Molecular evidence for sediment nitrogen fixation in a temperate New England estuary

Primary production in coastal waters is generally nitrogen (N) limited with denitrification outpacing nitrogen fixation (N2-fixation). However, recent work suggests that we have potentially underestimated the importance of heterotrophic sediment N2-fixation in marine ecosystems. We used clone libraries to examine transcript diversity of nifH (a gene associated with N2-fixation) in sediments at three sites in a temperate New England estuary (Waquoit Bay, Massachusetts, USA) and compared our results to net sediment N2 fluxes previously measured at these sites. We observed nifH expression at all sites, including a site heavily impacted by anthropogenic N. At this N impacted site, we also observed mean net sediment N2-fixation, linking the geochemical rate measurement with nifH expression. This same site also had the lowest diversity (non-parametric Shannon = 2.75). At the two other sites, we also detected nifH transcripts, however, the mean N2 flux indicated net denitrification. These results suggest that N2-fixation and denitrification co-occur in these sediments. Of the unique sequences in this study, 67% were most closely related to uncultured bacteria from various marine environments, 17% to Cluster III, 15% to Cluster I, and only 1% to Cluster II. These data add to the growing body of literature that sediment heterotrophic N2-fixation, even under high inorganic nitrogen concentrations, may be an important yet overlooked source of N in coastal systems

DEVELOPMENT AND APPLICATION OF MASS SPECTROMETRY-BASED PROTEOMICS TO GENERATE AND NAVIGATE THE PROTEOMES OF THE GENUS POPULUS

Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as plants or humans, remains challenging because of the incongruity between the complexity of the biological samples and the analytical techniques available. In this dissertation, analytical methods utilizing state-of-the-art MS instrumentation have been developed to address challenges associated with both qualitative and quantitative characterization of eukaryotic organisms. In particular, these efforts focus on characterizing Populus, a model organism and potential feedstock for bioenergy. The effectiveness of pre-existing MS techniques, initially developed to identify proteins reliably in microbial proteomes, were tested to define the boundaries and characterize the landscape of functional genome expression in Populus. Although these approaches were generally successful, achieving maximal proteome coverage was still limited by a number of factors, including genome complexity, the dynamic range of protein identification, and the abundance of protein variants. To overcome these challenges, improvements were needed in sample preparation, MS instrumentation, and bioinformatics. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: 1) network-wide, 2) pathway-specific, and 3) protein-level viewpoints. In addition, we implemented two bioinformatic approaches that were capable of decoding the plasticity of the Populus proteome, facilitating the identification of single amino acid polymorphisms and generating a more accurate profile of protein expression. Though the methods and results presented in this dissertation have direct implications in the study of bioenergy research, more broadly this dissertation focuses on developing techniques to contend with the notorious challenges associated with protein characterization in all eukaryotic organisms

The FIELDS Instrument Suite for Solar Probe Plus: Measuring the Coronal Plasma and Magnetic Field, Plasma Waves and Turbulence, and Radio Signatures of Solar Transients.

NASA's Solar Probe Plus (SPP) mission will make the first in situ measurements of the solar corona and the birthplace of the solar wind. The FIELDS instrument suite on SPP will make direct measurements of electric and magnetic fields, the properties of in situ plasma waves, electron density and temperature profiles, and interplanetary radio emissions, amongst other things. Here, we describe the scientific objectives targeted by the SPP/FIELDS instrument, the instrument design itself, and the instrument concept of operations and planned data products

GABA-modulating bacteria of the human gut microbiota.

The gut microbiota affects many important host functions, including the immune response and the nervous system1. However, while substantial progress has been made in growing diverse microorganisms of the microbiota2, 23-65% of species residing in the human gut remain uncultured3,4, which is an obstacle for understanding their biological roles. A likely reason for this unculturability is the absence in artificial media of key growth factors that are provided by neighbouring bacteria in situ5,6. In the present study, we used co-culture to isolate KLE1738, which required the presence of Bacteroides fragilis to grow. Bioassay-driven purification of B. fragilis supernatant led to the isolation of the growth factor, which, surprisingly, is the major inhibitory neurotransmitter GABA (γ-aminobutyric acid). GABA was the only tested nutrient that supported the growth of KLE1738, and a genome analysis supported a GABA-dependent metabolism mechanism. Using growth of KLE1738 as an indicator, we isolated a variety of GABA-producing bacteria, and found that Bacteroides ssp. produced large quantities of GABA. Genome-based metabolic modelling of the human gut microbiota revealed multiple genera with the predicted capability to produce or consume GABA. A transcriptome analysis of human stool samples from healthy individuals showed that GABA-producing pathways are actively expressed by Bacteroides, Parabacteroides and Escherichia species. By coupling 16S ribosmal RNA sequencing with functional magentic resonance imaging in patients with major depressive disorder, a disease associated with an altered GABA-mediated response, we found that the relative abundance levels of faecal Bacteroides are negatively correlated with brain signatures associated with depression

Research and Development of a General Purpose Instrument DAQ-Monitoring Platform applied to the CLOUD/CERN experiment

The current scientific environment has experimentalists and system administrators allocating large amounts of time for data access, parsing and gathering as well as instrument management. This is a growing challenge since there is an increasing number of large collaborations with significant amount of instrument resources, remote instrumentation sites and continuously improved and upgraded scientific instruments. DAQBroker is a new software designed to monitor networks of scientific instruments while also providing simple data access methods for any user. Data can be stored in one or several local or remote databases running on any of the most popular relational databases (MySQL, PostgreSQL, Oracle). It also provides the necessary tools for creating and editing the metadata associated with different instruments, perform data manipulation and generate events based on instrument measurements, regardless of the user’s know-how of individual instruments. Time series stored in a DAQBroker database also benefit from several statistical methods for time series classification, comparison and event detection as well as multivariate time series analysis methods to determine the most statistically relevant time series, rank the most influential time series and also determine the periods of most activity during specific experimental periods. This thesis presents the architecture behind the framework, assesses the performance under controlled conditions and presents a use-case under the CLOUD experiment at CERN, Switzerland. The univariate and multivariate time series statistical methods applied to this framework are also studied.O processo de investigação científica moderno requer que tanto experimentalistas como administradores de sistemas dediquem uma parte significativa do seu tempo a criar estratégias para aceder, armazenar e manipular instrumentos científicos e os dados que estes produzem. Este é um desafio crescente considerando o aumento de colaborações que necessitam de vários instrumentos, investigação em áreas remotas e instrumentos científicos com constantes alterações. O DAQBroker é uma nova plataforma desenhada para a monitorização de instrumentos científicos e ao mesmo tempo fornece métodos simples para qualquer utilizador aceder aos seus dados. Os dados podem ser guardados em uma ou várias bases de dados locais ou remotas utilizando os gestores de bases de dados mais comuns (MySQL, PostgreSQL, Oracle). Esta plataforma também fornece as ferramentas necessárias para criar e editar versões virtuais de instrumentos científicos e manipular os dados recolhidos dos instrumentos, independentemente do grau de conhecimento que o utilizador tenha com o(s) instrumento(s) utilizado(s). Séries temporais guardadas numa base de dados DAQBroker beneficiam de um conjunto de métodos estatísticos para a classificação, comparação e detecção de eventos, determinação das séries com maior influência e os sub-períodos experimentais com maior actividade. Esta tese apresenta a arquitectura da plataforma, os resultados de diversos testes de esforço efectuados em ambientes controlados e um caso real da sua utilização na experiência CLOUD, no CERN, Suíça. São estudados também os métodos de análise de séries temporais, tanto singulares como multivariadas aplicados na plataforma