CHARACTERIZATION OF THE ENVIRONMENTAL RESISTOME IN THE GALAPAGOS ISLANDS, ECUADOR: A ONE HEALTH PERSPECTIVE

Antibiotic resistance represents one of our generation’s most pressing public health challenges, with some exports warning of an approaching post-antibiotic era. Mitigating this threat requires an understanding of the evolutionary ecology of resistance, including the unique ability of microorganisms to move between humans, animals, and the environment. However, significant questions remain regarding the role of the environment as a source and reservoir for antibiotic resistance. Moreover, there few environments left on earth where we can study background antibiotic resistance in the absence of significant anthropogenic influence. The Galapagos Islands of Ecuador, where the human population is restricted to 3% of the landmass, represent a unique model system to study how human activity influences antibiotic resistance patterns in wildlife and the environment in a largely protected ecosystem. With samples from humans, animals, and the environment, we designed a One Health study aimed at answering what, where, and who: what antibiotic resistance genes are present, where are they located in regards to mobile genetic elements, and who may be the presumptive bacterial host? We employed shotgun metagenomic sequencing to achieve a broad characterization of 90 environmental, wildlife, and human resistomes and mobilomes, and paired this data with targeted detection of the class I integron-integrase gene using a novel ddPCR assay in > 250 Galapagos samples. Additionally, we used a combination of 16S rRNA amplicon sequencing and taxonomic inference from metagenomes to profile the microbial communities associated with these samples. Our results suggest that human, environmental, and wildlife reservoirs are characterized by distinct resistomes and mobilomes, with overall abundance and diversity of antibiotic resistance genes (ARGs) increasing along a gradient of anthropogenic influence. Overall, we found wildlife to harbor fewer ARGs than wastewater and humans, though some exceptions were noted among land iguanas. Differential abundance analysis revealed ARGs unique to each wildlife species with possible bacterial hosts identified in taxonomic assignments in some cases. We recorded overall agreement between resistome and mobilome data sets, and correlation between taxa, ARGs, and MGEs pointed to a key relationship with Enterobacteriaceae.Doctor of Philosoph

The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues

<p>Abstract</p> <p>Background</p> <p>Expansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation.</p> <p>Results</p> <p>Here, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly explain the observed expression pattern similarities.</p> <p>Conclusions</p> <p>The ZNF genes at human 8q24.3 form a relatively old mammalian paralog group conserved in eutherian mammals for at least 130 million years. The members persisted after initial duplications by undergoing subfunctionalizations in their expression patterns and target site recognition. KRAB-ZNF mediated repression of transcription might have shaped organogenesis in mammalian ontogeny.</p

Life cycle studies of the red tide dinoflagellate species complex Alexandrium tamarense

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2011Blooms of toxic species within the algal dinoflagellate species complex Alexandrium tamarense may cause Paralytic Shellfish Poisoning, a significant and growing environmental threat worldwide. However, blooms of closely related nontoxic A. tamarense also occur, sometimes in close geographical proximity to toxic blooms. This thesis explores the interactions between toxic and nontoxic blooms by examining sexual crosses between each of five ribosomal clades within the A. tamarense complex (termed Groups I-V). Several lines of evidence argue that these clades represent separate species. Particular emphasis was given to interactions between toxic Group I and nontoxic Group III species because they are among the most closely related A. tamarense clades and because they share a natural range boundary in several parts of the world. Interspecies hybridization appeared widespread between different clades and between geographically dispersed isolates. However, subsequent germination studies of hypnozygotes produced from combinations of Group I and Group III clones failed to yield new vegetative cultures in multiple trials. The possibility that these hypnozygotes were actually inbred (i.e. the result of pairs of only Group I or only Group III gametes) was considered and rejected using a nested PCR assay that was developed to assess the parentage of individual cysts. The assay was also suitable for analyzing cysts collected from the field and was applied to individual cysts collected from Belfast Lough, an area where both Group I and Group III blooms were known to occur. Two Group I/Group III hybrids were detected in fourteen successful assays from the Belfast sample, showing that hybridization does occur in nature. These findings have several important implications. First, the failure of Group I/Group III hybrids to produce new vegetative cultures serves as a proof that the A. tamarense clades represent cryptic species because they are unable to produce genetic intermediates. Second, the presence of hybrid cysts in Belfast Lough indicates ongoing displacement of a nontoxic population by a toxic one (or vice versa) in that region. Third, the inviability of toxic/nontoxic hybrids suggests a remediation strategy whereby the recurrence of toxic A. tamarense blooms might be combated through the introduction of nontoxic cells.research support from NSF (grants OCE-0402707 and OCE- 9808173 awarded to Don Anderson), the Woods Hole Center for Oceans and Human Health (NSF Grant no. OCE-0430724 and NIEHS Grant no. P50ES012742-0), an EPA STAR graduate fellowship (FP-91688601), and the Coastal Ocean Institute at WHOI. I’ve also benefitted greatly from conference travel funds provided by the NOAA Center for Sponsored Coastal Ocean Research, the MIT Student Assistance Fund, and the Academic Programs Office at WHOI

One health toxicology: expanding perspectives and methods to assess environmental contaminants

Thesis (Ph.D.) University of Alaska Fairbanks, 2017The discipline of One Health is founded on the principal that environmental health, animal health, and human health are interconnected. Although the field has been largely focused on zoonotic diseases, examining concepts such as toxicology under a One Health lens can offer a more holistic and preventative approach to research and implementation and, in particular, how fish-based diets may be involved with One Health outcomes. Here we present three general case studies that demonstrate new approaches to investigating One Health toxicology. In Chapter One we show how Arctic canids can be used as environmental sentinels for human health. We discuss three separate canid studies; in the first we find that Arctic foxes can act as sentinels of Arctic contaminants due to their foraging plasticity, in the second we examine the use of fish-fed sled dogs as a model for the effects of a fish-based diet on contaminants exposure and gene transcription, and in the third we develop the sled dog as a model for particulate matter air pollution in the Fairbanks North Star Borough. In Chapter Two we utilize the Steller sea lion, a nonmodel organism, as a sentinel for the effects of fish-based diet mercury exposure induced whole-genome changes in gene transcription (RNA-Seq). Using newly developed informatics tools we assemble a de novo transcriptome and examine large scale changes in gene expression related to mercury exposure and other One Health uses. This approach is extremely adaptable and has the potential to be applied across numerous non-model organisms and contaminants. We also applied a microbial mining algorithm to our RNA-Seq data and found evidence for a hemotropic Mycoplasma spp. in one of our samples. In Chapter Three we examine sources of mercury exposure for pregnant women from La Paz, Baja California Sur, Mexico. We found mercury concentrations to be generally low among the examined fish species and staple foods. While typical dietary assessments rely on recall surveys and questionnaires, we found that examining chemical biomarkers of diet including stable isotopes of carbon and nitrogen are critical in dietary risk assessment. Taken together these three investigations offer valuable lessons and techniques which can be applied to the field of One Health toxicology; especially to those fish diet based systems.Chapter 1 - One Health Toxicology - Review of concepts, tools and scope of an emerging discipline -- Chapter 2 - Using domestic and free ranging Arctic canid models for environmental molecular toxicology research -- Chapter 3 -Novel applications of next generation sequencing tools to assess the health of Steller sea lion (Eumetopias jubatus) populations -- Chapter 4 - Using carbon and nitrogen stable isotope modeling to assess mercury exposure for pregnant women in Baja California Sur, Mexico -- Chapter 5 - What does One Health want? -- Chapter 6 (Appendix) - Validation of an acidic digestion method for the determination of methylmercury in hair samples

Identifying Regulators from Multiple Types of Biological Data in Cancer

Cancer genomes accumulate alterations that promote cancer cell proliferation and survival. Structural, genetic and epigenetic alterations that have a selective advantage for tumorigenesis affect key regulatory genes and microRNAs that in turn regulate the expression of many target genes. The goal of this dissertation is to leverage the alteration-rich landscape of cancer genomes to detect key regulatory genes and microRNAs. To this end, we designed a feature selection algorithm to identify DNA methylation signals around a gene that would highly predict its expression. We found that genes whose expression could be predicted by DNA methylation accurately were enriched in Gene Ontology terms related to the regulation of various biological processes. This suggests that genes controlled by DNA methylation are regulatory genes. We also developed two tools that infer relationships between regulatory genes and target genes leveraging structural and epigenetic data. The first tool, ProcessDriver integrates copy number alteration and gene expression datasets to identify copy number cancer driver genes, target genes of these drivers and the disrupted biological processes. Our results showed that driver genes selected by ProcessDriver are enriched in known cancer genes. Using survival analysis, we showed that drivers are linked to new tumor events after initial treatment. The second tool was developed to leverage structural and epigenetic data to infer interactions between regulatory genes and targets on a network-level. Our canonical correlation analysis-based approach utilized the DNA methylation or copy number states of potential regulators and the expression states of potential targets to score regulatory interactions. We then incorporated these regulatory interaction scores as prior knowledge in a dynamic Bayesian framework utilizing time series gene expression data. Our results indicated that the canonical correlation analysis-based scores reflect the true interactions between genes with high accuracy, and the accuracy can be further increased by using the scores as a prior in the dynamic Bayesian framework. Finally, we are developing an algorithm to detect cancer-related microRNAs, associated targets and disrupted biological processes. Our preliminary results suggest that the modules of miRNAs and target genes identified in this approach are enriched in known microRNA-gene interactions

Diversity, specificity and evolutionary history of marine invertebrate symbioses and functions of the sulfur-oxidizing symbionts

Many marine invertebrates have established symbioses with chemosynthetic bacteria that metabolize reduced sulfur compounds and provide nutrition to their host. Two key questions in the field of symbiosis are: (1) How specific and evolutionarily stable are these symbioses? Chapters II, III and IV of this thesis contribute to a more comprehensive understanding of this question by investigating the diversity, specificity and evolutionary history of three sulfur-oxidizing symbioses: deep-sea vestimentiferan tubeworm endosymbioses, shallow water gutless phallodriline oligochaete endo- and stilbonematine nematode ectosymbioses. The studies emphasize the power of molecular analyses to uncover hidden symbiont diversity and highlight the remarkably stable and specific stilbonematine ectosymbioses. (2) What are the benefits for the symbiotic partners? Chapter V strengthens the hypothesis of stilbonematine ectosymbionts' nutritional role for their host and the genomic study in Chapter VI discusses potential additional functions of the ectosymbionts for their nematode host

Glassy dynamics of kinetically constrained models

We review the use of kinetically constrained models (KCMs) for the study of dynamics in glassy systems. The characteristic feature of KCMs is that they have trivial, often non-interacting, equilibrium behaviour but interesting slow dynamics due to restrictions on the allowed transitions between configurations. The basic question which KCMs ask is therefore how much glassy physics can be understood without an underlying ``equilibrium glass transition''. After a brief review of glassy phenomenology, we describe the main model classes, which include spin-facilitated (Ising) models, constrained lattice gases, models inspired by cellular structures such as soap froths, models obtained via mappings from interacting systems without constraints, and finally related models such as urn, oscillator, tiling and needle models. We then describe the broad range of techniques that have been applied to KCMs, including exact solutions, adiabatic approximations, projection and mode-coupling techniques, diagrammatic approaches and mappings to quantum systems or effective models. Finally, we give a survey of the known results for the dynamics of KCMs both in and out of equilibrium, including topics such as relaxation time divergences and dynamical transitions, nonlinear relaxation, aging and effective temperatures, cooperativity and dynamical heterogeneities, and finally non-equilibrium stationary states generated by external driving. We conclude with a discussion of open questions and possibilities for future work.Comment: 137 pages. Additions to section on dynamical heterogeneities (5.5, new pages 110 and 112), otherwise minor corrections, additions and reference updates. Version to be published in Advances in Physic

Patterns in richness and community structure: From bacteria to apex predators

Patterns of community structure and richness provide context for studies from microbial ecology, global macroecology, languages, to Bayesian statistics. Diversity patterns for animals on land and their predictor variables are well studied. However, diversity patterns for bacterial communities and marine macroorganisms are not well studied or understood. Here I examine diversity patterns in caves, on the external surface of Chiroptera (bats), and in marine ecosystems. At the local to regional scale we investigate factors that drive bacterial community patterns in richness and composition in lava cave microbial mats and microbes on bats. Lastly, out of the cave and into the surface world, a global picture emerges of factors that drive community structure and richness from bacteria to apex predators in marine environments. I hypothesize that for cave microbial mats found in lava caves, local factors (i.e. sample site temperature and relative humidity) are important factors for determining community structure and richness. For bacteria on bats, a mix of local factors (bat species, bat body mass, location of capture) and regional factors (net primary productivity (NPP), annual mean rainfall) explain richness and structure of the microbial communities. In addition, the predictor variables for richness and community structure will vary with spatial scale (local to regional to landscape). In the global marine data set, richness and community structure will be dependent on net primary productivity, temperature, thermal lifestyle, and foraging behavior. At small scales, temperature and NPP will be variable in their predicting power, while at large scales they will be positivity correlated with species richness. Local factors likely drive the larger scale patterns in community structure and richness