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The evolution of genes and genomes has attracted great interest. The research presented here is an examination of genomes at three distinct levels, protein evolution, gene family evolution, and TE content regulation. First at a genetic level, I conducted an analysis of the salivary androgen-binding proteins (ABPs). I focused on comparing patterns of molecular evolution between the Abpa gene expressed in the submaxillary glands of species of New World and Old World muroids and found that in both sets of rodents, the Abpa gene expressed in the submaxillary glands appear to be evolving under sexual selection, suggesting ABP might play a similar biological role in both systems. Thus, ABP could be involved with mate recognition and species isolation in New World as well as Old World muroids. Second I examined the largest gene family in vertebrate olfactory receptors (ORs) among birds and reptiles. I found that the number of intact OR genes in sauropsid genomes analyzed ranged over an order of magnitude, from 108 in the lizard to over 1000 in turtles. My results suggest that different sauropsid lineages have highly divergent OR repertoire compositions. These differences suggest that varying rates of gene birth and death, together with selection related to diverse natural histories, have shaped the unique OR repertoires observed across sauropsid lineages. Lastly, I studied the interactions between transposable elements (TEs) and PIWI-interacting RNAs (piRNAs) among laurasiatherian mammals. piRNAs are predominantly expressed in germlines and reduce TE expression and risks associated with their mobilization. I found that within TE types, families that are the most highly transcribed appear to elicit the strongest ping-pong response. This was most evident among LINEs, but the relationships between expression and PPE was more complex among SINEs. I also found that the abundance of insertions within piRNAs clusters strongly correlated with genome insertions and there was little evidence to suggest that piRNA clusters regulated TE silencing. In summary, the piRNA response is efficient at protecting the genome against TE mobility, particularly LINEs, and can have an evolutionary impact on the TE composition of a genome.

The butterfly genus Heliconius has an unusually long lifespan (six to eight months compared to only several weeks) and the unique ability to feed on pollen. The additional amino acids provided by a pollen diet seem intrinsically tied to longer lifespan in Heliconius. Because of these traits, Heliconius may be a valuable model species for future aging studies. As a preliminary analysis, we investigated differences in gene evolution between Heliconius and seven short-lived butterflies. We observed that the Heliconius genes had significant differences in evolutionary rates, in the direction of selection, and in the amounts of site-specific positive selection compared to the short-lived butterflies. We also investigated functional differences in gene complements between the Heliconius and short-lived butterflies but found only small differences. With this research, we present exploratory evidence supporting differences in gene evolution of the Heliconius to establish targets for future analysis of Heliconius as models for aging.

Haloxylon persicum is a xerophytic desert tree that grows mostly in deserts in West and Central Asia. This tree is very tolerant to the harsh conditions of deserts, mainly drought and heat. As a part of the Kingdom of Saudi Arabia Native Genome Project, a voucher specimen was identified, and the genome of this plant was sequenced, assembled, and annotated. The chromosome level assembly was performed using the integration of PacBio Hifi reads and Bionano optical maps, resulting in 9 chromosome-sized molecules that only exhibit 3 gaps located in highly repetitive regions. The annotation of the transposable elements in the genome shows that more than 55% of the genome consists of transposable elements. Moreover, genes were predicted using Iso-seq and RNA-seq and annotated using publicly available protein databases, resulting in the identification of more than 45,000 predicted genes, of which ≈ 10,000 have RNA evidence. The genome assembly and annotation of Haloxylon persicum will: provide valuable insight on the evolutionary history of desert plants, aid in discovering the mechanisms developed by this species to cope with the extreme desert conditions and unveil the possibilities and opportunities of neo-domesticating this plant. Furthermore, this assembly can serve as a reference for assembling other plant species in the KSA Native genome project or any other project worldwide.

The advent of large-scale genomic sequencing is providing researchers with an unparalleled wealth of information which can be used to elucidate the evolutionary relationships of living organisms. The newly available genome sequence data have enabled the use of comparative genomic techniques for the identification of novel molecular signatures, shared uniquely by evolutionarily related groups of organisms: conserved signature indels (CSIs) and conserved signature proteins (CSPs). These signatures allow for the unambiguous delineation of the prokaryotic taxa, independent of gene and genome based phylogenetic trees, and provide insights into novel aspects of their evolutionary relationships. The phylum Spirochaetes and the class Betaproteobacteria are large, diverse groups of bacteria, containing many important pathogenic and environmental organisms, which are classified primarily on the basis of 16S rRNA gene analysis. Here, I describe phylogenetic analyses of the phylum Spirochaetes based on genome derived molecular signatures. These analyses have yielded substantial evidence for differentiation between the three main sequenced groups of organisms within the phylum Spirochaetes and between the genus Borrelia from other closely related Spirochaetes. These findings have prompted a proposal to create three new orders and a new family within the phylum. These analyses have also supported the differentiation of two clinically distinct groups within the genus Borrelia and a proposal to divide the genus Borrelia into two genera. The use of molecular signatures and phylogenetic analysis of major groups within the class Betaproteobacteria are also described. The analyses of the order Neisseriales within this class resulted in a division of the order into two families, while the analyses of the genus Burkholderia supported the differentiation of the clinically relevant members of the genus Burkholderia from the plant-beneficial and environmental Burkholderia and a proposal to divide the genus into two genera. I also describe the use of phylogenomic techniques and molecular signatures to differentiate the seven main groups within the order Enterobacteriales and the integrated software pipeline used to produce the supermatrix based phylogenomic tree and genome distance calculations in the analysis of the order Enterobacteriales. The molecular signatures described in this thesis represent powerful new tools for evolutionary and systematic studies. Additionally, due to their taxon specificity, these molecular signatures are novel diagnostic markers for their specified group. Further analyses of these molecular signatures should lead to the discovery of novel functions and biological characteristics, mediated by CSIs and CSPs, which will provide important insights into the physiology, evolution, and adaptations of these groups. / Thesis / Doctor of Philosophy (PhD)

Despite significant advances in chemotherapeutics since their initial application in the early 1900s, cancer remains a leading cause of death worldwide. Furthermore, issues encountered with the first chemotherapeutics, off-target toxicity, limited effective dosing time in the therapeutic window, multidrug resistance, and poor uptake in solid tumors persist today. This dissertation investigates two platforms to enhance chemotherapy efficacy, an implantable drug delivery depot affording tumor drug levels unachievable through systemic delivery, and a platform utilizing CRISPR knockout screening (CRISPRKO) to identify microRNA (miRNA) targets that synergize with chemotherapeutics using RNA interference (RNAi). First, we developed a flexible, implantable surgical buttress coated with a free and covalently-bound paclitaxel polymer blend with poly(1,2-glycerol carbonate)-graft-succinic acid-paclitaxel (PGC-PTX). Drug release is tunable between burst release of free paclitaxel and delayed, extended release of paclitaxel from PGC-PTX, delivering supratherapeutic levels of PTX locally at the tumor resection bed while avoiding systemic toxicity. Fabrication of paclitaxel-loaded film is scalable up to 8”x11” and well-tolerated in a porcine model wherein surgical technique and optimized film formulations reduce foreign body response and bowel adhesions. We test a similarly-designed film formulation with the novel chemotherapeutic, eupenifeldin, for treatment of lung cancer. Eupenifeldin-loaded films significantly prolong mice survival, although 60% of mice present issues with drug-related toxicity and wound healing. Second, despite the formidable ability of pooled CRISPRKO libraries to screen thousands of single-gene knockouts against a selection agent, minimal has been published on their use to determine novel targets for RNAi-based sensitization of chemotherapeutics. We utilize whole-genome CRISPRKO screening against a panel of lung cancers to identify miRNAs capable of synergizing with paclitaxel as well as two novel chemotherapeutics, eupenifeldin and verticillin A. Identified targets are validated for synergy with their respective chemotherapeutic in vitro, demonstrating a 50% or greater increase in cell death compared to drug-only treatment. In summary, this work presents two successful treatment platforms that address key issues preventing translation of chemotherapeutics to the clinic: 1) dose-limiting toxicity and 2) limited efficacy with poor safety/benefit ratio. Implantable, drug-loaded films serve as a platform to deliver increased doses of chemotherapeutic to tumor while avoiding off-target toxicity. As a second platform, we enhance tumor-specific chemotherapy efficacy utilizing miRNA targets identified in a CRISPRKO screening pipeline.

This thesis proposal shows the efficiency of different methods of Myostatin inhibition by using CRISPR/CAS9. With the data cataloged by this thesis, researchers will have a better understanding of what methods are better to achieve their goals. The data was collected by reading multiple scientific articles involving the usage of CRISPR/CAS9 to inhibit the Myostatin protein. The data collected from all the different studies were analyzed in the same categories. The experiment that used CRIPSR/CAS9 on in vitro specimens had a superior Myostatin inhibition overall, therefore presenting higher muscle mass. The method using CRISPR/CAS9 to inhibit the Myostatin in vivo and in vitro depends on what the researcher is trying to accomplish. By reading this thesis researchers can understand that if they choose to use an in vitro treatment the results would be way more severe than using an in vivo application treatment.

Fetal Alcohol Spectrum Disorders (FASDs), are caused by maternal alcohol consumption during pregnancy [3]. FASD encompasses a wide variety of cardiac and neural anomalies, while also associated with improper limb development, abnormal craniofacial features, problems within the central nervous system (CNS), and disabilities in learning and communication. Gene-regulating FASDs have not been well studied during the crucial phases of early embryonic development. Genes within the Wnt/Beta-catenin pathway control a vast amount of embryonic developmental processes. Among them is the Wnt7a gene, a significant downstream gene regulator which positively controls neural stem cell proliferation and cardiomyocyte differentiation on a large scale during early embryonic development. This project will serve to provide potential insight into the genes involved in FASD. We hypothesize that ethanol administration to early embryonic mice will suppress Wnt7a expression in the heart and brain, leading to FASD development. RNA-sequencing (RNA-Seq) and real-time quantitative PCR (qPCR) were used to measure Wnt7a gene expression within the early embryonic mouse heart and brain. After evaluation of RNA-Seq data and a comparative analysis using the 2-ΔΔCTmethod, it is evident Wnt7a is present in embryonic mouse age E10.5 heart and brain samples, and Wnt7a is suppressed at age E10.5 in embryonic mouse heart, but not brain, when induced with ethanol.

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