EPIGENETIC MODIFICATIONS TO CYTOSINE AND ALZHEIMER’S DISEASE: A QUANTITATIVE ANALYSIS OF POST-MORTEM TISSUE

Alzheimer’s disease (AD) is the most common form of dementia and the sixth leading cause of death in the United States, with no therapeutic option to slow or halt disease progression. Development of two characteristic pathologic lesions, amyloid beta plaques and neurofibrillary tangles, in the brain are associated with synaptic dysfunction and neuron loss leading to memory impairment and cognitive decline. Although mutations in genes involved in amyloid beta processing are linked to increased plaque formation in the inherited familial form of AD, the more common idiopathic form, termed sporadic AD, develops in the absence of gene mutations. In contrast, alterations in gene expression and transcription occur in plaque and tangle susceptible brain regions of sporadic AD subjects, even in the earliest stages of development of pathologic burden, and may give insight into the pathogenesis of AD. Epigenetic modifications to cytosine are known to alter transcriptional states and gene expression in embryonic development as well as in cancer studies. With the discovery of enzymatically oxidized derivatives of 5-methylcytosine (5-mC), the most common epigenetic cytosine modification, a probable demethylation pathway has been suggested to alter transcriptional states of DNA. The most abundant 5-mC derivative, 5-hydroxymethylcytosine (5-hmC), while expressed at low concentrations throughout the body, is expressed at high concentrations in brain cells. To determine the role cytosine modifications play in AD, this study was directed at the quantification of epigenetic modifications to cytosine in several stages of AD progression using global, genome-wide, and gene-specific studies. To determine global levels of each cytosine derivative in brain regions relevant to AD progression, a gas chromatography/mass spectrometry quantitative analysis was utilized to analyze cytosine, 5-mC, and 5-hmC in tissue specimens from multiple brain regions of AD subjects, including early and late stages of AD progression. To determine the genome-wide impact of 5-hmC on biologically relevant pathways in AD, a single-base resolution sequencing analysis was used to map hydroxymethylation throughout the hippocampus of late stage AD subjects. Finally, to determine gene-specific levels of cytosine, 5-mC, and 5-hmC, a quantitative polymerase chain reaction (qPCR) protocol was paired with specific restriction enzyme digestion to analyze target sequences within exons of genes related to sporadic AD. Results from these studies show epigenetic modifications to cytosine are altered on the global, genome-wide, and gene-specific levels in AD subjects compared to normal aging, particularly in early stages of AD progression, suggesting alterations to the epigenetic landscape may play a role in the dysregulation of transcription and the pathogenesis of AD

Shape Analysis of High-throughput Genomics Data

RNA sequencing refers to the use of next-generation sequencing technologies to characterize the identity and abundance of target RNA species in a biological sample of interest. The recent improvement and reduction in the cost of next-generation sequencing technologies have been paralleled by the development of statistical methodologies to analyze the data they produce. Coupled with the reduction in cost is the increase in the complexity of experiments. Some of the old challenges still remain. For example the issue of normalization is important now more than ever. Some of the crude assumptions made in the early stages of RNA sequencing data analysis were necessary since the technology was new and untested, the number of replicates were small, and the experiments were relatively simple. One of the many uses of RNA sequencing experiments is the identification of genes whose abundance levels are significantly different across various biological conditions of interest. Several methods have been developed to answer this question. Some of these newly developed methods are based on the assumption that the data observed or a transformation of the data are relatively symmetric with light tails, usually summarized by assuming a Gaussian random component. It is indeed very difficult to assess this assumption for small sample sizes (e.g. sample sizes in the range of 4 to 30). In this dissertation, we utilize L-moments statistics as the basis for normalization, exploratory data analysis, the assessment of distributional assumptions, and the hypothesis testing of high-throughput transcriptomic data. In particular, we introduce a new normalization method for high-throughput transcriptomic data that is a modification of quantile normalization. We use L-moments ratios for assessing the shape (skewness and kurtosis statistics) of high-throughput transcriptome data. Based on these statistics, we propose a test for assessing whether the shapes of the observed samples differ across biological conditions. We also illustrate the utility of this framework to characterize the robustness of distributional assumptions made by statistical methods for differential expression. We apply it to RNA-seq data and find that methods based on the simple t-test for differential expression analysis using L-moments statistics as weights are robust. Finally we provide an algorithm based on L-moments ratios for identifying genes with distributions that are markedly different from the majority in the data

Role of post-transcriptional regulation in human liver

Indiana University-Purdue University Indianapolis (IUPUI)My thesis comprises of two individual projects which revolve around the importance of post-transcriptional regulation in liver. My first project is studying the integrated miRNA – mRNA network in NAFLD. For fulfillment of the study we conducted a genome-wide study to identify microRNAs (miRs) as well as the miR-mRNA regulatory network associated with hepatic fat and NAFLD. Hepatic fat content (HFC), miR and mRNA expression were assessed in 73 human liver samples. Liver histology of 49 samples was further characterized into normal (n=33) and NAFLD (n=16). Liver miRNome and transcriptome were significantly associated with HFC and utilized to (a) build miR-mRNA association networks in NAFLD and normal livers separately based on the potential miR-mRNA targeting and (b) conduct pathway enrichment analyses. We identified 62 miRs significantly correlated with HFC (p < 0.05 with q < 0.15), with miR-518b and miR-19b being most positively and negatively correlated with HFC, respectively (p < 0.008 for both). Integrated network analysis showed that six miRs (miRs-30b*, 612, 17*, 129-5p, 204 and 20a) controlled ~ 70% of 151 HFC-associated mRNAs (p < 0.001 with q < 0.005). Pathway analyses of these HFC-associated mRNA revealed their key effect (p<0.05) in inflammation pathways and lipid metabolism. Further, significant (p<2.47e-4, Wilcoxon test) reduction in degree of negative associations for HFC-associated miRs with HFC-associated mRNAs was observed in NAFLD as compared to normal livers, strongly suggesting highly dysfunctional miR-mRNA post-transcriptional regulatory network in NAFLD. Our study makes several novel observations which provide clues to better understand the pathogenesis and potential treatment targets of NAFLD. My second project is based on uncovering important players of post-transcriptional regulation (RBPs) and how they are associated with age and gender during healthy liver development. For this study, we performed an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Experimental verification of several of the predicted associations in the mouse model confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are likely to be up-regulated in males. Altogether, these observations show that several of these RBPs are important developmentally conserved regulators. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in liver development and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. The compendium of RBPs and this study will help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes

Generation and Characterisation of Novel Monoclonal Antibodies towards Ovarian Tumour Stem Cells

Tumour stem cells (TSCs) are hypothesised to be a rare population of tumour cells which possess stem cell-like properties and are resistant to conventional therapy. Although cell surface markers have been widely used to characterise TSCs, previous literature suggests that no specific marker has been found for ovarian TSCs. We aimed to identify and characterise novel antibodies specific to ovarian epithelial TSCs, particularly towards populations with Hoechst efflux (Side Population, SP) and aldehyde dehydrogenase activity which are associated with a stem cell phenotype and drug resistance. Putative TSC subpopulations from ovarian tumour cell lines isolated by fluorescence activated cell sorting (FACS) using differential Hoechst dye uptake and Aldefluor activity assays displayed stem cell-like characteristics, including the upregulation of stem cell markers, increased anchorage-independent growth and increased invasive properties. A panel of monoclonal antibodies (mAbs) was then generated by injecting Aldefluor-positive ovarian tumour IGROV1 cells into female BALB/c mice. 34 antibodies were found to be specific to Aldefluor-positive cells and 2 of these enriched for SP cells. The 2 mAbs demonstrated cross-reactivity on human embryonic stem cells but no cross-reactivity to normal ovarian cell lines. Subpopulations of ovarian cell lines positive for the mAbs displayed stem cell-like characteristics, including upregulation of stem cell markers - CD133, ABCB1 and ALDH1A1. Sorted mAb populations were injected into non-obese diabetic/severe combined (NOD/SCID) mice and differential in vivo tumour formation was observed. Finally, the target antigen which both mAbs recognised was identified by mass spectrometry to be clathrin heavy chain (CHC1). We conclude that rare subpopulations with tumour-sustaining capability and stem cell-like characteristics can be identified in ovarian cancer using the 2 novel antibodies generated. Both mAbs target CHC1 on tumour-sustaining populations which are enriched for multiple stem cell markers and are therefore potential novel diagnostic markers and/or therapeutic agents

Using genetic analysis to determine the effects of disease on the spatial dynamics of the invasive fall armyworm, Spodoptera frugiperda, in Africa.

Invasive crop pests are a global problem, resulting in food shortages, severe economic burdens, and devastating environmental losses. Many invasive insects are migratory with strong flight ability which further enables their rapid spread. Disease can control pests through occurring naturally and as biocontrol agents. However, the presence of naturally occurring diseases and potential flight routes of invasive pests are unknown when an invasive species first reaches an area. This thesis uses a combination of field collections, molecular biology, and behavioural experiments to fill some key knowledge gaps in our understanding of the invasive fall armyworm (Spodoptera frugiperda) in Africa. The fall armyworm is a migratory crop pest that first arrived in Africa in 2016 and rapidly spread across the continent. Chapter One provides a general introduction to the topics covered in this thesis, focusing on migratory insects to explore what is currently known about pest control, how disease interacts with migration and what genetic analysis can tell us about these topics. Chapter Two, uses fall armyworm larvae collected in six African countries to identify the presence of microbial natural enemies. It then models how environmental factors may influence the distribution and prevalence of the viral natural enemy, Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). To improve current understanding of population structure of the fall armyworm in Africa, Chapter Three uses a combination of molecular approaches, and investigates whether this can explain variation in the distribution of SfMNPV. Chapter Four takes an in-depth look at the effects SfMNPV has on the fall armyworm during flight, using RNA sequencing to reveal what occurs at the molecular level that could lead to changes in migratory capacity. It compares the molecular responses of males and females, highlighting similarities and differences across many key areas including metabolism, immunity, and reproduction. Using four genes identified by RNA sequencing, Chapter Five focuses on gene expression of the immunity related Toll-pathway. This furthers our knowledge of how the fall armyworm responds to SfMNPV and flight and helps to unravel why males and females respond differently. Finally, Chapter Six is a general discussion chapter that brings these findings together to discuss and consolidate the knowledge that this research has contributed to the field. Overall, this thesis greatly contributes to current understanding of fall armyworm in Africa, and what molecular changes are triggered by flight and disease in a migratory insect pest

Toxicity of TCDD in the mouse antral follicle

The persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent ovarian toxicant. These studies were designed to characterize the actions of TCDD on steroidogenesis, growth, and atresia of intact mouse antral follicles in vitro. Interestingly, TCDD has been shown to act mainly through binding to the aryl hydrocarbon receptor (AHR) in other tissues, and the AHR has been identified as an important factor in ovarian function. Thus, these studies were also designed to test the hypothesis that TCDD activates the AHR in antral follicles. Specifically, these studies were designed to test the hypothesis that TCDD exposure leads to decreased sex hormone production/secretion by antral follicles as well as decreased growth of antral follicles in vitro. Mouse antral follicles were exposed for 96 hours to a series of TCDD doses previously shown to have effects on ovarian tissues and cells in culture and to encompass environmentally relevant and pharmacological exposures (0.1-100nM). The results indicate that TCDD decreases progesterone (P), androstenedione (A4), testosterone (T), and estradiol (E2) levels in a dose response manner without altering growth of antral follicles. The 1nM dose of TCDD consistently lowered all four hormones measured. Thus, this dose was chosen to study more closely the mechanism of TCDD toxicity in the antral follicle. Interestingly, the addition of pregnenolone substrate (10μM) restores hormone levels to control levels, suggesting that TCDD has actions upstream of progesterone production. Thus, the next goal of these studies was to determine the mechanism by which TCDD inhibits steroidogenesis and whether this occurs in a time dependent manner. Specifically, experiments were designed to compare the effects of 48 and 96 hours TCDD exposure on hormone production/secretion, steroidogenic enzymes, atresia ratings, expression of members of the apoptotic pathway, and expression of the AHR and cytochrome P450, family 1, subfamily b, polypeptide 1 (Cyp1b1). TCDD exposure for 48 hours increased levels of A4, without changing HSD3B1 protein, HSD17B1 protein, estrone (E1), T or E2 levels. Further, TCDD did not alter atresia rating, but it did down-regulate the AHR protein compared to vehicle. TCDD exposure for 96 hours decreased transcript levels for Cyp11a1, Cyp17a1, Hsd17b1, and Cyp19a1, but increased Hsd3b1 transcript. TCDD exposure particularly lowered both Hsd17b1 transcript and HSD17B1 protein. However, TCDD exposure did not affect levels of E1 in the media. The down regulation of the AHR protein in TCDD exposed follicles persisted and Cyp1b1 transcripts were increased by 3-4 fold. Interestingly, levels of the proapototic factor, BCL2-associated X protein (Bax) were significantly reduced without affecting atresia ratings in antral follicles following TCDD exposure, suggesting an altered apoptotic pathway in exposed antral follicles. Finally, because Cyp1b1 is an important enzyme that is implicated in both biotransformation of TCDD and also in E2 catabolism to catecholestrogens and the highly reactive quinones, increased levels of Cyp1b1 could be an indication of a system under stress. Quinones are known to cause DNA damage, and if the DNA is left unrepaired, it can lead to mutations that propagate into daughter cells, leading to cancer. Besides its effects on E2 levels, chronic TCDD exposure causes promotion of ovarian tumors in rats and acute exposure is associated with an increased incidence of ovarian cancer in young woman. The enzyme catechol-o-methyltransferase (COMT) is regulated by E2 and is an important enzyme for inactivating the catecholestrogens. Thus, we hypothesized that TCDD exposure affects the levels of COMT. TCDD exposure reduced COMT protein levels, while there was no change in transcript levels for COMT after 96 hours exposure in antral follicles. Overall, these data indicate that TCDD exposure perturbs steroidogenesis by impairing the steroidogenic enzymes, and that it alters apoptotic pathways, and potentially decreases the ability to neutralize reactive metabolites in antral follicles. Finally, the activation and proteasomal degradation of the AHR in the antral follicle likely plays a central role in these processes, contributing to endocrine disruption, cancer promotion, and ovarian toxicity by TCDD

Analysis of microbial populations in wines through NGS methodologies.

La vinificación es un proceso complejo que involucra varias etapas hasta el embotellado y comercialización del vino. Durante este proceso, la cantidad limitada de nutrientes provoca la competencia microbiana, que resulta en la producción de metabolitos que modulan el producto final del vino. Esta actividad microbiana puede conferir características organolépticas beneficiosas o indeseables a la calidad del vino. En los últimos años, el enfoque principal se ha centrado en la detección y el seguimiento de microorganismos determinados, que supuestamente estropean el vino, y la aplicación de metodologías empíricas para la prevención del crecimiento microbiano indeseable. Sin embargo, los hallazgos de las investigaciones han mostrado una base multifactorial del deterioro del vino, y han subrayado la necesidad de una estrategia innovadora que permita el estudio de la diversidad microbiana en su totalidad. La secuenciación de última generación parece un enfoque adecuado y prometedor para este propósito, ya que parece capaz de superar las limitaciones de las metodologías convencionalesevaluación de los resultados derivados en función de su alineación con hallazgos anteriores y su capacidad para proporcionar nuevos conocimientos. En general, el trabajo actual ha logrado corroborar estudios previos, sugerir mejoras sobre las implementaciones relacionadas con la bioinformática y la estadística y ampliar nuestro conocimiento sobre varios factores que influyen en la vinificación. Winemaking is a intricate process, involving various stages until the wine bottling and commercialization. During this process, the limited amount of nutrients leads to microbial competition, which in turn results in the production of metabolites that modulate the final wine product. This microbial activity may confer beneficial or undesirable organoleptic characteristics to the wine quality. The past years, the main focus has been given to the detection and monitoring of specific putative wine-spoiling microorganisms and the application of empirical methodologies for the prevention of unwanted microbial growth. Nevertheless, research findings have shown a multifactorial basis of the wine spoilage and underlined the need for an innovative strategy that will allow the study of the microbial diversity in its entirety. Next-generation-sequencing appears a suitable and promising approach for this purpose, as it seems able to overcome the limitations of conventional methodologies. In this work, various aspects associated to the NGS-based metataxonomic analysis have been studied, in relation to the performance of the NGS technology against conventional applications, and the establishment of a bioinformatic and statistical framework for the analysis of metataxonomic data

The role of the Hippo pathway in etiology and progression of pancreato-biliary tumors and clear cell renal cell carcinoma

In the quest for the discovery of novel targets for cancer therapy, aberrantly reactivated embryonic signaling pathways have proven to be a rich mine and recent years have seen the introduction of the hedgehog-inhibitor cyclopamine into clinical practice. Dysfunctional signaling via the growth inhibitory embryonic signaling pathway clustered around the Hippo kinase and aberrant expression of its main target Yes-associated protein (YAP) is likewise emerging to be involved in maintenance and progression of various human cancers. The purpose of this dissertation is to identify the incidence, functional relevance and mechanistic significance of aberrant Hippo signaling in etiology and progression of different solid tumor entities that vary in their biology as well as tissue of origin, but share an unfavorable clinical prognosis and the dire need for novel therapeutic approaches. Specifically cancers of the pancreato-biliary tract, more precisely pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCC), as well as clear cell renal cell carcinoma (ccRCC) have been analyzed in the course of this dissertation. For these tumor entities initial evidence of aberrant Hippo signaling has been published. In a first step, aberrant expression of the transcriptional co-activator YAP, which constitutes the principal target of the growth inhibitory Hippo-pathway, was confirmed in human tumor tissue samples by immunohistochemistry. Nuclear YAP expression correlated with nodal stadium in PDAC and was also more frequent in ccRCC patients with tumor-positive lymph nodes. A common feature in all three tumor entities were solitary, fibroblast-like cells residing inside the tumor adjacent stroma that were highlighted by robust nuclear YAP-staining, suggesting that YAP might be a mediator of tumor-stroma crosstalk. In a second step, the role of aberrant YAP activity in tumor cell lines was examined by shRNA-mediated knockdown of the protein in selected, highly YAP-expressing cell lines. Phenotypic consequences of the knockdown included inhibition of cell proliferation as measured by the MTS-assay as well as impairment of colony formation in vitro for PDAC, CCC and ccRCC cell lines and decreased tumorigenicity in vivo for ccRCC cells in tumor xenograft experiments. However, shRNA-mediated YAP knockdown impaired in vitro cancer cell migration only in ccRCC cell lines. In a third and final step, the transcriptomic output of YAP in the three different tumor entities was compared by gene expression analysis of YAP-knockdown vs. mock transfected cancer cell lines. Although the phenotype of YAP-knockdown cells was functionally similar in PDAC, CCC and ccRCC cell lines, the transcriptional output of the Hippo-YAP axis as determined by transcriptomic profiling appeared to be fundamentally different and seemed to be highly tissue context-dependent. In fact, certain pathways were activated by YAP-knockdown in one cell line while they were inhibited in another (e.g. MAPK in PK9 v. MZ1774 cells). As no single, clearcut mechanism of YAP-mediated tumorigenicity could be observed, mechanistic considerations were made for each of the three tumor entities individually and feature CTGF and Wnt-signaling for pancreatic cancer as well as c-Myc and endothelins in ccRCC. The observation of solitary cells that exhibit robust nuclear YAP-expression was common in all three tumor entities analyzed, suggesting a role of Hippo-YAP signaling in tumor-stroma crosstalk which is certainly a highly interesting starting point for future research