The use of the MinION sequencer (Oxford Nanopore) was tested on samples prepared to simulate infectious samples. The tested procedure is to simulate work with a sample with an unknown pathogen. Therefore, a metagenomic approach was chosen. Three kits were tested: Rapid Barcoding Sequencing, PCR Barcoding and Premium whole genome amplification. Each kit differed in duration, difficulty to prepare and in amplification of nucleic acids. In total it was chosen eight viruses with different genome lengths and with varying types of the genome (5,6 - 152 kb, ss/ds RNA, dsDNA). Ten samples were prepared to simulate different types of infection (respiratory, gastrointestinal tract and urine), and one sample contained pure water as a negative control. Before preparation of the library with Oxford Nanopore's kits, DNase/RNase treatment was used. The viral RNA was transcribed into DNA and in chosen samples were amplificated to reach a higher concentration of nucleic acids. Rapid barcoding sequencing kit detected all selected viruses with the highest number of viral reads (4403) with a length between 100 and 250 nt and quality coverage of viral genomes. PCR Barcoding kit detected five out of eight viruses, and the number of identified reads with a length of 100-200 nt distinctly decreased. Premium whole genome...

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MSc thesis - Faculty of Engineering / Service creation in the Next Generation Network (NGN) is focused around software creation and borrows heavily from the Software Engineering community. In the NGN, telecommunication companies demand simple, rapid and economical service creation. The key to this type of service creation is software re-use. Software re-use is a conundrum where limited, dedicated solutions exists. These solutions include amongst others Enterprise JavaBeansTM (EJBs), design patterns and object-oriented programming. The Telecommunications Information Networking Architecture- Conformance And Testing (TINA-CAT) workgroup has done work on a functionality centric concept called RP-facets. This report proposes a redefinition of RP-facets, as Facets, for software re-use across the design and code level. We redefine Facets as functionality centric reusable components. A Facet is independent of the implementation language and the execution platform. Facets allow containment in a structured manner via a user defined Facet Hierarchy. Facets are resource, context and data agnostic. They also introduce a structured way to allow source code to be changed based on design level decisions. Also, possessing the ability to allow the simultaneous use of other reuse solutions and programming paradigms. Abstraction of detail from developers and platform migration can be achieved by using Facets. Facets are composed of a Generic definition and any number of Implementation definitions. The definitions are supported by an underlying informational model called meta-¼. Meta- ¼ is a model at the M3 meta-level that focuses on describing entities. Most of the Facet’s capabilities are enabled by the meta-¼ model. An environment for developing Facets is created, called the Facet Development Environment (FDE). The Facet Developer (FD) role is introduced to develop and maintain Facets. The FD verifies programmes from programmers to be included into the catalogue of Facets via the FDE. The FD interacts with service creation teams to determine which Facets can be used in the service they wish to develop. Facets prove their capability in targeted areas, yet lack in other categories. It is recommended that the underlying informational model should be revised to form a more robust and flexible entity describing model. In addition, a cataloging capability to easily find Facets with particular functionality should be appended to the capabilities of the facet. It is proposed, for future work, that a development environment be created that encompasses a process for using Facets to create services.

Ovarian cancer (OC) is the deadliest gynecologic malignancy with a substantial proportion of hereditary cases and a frequent association with breast cancer (BC). Genetic testing facilitates preventive management for carriers of mutations in OC-susceptibility genes. However, the prevalence of germline mutations varies among populations and many rarely mutated OC predisposition genes remain to be identified. We analyzed 219 genes in 1333 Czech OC patients and 2278 population-matched controls (PMC) using next-generation sequencing. Altogether, 427/1333 (32%) patients and 58 /2278 (2,5%) PMC carried pathogenic mutations in 18 known/anticipated OC predisposition genes. Mutations in BRCA1, BRCA2, RAD51C, RAD51D, BARD1 and mismatch repair genes conferred a high OC risk (with OR>5). Mutations in BRIP1 and NBN were associated with moderate risk (both OR ≥2 - <5). BRCA1/2 mutations dominated in almost all clinicopathological subgroups including sporadic borderline tumors of ovary (BTO). Analysis of remaining 201 genes revealed somatic mosaics in PPM1D and germline mutations in SHPRH and NAT1 associating with a high/moderate OC risk significantly; however, further studies are warranted to delineate their contribution to OC development in other populations. Results of this study demonstrate the high proportion...

In this study we build solutions to three common challenges in the fields of bioinformatics through utilizing statistical methods and developing computational approaches. First, we address a common problem in genome wide association studies, which is linking genotype features within organisms of the same species to their phenotype characteristics. We specifically studied FHA domain genes in Arabidopsis thaliana distributed within Eurasian regions by clustering those plants that share similar genotype characteristics and comparing that to the regions from which they were taken. Second, we also developed a tool for calculating transposable element density within different regions of a genome. The tool is built to utilize the information provided by other transposable element annotation tools and to provide the user with a number of options for calculating the density for various genomic elements such as genes, piRNA and miRNA or for the whole genome. It also provides a detailed calculation of densities for each family and subamily of the transposable elements. Finally, we address the problem of mapping multi reads in the genome and their effects on gene expression. To accomplish this, we implemented methods to determine the statistical significance of expression values within the genes utilizing both a unique and multi-read weighting scheme. We believe this approach provides a much more accurate measure of gene expression than existing methods such as discarding multi reads completely or assigning them randomly to a set of best assignments, while also providing a better estimation of the proper mapping locations of ambiguous reads. Overall, the solutions we built in these studies provide researchers with tools and approaches that aid in solving some of the common challenges that arise in the analysis of high throughput sequence data.

Experiential, outdoor education supports improvement in students’ problem-solving skills; collaboration and communication skills; and enjoyment in learning in the outdoors. Outdoor instruction is becoming increasingly underutilized. A residential environmental education center, located in Tennessee has conducted professional development programs in effort to increase teacher implementation of instruction in outdoor spaces. This institute revealed concern for low implementation rates to past professional development opportunities. Their newly designed, long-term professional development explored teacher’s perceived challenges and needs, then combined effective experiential pedagogy in outdoor spaces with pre-established communities of support from the participating schools in effort to contribute to experiential, outdoor instruction reform. This program entailed four workshop meetings over a seven-month time span, producing over 50 hours of face-toace contact during the training. Program leaders designed the learning experience to include effective professional development strategies; reflective assignments; and activities that related to citizen science, experiential learning, and science and engineering practices found in the recently adopted Tennessee State Science Standards. This study identified concepts of the planned, delivered, and received curricula of the workshop series to define the intentions, methodologies, and impact of the experience. The intentions of the program were aligned to the delivered curricula then the impact of the program was considered. Data collected during this qualitative study included over 15 hours of interviews; over 110 hours of observation field notes; and various artifacts including journals, handouts and applications. This long-term professional development provided a pre-established community of practice and advocated for experiential instruction in outdoor spaces; eliminating barriers; improving teacher confidence and implementation of knowledge gained; and reinforcing the professional development experience.

Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancers (TNBCs) are negative for the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. TNBC accounts for 15% of all breast cancers and results in disproportionally higher mortality compared to ER & HER2-positive tumours. Moreover, there is a paucity of therapies for this subtype of breast cancer resulting primarily from an inadequate understanding of the transcriptional differences that differentiate TNBC from normal breast. To this end, we embarked on a comprehensive examination of the transcriptomes of TNBCs and normal breast tissues using next-generation whole transcriptome sequencing (RNA-Seq). By comparing RNA-seq data from these tissues, we report the presence of differentially expressed coding and non-coding genes, novel transcribed regions, and mutations not previously reported in breast cancer. From these data we have identified two major themes. First, BRCA1 mutations are well known to be associated with development of TNBC. From these data we have identified many genes that work in concert with BRCA1 that are dysregulated suggesting a role of BRCA1 associated genes with sporadic TNBC. In addition, we observe a mutational profile in genes also associated with BRCA1 and DNA repair that lend more evidence to its role. Second, we demonstrate that using microdissected normal epithelium maybe an optimal comparator when searching for novel therapeutic targets for TNBC. Previous studies have used other controls such as reduction mammoplasties, adjacent normal tissue, or other breast cancer subtypes, which may be sub-optimal and have lead to identifying ineffective therapeutic targets. Our data suggests that the comparison of microdissected ductal epithelium to TNBC can identify potential therapeutic targets that may lead to be better clinical efficacy. In summation, with these data, we provide a detailed transcriptional landscape of TNBC and normal breast that we believe will lead to a better understanding of this complex disease.

The goal of this dissertation in practice was to create professional development and curriculum modules focused on historical literacy in order to help teachers fully engage students in learning historical literacy skills. Historical literacy is the ability to understand the importance of the source of a document, being able to close read a text, to place a source within its proper context, and to corroborate the information from one source to another. The implementation of a program of this nature is designed to help teachers and students develop these skills with the hope that it positively impacts not only student learning in the social studies classroom, but will also have a positive impact on student test scores, student college experiences, students* future careers, and students* role within our civic society. Included within this dissertation in practice is a model for how to facilitate an effective professional development program that helps increase teacher efficacy, teacher skill level, and teacher use of historical literacy. This model pays special attention to ensuring that teachers also see how the demands of various standards and teacher evaluation systems can be addressed through the use of historical literacy. Suggested use for this dissertation in practice is the creation of professional development programs that help schools implement best practices throughout the learning organization.

<p>  </p> <p>Plant complex traits are controlled by multi-layer of dynamic and complicated gene networks regulated at different levels. To better inform crop breeding to promote desired traits, a comprehensive and fundamental understanding of their genetic basis is much needed. With the rapid developments of <em>omics</em> planforms and next generation sequencing technology, we now have large-scale data from genome, epigenome, transcriptome, metabolome, and others for the crop plants. Integration of those multiple <em>omics</em> data together with computational approaches led to the establishment of a novel science known as system biology. Research described in this thesis used system biology approaches to dissect complex crop traits such as disease response of tomato (Chapter2 and Chapter3) and the heterosis of nitrogen use efficiency of maize (Chapter4).</p> <p>Plant disease response is an elaborate, multilayered complex trait involving several lines of defense signaling. In the past decades, progress in molecular analyses of plant immune system has revealed key elements of a complex response network in Arabidopsis, a model species. Histone modifications, a type of epigenetic regulation, have emerged as key modulators that regulate defense responses, while our understanding of the role of histone-modifying enzymes in this process is still in its infancy. Here, we described the immune function of two histone methyltransferases SDG33 and SDG34 in tomato. We found the single mutants in <em>sdg33</em> and <em>sdg34</em> showed increased susceptibility to hemibiotrophic bacterial pathogen <em>Pseudomonas syringae</em> whereas the double mutant <em>sdg33sdg34</em> is comparable to wild type. Using RNA-seq and histone ChIP-seq approaches, we investigated the possible underlying mechanisms and found that the expression of a set of immune-related genes is misregulated by <em>P. syringae</em> only in the single mutants but not in the double mutant. Integrating with epigenomic data, we found that the misexpression of those SDG33/SDG34 dependent immune-response genes was associated with altered histone methylation status in the single mutant. Intriguingly, the double mutant also showed altered histone methylation but unaffected gene expression, suggesting a compensating regulatory mechanism at play. The function of SDG33 and SDG34 in immune response seems to be specific for the pathogen, as the double mutants exhibited enhanced resistance the single mutants showed no altered responses when treated with necrotrophic fungal pathogen <em>Botrytis cinerea</em>. Network analysis found the most regulatory gene by <em>B. cinerea</em> in a SDG33/SDG34 dependent manner have been implicated in biotic stress response such as <em>ERF4, TOPLESS, PUB23 </em>and<em> RCD1</em>. Comparing the immune response of double mutant against <em>P. syringae</em> and <em>B. cinerea</em>, we found that the disease related genes are only mis-regulated in the interaction of <em>B. cinerea</em> treatment not in the <em>P. syringae</em> treatment, which could be the reason of enhanced resistance to <em>B. cinerea</em> but not for <em>P. syringae</em> in the double mutants. In summary, we found the histone methyltransferases SDG33 and SDG34 has different functions in the immune response against <em>P. syringae</em> and <em>B. cinerea</em>, which might be direct or indirect relevant to the histone methylation level of the expression of downstream immune related gene.</p> <p>In addition to biotic stress, another complex trait studied in this thesis is the heterosis of nitrogen use efficiency (NUE) in Maize. NUE is another complex trait associated with multiple physiological processes including N sensing, uptake, assimilation, transport, and storage. Heterosis refers to a phenomenon where the progeny generated by crossing two different cultivars of the same species exhibit superior fitness than the inbred parents. Even though, heterosis has been exploited to improve complex traits including NUE, the underlying molecular mechanisms is not completely understood. Here, we analyzed N-responsive transcriptomes and physiological traits of a panel of six maize hybrids and their corresponding inbreds grown in the field at two different N levels. We observed diverse levels of trait heterosis that are dependent on the N conditions and organ types. We discovered dramatic pattern shift of beyond-parental-range gene expression in hybrids in response to varying N levels. We identified through integrative analyses a set of genes whose expression heterosis are quantitatively correlated to trait heterosis. These genes are involved in response to stimulus, photosynthesis, and N metabolism, and likely mediate the heterosis phenotype of N-use and growth traits in maize. In summary, our integrated analysis provided insights into the mechanistic basis of the heterosis of NUE. </p> <p>Together, applying systems and functional genomics approaches to investigate important agricultural traits could lead to a comprehensive understanding of plant complex traits to inform future engineering and breeding for better crops.</p>

My dissertation comprises three related topics of evolutionary and computational biology, which correspond to the three Chapters. Chapter 1 focuses on tempo and mode of evolution in visual genes, namely opsins, via duplication events and subsequent molecular adaptation in Odonata (dragonflies and damselflies). Gene duplication plays a central role in adaptation to novel environments by providing new genetic material for functional divergence and evolution of biological complexity. Odonata have the largest opsin repertoire of any insect currently known. In particular our results suggest that both the blue sensitive (BS) and long-wave sensitive (LWS) opsin classes were subjected to strong positive selection that greatly weakens after multiple duplication events, a pattern that is consistent with the permanent heterozygote model. Due to the immense interspecific variation and duplicability potential of opsin genes among odonates, they represent a unique model system to test hypotheses regarding opsin gene duplication and diversification at the molecular level. Chapter 2 primarily focuses on reconstruction of the phylogenetic backbone of Odonata using RNA-seq data. In order to reconstruct the evolutionary history of Odonata, we performed comprehensive phylotranscriptomic analyses of 83 species covering 75% of all extant odonate families. Using maximum likelihood, Bayesian, coalescent-based and alignment free tree inference frameworks we were able to test, refine and resolve previously controversial relationships within the order. In particular, we confirmed the monophyly of Zygoptera, recovered Gomphidae and Petaluridae as sister groups with high confidence and identified Calopterygoidea as monophyletic. Fossil calibration coupled with diversification analyses provided insight into key events that influenced the evolution of Odonata. Specifically, we determined that there was a possible mass extinction of ancient odonate diversity during the P-Tr crisis and a single odonate lineage persisted following this extinction event. Lastly, Chapter 3 focuses on identification of erroneously assigned sequence homology using the intelligent agents of machine learning techniques. Accurate detection of homologous relationships of biological sequences (DNA or amino acid) amongst organisms is an important and often difficult task that is essential to various evolutionary studies, ranging from building phylogenies to predicting functional gene annotations. We developed biologically informative features that can be extracted from multiple sequence alignments of putative homologous genes (orthologs and paralogs) and further utilized in context of guided experimentation to verify false positive outcomes.