Chapter 1. The family Pteronarcyidae (Plecoptera) is a highly studied group of stoneflies and very important to a wide variety of aquatic studies. Several phylogenies have been proposed for this group recent decades, however there is little congruence between the various topologies. The present study revises the phylogeny of the group by combining molecular data from mitochondrial cytochrome oxidase subunit II, ribosomal subunit 12S, ribosomal subunit 16S, and nuclear loci ribosomal subinit 18S and Histone H3, with published morphological data in a parsimony-based total evidence analysis. The analysis produced a well-supported phylogeny with novel relationships within the genus Pteronarcys. Maximum Likelihood and Bayesian analyses produced topologies congruent with parsimony analysis. Character mapping revealed several homoplasious morphological characters that were previously thought to be homologous. Chapter 2. Phylogeographic studies in aquatic insects provide valuable insights into mechanisms that shape the genetic structure of aquatic communities. Yet studies that include broad geographic areas are uncommon for this group. We conducted a broad scale phylogeographic analysis of P. badia across western North America. In order to allow us to generate a larger mitochondrial data set, we used 454 seqeuncing to reconstruct the complete mitochondrial genome in the early stages of the project. Our analysis reveals what appears to be a complex history of isolation and multiple invasions among some lineages. The study provides evidence of multiple glacial refugia and suggests that historical climactic isolations have been important mechanisms in determining genetic structure of insects in western North America. Our ability to generate a large mitochondrial data set through mitochondrial genome reconstruction greatly improved nodal support of our mitochondrial gene tree, and allowed us to make stronger inference of relationships between lineages and timing of divergence events.

The grain amaranths (Amaranthus sp.) and quinoa (Chenopodium quinoa Willd.) are important seed crops in South America. These crops have gained international attention in recent years for their nutritional quality and tolerance to abiotic stress. We report the identification and development of functional single nucleotide polymorphism (SNP) assays for both amaranth and quinoa. SNPs were identified using a genome reduction protocol and next generation sequencing. SNP assays are based on KASPar genotyping chemistry and were detected using the Fluidigm dynamic array platform. A diversity screen consisting of 41 amaranth accessions showed that the minor allele frequency (MAF) of the amaranth markers ranged from 0.05 to 0.5 with an average MAF of 0.27. A diversity screen of 113 quinoa accessions showed that the MAF of the quinoa markers ranged from 0.02 to 0.5 with an average MAF of 0.28. Linkage mapping in amaranth produced a linkage map consisting of 16 linkage groups, presumably corresponding to each of the 16 amaranth haploid chromosomes. This map spans 1288 cM with an average marker density of 3.1 cM per marker. Linkage mapping in quinoa resulted in a linkage map consisting of 29 linkage groups with 20 large linkage groups, spanning 1,404 cM with a marker density of 3.1 cM per SNP marker. The SNPs identified here represent important genomic tools needed for genetic dissection of agronomically important characteristics and advanced genetic analysis of agronomic traits in amaranth and quinoa. We also describe in detail the scalable and cost effective SNP genotyping method used in this research. This method is based on KBioscience's competitive allele specific PCR amplification of target sequences and endpoint fluorescence genotyping (KASPar) using a FRET capable plate reader or Fluidigm's dynamic array high throughput platform.

In 2013, a new set of science standards was introduced for K-12 science education, called the Next Generation Science Standards (NGSS), which focused on three dimensions of science learning that work together: disciplinary core ideas, crosscutting concepts, and science and engineering practices. These standards are novel in their emphasis on students needing more than only content knowledge to learn science and engineering. The NGSS science and engineering practices (SEPs) stress the importance of students engaging in the authentic practices of scientists and engineers to help them think like a scientist, practice science themselves, and overcome the misconception that science is a collection of isolated facts (NRC, 2012a). These SEPs include: asking questions and defining problems, developing and using models, planning and carrying out investigations, analyzing and interpreting data, using mathematics and computational thinking, constructing explanations and designing solutions, engaging in argument from evidence, and obtaining, evaluating, and communicating information. We performed a qualitative study to determine the conceptions of undergraduate students, the majority of whom were enrolled in STEM majors, about the SEPs. With a theoretical framework of phenomenography guiding our study's design, we conducted deep, open-ended interviews with 59 undergraduate students. The analysis consisted of transcribing interviews, coding transcriptions, writing descriptions of the codes to identify a limited number of distinct categories that describe how students viewed individual SEPs, and analyzing relationships among these categories to create outcome spaces for the ways students viewed the SEPs. In the current dissertation, we present the students' specific conceptions of each of the SEPs. Although the students had a better understanding of some of the SEPs than others (e.g., they understood planning and carrying out investigations and using mathematics and computational thinking best), we found that undergraduates did not have a high level of understanding of any SEP. This is similar to conceptions of undergraduates in the literature, which mostly consisted of preservice teachers. The undergraduates in this study conflated many of the SEPs, misinterpreted SEPs based on the everyday meaning of terms in the practices (i.e., they discussed an everyday meaning of words like "questions" instead of focusing on the unique meaning of "questions" in a scientific context), and perceived that the structured learning activities in which they engage in their current coursework limit their abilities to engage in the SEPs. These results suggest that STEM students need more opportunities to authentically engage in the SEPs in open-ended environments, coupled with explicit instruction that emphasizes the difference between everyday usages of words in the SEPs and their scientific meanings.

Next-generation Internet will be governed by the need for flexibility. Heterogeneous end-systems, novel applications, and security and manageability challenges require networks to provide a broad range of services that go beyond store-and-forward. Following this trend, a service-centric network architecture is proposed for the next-generation Internet. It utilizes router-based programmability to provide packet processing services inside the network and decompose communications into these service blocks. By providing different compositions of services along the data path, such network can customize its connections to satisfy various communication requirements. This design extends the flexibility of the Internet to meet its next-generation challenges. This work addresses three major challenges in implementing such service-centric networks. Finding the optimal path for a given composition of services is the first challenge. This is called "service routing" since both service availability and routing cost need to be considered. Novel algorithms and a matching protocol are designed to solve the service routing problem in large scale networks. A prototype based on Emulab is implemented to demonstrate and evaluate our design. Finding the optimal composition of services to satisfy the communication requirements of a given connection is the second challenge. This is called "service composition." A novel decision making framework is proposed, which allows the deduction of the service composition problem into a planning problem and automates the composition of service according to specified communication requirements. A further investigation shows that extending this decision making framework to combine the service routing and service composition problems yields a better solution than solving them separately. Run-time resource management on the data plane is the third challenge. Several run-time task mapping approaches have been proposed for Network Processor systems. An evaluation methodology based on queuing network is designed to systematically evaluate and compare these solutions under various network traffic scenarios. The results of this work give qualitative and quantitative insights into next-generation Internet design that combines issues from computer networking, architecture, and system design.

Next Generation Networks (NGNs) aim to provide a unified network infrastructure to offer multimedia data and telecommunication services through IP convergence. NGNs utilize multiple broadband, QoS-enabled transport technologies, creating a converged packet-switched network infrastructure, where service-related functions are separated from the transport functions. This requires significant changes in the way how networks are managed to handle the complexity and heterogeneity of NGNs. This thesis proposes a Service Oriented Architecture (SOA) based management framework that integrates heterogeneous management systems in a loose coupling manner. The key benefit of the proposed management architecture is the reduction of the complexity through service and data integration. A network management middleware layer that merges low level management functionality with higher level management operations to resolve the problem of heterogeneity was proposed. A prototype was implemented using Web Services and a testbed was developed using trouble ticket systems as the management application to demonstrate the functionality of the proposed framework. Test results show the correcting functioning of the system. It also concludes that the proposed framework fulfils the principles behind the SOA philosophy.

With the rapid progress of next-generation sequencing (NGS) technologies, new tools and methods have emerged to investigate the transcriptomics of various organisms. RNA sequencing (RNA-seq) employs NGS to evaluate the presence and abundance of RNA transcripts in biological samples. This technique offers a comprehensive snapshot of the RNA dynamics within cells. With the ability to profile the entire transcriptome of organisms rapidly and accurately, RNA-seq has become the state-of-the-art method for transcriptome profiling, surpassing the traditional microarray approach. Single-cell RNA sequencing (scRNA-seq) was introduced in 2009 to profile the single-cell gene expression in highly heterogeneous samples such as brain tissue and tumors. The advancement of scRNA-seq technologies enables the in-depth transcriptomic study in each cell subtype. When selecting an scRNA-seq method, researchers must weigh the trade-off between profiling more single cells versus obtaining more comprehensive transcripts per cell, while considering the overall costs. The throughput of full-length scRNA-seq methods is usually lower, as each single cell needs to be processed separately to produce scRNA-seq libraries. However, full-length methods enable the researchers to investigate the splicing variants and allele-specific expression. Non-full-length methods only capture the 3' or 5' ends of transcripts, which limits their application in isoform detection, but as cells are pooled after barcoding for cDNA synthesis, the throughput is 2–3 orders of magnitude higher than full-length methods. We developed a droplet-based platform for full-length single-cell RNA-seq, which enabled the efficient recovery of full-length mRNA from individual cells in a high-throughput manner. The developed platform can process ~8,000 single cells within 2 days and detect ~20% more genes compared to Drop-seq. Besides scRNA-seq technology development, we also applied a low-input RNA-seq method to study the transcriptomics in different biological samples. When handling precious biological samples, a low-input method is necessary to profile the transcriptome of homogeneous cell populations. We first studied the epigenomic and transcriptomic regulations in colorectal cancer (CRC) using MOWChIP-seq, a low-input high-throughput method, in conjunction with our low-input RNA-seq approach. Fusobacterium nucleatum (Fnn) is closely related to the progression of cancers like CRC and pancreatic cancer. However, the molecular mechanisms of how Fnn adjusts the tumor microenvironment (TME) and leads to poor clinical outcomes are still unclear. In this in-vitro study, we characterized how hypoxia, an important TME ignored by previous research, facilitates Fnn infection of CRC and corresponding alterations of global epigenome and transcriptome. We infer that hypoxia has similar effects as Fnn infection alone on the CRC cells. The Fnn infection under hypoxia further boosts the proliferation and progression of CRC. We then applied our low-input RNA-seq method to study brain neuroscience and immunology. Psychedelics like DOI show promising clinical efficacy in patients with psychiatric conditions. Although psychedelics exhibit rapid antidepression action and long-lasting effectiveness compared to conventional treatment, their acute psychotic symptoms and potential for drug abuse discourage their application in clinical practice. In this case, it is important to comprehend the molecular mechanisms responsible for psychedelics' clinical efficacy. This understanding can pave the way for the development of improved treatments that do not rely on psychedelics. After profiling the transcriptome of mouse brain samples exposed to psychedelics with different post-exposure times, we concluded that the psychedelic-induced transcriptomic variations are more transient than epigenomic changes. In the second brain neuroscience project, we first applied 3-color FACS sorting to differentiate four neuron and non-neuron subtypes in human postmortem prefrontal cortex tissues. Then we profiled the gene expression of the four subtypes and validated the FACS sorting by examining the expression of marker genes. Differentially expressed genes between each subtype and the others were extracted and proceeded to gene ontology analysis. We identified unique altered biological pathways related to each subtype. The immunology research focuses on revealing the difference between low-grade inflammation and monocyte exhaustion, as well as the unique biological pathways they regulate. Therefore, we profiled the transcriptome of bone marrow-derived monocytes stimulated by PBS control, a low- or high-dose LPS. In addition to wild-type mice, we also included TRAM-deficient and IRAK-M-deficient mice. We concluded that low-dose LPS specifically regulates the TRAM-dependent pathway of TLR4 signaling, and high-dose LPS exclusively upregulates exhaustion markers by impacting metabolic and proliferative pathways. / Doctor of Philosophy / Transcriptomics is the comprehensive study of RNA transcripts derived from an organism's genome. RNA plays a vital role in maintaining the fundamental functions of cells and organisms. In eukaryotes, the genetic information stored in the DNA of cells is transferred to messenger RNA (mRNA) molecules through a process called transcription. These mRNA molecules serve as a bridge between DNA and proteins, as they carry the instructions encoded in genes to ribosomes for protein synthesis. Studying mRNA transcripts reveals various cellular mechanisms and their impact on overall organism function, gene regulation, and disease pathways. With the aid of next-generation sequencing, various RNA-seq approaches have been developed to study mRNA transcripts quantitatively in the past decades. To better understand the gene expression regulations in biological samples, we first applied bulk RNA-seq to profile the transcriptome of various samples under different conditions. Our in-house bulk RNA-seq protocol has been proven to be both high-performance and cost-effective compared to commercial kits. To better understand cellular diversity and uncover rare cell types in heterogeneous biological samples, we developed a droplet-based scRNA-seq platform that can recover full-length mRNA transcripts in a high throughput manner. It can profile the transcriptome of thousands of single cells within two days. It combines the advantages of the droplet-based scRNA-seq method (high throughput) and the well plate-based scRNA-seq method (full-length mRNA recovery).

For over thirty years, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has been a major contributor to morbidity and mortality in the commercial swine industry across the globe. This highly mutagenic RNA virus causes significant economic losses wherever it is prevalent, leading to $664 million in annual losses in the United States. Unfortunately, the current prevention and diagnostic techniques available have proven to be insufficient in controlling the spread of this disease. We describe an alternative diagnostic method exploiting the rapid turnaround time and long-read capacity of Oxford Nanopore Technology's MinION next-generation sequencer. We have developed a novel primer set designed to span Open Reading Frames 3 through 7 of the PRRSV genome, which has allowed for multiplexing of samples, thus reducing individual cost of testing, while yielding significantly more information than previously available. This novel primer pair and sequencing technique have distinguished mixed infections within individual animals and may be used to determine vaccination status. This new approach will help producers and veterinarians make better-informed decisions about co-mingling of animals and vaccination strategies, thus reducing the emergence of new, pathogenic strains of PRRSV. / Master of Science / Porcine reproductive and respiratory syndrome virus (PRRSV) is a common, economically important pathogen in commercial swine production. The virus was first identified in the late 1980's during outbreaks in the United States and Europe. In female pigs, the disease is characterized by abortion storms, and the delivery of mummified fetuses or very weak, ill piglets. Neonates often display signs of pneumonia, respiratory distress, and many die from hypoxia. Surviving piglets are highly susceptible to other diseases and are poor growers compared to other, unaffected piglets. Boars may show signs of respiratory disease and can also have decreased libido and reproductive success for months at a time. The virus is prone to mutating once a pig is infected, preventing herds from mounting sufficient immunity to protect against new, mutant strains. Identifying infected pigs early and accurately is crucial to managing PRRSV outbreaks. Currently available diagnostic tests for PRRSV have many limitations, thus we have developed a new diagnostic test using next-generation sequencing technology. Oxford Nanopore Technology provides a commercially available nanopore sequencer, the MinION, that can read long DNA strands in real-time. With this technology we have expanded the area of the PRRSV genome that can be sequenced, which allows us to better identify and distinguish strains of PRRSV in infected, and vaccinated pigs. This new testing method will allow veterinarians and practitioners across the country to better identify and predict outbreaks in their herds, helping them develop better management strategies against PRRSV.

As zoos and aquariums have become increasingly focused on conservation education, their menageries of unique and diverse learning opportunities have been underutilized. Through a new postsecondary-level animal behavior laboratory experience at an aquarium (“ZooU”), this study demonstrates that active learning pedagogy aligned with the Next Generation Science Standards (NGSS) could facilitate expansion of education at zoos and aquariums beyond their conservation education niche. Generally, students indicated that ZooU provided new opportunities for them to explore their own interests, demonstrate their learning, and augment their previous laboratory and aquarium experiences. Following both self- and researcher assessments of the students’ work, integrated analyses revealed that students who engaged in more active learning activities at the aquarium demonstrated a greater increase in skills aligned with the NGSS. Additionally, a novel intra-individual analysis was utilized to embrace the variation between learners that typically confounds the results of education studies with repeated measures design. Common challenges for education at zoos and aquariums are discussed through the context of ZooU as a foundation for future investigations. A practical NGSS-aligned guide to field trips at zoos and aquariums—written specifically for science teachers—is also included to support broader utilization of zoos and aquariums as educational resources.

The maritime industry is growing more and more for every year that passes. As the industry grows it also becomes a more attractive target for cyber criminals. The amount ofcyberattacks in the industry are few, but it is growing at an alarming rate. This literaturestudy identifies the most common datacom systems and infrastructure in the maritimeindustry and their vulnerabilities. This paper also identifies possible solutions and improvements that can be made to existing datacom systems to make them less susceptible tocyber attacks. The results show that there are many solutions that could be implementedthat would increase the cyber security in the industry, but many of them require international cooperation to implement. Therefore standards are suggested to be implemented inorder to push organisations to update their systems. Additionally, this paper delves intothe aviation industry to examine how the datacom infrastructure utilized in the maritimeindustry could be adopted to enhance both efficiency and security

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24241号 / 農博第2520号 / 新制||農||1094(附属図書館) / 学位論文||R4||N5412(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 宮川 恒, 教授 三芳 秀人, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM