UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
136 results
Search Results
Item MEDIATION OF CORTICOSTERONE-INDUCED GROWTH HORMONE GENE EXPRESSION IN CHICKEN EMBRYONIC PITUITARY CELLS: IDENTIFICATION OF TRANS-ACTING FACTORS AND A NOVEL PITUITARY CELL TYPE(2024) Liu, Kuan Ling; Porter, Tom E.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) is responsible for up to 30% of growth in broiler chickens. Somatotrophs, or GH secreting cells, begin to differentiate around embryonic day (e)14, in conjunction with an increase in the primary plasma glucocorticoid (GC) corticosterone (CORT). CORT treatment of e11 chicken embryonic pituitary (CEP) cells induces premature GH secretion. This GC-induced process involves trans-acting factors because the GH gene lacks a canonical GC response element (GRE). In addition to the binding of ETS1 and the GC receptor (GR) to the GC-responsive region (GCRR; 1045/ 964), we hypothesize that there are other regulatory factors necessary for CORT responsiveness. By modifying the pGL3_-1742/+25 GH-luciferase reporter, we have constructed various other GH-luciferase reporters and analyzed them for promoter activity in response to CORT treatment. We identified a putative distal (d) ETS-Like 1 (ELK1) binding site that is necessary. The proximal (p)PIT1 site and pTATA box were also identified to be critical for CORT induction of the GH gene. Interestingly, cloning multiple copies of the extended GCRR (eGCRR; -1067/-900) further increased promoter activity in an additive manner under both basal and CORT treated conditions. Through single-cell RNA sequencing (scRNAseq), 8 members of the ETS family of transcription factors were identified in > 5% of the somatotroph population. Commercial antibodies were validated, and human (h)ETV1, hELF2, hELK3, and hETV6 antibodies were confirmed to recognize their recombinant chicken ortholog and to identify their corresponding protein in e11 CEP cells. Results from chromatin immunoprecipitation quantitative PCR suggest that multiple ETS members are involved in CORT induction of the GH gene with more evidence pointing towards ELF2 and ELK3. Identifying trans-acting factors for the GH gene inducible by CORT allows for better understanding of endogenous GH regulation in chickens. Further analysis of the scRNAseq data from e11 CEP cells revealed a cluster of cells expressing genes for more than one hormone-producing cell type (“premature nebulous” cluster). Within the premature nebulous cluster, a large population (~30%) was co-expressing proopiomelanocortin (POMC) and growth hormone (GH). We named this novel cell population the cortico-somatotrophs. Through RNA fluorescent in-situ hybridization (RNA-FISH) and dual label immunofluorescence, we verified the existence of the cortico-somatotrophs at both the mRNA and protein level, respectively. Cortico-somatotrophs were also shown to share genes for receptors normally specific to both corticotrophs (CRH-R1) and somatotrophs (GHRHR). Additionally, in response to CORT treatment, the cortico somatotrophs showed an increase in GH as well as a decrease in POMC mRNA levels. The discovery of the cortico-somatotrophs suggests a modification to the current dogma on pituitary cell lineages, where corticotrophs and somatotrophs may have overlapping developmental pathways. In conclusion, our discovery of the cortico somatotrophs has furthered our understanding of CEP development and opened the door for further exploration of the cell lineages during pituitary development.Item Cell Population Shifts and Clinical Heterogeneity in Sjögren's Disease(2024) Pranzatelli, Thomas J; Johnson, Philip L.F.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sjögren's disease (SjD) is a systemic autoimmune disease that causes loss of function of the salivary and lacrimal glands. Those with the disease, overwhelmingly female with an onset of disease in the fourth or fifth decade of life, commonly suffer from dry mouth, cavities and damage to the eyes. Patients present with a wide variety of clinical phenotypes, with variation in degree of immune infiltration and glandular damage as well as positivity for autoantibodies. This thesis uncovers the changes in cell population and gene expression in the gland that underpin diversity in disease severity. SjD patients lose the majority of a specific epithelial population in their labial salivary glands and, as the number of immune infiltrates grows the surviving members of this population can be found colocalizing with invading GZMK+ T cells and expressing markers of increased proliferation. Standard differential gene expression analysis highlighted gene markers of cell types changing in proportion with disease; an unenlightening result when the cell population changes are well-characterized. To avoid this pitfall an ensemble of random forests was trained to find genes predictive of patient subtypes without being correlated with diagnosis. Genes with high importance for autoantibody positivity were enriched for GO terms related to antigen processing and presentation. A master regulator of salivary gland identity, ZBTB7B, was identified from chromatin accessibility data. Mice with this transcription factor knocked out lose salivary flow and develop pockets of tissue in their glands that resemble other glands, eg., labial gland epithelium inside of parotid glands. This work supports a clinical presentation-specific approach to therapy and paves the path for reengineering the glands to correct the effects of disease.Item Phenotypic and Genetic Analysis of Reasons for Disposal in Dairy Cattle(2024) Iqbal, Victoria Audrey; Ma, Li; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Reasons for disposal are defined as why a cow has left the herd during lactation and are documented as termination codes. Dairy cattle termination codes were collected by Dairy Records Processing Centers and stored in the National Cooperator Database maintained by the Council on Dairy Cattle Breeding for analysis. The list of possible termination codes is as follows: code 0 is cow lactation that ended typically without an abortion, code 1 is locomotion problems, code 2 is female transferred or sold, code 3 is low milk yield, code 4 is reproductive problems, code 5 is unspecified reasons, code 6 is death, code 7 is the presence of mastitis, code 8 is abortion, code 9 is udder problems, code A is an unfavorable phenotype, and lastly code B is undesirable temperament. Understanding termination codes is the key to understanding and improving farm management. Unfortunately, the secondary termination codes are not utilized, despite studies saying one reason is too limited. Heifer termination codes should be more utilized, and studies show that this could improve heifer management. The four processing centers' principal termination codes deviated a little from year to year, but processing center D had the most variation in principal termination codes. There were few records with termination codes 9, A, and B. There was low lameness found for Jersey cattle but more fluctuations for their termination codes 6, 7, and 8. Jersey's main reason for disposal was sold and low milk yield. As for Holstein, the main reasons for disposal were low milk production and death. Recommendations include removing termination code 5 (other reasons) and enforcing a secondary termination code for code 2 (sold). Also, including the percentage of animal records used in traits developed at the CDCB was recommended to encourage farmers to add more records to improve breeding selections. Overall, the top main reasons for disposal were low milk yield, death, and reproduction across breeds from 2011 to 2022. To determine whether health traits correlate to termination codes and how health traits change the probability of survival, a multinomial logistic regression was developed, where twelve health traits, breeds, and other factors were used as an independent variable for the termination code, the dependent variable. The output is a regression coefficient list that conveys the effect of each health trait for each termination code. The results show the apparent impacts of animal breeds on different termination codes, such as dairy crossbreeds negatively affecting termination due to reproductive advantages that follow the literature. Lastly, using termination codes as phenotype, this study focuses on developing a genome-wide association study (GWAS) using the Weighted single-step Genomic Best Linear unbiased prediction (WssGBLUP) model to find significant SNPs related to survival in Holstein cows. In summary, this study provided an understanding of reasons for disposal trends, modeled the reasons for disposal, determined the likelihood of termination post-incidence, and found the heritability and important SNPs of each termination code.Item INTEGRATING FORWARD AND REVERSE GENETIC TOOLS FOR FAST FORWARDING WHEAT IMPROVEMENT(2024) Schoen, Adam William; Tiwari, Vijay K; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bread wheat (Triticum aestivum) provides roughly 20% of the human daily caloric intake and is an important crop for global food security. Changing climatic conditions as well as biotic and abiotic stresses are threatening wheat production worldwide. Sustainable and continuous improvement of wheat using novel genes and alleles is critical to tackle wheat’s imminent challenges. Recent advances in wheat genomics have allowed researchers now to fast-track gene discovery pipelines by implementing strategies first developed in less complex model species. This thesis explores the use of forward and reverse genetic approaches to efficiently discover, map, and validate genes controlling important agronomic traits in bread wheat as well as describes a robust protocol to reduce the generation time in winter wheat. Speed breeding is an important tool that utilizes an increased photoperiod and growing temperature to increase vegetative growth and reduce the time from sowing to harvest. Chapter 1 of this thesis outlines a reproducible method to significantly reduce the generation time in winter wheat from over 120 days based on what has been previously reported to 93 days regardless of vernalization requirements or photoperiod sensitivity and provide a useful tool to increase the pace of the genetic gains in the winter wheat breeding programs. Tillering in wheat directly influences the major yield-related trait, spikes per unit area. Using the forward genetics approach, chapter 2 of thesis reports the identification of a novel tiller inhibition gene (tin6) to a small physical region of 2.1 Mb region on chromosome 2DS. This was the first example of using a genome coming from the pan-genome of wheat to perform MutMap. Using reverse genetics also has the potential to improve the end-use properties of wheat by knocking out genes which result in an increase of the nutritional value of the flour. Chapter 3 of this thesis, TILLING was used to identify knockouts in all three homeologous copies of the starch synthase gene SSIIa, which has been shown to increase the amount resistant starch in the endosperm of wheat which is known to have health benefits in humans. The grains coming from triple knockouts of SSIIa contained 118% higher resistant starch, and though they showed a decrease in thousand kernel weight, they did not have a shriveled phenotype which had been seen in other ssiia mutants. Chapter 4 of the thesis demonstrate reference genome enabled positional cloning of a tiller inhibition gene (tin3) in diploid wheat species Triticum monococcum. A MutMap population generated from a cross between tin3 and wildtype T. monococcum resulted in the identification of a single candidate gene, encoding a BLADE-ON-PETIOLE-Like protein, containing a splice-variant mutation. To show the power of using a diploid species for translational research in hexaploid wheat, the reverse genetics approach TILLING (Targeting Induced Local Lesions IN Genomes) was used to identify mutations in all three homeologous copies of tin3 in the Jagger mutant population. The full null mutant for the tin3 locus in wheat showed significantly reduced tillering in comparison to wildtype providing concrete evidence that genetic discoveries that are found in diploid wheat can be effectively translated to hexaploid wheat. There are some genes and QTLs have been identified that increase spike length, spikelets per spike, and grain size, very few studies have focused on increasing the number of grains per floret. Chapter 5 of the thesis was focused on positional cloning of the Mov-1 locus which is the underlying gene responsible for the multiovary (MOV) phenotype. The Mov-1 locus dominantly expresses as three ovaries per wheat floret, each of which develop into a grain. Using high resolution genetic mapping with the MOV-reference genome and gene expression data, we identified a single candidate gene that was localized to a small 144kb region on the Mov-1 physical region. To validate the role of the Mov-1 candidate gene in the MOV phenotype, ethyl methanesulfonate (EMS) and gamma radiation mutagenesis was performed to create deleterious point and deletions mutations, respectively. Using 5 independent TILLING and 5 deletion mutants this study demonstrate that Mov-1 candidate gene is required for the MOV phenotype in wheat. It is an exciting time to work in wheat research as the growing wheat genomic toolbox allows for researchers to efficiently identify and validate genes that have potential to improve wheat performance. The methods and findings in this body of work provide a breadth of knowledge that can be implemented in additional genetic studies in wheat in order to fast-track gene and trait discovery for the benefit of wheat geneticists and breeders alike.Item CHARACTERIZING THE ROLES AND MECHANISMS OF CYTONEMES IN ASYMMETRIC SIGNALING AND ORGANIZATIONS IN THE DROSOPHILA MUSCLE PROGENITOR NICHE.(2024) Patel, Akshay Jitendrakumar; Roy, Sougata; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tissue development and homeostasis rely on the ability of embryonic or stem cells to efficiently determine whether to multiply for self-renewal or differentiate to generate a wide range of cell types that constitute an adult body. Stem cells determine these fates in the context of a specialized microenvironment or the niche that they occupy. All stem cell niches characterized to date are known to function using two key processes - adhesive interactions and asymmetric growth factor signaling between the niche and stem cells. While adhesion to the niche maintains niche occupancy and stemness, the loss of niche adhesion and occupancy initiates stem cell differentiation. Moreover, niche cells produce secreted growth factors to support stem cell self-renewal. Despite the ability of secreted growth factors to disperse across tissues over a long range, only the niche-adhering stem cells receive the self-renewal signals. The genetically identical daughter cells that lack adhesion to the niche fail to receive self-renewal signals, even when located within one or two cell diameters away, leading to the activation of their post-mitotic fates. Therefore, understanding how asymmetric signal distribution and adhesive interactions are produced and coordinated within the niche is critical to understanding how stem cells determine their identity and prime differentiation to generate or regenerate tissues. This thesis investigated and characterized a new mechanism of asymmetric signaling and cell organization in the Drosophila Adult Muscle Progenitor (AMP) niche. By employing genetic, cell-biological, and high-resolution microscopy techniques, this work discovered that AMPs extend thin polarized actin-based filopodia, called cytonemes, by orienting toward the wing disc niche. Cytonemes play a dual role. Cytonemes help AMPs to physically adhere to the wing disc niche and also directly receive a self-renewal Fibroblast Growth Factor (FGF) through the cytoneme-niche contact sites. AMP cytonemes localize the FGF-receptor (FGFR), called Heartless (Htl), and selectively adhere to the wing disc areas that express two different Htl ligands, Pyramus and Thisbe, both mammalian FGF8 homologs. Htl on these cytonemes directly receives Pyramus and Thisbe through the cytoneme-niche contact sites. Although FGFs are long-range secreted paracrine signals and Htl is the only receptor shared by Pyramus and Thisbe, these FGFs are received and restricted only to the niche-adhering AMPs due to the contact-dependent cytoneme-mediated asymmetric delivery of the signals. Moreover, despite employing a common FGF signal transduction pathway, Thisbe- and Pyramus-signaling initiates divergence of AMP fates into two distinct muscle-specific lineages. These experiments showed that cytoneme-mediated signal communication forms the basis of asymmetric signaling and organization within the AMP niche. We next asked how AMPs determine the niche-specific polarity and affinity of cytonemes. This research discovered that FGF reception and signaling activation in AMPs are required to activate polarized cytoneme formation orienting toward the wing disc niche. Without FGF signaling, AMPs cytonemes fail to polarize and adhere to the FGF-producing niche, causing them to exit the niche and start to differentiate. Thus, while target-specific asymmetric FGF distribution relies on cytonemes, activation of FGF signaling feedback maintains the polarity and adhesion of the signaling cytonemes toward the FGF-producing niche. A consequence of this interdependent relationship between niche adhesion, polarized FGF-reception, and stimulation of FGF signaling feedback is the maintenance of the self-organized niche-specific asymmetric signaling and organization via cytonemes. We next investigated whether the niche-adhering cytonemes receive additional fate-specifying cues, particularly the mechanical cues from the niche. Recent evidence suggests a critical role of mechanical and physical cues in determining stem cell fates. This work discovered that the AMP cytonemes are enriched with a common mechano-transducer, named Talin. AMP-specific genetic manipulation of talin indicates that Talin is critical for cytoneme-mediated niche occupancy and FGF signaling. Using a Talin-based force probe expressed at the physiological levels and FLIM-FRET microscopy, we discovered that Talin experiences pN level force within the cytonemes. These findings suggest that AMPs employ cytonemes not only for receiving FGFs in a restricted polarized manner but also for a mechanosensory function. In conclusion, these results strongly suggest a critical role of cytonemes in coordinating asymmetric signaling and organization in the stem cell niche. In addition, the work provides evidence that the stem cell cytonemes are critical organelles for integrating the inputs and outputs of both growth factor signaling and mechanical cues to sculpt tissues.Item Using CRISPR/Cas9 to functionally dissect Blimp1, a newly identified pair-rule gene in the hemipteran Oncopeltus fasciatus(2024) Reding, Katie; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genetic screens in the fruit fly Drosophila melanogaster identified a class of mutants displaying half as many segments as seen in wild type fly larvae (Nüsslein-Volhard and Wieschaus 1980). Careful examination of the larval cuticle revealed that one out of every two segments were deleted across the anterior-posterior (AP) axis, an unexpected phenotype suggesting that segmentation in Drosophila follows a ‘pair rule’. Thanks in part to the many genetic tools available for this model species, we now have a clear picture of how the AP axis of the Drosophila embryo is polarized and subsequently divided into distinct segments, and how the pair-rule genes (PRGs) define alternate segments during this process. Since all insects share a similar body plan, it is reasonable to expect that the processes regulating establishment of this body plan would be conserved. However, studies of the Drosophila segmentation gene orthologs in non-model insects suggest that this is not always the case. While the use of model organisms enables an unmatched depth of understanding of the mechanisms underlying development, it comes at the expense of understanding the diversity of these mechanisms across taxa. The milkweed bug Oncopeltus fasciatus (Ofas) (Hemiptera) is a particularly useful insect to study in this regard, as none of the orthologs of the Drosophila PRGs have clear PR-function in this species (Liu and Kaufman 2005b; Auman and Chipman 2018; Reding et al. 2019), while the gene E75A, which has no role in segmentation in Drosophila, is expressed in a PR pattern and its knockdown yields PR segmentation defects (Erezyilmaz et al. 2009). These results suggested that PR-regulation of segmentation in Oncopeltus might require a different set of factors than those discovered in Drosophila. To identify other non-canonical PRGs in this species, I conducted an expression pattern-based screen of transcription factor-encoding genes that are co-expressed with E75A during embryogenesis, and have identified the gene Blimp1 as an Oncopeltus PRG. Like the Drosophila PR mutants, Ofas-Blimp1 mutants display loss of alternate segments across the AP axis. No roles of Blimp1 in insect segmentation had been identified prior to this finding. This result suggests that while insect segmentation may be constrained to follow a pair rule, the genes responsible for regulating PR-segmentation are evolutionarily labile. Further, a major barrier to studying gene function in non-models is the lack of genetic tools such as visible markers and established methods for gene editing. Here I will describe deployment of CRISPR/Cas9 technology in Oncopeltus for targeted mutagenesis. While mutation of the ABC transporter-encoding gene white proved to be recessive lethal, I was able to generate a viable visible marker line by disrupting the X-linked gene Ofas-vermilion (v). Of-v is required for production of dark brown eye pigments, thus Ofas-v mutants have bright red eyes, easily discernible from the black eyes of wild type bugs. I show that a co-CRISPR approach using Of-v as a marker of germline mutation is a helpful strategy to identify mutations of interest at unlinked loci, enabling many future genetic manipulations in this species.Item STUDIES ON VARIABILITY IN CANCER GENE EXPRESSION: FROM SINGLE PROTEINS TO POPULATIONS(2023) Crawford, David Robert; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this dissertation I describe four projects investigating different aspects of the variability of gene expression in human cancers. In the first chapter, we analyze epidemiological incidence rates for autoimmune diseases and cancers across numerous populations and find that sex biases in incidence rates are positively correlated between autoimmune diseases and cancers arising from the same tissue. We find that across these tissues the expression of protein-codingmitochondrial genes is positively correlated with both autoimmune disease and cancer incidence rate sex biases, suggesting a possible direction for further investigation. In the second chapter, I construct a computational pipeline to conduct unbiased searches in large databases for possible events accounting for cancer neopeptides predicted by mass spectrometry. I identify several ribosomal frameshift-derived neopeptides from HLA-peptidomics data and discuss future approaches for further improving the accuracy and flexibility of our approach. In the third chapter, I compare the power of different multivariate Cox proportional hazards survival models based on gene- and below-gene-level expression measures to predict genes whose expression in tumor samples at diagnosis affects subsequent survival of cancer patients. I find that models based on both gene-level expression and isoform-level expression (whether transcript abundance or relative transcript abundance) identify the greatest number of statistically significant genes of interest. Finally, in the fourth chapter I briefly explore how heteroformity and entropy measures can be used to examine differences in mRNA splicing diversity at numerous levels of comparison. I propose some simple visualizations that harness these measures to display patterns in splicing diversity.Item A WIDE SCALE INVESTIGATION INTO LNCRNA IN BOS TAURUS(2023) Marceau, Alexis; Ma, Li; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although the history of genetic research has focused on genes and gene products, there is an interesting emerging subclass of genetic elements: long noncoding RNAs (lncRNAs). These are portions of the genome that are longer than 200 base pairs in length and are transcribed from DNA to RNA but do not yield a protein. The function of lncRNA is wide reaching and difficult to define; however, they are predominantly linked to the regulation of gene expression. This is done via transcriptional control, translation control, pre- and post- transcriptional and translational control, epigenetic modifications, RNA processing,as well as other methods. In this dissertation, multiple Bos taurus tissues across various life conditions were investigated in order to identify lncRNA and to begin making predictions about the role and function of identified transcripts. First, lncRNA were identified and analyzed in Bos taurus rumen tissue in pre-weaning and post-weaning cattle. lncRNA were implicated in the weaning process and demonstrated enrichment in complex traits, indicating the continued impact rumen-associated lncRNA have on dairy cattle. Following this study, mammary tissues from dry and lactating cattle were used for lncRNA analysis, in relation to the lacta-tion processes. This study revealed both the presence and impact of mammary lncRNA, and identified lncRNA associated with genes and biological processes that are strongly linked to lactation and mammary tissue function. Subsequently, immune system related tissues were analyzed for lncRNA and their roles. This investigation demonstrated lncRNA to be present in all investigated tissues, including transcripts being repeatedly present. Further analysis into identified lncRNA associated transcripts with genes and functions that are crucial to immune response. Finally, a tutorial was created to make lncRNA identification research more easily accessible to future researchers. The findings and creations of this dissertation increase the knowledge base of lncRNA and their role, allowing for further research endeavors and improvements in Bos taurus husbandry.Item Investigation of progerin expression in non-Hutchinson-Gilford Progeria Syndrome individuals(2023) Yu, Reynold; Cao, Kan; Mount, Steve; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hutchinson-Gilford Progerin Syndrome (HGPS) is a premature aging disease caused by a point mutation in the LMNA gene, which encodes A-type lamins. This mutation activates a cryptic splice donor in exon 11 and leads to the production of a toxic lamin variant called progerin. Interestingly, small amounts of progerin have also been found in cells and tissues of normal individuals. Here we examine the expression of progerin in publicly available RNA-seq data from normal individuals of the GTEx project. Among the 30 available tissues, progerin expression in normal individuals is highest in sun-exposed skin samples, and its expression in different tissues of the same donor is correlated. In addition, telomere shortening is significantly correlated with progerin expression. Transcriptome-wide correlation analyses suggest that the level of progerin expression is highly correlated with switches in gene isoform expression patterns, perhaps reflecting widespread isoform shifts in these samples. Differential expression analyses show that progerin expression is correlated with significant changes in the level of transcripts from genes involved in splicing regulation and a significant reduction of mitochondrial transcripts. Interestingly, 5’ splice sites whose use is correlated (either positively or negatively) with progerin expression have significantly altered frequencies of consensus trinucleotides within the core 5’ splice site. Furthermore, introns whose alternative splicing is correlated with progerin have reduced GC content. Together, our study suggests that progerin expression in normal individuals is part of a global shift in splicing patterns and provides insight into the mechanism behind these changes.Item The genomics of species divergence in drosophila(2023) Carpinteyro Ponce, Javier; Machado, Carlos A; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)How do new species arise and diverge? Has been a fundamental question in evolutionary biology. The process of species divergence can be studied at many different levels of biological organization. However, it is until the recent advancements of genome sequencing technologies that genome-wide signatures of species divergence have started to unveil the complex genomic landscape of speciation. In this dissertation we investigate the landscape of genomic divergence using a classic pair of Drosophila species. We generated four new high quality genome assemblies for Drosophila pseudoobscura and D. persimilis to explore the genomic differences at three different levels. We first characterized the structural variation landscape between D. pseudoobscura and D. persimilis and stablished its association with transposable elements and tested how intrinsic genomic factors, such as recombination, influence the accumulation ofstructural variation associated with transposable elements in both species. With a combination of high-quality genome assemblies and a comprehensive population genomics data set, we also explored how the contribution of recombination rate and introgression promote sequence divergence with the potential of forming species barriers. Moreover, we investigated how gene co-expression networks potentially rewiring between species contribute to the divergence landscape between D. pseudoobscura and D. persimilis. Our work highlights the complex landscape of species divergence occurring at multiple levels of organization. Moreover, the integration of potential species drivers identified at different scales shed lights on the molecular mechanisms involved in speciation.