Both technological and experimental advancements in the field of biotechnology have allowed scientists to make leaps in areas such nucleic acid, antibody, and recombinant protein technologies. Here we focus on the use of recombinant proteins as molecular recognition motifs, wound healing biomaterials, and agents for cell cycle pathway elucidation are discussed. The author's primary project is described in chapters 2 and 3, and is focused on designed leucine-rich repeat proteins which offer increased stability, modularity, and surface area for binding interactions. These proteins bind at least two muramyl dipeptide ligands with picomolar to nanomolar affinity (Kd1 = 0.04 – 3.5 nM); as measured by fluorescence quenching experiments and ITC. The longest designed repeat, CLRR8, has a Kd app value of 1.0 nM which is comparable to full length native NOD2 protein. Molecular docking simulations revealed the locations of two potential binding sites and their respective interactions. The series of proteins represents a foundation for a high affinity and highly specific molecular recognition scaffold that has the potential to bind a variety of ligands. Previously the author contributed to the design of recombinant keratin proteins, and the work in Chapter 4 builds on the original design to allow for controlled degradation in wound healing systems. Site-directed mutagenesis was utilized to introduce these degradation sites, and modified keratin proteins were expressed with no differences to native recombinant keratin proteins. Success in engineering a variation of native keratin protein with no issues in expression lay the foundation for further engineering of native keratin or other relevant proteins for improved functionality. Chapter 5 describes steps towards producing human Aurora borealis (Bora) protein, an important substrate in cell cycle regulation, by in vitro transcription-translation with locked Ser–Pro analogues. This will allow for the elucidation of the active isomerization form to ensure proper cell division. Site-directed mutagenesis successfully introduced the amber codon to relevant Ser-Pro sites at positions 274 and 278. These mutated Bora genes along with modified ribosomes and aminoacyl tRNA will allow for the incorporation of locked dipeptide analogues. Expression of native Bora was carried out as a control, and appeared to express in dimeric form. The experiments carried out in Chapter 5 describe and outline all the molecular biology work completed and to be completed for this novel method of studying cis-trans isomerization in living cells. / Doctor of Philosophy / Sequencing of the human genome and the rapid development of gene editing and recombinant DNA technologies paved the way for a massive shift in the pharmaceutical industry. The first pharmaceutical companies in the 19th century started as fine chemicals businesses. The discovery of penicillin introduced antibiotics, and improved synthetic techniques led to the giants we know as big pharma today. Today, in the 21st century both computing and biotechnology has allowed for great leaps forward in precision medicine. Biotechnology refers to the manipulation of living organisms or their components to produce useful commercial products. In the pharmaceutical industry this refers to genetic engineering for novel pharmaceuticals. Here, we focus on the use of recombinant technology to create proteins for use in biomedical applications. Recombinant proteins are proteins formed by laboratory methods of molecular cloning. Through this technology, we are able to elucidate sequence-structure-function relationships of proteins, and determine their specific functions. Additionally, recombinant methods allow us to fine tune or modify the sequences of natural proteins to be more effective scaffolds or reagents. Chapter 3 focuses on the development of synthetic proteins for medical diagnostics. We designed a protein scaffold, based on natural innate immunity proteins, to detect bacteria cell wall components. Chapter 4 focuses on the engineering of keratin protein with applications in wound healing. We introduce controlled degradation of the biomaterial for use in potential drug delivery systems at the wound site. Chapter 5 focuses on the use of recombinant technologies aiding in the elucidation of a regulatory protein's function in cell division.

Soybean is a dominant oilseed cultivated worldwide for its use in multiple sectors such as food and feed industries, animal husbandry, cosmetics and pharmaceutical sectors, and more recently, in production of biodiesel. Increasing demand of soybean, changing environmental conditions, and evolution of pathogens pose challenges to soybean production in limited acreage. Genetic research is the key to ensure the continued growth in soybean production, with enhanced yield and quality, while reducing the losses due to diseases and pests. This research is focused on the understanding of transcriptional regulation of two economically important agronomic traits of soybean: low seed phytic acid and resistance to Soybean mosaic virus (SMV), using the 'transcriptomics' and 'genomics' approaches. The low phytic acid (lpa) soybean is more desirable than conventional soybean, as phytic acid is an anti-nutritional component of seed and is associated with phosphorus pollution. Despite the eco-friendly nature of the lpa soybean, it shows poor emergence, which reduces soybean yield. This research is mainly focused on addressing the impact of lpa-causing mutations on seed development, which is suspected to cause low emergence in lpa soybeans. The differences in transcriptome profiles of developing seeds in lpa and normal phytic acid soybean are revealed and the biological pathways that may potentially be involved in regulation of seed development are suggested. The second research project is focused on Rsv3-type resistance, which is effective against most virulent strains of Soybean mosaic virus. The Rsv3 locus, which maps on to soybean chromosome 14, contains 10 genes including a cluster of coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) protein-encoding genes. This dissertation employed a comparative sequencing approach to narrow down the list of Rsv3 gene candidates to the most promising CC-NB-LRR gene. The evidence provided in this study clearly indicates a single CC-NB-LRR gene as the most promising candidate to deliver Rsv3-type resistance. / Ph. D.

Soybean mosaic virus (SMV) is the most common member of the viral genus Potyvirus to infect soybeans (Glycine max [L.] Merr.) worldwide. SMV has been traditionally controlled by the deployment of single dominant, strain specific resistance genes, referred to as Rsv genes. Rsv1 is the most widely used form of SMV resistance with nine different alleles conferring resistance only to the lower numbered less virulent strains, G1 to G3. Rsv3 gives resistance to higher numbered more virulent strains G5 to G7. Soybean lines containing Rsv4, are resistant to all seven currently recognized North American SMV strains. In this study, the recently released soybean whole genome sequence was used to design molecular markers for fine mapping Rsv3 to a ~150 kb genomic region containing four coiled-coil nucleotide-binding leucine-rich repeat proteins. In a related study a large population segregating at the Rsv3 locus was screened for resistance to facilitate future characterization of this region. The markers identified in this study will allow for more accurate marker-assisted selection of Rsv3. / Master of Science

Over the past several decades, naturally occurring populations of Fraser fir (Abies fraseri) in the Black Mountains of North Carolina have been heavily impacted by both direct and indirect anthropogenic disturbances, including logging and logging- associated fires, and high mortality rates due to the introduction of the exotic insect, balsam woolly adelgid (BWA) (Adelges piceae). The decline in Fraser fir is particularly concern because it serves as a foundation species within the spruce-fir forests of the Southern Appalachian Mountains. Our objectives for this research were to 1) use current stand structure to infer whether Fraser fir trees are experiencing a cycle of regeneration-mortality that will lead to eventual decline of the population, 2) determine what role, if any, the site-specific geographic variables of slope, elevation, aspect, and land use history have on stand structure, mortality, and BWA infestation level, and 3) analyze repeat aerial photography to examine broad trends of spruce-fir forest cover change caused by anthropogenic disturbance and the BWA. In order to understand stand structure, mortality, and infestation levels, we conducted detailed field surveys of Fraser fir trees throughout the Black Mountains using 44, fixed-radius circular sampling plots. These plots were placed throughout a series of aspects, elevations, and disturbance types in order to understand geographic variability among these variables. An analysis of 4 repeat aerial photographs and corroborating ground photographs revealed broad spatio-temporal trends of spruce-fir regeneration and mortality from 1954 to 2006. Our results indicate that Fraser fir stands at higher elevations are currently in a state of recovery; whereas stands at lower elevations appear to be more susceptible to BWA-induced mortality. Changes in forest cover area from 1954 to 2006 were influenced greatly by direct and indirect anthropogenic disturbance. Our results call attention to the significant impact that direct and indirect anthropogenic disturbance has had on Fraser fir stand structure, but also provide evidence for the ability of an imperiled ecosystem to recover from high rates of insect caused mortality. / Master of Science

Even two years after the COVID-19 pandemic, the meeting and event industry's efforts must focus on understanding attendees' purchase behaviors and event modality preferences. This study aims to address these factors by using a 2 (Event modality: in-person or virtual) × 6 (six dimensions of perceived value: social value, emotional value, monetary value, functional value, novelty value, and convenience value) between-subject experiment design and a 6 (Six dimensions of perceived value (PV): social value, emotional value, monetary value, functional value, novelty value, and convenience value) x 2 (PV effect on word-of-mouth (WOM) intent and repeat purchase intent (RPI)) between-subject experimental design. Specifically, it applies the perceived value theory in investigating the effects of different value dimensions (social, emotional, monetary, functional, novelty, and convenience) based on attendee event modality (in-person or virtual) and their impact on WOM intent and RPI. The data was collected via Prolific. Different ANOVA and t-tests were conducted. The results suggest that event modality had little bearing on the six dimensions of PV. Results also showed that PV positively impacts WOM intent and RPI.

本研究運用多元資料，自不同面向揭露臺灣原住民的遷徙樣貌，並連結遷徙與原住民個人社會地位取得的關連。藉遷徙多層次社會鑲嵌的特質，以解開原住民頻繁遷徙但並未對等呈現向上社會流動的弔詭。本研究同時檢視原住民族於臺灣大社會的位置，包含其空間分布、流動趨勢及其社會經濟地位。研究主要發現如下：（1）歷經卅年的遷移，原住民族大量移徙西半部，並集中在三大都會區，不同遷徙類型在各區域形成流動體系，以北部體系及東部體系擁有較大的遷徙流量；（2）原住民族較一般民眾更易集中在中低度現代化區域，主要係往都會區周邊移動，臺北縣與桃園縣對初級與連續遷徙有極大的拉力；（3）原住民族與一般民眾的遷徙模式相近，遷徙主要是朝鄰近區域與核心縣市移動，但原住民族重複遷徙行為較為獨特，連續遷徙與回流遷徙呈相反的流動；（4）自遷徙決策模型可發現，遷徙受多重因素影響，除工作要素外，家庭居住安排、生命階段的居住區位、區域性資本、社會網絡與遷徙成本及預算皆會影響其遷徙決策；長遠而言，遷徙有助於個人取得教育資源、提升社經地位，無力遷徙者與遷徙者間貧富差距逐漸拉大；（5）與理論預期相反，初級與回流遷徙對個人地位取得具正面效益，連續遷徙則為負向作用，此與原住民族社會網絡有限鑲嵌及累積資本困難所致；（6）隨著人口移動，原居地與移入地社群重組，原居地經歷了人口老化、祖孫家庭增加、傳統部落秩序瓦解，移入地蓬勃的制度化社群組織、族群聚落、同鄉會與協進會扮演都市原住民與原鄉的橋樑，遷徙所生成的脈絡亦將影響後續移動者的社會處境。奠基以上研究發現，提出政策建議與未來研究方向。 / Mainly based on a variety of data, this research aims to study several aspects of migration of Taiwan aborigines and to explore the association and causal relationship between migration and the advance of socioeconomic status. This study is originally inspired from an observed paradox that, according to the theoretical expectation and a body of existing empirical evidences, it has long been confirmed that migration is an effective means of promoting individual social mobility and lifetime wellbeing; nevertheless, the fact that the Taiwan aborigines are associated with lower socioeconomic status does not fit the fact of Taiwan aborigines being more mobile than the ordinary people. The purposes of this dissertation are (1) to characterize migration types and pattern of Taiwan aborigines, including spatial pattern, migration and mobility tendency and likelihood, and their social economic status, (2) to distinguish determinants of aborigine migration, and (3) to examine the outcome of migration whether it helps or stumbles the advance of aborigine’s socioeconomic status and mobility. Main findings are as follows: (1) in the past three decades, voluminous aborigines migrated to the western urbanized area, with the three major metropolitan areas of Taiwan as the major destination for aborigine migrants; it also forms migratory system in each area, with northern Taiwan and eastern Taiwan gaining the most number of migrants; (2) Although metropolitan areas serve as major destination for aborigine migrants, the study finds that they tend to concentrate more on the periphery than on the core area. Both counties of Taipei and Taoyuan are very attractive for primary and onward migrants; (3) the migration pattern of ordinary people is similar to that of aborigines. People usually tend to move to neighborhood and the core city. In addition, repeat migration is much more noteworthy than its primary counterpart, and onward migration is totally opposite to return migration; (4) The model of aboriginal migration indicates that migration is affected by various factors. The most salient ones include work status, living arrangement, attributes of residential location, location-specific capital, ethnic network, and availability of migration budget. Because migration help acquire educational resources and improve one’s socioeconomic status, the gap between migrants and people who are not capable of making migration will become exaggerated; (5) in opposition to theoretical expectation, primary and return migrations exhibit positive effect on the improvement of individual socioeconomic status, whereas onward migration should have negative effect. This finding is not counter to various schools of migration theory, rather, it reflects a result of limited embedded inter- and intra-ethnic network and barriers of capital accumulation; (6) migration affects both communities of origin and destination. Aging population, increasing grandparent-grandchild family, collapsing tribal authority become prevalent in original community; on the other hand, flourishing ethnic enclaves, associations, and institutionalized organizations connect urban and hometown in destination community. The context which migration results from is changed by migration itself and further affects the situation of subsequent migrants. According to empirical findings, the dissertation further suggests corresponding policy implications and proposes future research direction.

The purpose of this study is to explore the differences and characteristics between first time felony probationer and recidivist felony probation offender. The importance of said studies grows significantly, given current trends of sentencing offenders to probation. Using archived data on random sample of felony offenders in 2000 and based on information acquired and maintained by the Denton County Community Supervision and Corrections Department (CSCD), the study consists of 40 first time felony offenders and 40 recidivist felony offender placed on probation during the year 2000. The method consists of a longitudinal comparison model. To examine the research question, descriptive statistics are used to compare basic demographics. Then, in order to answer the research question bi-variate significant tests, Chi-square and Independent Sample T-tests were employed when appropriate. Results indicate differences between first time felony probation offenders and recidivist felony probationers.

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres. / Dissertation

This study used a comparison of one hundred and forty-eight historical (1888-1913) and current (2008-2014) oblique photographs from thirty-two stations to identify land cover changes that have occurred in portions of the Bow and Elbow valleys as well as surrounding Kananaskis Country region. Implications of these changes for flooding and flood management were explored. Forest cover was found to have drastically increased over the past century, particularly in the Bow valley, as did areas of direct human development. In the same time period, grasslands increased in the Elbow valley but decreased in the Bow, while regenerating areas decreased uniformly throughout both valleys. An analysis of pre (2008)-and-post (2014) flood conditions demonstrated no change in coniferous forest cover in both valleys over the 6-year period, but uncovered a decline of 20% in the Elbow and 3% in the Bow in the broadleaf/mixedwood category. The Elbow’s channel zone was larger in 2014 compared to 2008, whereas the extent of the Bow’s channel zone remained constant. However, both the Bow and Elbow’s bare exposed bars increased substantially, most likely as a result of the 2013 flood. The major source of water flows that contributed to the 2013 flood event originated in high elevation rock and scree areas, which, unlike floodplains, are elements of the watershed that cannot be manipulated over time. It is now recognized that forest cover should act as a buffer to floods. Nevertheless, the 2013 flood event occurred despite the massive buffering effect of a huge increase in older forest stands across the study area. The final discussion includes recommendations for improving flood management in the area. / Graduate / 0329, 0768, 0478 / tanya.taggarthodge@gmail.com

Le paludisme, première endémie parasitaire mondiale ayant engendré près d’un demi-million de morts en 2017 (d’après l’OMS), est due à une infection par un parasite du genre Plasmodium. Cet apicomplexe infecte, au cours de son cycle de vie, un hôte définitif, un moustique femelle du genre Anopheles, et un hôte intermédiaire homéotherme (l’Homme pour au moins 6 espèces). Chez ce dernier, après une phase de développement hépatique, le parasite envahit puis lyse les érythrocytes. L’accroissement exponentiel de la parasitémie engendre les symptômes du paludisme et permet la production de formes sexuées (gamétocytes) qui seront transmises au vecteur arthropode, permettant ainsi la complétion du cycle de vie du parasite.Plasmodium a co-évolué avec ses hôtes et mis en place divers modes de régulation de l’expression de ses gènes. La phosphorylation est l’une des modifications post-traductionnelles majeures et rapides qu’il utilise pour répondre aux changements environnementaux auxquels il est confronté au cours de son cycle de vie. Nombre de ses kinases et phosphatases jouent un rôle essentiel dans l’invasion de cellules hôtes, la croissance et la division cellulaires, ainsi que la motilité de certains stades. En revanche, le rôle des cinq pseudokinases de Plasmodium dans son développement n’a jusqu’ici pas été exploré.Durant ma thèse, j’ai caractérisé l’unique pseudo-Tyrosine Kinase-like (pTKL) de Plasmodium et étudié son rôle au cours du cycle intra-érythrocytaire du parasite.L’annotation de la pTKL de P. falciparum (PfpTKL) m’a permis d’identifier différents domaines et motifs, et notamment un domaine SAM (Sterile Alpha Motif), deux motifs RVxF (connus pour leur capacité d’interaction avec la Protéine Phosphatase de type 1, PP1) et un pseudo-domaine kinase appartenant à la famille des Tyrosine Kinases-like (TKL). Nous avons montré que ce pseudo-domaine kinase est capable de lier l’ATP de manière cation-indépendante, mais est dépourvu d’activité enzymatique. Des études d’interaction in vitro couplées à l’utilisation de modèles hétérologues (Levure, ovocytes de Xénope) m’ont permis d’identifier deux protéines parasitaires partenaires de PfpTKL : le domaine SAM de PfpTKL interagit directement avec la pseudo-protéase PfSERA5 (SErine Repeat Antigen 5), alors que les deux régions de la protéine contenant les motifs RVxF de PfpTKL interagissent avec PfPP1c (phosphatase majeure de Plasmodium). De façon intéressante, le deuxième motif RVxF est directement impliqué dans l’interaction avec PP1c et serait capable de moduler l’activité de cette dernière de manière allostérique.La localisation de la pTKL de P. berghei (PbpTKL) a ensuite été étudiée par immunofluorescence et confirmée par des expériences de fractionnement cellulaire. Nous avons ainsi observé que PbpTKL est exportée dans l’érythrocyte infecté au stade trophozoïte, puis retenue dans le parasite et la vacuole parasitophore au stade schizonte. L’étude de l’interactome de PbpTKL par IP/MS au stade trophozoïte a montré que PbpTKL s’associe à diverses protéines impliquées dans l’organisation du cytosquelette de l’érythrocyte, ainsi que dans l’érythropoïèse et l’homéostasie cellulaire. Ces observations suggèrent que pTKL joue un rôle, direct ou via ses partenaires, à l’interface entre le parasite et sa cellule hôte.Enfin, afin d’approcher la fonction de pTKL chez le parasite, nous avons généré différentes lignées génétiquement modifiées. L’étude phénotypique des souches de P. berghei KO et iKD pour pTKL a montré qu’elle était dispensable pour la complétion du cycle intra-érythrocytaire, l’expression des gamétocytes ainsi que l’activation des gamétocytes mâles. Ces données suggèrent que pTKL est dispensable pour ces stades de développement ou que l’expression de gènes redondants compense son absence. Quoi qu’il en soit, il est important de poursuivre les recherches sur le rôle de cette protéine aux autres stades de développement du parasite, notamment du zygote aux stades hépatiques. / Malaria is the first endemic parasitic disease in the world with nearly half million deaths in 2017 according to the WHO. This disease is the result of an infection by an agent belonging to the Plasmodium genus. This apicomplexan parasite infects two hosts over its complex life cycle: a definitive one – a mosquito belonging to the Anopheles genus – and a homoeothermic intermediate host. At least six Plasmodium species can infect humans. In its intermediate host, Plasmodium first replicates in hepatocytes before releasing erythrocyte-infectious stages in the bloodstream. Once there, parasites invade and replicate within erythrocytes, before lysing them to release other infectious stages. This triggers an exponential rise in the parasitemia, as well as malaria symptoms. Sexual stages, called gametocytes, are produced over this intra-erythrocytic cycle to be transmitted to the arthropod vector, thus allowing the completion of the parasite life cycle.Plasmodium co-evolved with its hosts and set up diverse gene expression regulation pathways accordingly. Phosphorylation is one of the major and fastest post-translational modifications used by the parasite to respond to environmental changes. Many of its kinases and phosphatases play key roles in host cell invasion, cellular growth and division, as well as motility of specific developmental stages. However, the role of the five pseudo-kinases expressed by Plasmodium has not been explored yet.During my PhD project, I have performed the characterization of the unique Plasmodium pseudo-Tyrosine Kinase-like (pTKL) and explored its role over the parasite intra-erythrocytic cycle.P. falciparum pTKL (PfpTKL) in silico annotation allowed the delineation of the protein domains. Notably, a SAM (Sterile Alpha Motif) domain, two RVxF motifs (known for their binding potential with the major protein phosphatase type 1, PP1) and a pseudo-kinase domain belonging to Tyrosine Kinase-like (TKL) family were found. This pseudo-kinase domain was found to be able to bind ATP in a cation-independent way although devoid of kinase activity. Two parasite protein partners of PfpTKL have been identified using in vitro protein-protein interaction studies together with heterologous models (yeast, Xenopus ovocytes). First, PfSERA5 (SErine Repeat Antigen 5) specifically and strongly interacts with PfpTKL SAM domain and second, PfPP1c binds the two RVxF-containing regions of PfpTKL. Interestingly, the second RVxF motif, which is located within the pseudo-kinase domain, directly binds PfPP1c and seems to be involved in the allosteric regulation of the phosphatase activity. The subcellular localization of P. berghei pTKL (PbpTKL) was studied by IFA as well as sequential lysis of erythrocytes followed by immunoprecipitation assays. PbpTKL was shown to be exported to the host cell cytosol at the trophozoite stage, but retained in the parasitophorous vacuole and the parasite cytosol at the schizont stage. Furthermore, our interactome analysis conducted at the trophozoite stage by IP/MS showed that PbpTKL binds many host cell proteins involved in erythrocyte cytoskeleton organization, as well as erythropoiesis and cell homeostasis. These data suggest that pTKL plays a role at the parasite/host interface, either directly or via its protein partners.Finally, in an attempt to understand the role of pTKL for the parasite development, we generated genetically modified P. berghei strains. The phenotypic study of PbpTKL KO and iKD strains did not show any difference between the defective parasites and the parental wild type ones during the intra-erythrocytic cycle, gametocyte expression and male gametocyte activation. These data suggest the dispensability of pTKL or the expression of redundant gene(s) with similar functions in these parasite stages. Whatever the explanation, it is still important to follow up this investigation in other parasite stages, from zygotes to hepatic stages.