Search

Biotechnology has the potential to deliver solutions to many global problems in medicine, materials science, nutrition, agriculture, natural resource preservation, and energy. Engineered cells and enzymes can perform chemical transformations that are rare or unknown in nature, and even catalyze reactions not accessible to traditional synthetic chemistry while also operating at gentler, more environmentally friendly conditions. Decreases in the price of DNA sequencing and synthesis has led to generation of vast databases that can be screened for any imaginable function. These sequence databases are even more powerful now due to the development of software to enable rapid generation of 3D protein structures, like AlphaFold2. However, tools to predict the function of these proteins or their performance in engineered cells are not yet robust, leading to long development times and limited successful applications to date. New tools and methods must be developed for the true potential of biotechnology to be unlocked.For my thesis, I explore metabolic pathway construction and screening, protein design, and protein sequence-structure-function relationships across broad contexts with the objective of tool and knowledge development for future efforts in biotechnology. My first chapter discusses the introduction of the triazine degradation pathway, of interest for remediation of contaminated sites, into E. coli and methods for characterizing pathway flux and identifying bottlenecks to guide engineering efforts and limit accumulation of metabolic intermediates. My second chapter focuses on methods for the design of supercharged proteins, which have many interesting potential applications, and parameters that increase the likelihood of successful design of these proteins. My third chapter regards the generation and characterization of a library of single point mutations in the enzyme B-glucosidase B and use of kinetic data to predict the effects of changes in sequence on enzyme function. These seemingly disparate topics all serve to improve tools for protein screening, production, functional prediction, and application, addressing several gaps toward improved development timelines and success rates for biocatalysts.

Owing to the fact that invasive species have a strong impact on native communities, much effort has been made to understand why they become problematic following their introduction to novel habitats. My thesis examined Silene latifolia, an invasive weed in North America that was introduced from Europe about 200 years ago. During the introduction process this plant species escaped its natural enemies and evolved a weedier phenotype in North America. However, adaptive evolution in an invasive species may seem counterintuitive. A bottleneck occurred in the colonization of Silene in North America involving only a small subset of the variation present in native Europe, and this founder effect could have theoretically reduced the amount of genetic (and therefore phenotypic) variation present in the introduced range. My thesis explored if reduced genotypic variation in North American Silene has resulted in a decrease in the expression of phenotypic variation. Furthermore, I examined whether the magnitude of variation was sensitive to plant density. To test this, a common garden experiment was conducted comparing the variation (coefficient of variation) in life history and morphological traits of native and introduced populations (25 each from Europe and North America) in different densities (1, 3, and 9 plants per pot). There were two major results. First, there was significant among-population variation in most life history and morphological traits on each continent despite the bottleneck event. Second, there was a pattern of decreased phenotypic variation among traits with increasing density and the amount of variation was greater in Europe than in North America; however, these differences were not statistically significant. The results of my research demonstrate that despite undergoing a founder event during its colonization, significant levels of genetic variation exist in important life history and morphological traits in introduced populations of Silene latifolia.

This dissertation outlines research quantifying the entering and exiting the quinone reaction chamber in NADH dehydrogenase or respiratory complex I. Respiratory complex I, the first complex in the respiratory electron transport chain. The respiratory electron transport is essential for all aerobic life. The methods used to quantify the entrance and exit process are geometric modeling, steered molecular dynamics, and singular value decomposition of the process. Five structures were analyzed: bacterial, yeast, ovine mt, mice mt, and human complex I. The structures reveal an almost 30 angstrom tunnel-like chamber for quinone binding in the core part of the enzyme, at the joint between the membrane and hydrophilic arms of the enzyme. The entrance of this quinone chamber located in ND1 subunit and has an apparent bottleneck of quinone/quinol passage. The first chapter introduces complex I and how transition state theory using diffusion kinetics gives an approximate maximum for the energy of crossing the bottleneck for quinone/quinol passage. Chapter 2 introduces the techniques used to quantify the difficulty of passage as well as methods to identify modes for collective confirmational changes for bottleneck opening. Chapters 3 and 4 are reproductions of the published papers based on this work. The appendices are reproductions of the supplemental information for those two papers.

The complexity and redundancy in the network topology of the cell hinder our understanding of the natural system, making it challenging to engineer living organisms with the desired functionality. The long-term goal of synthetic biology and bioengineering is to engineer cells to perform specific functions with increased robustness. The robust engineered systems can be easily applied to different species with the same outcome. An effective approach is a bottom-up approach where we tease apart the biological pathways and study them independently. Understanding the underlying mechanisms of biological pathways can be further applied to construct complex biological circuits that function similarly to natural systems. In this study, I have used the Design-Build-Test-Learn cyclic approach using a widely used organism, E. coli, to engineer synthetic circuits that relied on post-translational, degradation, and transcriptional control. This work is novel because traditional synthetic circuits often rely only on transcriptional control. Synthetic circuits contain engineered genetic frameworks that rely on basic principles of the natural system. I took advantage of the vast knowledge of biological queueing theory to develop robust synthetic circuits. Queueing theory is the study of waiting lines caused due to the bottleneck in the system. It is classically applied to common human activities such as line form (queues) and dissipation at grocery stores, roadways, at the airport, the flow of traffic at traffic signals, etc. Our approach takes the available knowledge we have gained in the macro-world and applies it to the micro (bacteria) and nano-world (proteins, RNA, etc.). The use of queueing theory is explained in detail in Section 1.4. In this study, I examined and utilized the queue formed with proteins at the proteolytic system. Proteases in the cell are limited in numbers (bottleneck) where the queue forms when proteins targeted to proteolysis are waiting in the line to be degraded. I used the understanding of proteolytic queueing and constructed circuits that uses proteolytic queues as a core mechanism. In Chapter 2, I have reviewed the application of proteolytic queueing in synthetic biology, especially in dynamic synthetic oscillators (a review chapter with the most up-todate information). ClpXP proteolytic system is the most explored proteolytic pathway and has been employed in synthetic biology using a well-studied SsrA degradation tag from E. coli. The SsrA tag is a known substrate for ClpXP, and the affinity of the SsrA tag is relatively constant for the ClpXP complex. To acquire a faster degradation rate, I deconstructed and modified the SsrA tag to develop an ultra-high affinity degradation tag (Chapter 3). Faster degradation of tagged protein increases the turnover rate; thus, the processing at the proteolytic queue is faster. However, the specific component of the SspBClpXP proteolytic system causing the queue was unknown, limiting our understanding of the queue complexity. But here, I provide substantial evidence that the ClpX chaperone likely causes queue formation (Chapter 3). I engineered a new synthetic dual-feedback oscillator using this information and the ultra-high affinity tag. This new oscillator dynamic output is unique from previously developed ones (Chapter 4). The understanding of ClpXP proteolytic queueing and synthetic circuits developed in this study can be used to engineer complex circuits for future biotechnology applications.

Population genomic data are becoming an essential component of the best available science used by conservation professionals to make management decisions to preserve biodiversity in a rapidly changing world. The proliferation of high-throughput sequencing technologies has made generating genome scale data feasible for non-model organisms that are of conservation concern. In addition to applicability in conservation science, genomic data enable the exploration of basic questions related to species evolution. To that end, this body of research exemplifies the applied and basic utility of genomic data and is focused on two federally-protected minnows endemic to the Gila River basin, Loach Minnow (Tiaroga cobitis) and Spikedace (Meda fulgida). Through developing reference genome assemblies, I explore questions related to gene family expansion, structural variation and selection through a comparative framework. Further, I detail the range-wide population genomics of both species. Lastly, I explored how factors related to processes occurring over historical and ecological timescales act synergistically to influence contemporary patterns of genomic diversity in Spikedace. This dissertation provides a novel perspective on population dynamics and evolution of these fishes, and provides information required for data-driven conservation and management of two members of the native fish community within the Gila River basin.

Although several studies have documented the genetic effects of demographic bottlenecks on the overall genetic diversity of natural populations, there is conflicting evidence on the potential roles that genetic drift and selection may play in driving changes in genetic variation at neutral and adaptive loci. In this study, genetic variation at neutral microsatellite and mtDNA loci in conjunction with an adaptive MHC class II locus was analyzed in the Galapagos penguin (Spheniscus mendiculus), a species that has undergone serial demographic bottlenecks associated with El Niño events through its evolutionary history. Levels of variation in the Galapagos penguin were compared to those of its congener, the Magellanic penguin (Spheniscus magellanicus), a closely related species that has consistently maintained a large populations size, and thus was used a non-bottlenecked control. The comparison of neutral and adaptive markers in these two demographically distinct species allowed assessment of the potential role of balancing selection in maintaining levels of MHC variation during bottleneck events. This analysis highlights the dearth of genetic diversity at all loci in the Galapagos penguin compared to the Magellanic penguin due to its history of demographic bottlenecks. There were two MHC alleles, one mtDNA haplotype, and six alleles across all five microsatellite loci assessed in the Galapagos penguin, a small fraction of the allelic and haplotype diversity detected in the Magellanic penguin. Despite the decreased genetic diversity in the Galapagos penguin, results revealed signals of current balancing selection at the MHC, which suggest that selection can counteract the effects of genetic drift through bottleneck events, maintaining current levels of polymorphism at this locus. Although Galapagos penguin populations have persisted for a long time, increased frequency of El Niño events due to global climate change, as well as the low diversity exhibited at immunological loci, may put this species at further risk of extinction.

O-GlcNAc is a ubiquitous monosaccharide posttranslational modification analogous to phosphorylation found on thousands of nuclear and cytoplasmic proteins involved in every major cellular process. Dysregulation of O-GlcNAc signaling contributes to the development and progression of several chronic diseases, though the exact molecular mechanisms driving these effects have remained elusive. A major bottleneck towards progress in the O-GlcNAc field is the absence of a facile method to increase O-GlcNAc on select proteins to determine the functional outcomes of O-GlcNAc modification. In this dissertation, I describe my efforts to generate a new protein tool that employs nanobodies and protein engineering of the O-GlcNAc Transferase (OGT) to control O-GlcNAc signaling in live cells. I begin in chapter 1 by providing a historical overview of the key concepts driving the O-GlcNAc field from before its inception to the present day. This is followed by the major concepts, applications, and current developments in the single-chain antibody field in Chapter 2. In Chapter 3, I detail the development of a nanobody-OGT fusion to target tagged and endogenous proteins to increase their levels of O-GlcNAc through a proximity-directed mechanism. Application of the nanobody-OGT technology to the methylcytosine dioxygenase TET3 reveals a scaffolding function for OGT in the regulation of TET3 localization. To further improve the nanobody-OGT system, advances in nanobody engineering are described to gain spatiotemporal control of O-GlcNAc signaling. In the fourth chapter, I study several structural features of OGT. I describe the effects of truncations of the tetratricopeptide (TPR) domain of OGT in substrate and glycosite selection in cells. I additionally explore mutations in the TPR domain, catalytic site, and intervening domain of OGT and their effects on activity, localization and selection of substrates and glycosites for future protein engineering of OGT. I conclude with a discussion of emerging concepts in the O-GlcNAc field and how the nanobody-OGT technology and single-chain antibodies could be harnessed to gain additional insight into this ubiquitous and important posttranslational modification for the advancement of basic discoveries in the O-GlcNAc field.

RNA binding proteins (RBPs) impact cellular protein levels by regulating messenger RNA (mRNA) levels. Assignment of function to hundreds of emerging, uncharacterized RBPs is a critical bottleneck to a complete understanding of gene expression control. Here, large-scale tethering of nearly a thousand RBPs discovers 50 RBPs that affect reporter RNA turnover and translation. Enhanced UV crosslinking and immunoprecipitation (eCLIP) identify hundreds of endogenous mRNA targets affected by manipulating levels of more than a dozen candidate RBPs. Among these candidates, we characterize the ubiquitin-associated protein 2-like (UBAP2L) gene. Polysome profiling assays indicate that UBAP2L enhances translation of target mRNAs, likely due to ribosome interactions as supported by eCLIP data. UBAP2L can also be found in complex with fragile X mental retardation protein FMRP, and 52% of UBAP2L mRNA targets are also FMRP targets. UBAP2L depletion in a cortical neuronal model of Fragile X Syndrome (FXS) corrects molecular, cellular and electrophysiological defects relevant to autism spectrum disorder. Reduction of the Drosophila ortholog of UBAP2L in a FXS fly model rescues the neurodevelopmental defects due to loss of FMRP. Our efficient and scalable method identifies proteins involved in RNA metabolism and detailed studies of UBAP2L provides a new therapeutic strategy into human disease.

Neurofilaments are abundant space-filling cytoskeletal polymers that accumulate in myelinated axons during development to drive the expansion of axon caliber, which is a key determinant of axonal conduction velocity [2, 39]. Neurofilaments are also cargoes of axonal transport that are assembled predominantly in the cell body and move out along axons on microtubule tracks powered by microtubule motor proteins [4]. This dual role of neurofilaments as space-filling structures and cargoes of axonal transport implies a complex relationship between axon caliber, neurofilament influx from the cell body, and neurofilament transport kinetics in the axon. Axonal injury present a good model system to study the role of neurofilament flux for transport velocity and axon caliber, because of the wealth of available morphometric and kinetic data [13, 23]. After injury, retrogradely transported injury signals trigger a transient reduction in neurofilament gene expression in the cell body and a corresponding transient reduction in neurofilament influx into the axon, resulting in a wave of axon thinning that propagates distally at a rate consistent with neurofilament transport [13]. Coincident with these changes, radioisotopic pulse labeling studies have revealed a transient increase the average neurofilament transport velocity [23]. Neurofilament kinetics near the nodes of Ranvier of myelinated axons is a model system probing the role of local changes of the transport machinery of neurofilaments for sculpting the characteristic nodal constrictions. Myelinated axons are wrapped by myelin sheaths, interrupted by short segments of a few microns where most ion channels important for the conduction of action potentials are located. Depending on the size of the axon, the diameter of the axon is significantly reduced at the nodes of Ranvier forming nodal constrictions, along which the number of neurofilaments is reduced, forming a bottleneck for axonal transport. The question addressed here is how neurofilaments navigate this bottle neck without forming traffic jams and causing axonal swelling.To answer this question, we developed a new computational model for axonal transport of neurofilaments in which neurofilament motility is related to the organization of the axonal cytoskeleton, in particular the microtubule tracks along which they are transported. Using this new model in the study of axonal injury, we can explain the time-course of post-injury decrease of axon caliber and increase in neurofilament transport velocity by a simple reduction of neurofilament flux, and the subsequent time-course of axonal recovery by a corresponding recovery of neurofilament flux. These findings demonstrate that proximity of neurofilaments to microtubules may be a key determinant of their motility. One important prediction of this model is that average neurofilament transport velocity and axon caliber are sensitive to small changes in neurofilament and microtubule density and organization. Applying our new model, we can predict the formation of a nodal constriction, where the local neurofilament kinetics and the morphology of the constricted axon are in good agreement with morphometric and kinetic data. In summary, our computational research has resulted in a predictive conceptual model framework to better understand the complex relationship between neurofilament kinetics and axon morphology. We believe that these insights have important implications for the mechanisms by which neurofilaments accumulate in axons during development, and contribute to pathological accumulations of neurofilaments observed in a host of neurodegenerative diseases (such as Charcot - Marie - Tooth disease).

Conservation genetics is a booming field focused on assessing the genetic structure and diversity among subpopulations of different species. However, the utilization of genetic analyses in management plans remains unclear because it is not known how often they are considered during creation of plans. Chapter one considers the question of how closely correlated the results from a genetic assessment on population structure are with the recommendations given at the conclusion of a study. Since conservation tends to have limited financial resources, it is imperative that the money spent on genetic studies is providing beneficial information for conservation. This analysis shows that genetic divergence is correlated with the recommendations, but different genetic markers (i.e. microsatellites) and divergence metrics (i.e. Fst) show different relationships between the recommendations and genetic divergence, possibly due to differences in the sample size associated with different markers.Small populations, such as those of conservation concern, that inbreed may lose alleles over time. This can be problematic if the alleles lost are beneficial, but making genetic assessments based on neutral genetic markers gives no information on the fitness of populations. Ideally, conservation programs should use quantitative genetics to assess population fitness. Chapter two looks at the change in additive genetic variance in populations following a dramatic decrease in population size (bottleneck). On average, populations experiencing a bottleneck showed an increase in additive genetic variance for populations that had levels of inbreeding equal to or less than that of sibling mating (0.25); above that a decrease in genetic variation was observed.Since genetic assessments using neutral markers can be costly, and the use of quantitative genetics may be impractical for informing conservation recommendations, chapter three looks at the use of statistical models to assess International Union for the Conservation of Nature Red list status using ecological characteristics. Here characteristics shared among species that have been assigned a threat status and those that have not been assigned a status, or those needing an update, are used in logistic regressions, regression tree analyses and discriminant function analyses to predict threat status and identify those species in most immediate need of attention. We found that logistic regression and discriminant function analysis are very good at predicting threat status. When resources are limited, using data on previously assessed species may be a good alternative to inform conservation recommendations.

The preservation of biodiversity is a pressing concern for many global taxa due to threats like climate change, habitat loss, and anthropogenic pressures. While in situ preservation of species in their habitats is ideal, it is not enough as threats often outpace our ability to conserve, and ex situ conservation methods must be used as a supplement to preserve species and their genetic diversity outside of their natural habitat. For exceptional species, those species ineligible for conventional seed banking due to production of little to no seeds or recalcitrant seeds, ex situ conservation must take the form of tissue culture and cryopreservation, the storage of seeds and tissues in liquid nitrogen (LN). The present study investigates the genetic consequences of these methods on exceptional species, from the initial conservation collection, to their tenure in ex situ collections, to their ultimate use in restorations and reintroductions. For Hedeoma todsenii, a federally endangered exceptional species, collections had been focused around populations located in the San Andres mountain range. A three-year study of populations in this range and the geographically distant Sacramento mountain range indicated low genetic diversity and high inbreeding in the species. However, the Sacramento population had a higher diversity and was genetically distinct from the San Andres populations, indicating that future collection efforts should include a greater focus on the Sacramento locality. Next, the genetic effects of long-term tissue culture and cryopreservation were assessed for 18 different plant species stored in LN for up to 23 years. No association was found between the survival of species after LN storage and RNA integrity, indicating that RNA may not be one of the key indicators of survival after storage. However, evidence of genetic change in DNA after cryopreservation and tissue culture was detected in three species, although this did not appear to have an effect on the survival and growth of species. It is recommended that species in long-term tissue culture or cryopreservation be regularly monitored for genetic stability. To understand the genetic effects of reintroduction using micropropagation, Minuartia cumberlandensis, a federally endangered exceptional species, was used as a case study. Seven genotypes of M. cumberlandensis were propagated using tissue culture and planted out into a suitable habitat in Daniel Boone National Forest (KY) in 2005. In 2015, 10 years after the initial outplanting, the population had more than tripled in size to over 200 plants. A genetic analysis indicated that the outplanting was genetically very similar to the natural source population despite the genetic bottleneck of only seven founding genotypes. This is likely due to low genetic diversity and clonal growth in the species. This case study shows that micropropagated plants can be successfully used to facilitate reintroductions. In sum, the presented research indicates that the ex situ conservation methods of tissue culture and cryopreservation are suitable for preserving exceptional species. These methods are key to preserving the biodiversity of often-overlooked exceptional plants for future generations.

Introduced populations of Eichhornia crassipes (Pontederiaceae) possess extremely low levels of genetic diversity due to severe bottleneck events and clonal reproduction. While populations elsewhere have been well studied, North American populations of E. crassipes remain understudied. We used Amplified Fragment Length Polymorphism markers to assess genetic diversity and population structure in North American E. crassipes populations. Patterns of diversity over the past fifty years were analyzed using herbarium specimens. Furthermore, we sampled populations across the Gulf Coast of the United States throughout a year to determine contemporary genetic diversity and assess potential seasonal effects. Genetic diversity was found to be scant in the United States without population structure, agreeing with previous studies from other regions. Genetic diversity has remained consistently low over the past fifty years despite significant changes in selection pressure. However, evidence for and against population structure between seasons was found and the consequences of this are discussed.

Production of a positron emission tomography (PET) tracer involves three stages: production of the radioisotope, radio-labeling and purification of the tracer, and quality control (QC) testing of the final product (if the tracer is used in humans). Though the production of the positron-emitting radioisotope requires expensive and complex instrumentation (e.g. cyclotron or generator), in many cases, the radioisotope can simply be obtained from a commercial source. The radio-labeling and purification step is performed by machines called radiosynthesizers, which perform chemical synthesis and purification processes in a remote-controlled or automated manner within radiation-shielded “hot cells”. Quality control testing is performed to ensure each batch of radiopharmaceutical is safe prior to use in human subjects, and requires several analytical chemistry instruments. Though a couple of PET tracers are routinely available in final form from commercial sources, most other tracers need to be specially manufactured and quality tested at the researcher’s facility.Advances in radiosynthesizer technology such as automation, programmability, and reagent kits simplify the production of diverse tracers. Furthermore, recent efforts in miniaturized synthesizers based on microfluidics aim to reduce equipment cost, shielding and lab space needs, and quantities of expensive reagents, to make it more affordable to produce custom batches of PET tracers on demand.In the context of these emerging technologies, quality control testing still remains a bottleneck due to the high cost of the many expensive instruments, specially-trained staff, and documentation needed to determine the purity and ensure the levels of all potential contaminants are below acceptable limits. Several companies are developing automated systems for quality control testing to alleviate some of these issues. However, high cost and large size remain obstacles. We have therefore explored the use of microfluidic analytical chemistry techniques to address these remaining critical issues. In this dissertation, the feasibility of using capillary electrophoresis (CE) to analyze the chemical purity of PET tracers is investigated as a compact and inexpensive replacement for high performance liquid chromatography (HPLC) and other techniques that are normally used for this purpose. After establishing that CE has sufficient performance (comparable to HPLC) using several example chemical systems, the design, development, and characterization of a microscale chemical purity testing system are presented, along with a discussion of its capabilities and limitations. Overall, it appears that microchip CE could be used instead of HPLC for performing many of the required QC tests of PET tracers.

Many natural products, including fatty acids, polyketides, and non-ribosomal peptides have pharmacologically valuable properties. These products are biosynthesized using diverse pathways, but they employ very similar machineries and chemical transformations. Central to these pathways are carrier proteins, which covalently tether a growing intermediate. Small, dynamic carrier proteins facilitate transport of intermediates to subsequent reaction partners within the biosynthetic pathway, and protect them from unfavorable reactions in the cytosol. This dissertation focuses on these carrier proteins, characterizing them both structurally and biophysically. Solution state protein NMR methods are ideal for studying carrier proteins, given their small size and high flexibility.Several studies of the Escherichia coli fatty acid synthase (FAS) pathway yielded significant information about the structure and dynamics of the acyl carrier protein (ACP). Increasing the basic understanding of how modular synthases function, interact, and maintain product specificity has been a driving goal; such information is critical to engineering de novo modular pathways. Identification of the proteins in the pathways is essential, but understanding how they move and interact remains an intellectual bottleneck. Using mechanism-based crosslinking, the E. coli ACP was trapped in functional interaction with the cognate dehydratase protein, FabA. Structures were determined crystallographically in collaboration with the Tsai lab at the University of California, Irvine and by NMR in collaboration with the Opella lab at the University of California, San Diego. Residual Dipolar Couplings from NMR, together with Accelerated Molecular Dynamics studies in collaboration with the McCammon lab at the University of California, San Diego led to the identification of key residues necessary for this protein-protein interaction, and quantification of the ACP's dynamics both as a free protein and during interaction with a partner. Furthermore, studies of the actinorhodin polyketide synthase (PKS) pathway led to insights into the sequestration and cyclization of polyketide intermediates. To prevent unwanted side-reactions of polyketide intermediates, geometrically appropriate atom replacement mimetic probes were synthesized and loaded onto the actinorhodin ACP. Chemical shift perturbations of the protein amide resonances allowed monitoring of sequestration, as probes of longer length demonstrated larger perturbation of more residues than shorter polyketide probes.Additionally, studies of the prodigiosin and pyoluteorin pathways give new information about sequestration. Traditional NOE methods were employed to solve the solution NMR structure of the pyoluteorin carrier protein, PltL, in multiple cargo states. For the first time, sequestration of intermediates was observed in a non-ribosomal peptide synthetase (NRPS) system. Further studies are underway to determine if sequestration is observed in other NRPS systems. Finally, a follow-up to the ACP FabA project is in an early manuscript stage, detailing the E. coli ACP interaction with another partner, the ketosynthase FabB. NMR titration experiments, demonstrating key residues in the ACP FabB interaction are reported. In vivo complementation of the wt ACP with mutated ACPs showed that perturbing the ACP FabB interaction reduces E. coli's ability to produce unsaturated fatty acids at lower temperatures, connecting in vivo observations to in vitro effects.While challenges and unknowns remain, the work of this dissertation provides significant progress towards the possibilities of engineering novel pathways to produce pharmaceutically relevant or biochemically valuable products.

Gravity is an important stimulus for plants. Gravitropism, the plants' response to gravity, can be divided into three phases: gravity perception, signal transduction and response. Various theories have been proposed to explain the process of gravitropism, yet more genes are needed to elucidate the mechanism of gravitropic signal transduction. A transcriptome analysis, in combination with the Gravity Persistent Signal treatment, was performed to specifically study the genes involved in signal transduction. Analysis generated a list of 318 transcripts that were differentially expressed in plants that were reoriented with respect to gravity as compared to vertical controls. Based on the expression profiles and gene function annotations, five transcription factors, WRKY18, WRKY26, WRKY33, BT2 and ATAIB, were selected for further study. In addition to the standard analysis of differentially expressed genes, a systems approach was adopted to uncover more gravity related genes. A semi-supervised learning method was developed to find additional novel genes. This learning method took a set of 32 known gravity genes from the literature as well as a collection of heterogeneous annotation features, such as existing protein-protein interactions, and co-expression profiles. The learning classifier predicted a list of 50 genes that are functionally related to gravity signal transduction. Based on this list of genes, an interaction network was predicted based two complementary approaches: a dynamic Bayesian network and a time-lagged correlation coefficient. To increase confidence in the predication, genes/interactions that appeared in both networks were selected. This 'intersected' network provided 20 hub and bottleneck genes, fourteen of which had not been previously identified as involved in gravitropism. Such an approach provides a framework to extend current research in a more comprehensive manner, and serves a complementary to the traditional mutant/gene discovery model.

The range and abundance of the Louisiana black bear (Ursus americanus luteolus) were greatly diminished during the 20th century. This subspecies was reduced to 3 small, isolated subpopulations in Louisiana as bottomland hardwood habitat was converted to agriculture. These bears were listed as threatened by the U.S. Fish and Wildlife Service in 1992 and a recovery plan was published in 1995. Recovery requires estimates of population parameters to evaluate current population status and future viability. I conducted a mark-recapture study from 2010 to 2012 to estimate demographic parameters of the coastal population of Louisiana black bears. Because inbreeding is a concern for small, isolated populations, I analyzed 23 microsatellite loci to investigate genetic structure and migration rates within the coastal population and between the coastal and other regional populations. Using non-invasive methods, I collected 3,698 hair samples during 3 summers and used DNA to identify 190 individuals. I analyzed encounter histories using Robust Design, a combination of open and closed mark-recapture models, with the full closed captures with heterogeneity model in Program MARK. I estimated density using spatially explicit capture-recapture. I used Akaike’s Information Criterion (AIC) to rank models and averaged across years according to AIC weight. The model-averaged abundance estimate for females was 77 (95% CI = 66–89) and for males was 61 (95% CI = 53–69). Population growth rate was negative from 2010 to 2011, positive from 2011 to 2012, and averaged 1.08. Apparent survival ranged from 0.83 to 0.89 depending on sex and year. Population density was 0.35 bears/km2 (95% CI = 0.30–0.41). Principal Coordinate Analysis and assignment tests revealed 2 genetic clusters within the population. Migration rates were male biased and higher than expected based on genetic structure. The population appears to be recovering from past fragmentation but evidence for a bottleneck was inconclusive. I conclude that genetic isolation and inbreeding within the coastal population pose less danger than isolation and demographic threats. My results will ultimately be used as part of a population viability analysis to estimate the sustainability of the Louisiana black bear population.

Recent improvements in mass spectrometry instrumentation have stimulated the fusion of this technology with protein crosslinking to advance the structural proteomics field. However, analysis of complex datasets from crosslinking experiments remains a bottleneck. The majority of crosslinking studies for structural characterization of protein- protein interactions have been conducted with reagents specific for discrete amino acids. While this approach simplifies data analysis, the requirement for specific functionalities to be present at the interaction interface limits resolution. Herein, we report the application of stapled peptides for the development of photoaffinity reagents for mass spectrometric characterization of BCL-2 family protein interactions. To validate this approach, we synthesized photoreactive stabilized alpha-helices of BH3 domains (pSAHBs) incorporating a benzophenone containing amino acid, and demonstrated that the photo crosslinking specificity of these reagents paralleled the interaction specificity of the native proteins. We show that the standard SEQUEST algorithm is effective at identifying specific amino acids crosslinked by pSAHBs, and that this information can be used to create distance restraints for characterizing interaction interfaces by in silico docking. The pSAHB approach is applied to characterize previously elusive activating interactions between BH3 domains and the proapoptotic proteins BAX and BAK. We demonstrate that full-length BAK requires a direct activation stimulus, and that this involves interaction at a canonical surface groove at the C-terminal face of BAK. We confirmed that initiation of direct BAX activation occurs at a geographically distinct site at the N-terminal face of BAX, but further find that induced release of its C-terminus from the canonical groove exposes these residues for secondary BH3 interaction. These data suggest that BAX may be subject to a two-step activation mechanism within distinct cytosolic and mitochondrial compartments. Finally, we report the structural characterization of an interaction between BAD and glucokinase, the first description of a BH3 domain interaction with a non-BCL-2 family member. We identify the active site region of glucokinase as the BAD interaction site, establishing this region as a novel target for development of glucokinase activators. We conclude that the pSAHB approach represents a rapid and powerful approach to protein interaction site identification that complements conventional structural biology techniques.

In recent years, the range of Peromyscus maniculatus gracilis in Michigan’s Lower Peninsula (LP) contracted simultaneously with the northward expansion of the closely related Peromyscus leucopus. Recent trapping shows that the range of P. m. gracilis has shrunk to two remaining areas: one in Cheboygan and Otsego Counties and another to the east in Alpena Co. (AP). I used a Geographic Information System habitat analysis to predict where P. m. gracilis would be found across the region, trapped at selected locations, and analyzed 11 microsatellite loci for all individuals. Genetic diversity and bottleneck analyses indicate that LP populations once had good gene flow and were continuous across the area; however, trapping data and assignment tests show that the AP population is in the process of becoming geographically and genetically isolated from all other populations. The AP population is currently genetically viable, but is showing signs of becoming genetically distinct from the western populations.

Despite having been studied for over 100 years, Erythronium americanum still has many aspects that remain relatively unknown or show conflicting results. To address this deficiency, multiple populations of various sizes were studied across a wide latitudinal range. Field observations were used to study pollinator abundance and activity and hand pollinations were performed to examine pollen limitation and self-incompatibility. Novel molecular markers were developed and used to quantify genetic diversity, clonal diversity, and population structure. Hand pollination treatments (natural, hand supplemented and bagged flowers) showed that smaller populations of E. americanum were more pollen limited and may be more susceptible to natural or anthropogenic factors affecting pollinators (p=0.0227). Based on self and outcross pollination treatments, smaller populations were more self-incompatible than larger populations (p=0.0011) and some populations were suffering from inbreeding depression (p < 0.0001). Pollen tube growth indicated that self-pollen could be more successful due to the mentor effect of outcross pollen (p < 0.0001). Genetic analysis of populations showed that populations of E. americanum that appeared larger and more dense actually had less clonal diversity. These results suggest that smaller populations of E. americanum are more reliant on sexual reproduction and that clonal recruitment increases as populations increase in size. Larger populations were dominated by fewer clones and were less genetically diverse than smaller populations. However, larger populations also displayed lower self-incompatibility, which could increase survivability of populations in cases of catastrophic bottleneck events. Implications from this research reach far beyond a common spring ephemeral and may be applicable to the many species which share some of the characteristics with E. americanum.

Humans have exerted strong selection pressures for the behavioral attributes which make dogs prized hunting companions, viz., superior sensory capabilities, social cognition, and tenacious pursuit of prey. With behavioral traits maintained in stable breed lines, and a unique genome organization, dogs are ideally suited for a genetic dissection of complex behavioral phenotypes. Despite studies employing substantial sampling regimes the promise of the canine model has yet to be realized, largely because of the dearth of robust quantitative behavioral metrics. Here we show that refined dissection of a complex behavior into component parts is instrumental in identifying genomic signatures associated with these elements. Using 11 spatially explicit measures we demonstrate the existence of four principal dispositions describing Search Eagerness, Linear Running, Handler Reliance, and Zigzagged Searching. A Single Nucleodide Polymorphic marker - based association study reveals that Search Eagerness significantly associates with genomic regions on chromosomes 1 and 27 (Chromosome wide significance, p [100,000 permutations] < 0.05). Adjacent are sites for brain specific Demethylase- (MBD2) and GABA transporter genes (BGT1, GAT2, GAT3) coding for transcriptional repressor proteins and regulating levels of extracellular neurotransmitters respectively. We anticipate that the wider application of such objective quantitative frameworks will improve the phenotypic characterization that remains the bottleneck in neurobehavioral genetic research.

The muskellunge (Esox masquinongy) is an important recreational fish species native to the northern and eastern United States. The species has a long history of stocking and hatchery propagation. In order for the best management practices to be implemented, the genetic variation and structure within and among stocks must be understood. Currently, muskellunge are classified into three subspecies corresponding to major drainages, but these designations are disputed. In this study, the genetic stock structure of muskellunge was investigated at two scales: at the species level and at the local population level for Ohio’s muskellunge stock. Using mitochondrial DNA (mtDNA) sequence variation, I examined the range-wide intraspecific phylogeographic patterns of muskellunge. Using three nuclear microsatellite loci, I assessed local stock structure of hatchery and natural populations in Ohio. Very little mtDNA sequence variation and a lack of genetic structure among populations from throughout the range do not support the subspecies designations. Further, the widespread distribution of two similar haplotypes suggests recent dispersal from a single glacial refugium. High levels of microsatellite variation within and between Ohio populations identified significant stock structuring among populations. Recent bottleneck events do not appear to have reduced genetic diversity in the sampled populations. Based on my data, Ohio contains at least five genetic stocks that should be maintained as separate management units.

Kearny Marsh located within the Hackensack Meadowlands is a freshwater wetland impacted by multiple environmental stressors from human activity. The marsh is located in the northeastern New Jersey metropolitan area. Chironomid larvae, aquatic larvae of midge fly, were analyzed from two Kearny Marsh sample sites, W9 and W22. A previous sediment analysis and toxicity study characterized heavy metal contaminants the marsh. The data results were applied in this thesis study to determine the correlation between environmental stressors and chironomid genetic diversity. Site W9 sediment was impacted by severe levels of total heavy metal contamination, whereas site W22 had just below to below effect levels of contamination. A primary thesis objective was to determine if chironomid hemoglobin genetic diversity is a suitable bioindicator of multiple environmental stressors. Prior to the genetic diversity evaluation, the thesis aim was to identify the genus of field chironomid with a molecular biomarker. SDS-PAGE gel electrophoresis was used to analyze hemoglobin protein band patterns. A molecular method was investigated because chironomid identification based purely on taxonomy is difficult, since the various chironomid species have similar morphological features. The hemoglobin protein measured by SDS-PAGE method demonstrated to be an appropriate identification method, because the hemoglobin protein band patterns, characterized by molecular weight and intensity, directly correlated with the respective head capsule morphology. Also, according to phylogenetic analysis with Hierarchical Cluster Analysis the three species evaluated: Glyptotendipes and Endochironomus clustered separately into two major clades. Chironomus and Glyptotendipes clustered separately within one major clade however diverged into two separate subclusters. These hemoglobin protein band patterns were applied to the genetic diversity study. The second objective was to determine if a correlation existed between multiple environmental stressors and genetic diversity. The thesis prediction was that a negative correlation exists between hemoglobin genetic diversity and heavy metal contamination, compounded by multiple environmental stressors. However, analysis of heavy metal concentration levels between the sample sites and Hb genetic diversity based on the SDS-PAGE gel band patterns indicated a positive correlation between Glyptotendipes hemoglobin protein diversity and heavy metal contamination levels. Site W9 with higher levels of total heavy metals had a greater Hb diversity level, in comparison to site W22 with lower levels of heavy metals. This result may indicate several possible endogenous and exogenous explanations for the genetic diversity difference between the sample sites. The thesis study suggested that a physiological adaptation response, population genetic dynamics (i.e., genetic drift and a in bottleneck), and food web cycle alterations played a key role in Glyptotendipes genetic diversity levels in response to varying levels of environmental stressors. The thesis study demonstrated that chironomid hemoglobin protein evaluated by the SDS-PAGE biomarker has potential for both identification purposes and to determine genetic diversity of the species which may be applied to bioassessment of wetland environmental health.