Fanconi anaemia (FA) is an inherited disorder characterized by bone marrow failure, cancer predisposition and congenital abnormalities. The 12 known FA genes have been implicated in homologous recombination (HR), a process involved in telomere maintenance. A complex of at least 7 FA proteins promotes FANCD2 monoubiquitination and nuclear foci formation. FANCD2 colocalizes and interacts with HR proteins, however the role of FANCD2 in HR is unclear. Telomeres in dividing human somatic cells shorten until they reach a critical length, triggering most cells to undergo senescence or apoptosis. Rare immortal cells escape this crisis by expressing telomerase, or activating the Alternative Lengthening of Telomeres (ALT) pathway, which involves HR. FA core complex proteins and FANCD2 colocalize with telomeric foci in ALT, but not telomerase positive cells. Localization of FANCD2 to ALT telomeric foci requires monoubiquitination by the FA core complex, but is independent of ATM and ATR. FANCD2 primarily colocalizes with ALT telomeric DNA within ALT-associated PML bodies (APBs). Electron spectroscopic imaging and FISH experiments show that APBs contain extra-chromosomal telomeric repeat (ECTR) DNA that is non-nucleosomal. Depletion of FANCD2 causes marked increases in ECTR in ALT, but not telomerase positive cells. Overexpression of BLM, the helicase mutated in Bloom syndrome, also causes an ALT-specific increase in ECTR DNA. FANCD2 coimmunoprecipitates with BLM in ALT cells, and FANCD2 localization to ALT telomeric foci requires BLM expression. FANCD2-depleted ALT cells have reduced viability, signs of mitotic catastrophe, and multiple types of telomeric abnormalities, including increases in telomeric recombination, entanglements, colocalization with DNA repair proteins, and expression of fragile site characteristics. SiRNA depletion of FANCD2 does not cause overexpression of BLM, however codepletion of BLM with FANCD2 suppresses the telomere phenotypes caused by FANCD2 knockdown. Together this suggests that FANCD2 regulates BLM-dependent recombination and amplification of telomeric DNA within ALT cells.

Fanconi anaemia (FA) is an inherited disorder characterized by bone marrow failure, cancer predisposition and congenital abnormalities. The 12 known FA genes have been implicated in homologous recombination (HR), a process involved in telomere maintenance. A complex of at least 7 FA proteins promotes FANCD2 monoubiquitination and nuclear foci formation. FANCD2 colocalizes and interacts with HR proteins, however the role of FANCD2 in HR is unclear. Telomeres in dividing human somatic cells shorten until they reach a critical length, triggering most cells to undergo senescence or apoptosis. Rare immortal cells escape this crisis by expressing telomerase, or activating the Alternative Lengthening of Telomeres (ALT) pathway, which involves HR. FA core complex proteins and FANCD2 colocalize with telomeric foci in ALT, but not telomerase positive cells. Localization of FANCD2 to ALT telomeric foci requires monoubiquitination by the FA core complex, but is independent of ATM and ATR. FANCD2 primarily colocalizes with ALT telomeric DNA within ALT-associated PML bodies (APBs). Electron spectroscopic imaging and FISH experiments show that APBs contain extra-chromosomal telomeric repeat (ECTR) DNA that is non-nucleosomal. Depletion of FANCD2 causes marked increases in ECTR in ALT, but not telomerase positive cells. Overexpression of BLM, the helicase mutated in Bloom syndrome, also causes an ALT-specific increase in ECTR DNA. FANCD2 coimmunoprecipitates with BLM in ALT cells, and FANCD2 localization to ALT telomeric foci requires BLM expression. FANCD2-depleted ALT cells have reduced viability, signs of mitotic catastrophe, and multiple types of telomeric abnormalities, including increases in telomeric recombination, entanglements, colocalization with DNA repair proteins, and expression of fragile site characteristics. SiRNA depletion of FANCD2 does not cause overexpression of BLM, however codepletion of BLM with FANCD2 suppresses the telomere phenotypes caused by FANCD2 knockdown. Together this suggests that FANCD2 regulates BLM-dependent recombination and amplification of telomeric DNA within ALT cells.

Although the completion of the Human Genome Project helps develop efficient treatment/prevention programs, it will raise new and non-trivial public health issues. Many of these issues fall under the professional purview of health educators. Yet, no studies have evaluated if health educators (HEs) are ready to adopt genomic competencies into health promotion. This dissertation addresses this issue by examining three research questions in three separate studies: 1) Why must HEs develop genomic competencies? 2) What are HEs’ knowledge of, and attitudes toward genomic competencies? And 3) what is HEs’ likelihood of adopting genomic competencies into health promotion? The first theoretical study proposed five arguments supporting the need for HEs to develop their genomic competencies and integrate public health genomics into health promotion. These arguments touched on various dimensions of HEs’ professional goals and ranged from professional responsibilities and competencies, to the availability of funding for genomic-related research or interventions and opportunities for future employment. For the second study, a web-based survey was developed and distributed to all members of four major health education organizations. A total of 1,925 HEs’ completed the survey and 1,607 responses were utilized in the final analysis. This study indicated that participants had deficient knowledge and unfavorable attitudes toward the CDCproposed genomic competencies. In the third study, a theoretical model was developed to predict HEs’ likelihood to incorporate genomic competencies into their practice. Using techniques from Structural Equation Modeling (SEM), the model was tested with the same data of the second study. Findings supported the proposed theoretical model. While genomic knowledge, attitudes, and self-efficacy were significantly associated with HEs’ likelihood to incorporate genomic competencies into their practice, attitudes was the strongest predictor of likelihood. In summary, these studies indicated that participating HEs had deficient genomic knowledge, unfavorable attitudes toward a set of CDC-proposed genomic competencies, and low likelihood to adopt genomic competencies into health promotion. Relevant training should be developed and advocated. As the SEM analysis results indicated the survey findings supported the proposed theoretical model, which can be utilized to steer future training for HEs.

No description available.

Genomic instability is commonly seen in breast cancers. To date, various chromosomal or segmental loss or amplification regions have been detected in primary tumors and cell lines. Hence, an intensive search for potent tumor suppressors or oncogenes located in these regions continues. MicroRNAs (miRNAs) are ~18-24 nt long non-coding RNAs that regulate protein expression either by target mRNA cleavage or translational repression. We hypothesized that miRNAs located in genomic instability regions in breast cancer cells may contribute to the initiation or maintenance of breast tumors. Here, we investigated genomic levels of miRNAs on frequent loss or gain regions of breast cancer cells. First, using bioinformatics resources we mapped known miRNAs and candidate miRNAs to reported genomic instability regions. Our extensive searches resulted with more than 30 known miRNAs and 35 candidate miRNAs. To further confirm loss or amplification of miRNA genes on these chromosomal regions in breast cancer cells, we designed specific primers for the known pre-miRNA DNA regions and performed semi-quantitative PCR in 20 breast cancer cell lines, 2 immortalized mammary cell lines, and 2 control samples. Densitometry results suggested that a striking 61 % (22/36) of selected miRNAs showed either loss or amplification in at least 3 different breast cancer cell lines. Interestingly most of these alterations were found to be amplifications even in regions reported to harbor losses in breast tumors. Genomic fold change results of these microRNAs provide a biologically relevant starting point for further expression and functional experiments of microRNAs in breast cancer studies. Genomic fold change analysis followed expression analysis of two significant microRNAs (hsa-miR-21 and hsa-miR-383) was done by qRT-PCR method. Our data provide a wide screen of genomic instability of 36 microRNA genes in 20 breast cancer cells and normal samples detected by semi-quantitative duplex PCR method as well as expression analysis of two microRNAs. To this date, such an extensive data on genomic status of microRNA genes in breast cancer cells did not exist. Therefore, our results are the first comprehensive investigation of many microRNA genes on genomic instability regions in breast cancers and provide further clues to the potential involvement of these microRNAs in breast tumorigenesis MicroRNA genomic instability may affect their expression and therefore their targets&rsquo / expressions. Understanding how these microRNAs regulate their targets and contribute to the neoplastic events will also contribute to the field by using this information for future diagnostic and threaupetical applications.

Genomic sequence databases, like GenBank, EMBL, are widely used by molecular biologists for homology searching. Because of the increase of the size of genomic sequence databases, the importance of indexing the sequences for fast queries grows. The DNA sequences are composed of 4 base pairs, and these genomic sequences can be regarded as the text strings. Similar to conventional databases, there are some approaches use indexes to provide efficient access to the data. The inverted-list indexing approach uses hashing to store the database sequences. However, the perfect hashing function is difficult to construct, and the collision in a hash table may occur frequently. Different from the inverted-list approach, there are other data structures, such as the suffix tree, the suffix array, and the suffix binary search tree, to index the genomic sequences. One characteristic of those suffix-tree-like data structures is that they store all suffixes of the sequences. They do not break the sequences into words. The advantage of the suffix tree is simple. However, the storage space of the suffix tree is too large. The suffix array and the suffix binary search tree reduce more storage space than the suffix tree. But since they use the binary searching technique to find the query sequence, they waste too much time to do the search. Another data structure, the word suffix tree, uses the concept of words and stores partial suffixes to index the DNA sequence. Although the word suffix tree reduces the storage space, it will lose information in the search process. In this thesis, we propose a new index structure, ACGT-Words tree, for efficiently support query processing in genomic databases. We define the concept of words which is different from the word definition given in the word suffix tree, and separate the DNA sequences stored in the database and in the query sequence into distinct words. Our approach does not store all of the suffixes in the database sequences. Therefore, we need less space than the suffix tree approach. We also propose an efficient search algorithm to do the sequence match based on the ACGT-Words tree index structure; therefore, we can take less time to finish the search than the suffix array approach. Our approach also avoids the missing cases in the word suffix tree. Then, based on the ACGT-Words tree, we propose one improved operation for data insertion and two improved operations for the searching process. In the improved operation for insertion, we sort the ACGT-Words generated and then preprocess them before constructing the tree structure. In the two improved operations, we can provide better performance when the query sequence satisfies some conditions. The simulation results show that the ACGT-Words tree outperforms the suffix tree and the suffix array in terms of storage and processing time, respectively. Moreover, we show that the improved operations in the ACGT-Words tree also require shorter time to construct or search than the original processes or the suffix array.

published_or_final_version / Statistics and Actuarial Science / Master / Master of Philosophy

Genetic association study is a useful tool to identify the genetic component that is responsible for a disease. The phenomenon that a certain gene expresses in a parent-of-origin manner is referred to as genomic imprinting. When a gene is imprinted, the performance of the disease-association study will be affected. This thesis presents statistical testing methods developed specially for nuclear family data centering around the genetic association studies incorporating imprinting effects. For qualitative diseases with binary outcomes, a class of TDTI* type tests was proposed in a general two-stage framework, where the imprinting effects were examined prior to association testing. On quantitative trait loci, a class of Q-TDTI(c) type tests and another class of Q-MAX(c) type tests were proposed. The proposed testing methods flexibly accommodate families with missing parental genotype and with multiple siblings. The performance of all the methods was verified by simulation studies. It was found that the proposed methods improve the testing power for detecting association in the presence of imprinting. The class of TDTI* tests was applied to a rheumatoid arthritis study data. Also, the class of Q-TDTI(c) tests was applied to analyze the Framingham Heart Study data. The human microbiome is the collection of the microbiota, together with their genomes and their habitats throughout the human body. The human microbiome comprises an inalienable part of our genetic landscape and contributes to our metabolic features. Also, current studies have suggested the variety of human microbiome in human diseases. With the high-throughput DNA sequencing, the human microbiome composition can be characterized based on bacterial taxa relative abundance and the phylogenetic constraint. Such taxa data are often high-dimensional overdispersed and contain excessive number of zeros. Taking into account of these characteristics in taxa data, this thesis presents statistical methods to identify associations between covariate/outcome and the human microbiome composition. To assess environmental/biological covariate effect to microbiome composition, an additive logistic normal multinomial regression model was proposed and a group l1 penalized likelihood estimation method was further developed to facilitate selection of covariates and estimation of parameters. To identify microbiome components associated with biological/clinical outcomes, a Bayesian hierarchical regression model with spike and slab prior for variable selection was proposed and a Markov chain Monte Carlo algorithm that combines stochastic variable selection procedure and random walk metropolis-hasting steps was developed for model estimation. Both of the methods were illustrated using simulations as well as a real human gut microbiome dataset from The Penn Gut Microbiome Project. / published_or_final_version / Statistics and Actuarial Science / Doctoral / Doctor of Philosophy

Malaria is a deadly disease that causes nearly one million deaths each year. Understanding the demographic history of the malaria parasite Plasmodium falciparum and the genetic basis of its adaptations to antimalarial treatments and the human immune system is important for developing methods to control and eradicate malaria. To study the long-term demographic history and recent effective size of the population in order to identify genes under selection more efficiently and predict the effectiveness of selection, in Chapter 2 we sequenced the complete genomes of 25 cultured P. falciparum isolates from Senegal. In addition, in Chapter 3 we estimated temporal allele frequencies in 24 loci among 528 strains from the same population across six years. Based on genetic diversity of the genome sequences, we estimate the long-term effective population size to be approximately 100,000, and a major population expansion of the parasite population approximately 20,000-40,000 years ago. Based on temporal changes in allele frequencies, however, the recent effective size is estimated to be less than 100 from 2007-2011. The discrepancy may reflect recent aggressive efforts to control malaria in Senegal or migration between populations.

Genomic imprinting, the parent-of-origin specific silencing of alleles, plays an important role in phenotypic plasticity and consequently evolution. The leading explanation for genomic imprinting is Haig's conflict theory, which suggests that alleles from each parent have evolved under different selectional pressures, resulting in the differential expression of patrigenes and matrigenes. Previous studies have mainly used mammals and flowering plants to test Haig’s theory. However, there is a lack of independent evidence to support the theory. My PhD thesis attempts to conduct an independent test of Haig’s conflict theory using buff tailed bumblebee Bombus terrestris. A methylation system to facilitate genomic imprinting has not been found in this species. Therefore the first aim of the study was to establish the presence of a functional methylation system in B. terrestris before testing Haig's conflict theory using worker reproduction in queen-less colonies. The initial finding is that a methylation system exists in B. terrestris. The next study, investigating the presence of methylated genes, revealed differential methylation patterns in caste and life stages. Finally, genes involved with worker reproduction in a range of social insects were identified, but distinguishing the matrigene and the patrigene for each gene was unsuccessful. Therefore the final study investigating the presence of imprinted genes in B. terrestris and whether they conform to the expression patterns hypothesised by Haig’s conflict theory could not be analysed. Although this study did not provide conclusive evidence to support Haig’s conflict theory, the presence of methylation in genes involved with worker reproduction in reproducing and non-reproducing B. terrestris workers suggests that further analysis is needed. With adequate evidence, proving Haig’s conflict theory will not only expand our knowledge of invertebrate methylation, but also our understanding of conflict within social insect societies and our knowledge of how genomic imprinting affects phenotypic plasticity.