leads to embryo abortion, directly affecting the grain yield production. We developed a working RNA-Seq pipeline to study maize (Zea mays) drought transcriptome sequenced by Illumina GSIIx technology to compare drought treated and well- watered fertilized ovary (1-2DAP) and basal leaf meristem tissue. The pipeline also identified novel splice junctions - splice variants of previously known gene models and potential novel transcription units. An attempt was also made to exploit the data to understand the drought mediated transcriptional events (e.g. alternative splicing). Gene Ontology (GO) enrichment analysis revealed massive down-regulation of cell division and cell cycle genes in the drought stressed ovary only. Among GO categories related to carbohydrate metabolism, changes in starch and sucrose metabolism-related genes occurred in the ovary, consistent with a decrease in starch levels, and in sucrose transporter function, with no comparable changes occurring in the leaf meristem. ABA-related processes responded positively, but only in the ovaries. GO enrichment analysis also suggested differential responses to drought between the two tissues in categories such as oxidative stress-related and cell cycle events. The data are discussed in the context of the susceptibility of maize kernel to drought stress leading to embryo abortion, and the relative robustness of actively dividing vegetative tissue taken at the same time from the same plant subjected to the same conditions. A hypothesis is formulated, proposing drought-mediated intersecting effects on the expression of invertase genes, glucose signaling (hexokinase 1-dependent and independent), ABA-dependent and independent signaling, antioxidant responses, PCD, phospholipase C effects, and cell cycle related processes. This work was supported by the National Science Foundation Plant Genome Research Pro- gram (grant no. DBI0922747), iPlant Collaborative (NSF DBI-0735191) and also NSF ABI1062472. / Master of Science in Life Sciences

La connaissance de la diversité microbienne des milieux ultramafiques est essentielle pour établir le fonctionnement écologique de ces milieux, qui présentent de fortes teneurs en Ni et sont caractérisés par une flore particulière, e.g. plantes hyperaccumulatrices de Ni. La rhizosphère des hyperaccumulateurs comporte une forte proportion de bactéries résistantes au Ni, qui peuvent aussi agir sur la nutrition des plantes et sur les propriétés physico-chimiques du sol. Le premier défi de cette thèse a été de cerner le déterminisme de la diversité bactérienne de la rhizosphère d’hyperaccumulateurs de Ni. Le second a été de tester l'intérêt de souches PGPR (Plant Growth Promoting Rhizobacteria) pour optimiser l'agromine à partir d'interactions entre les rhizobactéries et les hyperaccumulateurs de Ni. La démarche s'est appuyée sur un ensemble de prospections dans deux régions climatiques et sur des analyses de séquençage haut débit. Des tests de cultures de plantes hyperaccumulatrices inoculées ont également été conduits. Les résultats montrent que le déterminisme de la diversité bactérienne est variable selon l'échelle spatiale. A l'échelle mondiale, le type de végétation est le facteur majeur structurant les communautés bactériennes, elle-même contrôlée indirectement par le climat. L’influence directe du climat (température et humidité) sur la diversité est significative mais moindre. A l'échelle d'une région climatique, la physico-chimie des sols ultramafiques structure et détermine la diversité des communautés bactériennes rhizosphériques. Enfin, l'inoculation de souches PGPR fortement bioaccumulatrices de Ni modifie la dynamique du Ni dans le sol, ce qui démontre qu'il existe une compétition pour le Ni entre la plante et la bactérie inoculée. En conclusion, le déterminisme de la diversité des communautés bactériennes rhizosphériques est dépendant de l'échelle spatiale considérée. En outre, le choix de la souche PGPR à inoculer, dans un contexte d'amélioration de l'agromine du Ni, est primordial. / Knowledge of the microbial diversity in ultramafic areas is essential to establish the ecological functioning of these environments, which display high level of Ni and are characterized by the presence of particular plants, e.g. Ni hyperaccumulators. The rhizosphere of these plants promotes a high proportion of Ni resistant bacteria that can act on plant nutrition and soil physicochemical properties. The first challenge of this thesis was to understand the bacterial rhizosphere diversity of Ni hyperaccumulators. The second was to test the interest of PGPR (Plant Growth Promoting Rhizobacteria) strains in order to improve agromining based on rhizobacteria and Ni hyperaccumulators interactions. The approach was based on two-contrasted climatic areas prospection and on high-throughput sequencing analyzes. Tests on culture of hyperaccumulator plants inoculated were also conducted. The results show that the determinism of this bacterial diversity is variable according to the spatial scale. On a global scale, the vegetation type, indirectly influenced by the climate, is the major factor structuring bacterial communities. The direct influence of the climate (temperature and humidity) on bacterial diversity is significant but lower. At the scale of a climatic region, the physic-chemistry of ultramafic soils structures and determines the rhizosphere bacterial community diversity. Finally, the inoculation of highly Ni bioaccumulative PGPR strains modifies the Ni dynamic in the soil, demonstrating that there is a competition for this metal between the inoculated bacteria and the hyperaccumulator plant. In conclusion, the rhizosphere bacterial community diversity is dependent on the considered spatial scale. Furthermore, these results emphasize how the choice of the PGPR strain to inoculate is important in order to improve Ni agromining.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-11-23T13:00:29Z No. of bitstreams: 1 JoelmaMPL_DISSERT.pdf: 1767206 bytes, checksum: c5b6652a65db20c661f3ad0f021c09fe (MD5) / Made available in DSpace on 2017-11-23T13:00:30Z (GMT). No. of bitstreams: 1 JoelmaMPL_DISSERT.pdf: 1767206 bytes, checksum: c5b6652a65db20c661f3ad0f021c09fe (MD5) Previous issue date: 2017-07-21 / The coalho cheese is considered a cultural patrimony and has great social and economic importance for the northeastern Brazilian region. The evaluation of the microbiological conditions of this product becomes fundamental for the affirmation of food safety and investigation of the real characteristics that make this kind of cheese so peculiar. The objective of the study was to identify the microbial community of coalho cheese. Metagenomic target DNA was extracted from eight samples of coalho cheese, four of which were made from pasteurized milk and four from raw milk. The DNA of the samples was sequenced by the next generation sequencing technology using the 16S and 18S rDNA gene as the basis for the identification of the organisms. Sequencing analyzes revealed several prokaryotic pathogenic microorganisms such as Rothia dentocariosa, Elizabethkingia meningoseptica and Bacillus cereus, as well as bacterial genera belonging to the microbiota previously described as Lactobacillus, Lactococcus, Escherichia, Enterococcus. Several fungi, such as Candida tropicalis, Candida parapsilolis, Pichia membranifaciens, Tritirachium oryzae, Malassezia furfur and Kluyveromyces marxianus were identified in the eukaryotic research, as well as microorganisms that had not yet been described in rennet cheeses such as Vibrio rumoienses, Ruminococcus flavefaciens, Piscicoccus intestinalis and Dekkera bruxellensis / O queijo de coalho é considerado um patrimônio cultural e tem grande importância social e econômica para a região nordeste brasileira, por isso a avaliação das condições microbiológicas desse produto torna-se fundamental para a afirmação da segurança alimentar e investigação das reais características que fazem com que esse tipo de queijo apresente sabores, texturas e aromas tão peculiares. A partir disso, o objetivo do trabalho foi identificar a comunidade microbiana do queijo de coalho. Para isso, o DNA metagenômico do queijo foi extraído de oito amostras de queijo de coalho, sendo quatro feitas a partir do leite pasteurizado e quatro feitas a partir do leite cru. O DNA das amostras foi sequenciado pela tecnologia de próxima geração da Illumina utilizando como base para a identificação dos organismos o gene rDNA 16S e 18S. As análises do sequenciamento revelaram vários exemplares de microrganismos patógenos procarióticos como Rothia dentocariosa, Elizabethkingia meningoseptica e Bacillus cereus, assim como gêneros de bactérias próprias da sua microbiota previamente descrita como Lactobacillus, Lactococcus, Escherichia, Enterococcus. Na investigação dos eucarióticos foram identificados diversos fungos como a Candida tropicalis, Candida parapsilolis, Pichia membranifaciens, Tritirachium oryzae, Malassezia furfur e Kluyveromyces marxianus, além dos microrganismos que ainda não tinham sido descritos em queijos coalho como o Vibrio rumoienses, Ruminococcus flavefaciens, Piscicoccus intestinalis e Dekkera bruxellensis / 2017-11-23

Célèbres depuis l'accident de Seveso en 1976, les dioxines/furanes (PCCD/F) restent, malgré une forte baisse de leurs émissions, un sujet de préoccupation permanent en France et dans le monde. Le rémanence de ces composés organochlorés dans le sol et le risque toxique qu'ils représentent pour l'homme et l'environnement font que la gestion et la remédiation des sols contaminés par les PCDD/F sont devenues une priorité des industriels, législateurs et scientifiques. La phytoremédiation compte parmi les méthodes émergentes de dépollution des sols contaminés en raison de son adéquation avec le développement durable. Elle combine les capacités naturelles des plantes et de leur microbiote rhizosphérique à biodégrader les polluants organiques. Cependant, l'efficacité de cette phytotechnologie est encore souvent limitée, en particulier lorsqu'il s'agit de composés chlorés, à cause de leur récalcitrance, de leur phytotoxicité et leur faible biodisponibilité dans le sol. Ainsi, l'objectif de ce travail de thèse a consisté à étudier les performances de la phytoremédition assistée, en particulier par les champignons mycorhiziens arbusculaires, d'un sol agricole historiquement pollué par les PCDD/F prélevé sur une parcelle expérimentale située à proximité d'un ancien incinérateur. L'ensemble des résultats obtenus mettent en évidence, en particulier, le potentiel de deux espèces végétales, la luzerne et la fétuque, dans la rhizodégradation des PCCDD/F. La végétalisation du sol permet de moduler les communautés microbiennes du sol (bactéries, Archées et champignons) et notamment celles qui semblent impliquées dans la dissipation des PCCDD/F. En revanche, bien que la mycorhization agisse sur les communautés microbiennes du sol, celle-ci n'a pas eu d'impact, dans nos conditions expérimentales, sur la dissipation des PCCDD/F quelles que soit l'origine de l'inoculum utilisé et les espèces mycorhiziennes qui le compose. La dégradation de ces composés organochlorés est plus marquée dans un sol préalablement stérilisé, puis recolonisé par certaines communautés microbiennes spécifiques, impliquées dans la dissipation des PCCDD/F. L'utilisation combinée d'un mélange de rhamnolipides avec l'introduction dans le sol d'une bactérie Sphingomonas wittichii RWI, décrite pour ses capacités de dégradation des PCCDD/F, permet d'accroitre l'efficience de la rhizodégradation des PCDD/F qui se traduit par une baisse significative de la cytotoxicité du sol après phytoremédiation. / Famous since the Seveso accident in 1976, dioxins/furans (PCCD/F) remain, despite a sharp decline in emissions, a subject of permanent concern in France and in the world. The remanence of these organochlored compounds in soil and the toxic risk they represent for humans and the environment mean that the management and remediation of PCDD/F contaminated soil has become a priority for industrialists, legislators and scientists. Phytoremediation is one of the emerging depollution methods of contaminated soils due to Its suitability for sustainable development. It combines the natural capacities of plants and their rhizospheric microbiota to biodegrade organic pollutants. However, the effectiveness of this phytotechnology is still often limited, especially in the case of chlorinated compounds, due to their recalcitrance, phytotoxicity and low bioavailability in the soil. Thus, the thesis aims to study the performance of assisted phytoremission, in particular by mycorrhizal fungi, of an agricultural PCDD / F based-polluted soil from an experimental plot near an old incinerator. The results obtained show the potential of two plant species, alfalfa and tall fescue, in the rhizodegradation of PCCDD/F. Soil vegetation shows to modulate soil microbial communities (bacteria, archaea and fungi) includind those that appear to involved in the dissipation of the PCCDD/F. On the other hand, although mycorrhization affects soil microbial communities, it has not had any impact on the dissipation of PCCDD/F in our experimental conditions, whatever the inoculums origin and the mycorrhizal species which compose it. The degradation of these organochlorined compounds is more significant in a previously sterilized soil and then recolonized by specific microbial communities involved in the PCCDD/Fdissipation. The combined use of rhamnolipids mixture and Sphingomonas wittichii RWI bactrerium described for its degradation capabilities of PCCDD/F makes it possible to increase the efficiency of PCDD/F rhizodegradation which results in a significant decrease in soil cytotoxicity after phytoremediation.

Cystic fibrosis (CF) is genetically conditioned, autosomal recessive disease that occurs in the European population with a prevalence of about 1:2500 - 1:1800. In this disease we observe a mutation of the CTFR gene with subsequent fault in chloride channels. Such afflicted individuals usually suffer from chronic respiratory problems, pancreatic insufficiency, high concentration of chloride ions in sweat and obstructive azoospermia. Genetic testing of CFTR gene is indicated in individuals who meet the CF clinical picture and a positive sweat test (increased concentration of chlorides in the sweat). Genetic testing of the CFTR gene is usually done by using commercial kits detecting the most common mutations of the CFTR gene in the Czech Republic. If the testing results are negative, it is further performed an MLPA method that captures the larger deletions and duplications of gene, eventually a sequencing of all exons is. Despite the well-established algorithm of the testing, some patients suffering from symptoms of CF are left without genetic findings. Thanks to development of next generation sequencing, it is possible to make the diagnosis of CF more effective and uncover the variants that were not captured by previous methods.