Background: Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity. Results: Muscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 +/- 0.7 kg/m(2)) and obese (n = 10; BMI = 32.9 +/- 0.7 kg/m(2)) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P < 0.05) that were altered in the promoter and untranslated (5' and 3'UTR) regions in the obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P < 0.05) altered. Of these, 12 genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change -1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5' UTR (Chr. 8: 22,423,530-22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity. Conclusions: These results demonstrate that obesity alters the epigenome through DNA methylation and highlights novel transcriptomic changes in SORBS3 in skeletal muscle.

The last decade has seen considerable research focusing on understanding the factors underlying schizophrenia and bipolar disorder. A major challenge encountered in studying these disorders, however, has been the contribution of genetic, or etiological, heterogeneity to the so-called “missing heritability” [1-6]. Further, recent successes of large-scale genome-wide association studies (GWAS) have nonetheless seen only limited advancements in the delineation of the specific roles of implicated genes in disease pathophysiology. The study of microRNAs (miRNAs), given their ability to alter the transcription of hundreds of targeted genes, has the potential to expand our understanding of how certain genes relate to schizophrenia and bipolar disorder. Indeed, the strongest finding of one recent mega-analysis by the Psychiatric GWAS consortium (PGC) was for a miRNA, though little can be said presently about its particular role in the etiologies of schizophrenia and bipolar disorder [52]. Next generation sequencing (NGS) is a versatile technology that can be used to directly sequence either DNA or RNA, thus providing valuable information on variation in the genome and in the transcriptome. A variation of NGS, MicroSeq, focuses on small RNAs and can be used to detect novel, as well as known, miRNAs [26,125, 126]. The following thesis describes the role of miRNAs in schizophrenia and bipolar disorder in various experimental settings. As an index of the interaction between multiple genes and between the genome and the environment, miRNAs are great potential biomarkers for complex disorders such as schizophrenia and bipolar disorder.

An exciting new era has dawned for the prevention and management of CAD utilizing genetic risk variants. The recent identification of over 60 susceptibility loci for coronary artery disease (CAD) confirm not only the importance of established risk factors, but also the existence of many novel causal pathways that are expected to improve our understanding of the genetic basis of CAD and facilitate the development of new therapeutic agents over time. Concurrently, Mendelian randomization studies have provided intriguing insights on the causal relationship between CAD-related traits, and highlight the potential benefits of long-term modifications of risk factors. Lastly, genetic risk scores of CAD may serve not only as prognostic, but also as predictive markers and carry the potential to considerably improve the delivery of established prevention strategies. This review will summarize the evolution and discovery of genetic risk variants for CAD and their current and future clinical applications.

Thesis advisor: Gabor T. Marth / Partitioning an individual's phenotype into genetic and environmental components has been a major goal of genetics since the early 20th century. Formally, the proportion of phenotypic variance attributable to genetic variation in the population is known as heritability. Genome wide association studies have explained a modest percentage of variability of complex traits by genotyping common variants. Currently, there is great interest in what role rare variants play in explaining the missing heritability of complex traits. Advances of next generation sequencing and genomic enrichment technologies over the past several years have made it feasible to re-sequence large numbers of individuals, enabling the discovery of the full spectrum of genetic variation segregating in the human population, including rare variants. The four projects that comprise my dissertation all revolve around the discovery of rare variants from next generation sequencing datasets. In my first project, I analyzed data from the exon sequencing pilot of the 1000 Genomes Project, where I discovered variants from exome capture sequencing experiments in a worldwide sample of nearly 700 individuals. My results show that the allele frequency spectrum of the dataset has an excess of rare variants. My next project demonstrated the applicability of using whole-genome amplified DNA (WGA) in capture sequencing. WGA is a method that amplifies DNA from nanogram starting amounts of template. In two separate capture experiments I compared the concordance of call sets, both at the site and genotype level, of variant calls derived from WGA and genomic DNA. WGA derived calls have excellent concordance metrics, both at the site and genotypic level, suggesting that WGA DNA can be used in lieu of genomic DNA. The results of this study have ramifications for medical sequencing experiments, where DNA stocks are a finite quantity and re-collecting samples maybe too expensive or not possible. My third project kept its focus on capture sequencing, but in a different context. Here, I analyzed sequencing data from Mendelian exome study of non-sensorineural hearing loss (NSHL). A subset of 6 individuals (5 affected, 1 unaffected) from a family of European descent were whole exome sequenced in an attempt to uncover the causative mutation responsible for the loss of hearing phenotype in the family. Previous linkage analysis uncovered a linkage region on chr12, but no mutations in previous candidate genes were found, suggesting a novel mutation segregates in the family. Using a discrete filtering approach with a minor allele frequency cutoff, I uncovered a putative causative non-synonymous mutation in a gene that encodes a transmembrane protein. The variant perfectly segregates with the phenotype in the family and is enriched in frequency in an unrelated cohort of individuals. Finally, for my last project I implemented a variant calling method for family sequencing datasets, named Pgmsnp, which incorporates Mendelian relationships of family members using a Bayesian network inference algorithm. My method has similar detection sensitivities compared to other pedigree aware callers, and increases power of detection for non-founder individuals. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Existem vários sistemas de classificação para Annonaceae. No entanto, estudos recentes sugerem que esse grande número de classificações pode ser reduzido a um. O sistema elaborado por Fries (1959), por exemplo, apresenta vários grupos apoiados por revisões modernas. Um deles é o Grupo Trigynaea, sinonimizado como tribo Bocageeae Endlicher (Johnson e Murray, 1995). A análise cladística com dados morfológicos sugeriu uma hipótese sobre as relações intergenéricas na tribo (Johnson e Murray, 1995). A confiabilidade dessas relações foi verificada com o uso de dados fitoquímicos e sequências de DNA. Em ((Bocagea, Hornschuchia) (Trigynaea)), o clado (Bocagea, Hornschuchia, Trigynaea) foi detectado na análise Bayesiana do RPB2 e apoiado pela presença de apigenia (exceto em Bocagea) e síntese de quercetina via dihidrocampferol. No clado (((Cardiopetalum, Cymbopetalum) (Porcelia)) (Froesiodendron)) a relação (Cardiopetalum, Cymbopetalum, Porcelia) foi detectado nas análises de Máxima Parcimônia das regiões coxI, RPB2 e trnK-matK e nas análises Bayesianas das regiões coxI, trnL-trnL-F e trnK-matK. Esse clado foi apoiado pela presença de luteolina (exceto em Cardiopetalum e Porcelia) e síntese de quercetina pelas vias di-hidrocampferol e eriodictiol (exceto em Cymbopetalum que produziu luteolina). A presença, até o momento exclusiva em Annonaceae, de alcaloides pirrolizidínicos em Bocageeae e Annona foi congruente com as informações sobre a estreita relação desses grupos. A distribuição dos componentes das ceras delimitou grupos químicos em Bocageeae que não foram congruentes com as relações estabelecidas com os dados morfológicos. / There are several classification systems for Annonaceae. However, recent studies have suggested this high number of classifications can be reduced to one. The system of Fries (1959), for example, presents many groups supported by modern revisions. One of them is Trigynaea-Gruppe, synonym of tribe Bocageeae Endlicher (Johnson and Murray, 1995). The morphological cladistic analysis suggested one hypothesis of intergeneric relationships among the Bocageeae (Johnson and Murray, 1995). The consistency of these relationships was evaluated by the use of phytochemical data and DNA sequences. Within ((Bocagea, Hornschuchia) (Trigynaea)), the (Bocagea, Hornschuchia, Trigynaea) was detected in the Bayesian analysis of RPB2 and supported by presence of apigenin (except in Bocagea) and synthesis of quercetin via dihydrokaempferol. In the clade (((Cardiopetalum, Cymbopetalum) (Porcelia)) (Froesiodendron)), the relationship (Cardiopetalum, Cymbopetalum, Porcelia) was detected in the Maximum Parsimony analyses of coxI, RPB2 and trnK-matK regions, and Bayesian analyses of coxI, trnL-trnL-F e trnK-matK regions. This clade was supported by occurrence of luteolin (except in Cardiopetalum and Porcelia) and synthesis of quercetin via both dihydrokaempferol and eriodictyol (except in Cymbopetalum that produced luteolin). The presence of pyrrolizidine alkaloids in Bocageeae and Annona, unique in Annonaceae until now, was in agreement with information about the closest relationship of these two groups. The distribution of cuticular waxes components circumscribed chemical groups in Bocageeae that were not congruent with the relationships based in morphological data

The earthworm has long been of interest to biologists, most notably Charles Darwin, who was the first to reveal their true role as eco-engineers of the soil. However, to fully understand an animal one needs to combine observational data with the fundamental building blocks of life, DNA. For many years, sequencing a genome was an incredibly costly and time-consuming process. Recent advances in sequencing technology have led to high quality, high throughput data being available at low cost. Although this provides large amounts of sequence data, the bioinformatics knowledge required to assemble and annotate these new data are still in their infancy. This bottleneck is slowly opening up, and with it come the first glimpses into the new and exciting biology of many new species. This thesis provides the first high quality draft genome assembly and annotation of an earthworm, Lumbricus rubellus. The assembly process and resulting data highlight the complexity of assembling a eukaryotic genome using short read data. To improve assembly, a novel approach was created utilising transcripts to scaffold the genome (https://github.com/elswob/SCUBAT). The annotation of the assembly provides the draft of the complete proteome, which is also supported by the first RNA-Seq generated transcriptome. These annotations have enabled detailed analysis of the protein coding genes including comparative analysis with two other annelids (a leech and a polychaete worm) and a symbiont (Verminephrobacter). This analysis identified four key areas which appear to be either highly enhanced or unique to L. rubellus. Three of these may be related to the unique environment from which the sequenced worms originated and add to the mounting evidence for the use of earthworms as bioindicators of soil quality. All data is stored in relational databases and available to search and browse via a website at www.earthworms.org. It is hoped that this genome will provide a springboard for many future investigations into the earthworm and continue research into this wonderful animal.

Large-scale genomics projects such as the Cancer Genome Atlas (TCGA), and the Encyclopedia of DNA Elements (ENCODE) involve generation of data at an unprecedented scale, requiring new computational techniques for analysis and interpretation. In the three studies I present in this thesis, I utilize these data sources to derive biological insights or created visualization tools that enable others to obtain insights more easily. First, I examine the distribution of the lengths for copy number variations (CNVs) in the cancer genome. This analysis shows that a small number of genes are altered at a greater frequency than expected from a power law distribution, suggesting that a large number of genomes must be sequenced for a given tumor type to a comprehensive discovery of somatic mutations. Second, I investigate germline CNVs in thousands of TCGA samples using single nucleotide polymorphism (SNP) array data to find variants that may confer increased susceptibility to cancer. This CNV-based genome-wide association study resulted in many germline CNVs that potentially increase risk in brain, breast, colorectal, renal, or ovarian cancers. Finally, I apply several visualization techniques to create tools for the TCGA and ENCODE projects in order to help investigators better process and synthesize meaning from large volume of data. Seqeyes combines linear and circular genomic views to explore predicted structural variations to help guide experimental validation. The modEncode browser visualizes chromatin organization by integrating data from a multitude of histone marks and chromosomal proteins. These results present visualization as a useful strategy for rapid identification of salient genomic features from large, heterogeneous genomic datasets.

This thesis presents an investigation into the transcriptional regulation of TaxolTM biosynthsis in Taxus cuspidata cell suspension cultures. The potent anticancer drug TaxolTM has been shown to be successful in the treatment of breast, lung and ovarian cancer and the acquired immunodeficiency syndrome (AIDS) related Kaposi’s sarcoma. Produced by all species of yew, TaxolTM belongs to the class of taxane diterpenoids and is of huge pharmaceutical importance. The plant material utilised in this thesis is a cell suspension culture initiated from isolated procambium cells of T. cuspidata. The latter is a meristematic tissue giving rise to the conductive tissue of plants. This un-differentiated cell suspension culture exhibits an increased and stable production of TaxolTM in response to the plant hormone elicitor methyljasmonate, limited cell aggregation and fast growth when compared to a cell suspension culture initiated from differentiated cells (somatic) of T. cuspidata. In order to assess the stem cell characteristics of the employed procambium cell suspension culture, the transcriptome of T. cuspidata was sequenced utilising Roche/ 454 and Illumina/ Solexa NlaIII tag sequencing technoloxiv gies. Statistical analysis uncovered differential expression profiles of 563 genes present within the procambium cell derived transcriptome by comparison with the somatic cell derived transcriptome. Gene ontology analysis of the latter identified that genes associated with response to stress and defence response were upregulated in the differentially expressed portion within the procambium cell suspension culture. This is consistent with the characteristics of animal stem cells which exhibit robust defence strategies to environmental stress. Furthermore PHLOEM INTERCALATED WITH XYLEM (PXY ) and TRACHEARY ELEMENT DIFFERENTIATION 2 (TED2), which are essential for ordered procambium cell division and differentiation into trachaery elements respectively in A. thaliana and Z. elegans, are up-regulated in the T. cuspidata procambium cell suspension culture. Further T. cuspidata homologues of the jasmonate signalling components JASMONATE ZINC FINGER LIKE ZIM DOMAIN 2 (JAZ2) and JAZ3 were identified among up-regulated transcripts in response to jasmonate treatment in both the procambium and the somatic cell line. Blast analysis identified 211 transcription factors within the APETELA 2 (AP2), BASIC-HELIX-LOOPHELIX (bHLH), WRKY, MYB and BASIC-LEUCIN-ZIPPER (bZIP) families. Further characterisation established 21 transcription factors which are significantly up-regulated in response to jasmonate treatment and show a higher expression level in procambium cells. These provide promising targets for further functional characterisation to elucidate their involvement within TaxolTM biosynthesis. In order to investigate transcriptional regulation of the TaxolTM structural genes, a 513 bp fragment corresponding to the TAXADIENE SYNTHASE (TASY ) promoter was cloned by genome walking. In-silico analysis of the TASY and 3’-N-DEBENZOYLTAXOL N-BENZOYLTRANSFERASE (DBTNBT) promoter resulted in the identification of methyljasmonate and pathogen-responsive elements which may significantly contribute to jasmonate mediated accumulation of TaxolTM. Analysis of a chimeric promoter construct driving the reporter gene β-GLUCURONIDASE (GUS) in N. benthamiana confirmed jasmonate-responsiveness of the TASY promoter. Finally, comparison of the expression level of genes coding for potentially rate-limiting enzymes within the TaxolTM pathway established a significantly increased expression of BACCATIN II PHENYLPROPANOYLTRANSFERASE (BAPT) in response to jasmonate treatment within the procambium cell suspension culture. Furthermore transcripts of TASY, PHENYLALANINE AMINOMUTASE (PAM) and DBTNBT show an overall higher expression and prolonged transcript accumulation in procambium compared to somatic cells. In this thesis jasmonate-signalling components, jasmonate-responsive transcription factors and differential gene expression profiles of TaxolTM structural genes were identified which, may contribute to an increased TaxolTM production in the utilised procambium cell suspension culture. Furthermore the T. cuspidata procambium cell suspension culture was found to have an increased level of stress- and defence-response reflected by differential gene expression profiles and content of phenolic compounds and TaxolTM.

Genome sequencing is emerging as a powerful tool to identify the molecular mechanism of cancer progression. However, the software tools to define genomic and post-genomic mutations are just in its infancy. We have used a novel software algorithm to analyse the cancer genome by DNAseq and expressed cancer genome arising from transcription by RNAseq to define dominant sources of potentially expressed tumour-specific mutations and oncogenic targets. We focus primarily on the rare human pleomorphic sarcoma as a disease of high unmet clinical need but use a range of cancer models to accelerate the development of the pipeline. First, we applied next generation sequencing of whole exomes of tumour tissues and two matched normal tissues (blood and “normal” tumour adjacent tissue) from a small set of patients to define parameters for use of the new software. The approaches identified significant mutations in tumour relative to germline DNA, but also in normal adjacent tissue, relative to normal germline, consistent with known field cancerization. Thus, in setting up the larger sequencing screen in the subsequent set of twenty cancer pleomorphic sarcoma cancer patients, whole exome sequencing was performed on tumour tissue and their matched normal adjacent tissues, rather than germline blood derived DNA, to define truly tumour-specific mutations. This approach provided sets of recurrent non-synonymous mutations in tumour tissue such as a transmembrane protease and suggests potential therapeutic targets for future focus that are highly tumour specific in pleomorphic sarcoma. A major problem with using DNA genomics only to define drugable landscapes in cancer is that the tumour genome is static and the mutations do not reflect the expressed cancer landscape at the time of surgery. Thus, in a smaller subset of patients we also applied shotgun RNAseq to determine the number of expressed mutated genes. We defined within the parameters chosen, from 8-17% of the mutated genome is expressed as defined at the RNA level. However, to our surprise, there were an order of magnitude more RNA mutations that were not DNA encoded suggestive of RNA editing events. Each patient showed elevated RNA edits that were independent of each other suggesting a highly-patient, cancer-specific perturbation in the specificity of the RNA editing machinery. We thus developed a cancer cell model to validate the RNA-editing software and we found we could recapitulate some of the RNA edits observed in clinical tumour tissue, in particular the signalling kinase in the MAP kinase-kinase-kinase-kinase super-family. It was interesting that RNA edits can often cluster in exon-intron boundaries suggesting a link to splicing and allows us to begin to produce “rules” for RNA editing. These data provide future direction to understand the role of RNA editing, as well as DNA encoded mutations, as mutagenic events and possible drugable targets in cancer signalling. Lastly, novel or orphan mutant proteins observed in human cancers, whether from DNA encoded mutant proteins or from RNA-edited driven mutant protein synthesis require new tools and technologies to discover new oncogenic signalling mechanisms. We developed an SBP-tagged affinity purification method in combination with label-free SWATH mass spectrometry to identify a novel binding protein for the gain-of-function mutant protein in a key metastatic gene, ELMO1. This identified an elevated interaction with another oncogenic protein encoded by AGR2 gene and validates this proteomics discovery platform to further advance function of new mutated proteins. In conclusion, we have applied and validated newly emerging software to begin to interrogate cancer tissue from patients of unmet clinical need in order to define new mechanisms of cancer progression and to define possibly new or better drug targets for new therapies. The data identified highly recurrent genome encoded mutations in human pleomorphic sarcoma and a potentially novel, targetable landscape represented by RNA editing driven mutant protein production. This will provide a foundation for future work on making better choices to advance our ability to improve patient management in human pleomorphic sarcoma.

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Cigarette smoke creates a molecular field of injury in epithelial cells that line the respiratory tract. We hypothesized that transcriptome sequencing (RNA-seq) will enhance our understanding of the field of molecular injury in response to tobacco smoke exposure and lung cancer pathogenesis by identifying gene expression differences not interrogated or accurately measured by microarrays. We sequenced the high- molecular weight fraction of total RNA (>200 nt) from pooled bronchial airway epithelial cell brushings (n = 3 patients per pool) obtained during bronchoscopy from healthy never smoker (NS) and current smoker (S) volunteers and smokers with (C) and without ( C) lung cancer undergoing lung nodule resection surgery. RNA-seq libraries were prepared using two distinct approaches, one capable of capturing non-polyadenylated RNA (the prototype NuGEN Ovation RNA-seq protocol) and the other designed to measure only polyadenylated RNA (the standard Illumina mRNA-seq protocol) followed by sequencing generating approximately 29 million 36 nt reads per pool and approximately 22 million 75 nt paired-end reads per pool, respectively. The NuGEN protocol captured additional transcripts not detected by the Illumina protocol at the expense of reduced coverage of polyadenylated transcripts, while longer read lengths and a paired-end sequencing strategy significantly improved the number of reads that could be aligned to the genome. The aligned reads derived from the two complementary protocols were used to define the compendium of genes expressed in the airway epithelium (n = 20,573 genes). Pathways related to the metabolism of xenobiotics by cytochrome P450, retinol metabolism, and oxidoreductase activity were enriched among genes differentially expressed in smokers, whereas chemokine signaling pathways, cytokine-cytokine receptor interactions, and cell adhesion molecules were enriched among genes differentially expressed in smokers with lung cancer. There was a significant correlation between the RNA-seq gene expression data and Affymetrix microarray data generated from the same samples (P < 0.001); however, the RNA-seq data detected additional smoking- and cancer-related transcripts whose expression was were either not interrogated by or was not found to be significantly altered when using microarrays, including smoking- related changes in the inflammatory genes SIOOA8 and SIOOA9 and cancer-related changes in MUC5AC and secretoglobin (SCGB3Al). Quantitative realtime PCR confirmed differential expression of select genes and non-coding RNAs within individual samples. These results demonstrate that transcriptome sequencing has the potential to provide new insights into the biology of the airway field of injury associated with smoking and lung cancer. The measurement of both coding and non-coding transcripts by RNA-seq has the potential to help elucidate mechanisms of response to tobacco smoke and to identify additional biomarkers of lung cancer risk and novel targets for chemoprevention. / 2031-01-01