Browsing by Subject "Systematic biology"

An essential property of microbial communities is the ability to survive a disturbance. This is readily observed in bacteria, which have developed the ability to survive every antibiotic treatment at an alarming rate, considering the timescale at which new antibiotics are developed. Thus, there is a critical need to use antibiotics more effectively, extend the shelf life of existing antibiotics and minimize their side effects. This requires understanding the mechanisms underlying bacterial drug responses. Past studies have focused on survival in the presence of antibiotics by individual cells, such as genetic mutants. Also important, however, is the fact that a population of bacterial cells can collectively survive antibiotic treatments lethal to individual cells. This tolerance can arise by diverse mechanisms, including resistance-conferring enzyme production, titration-mediated bistable growth inhibition, swarming and interpopulation interactions. These strategies can enable rapid population recovery after antibiotic treatment and provide a time window during which otherwise susceptible bacteria can acquire inheritable genetic resistance.

To further explore bacterial antibiotic responses, I focused on bacteria producing β-lactamase, an enzyme that has drastically limited the use of our most commonly prescribed antibiotics: β-lactams. Through the characterization of clinical isolates and a computational model, my Ph.D. thesis has three implications:

First, survival can be achieved through resistance, the ability to absorb effects of a disturbance without a significant change, or resilience, the ability to recover after being perturbed by a disturbance. Current practices for determining the antibiotic sensitivity of bacteria do not characterize a population as resistant and/or resilient, they only report whether the bacteria can survive the antibiotic exposure. As resistance and resilience often depend on different attributes, distinguishing between these two modes of survival could inform treatment strategies. These concepts have long been applied to the analysis of ecological systems, though their interpretations are often subject to debate. This framework readily lends itself to the dissection of the bacterial response to antibiotic treatment, where both terms can be unambiguously defined.

Second, the ability to tolerate the antibiotic treatment in the short term corresponds to resistance, which primarily depends on traits associated with individual cells. In contrast, the ability to recover after being perturbed by an antibiotic corresponds to resilience, which primarily depends on traits associated with the population.

And finally, understanding the temporal dynamics of an antibiotic response could guide the design of a dosing protocol to optimize treatment efficiency for any antibiotic-pathogen combination. Ultimately, optimized dosing protocols could allow reintroduction of a repertoire of first-line antibiotics with improved treatment outcomes and preserve last-resort antibiotics.

The Polypodium vulgare complex (Polypodiaceae) comprises a well-studied group of fern taxa whose members are cryptically differentiated morphologically and have generated a confusing and highly reticulate species cluster. Once considered a single species spanning much of northern Eurasia and North America, P. vulgare has been segregated into approximately 17 diploid and polyploid taxa as a result of cytotaxonomic work, hybridization experiments, and isozyme studies conducted during the 20th century. Despite considerable effort, however, the evolutionary relationships among the diploid members of the P. vulgare complex remain poorly resolved, and several taxa, particularly allopolyploids and their diploid progenitors, remain challenging to delineate morphologically due to a dearth of stable diagnostic characters. Furthermore, compared to many well-studied angiosperm reticulate complexes, relatively little is known about the number of independently-derived lineages, distribution, and evolutionary significance of the allopolyploid species that have formed recurrently. This dissertation is an attempt to advance systematic knowledge of the Polypodium vulgare complex and establish it as a "model" system for investigating the evolutionary consequences of allopolyploidy in ferns.

Chapter I presents a diploids-only phylogeny of the P. vulgare complex and related species to test previous hypotheses concerning relationships within Polypodium sensu stricto. Analyses of sequence data from four plastid loci (atpA, rbcL, matK, and trnG-trnR) recovered a monophyletic P. vulgare complex comprising four well-supported clades. The P. vulgare complex is resolved as sister to the Neotropical P. plesiosorum group and these, in turn, are sister to the Asian endemic Pleurosoriopsis makinoi. Divergence time analyses incorporating previously derived age constraints and fossil data provide support for an early Miocene origin for the P. vulgare complex and a late Miocene-Pliocene origin for the four major diploid lineages of the complex, with the majority of extant diploid species diversifying from the late Miocene through the Pleistocene. Finally, node age estimates are used to reassess previous hypotheses, and to propose new hypotheses, about the historical events that shaped the diversity and current geographic distribution of the diploid species of the P. vulgare complex.

Chapter II addresses reported discrepancies regarding the occurrence of Polypodium calirhiza in Mexico. The original paper describing this taxon cited collections from Mexico, but the species was omitted from the recent Pteridophytes of Mexico. Originally treated as a tetraploid cytotype of P. californicum, P. calirhiza now is hypothesized to have arisen through hybridization between P. glycyrrhiza and P. californicum. The allotetraploid can be difficult to distinguish from either of its putative parents, but especially so from P. californicum. These analyses show that a combination of spore length and abaxial rachis scale morphology consistently distinguishes P. calirhiza from P. californicum and confirm that both species occur in Mexico. Although occasionally found growing together in the United States, the two species are strongly allopatric in Mexico, where P. californicum is restricted to coastal regions of the Baja California peninsula and neighboring Pacific islands and P. calirhiza grows at high elevations in central and southern Mexico. The occurrence of P. calirhiza in Oaxaca, Mexico, marks the southernmost extent of the P. vulgare complex in the Western Hemisphere.

Chapter III examines a case of reciprocal allopolyploid origins in the fern Polypodium hesperium and presents it as a natural model system for investigating the evolutionary potential of duplicated genomes. In allopolyploids, reciprocal crosses between the same progenitor species can yield lineages with different uniparentally inherited plastid genomes. While likely common, there are few well-documented examples of such reciprocal origins. Using a combination of uniparentally inherited plastid and biparentally inherited nuclear sequence data, we investigated the distributions and relative ages of reciprocally formed lineages in Polypodium hesperium, an allotetraploid fern that is broadly distributed in western North America. The reciprocally-derived plastid haplotypes of Polypodium hesperium are allopatric, with populations north and south of 42˚ N latitude having different plastid genomes. Biogeographic information and previously estimated ages for the diversification of its diploid progenitors, lends support for middle to late Pleistocene origins of P. hesperium. Several features of Polypodium hesperium make it a particularly promising system for investigating the evolutionary consequences of allopolyploidy. These include reciprocally derived lineages with disjunct geographic distributions, recent time of origin, and extant diploid progenitor lineages.

This dissertation concludes by demonstrating the utility of the allotetraploid Polypodium hesperium for understanding how ferns utilize the genetic diversity imparted by allopolyploidy and recurrent origins. Chapter IV details the use of high-throughput sequencing technologies to generate a reference transcriptome for Polypodium, a genus without preexisting genomic resources, and compare patterns of total and homoeolog-specific gene expression in leaf tissue of reciprocally formed lineages of P. hesperium. Genome-wide expression patterns of total gene expression and homoeolog expression ratios are strikingly similar between the lineages--total gene expression levels mirror those of the diploid progenitor P. amorphum and homoeologs derived from P. amorphum are preferentially expressed. The unprecedented levels of unbalanced expression level dominance and unbalanced homoeolog expression bias found in P. hesperium supports the hypothesis that these phenomena are pervasive consequences of allopolyploidy in plants.

Embryonic development occurs with precisely timed morphogenetic cell movements directed by complex gene regulation. In this orchestrated series of events, some epithelial cells undergo extensive changes to become free moving mesenchymal cells. The transformation resulting in an epithelial cell becoming mesenchymal is called an epithelial to mesenchymal transition (EMT), a dramatic cell biological change that occurs throughout development, tissue repair, and disease. Extensive in vitro research has identified many EMT regulators. However, most in vitro studies often reduce the complicated phenotypic change to a binary choice between successful and failed EMT. Research utilizing models has generally been limited to a single aspect of EMT without considering the total transformation. Fully understanding EMT requires experiments that perturb the system via multiple channels and observe several individual components from the series of cellular changes, which together make a successful EMT.

In this study, we have taken a novel approach to understand how the sea urchin embryo coordinates an EMT. We use systems level methods to describe the dynamics of EMT by directly observing phenotypic changes created by shifting transcriptional network states over the course of primary mesenchyme cell (PMC) ingression, a classic example of developmental EMT. We systematically knocked down each transcription factor in the sea urchin's PMC gene regulatory network (GRN). In the first assay, one fluorescently labeled knockdown PMC precursor was transplanted onto an unperturbed host embryo and we observed the resulting phenotype in vivo from before ingression until two hours post ingression using time-lapse fluorescent microscopy. Movies were projected for computational analyses of several phenotypic changes relevant to EMT: apical constriction, apical basal polarity, motility, and de-adhesion.

A separate assay scored each transcription factor for its requirement in basement membrane invasion during EMT. Again, each transcription factor was knocked down one by one and embryos were immuno-stained for laminin, a major component of basement membrane, and scored on the presence or absence of a laminin hole at the presumptive entry site of ingression.

The measured results of both assays were subjected to rigorous unsupervised data analyses: principal component analysis, emergent self-organizing map data mining, and hierarchical clustering. This analytical approach objectively compared the various phenotypes that resulted from each knockdown. In most cases, perturbation of any one transcription factor resulted in a unique phenotype that shared characteristics with its upstream regulators and downstream targets. For example, Erg is a known regulator of both Hex and FoxN2/3 and all three shared a motility phenotype; additionally, Hex and Erg both regulated apical constriction but Hex additionally affected invasion and FoxN2/3 was the lone regulator of cell polarity. Measured phenotypic changes in conjunction with known GRN relationships were used to construct five unique subcircuits of the GRN that described how dynamic regulatory network states control five individual components of EMT: apical constriction, apical basal polarity, motility, de-adhesion, and invasion. The five subcircuits were built on top of the GRN and integrated existing fate specification control with the morphogenetic EMT control.

Early in the EMT study, we discovered one PMC gene, Erg, was alternatively spliced. We identified 22 splice variants of Erg that are expressed during ingression. Our Erg knockdown targeted the 5'UTR, present in all spliceoforms; therefore, the knockdown uniformly perturbed all native Erg transcripts (∑Erg). Specific function was demonstrated for the two most abundant spliceoforms, Erg-0 and Erg-4, by knockdown of ∑Erg and mRNA rescue with a single spliceoform; the mRNA expression constructs contained no 5'UTR and were not affected by the knockdown. Different molecular phenotypes were observed, and both spliceoforms targeted Tbr, Tel, and FoxO, only Erg-0 targeted FoxN2/3 and only Erg-4 targeted Hex. Neither targeted Tgif, which was regulated by ∑Erg knockdown sans rescue. Our results suggest the embryo employs a minimum of three unique roles in the GRN for alternative splicing of Erg.

Overall, these experiments increase the completeness and descriptive power of the GRN with two additional levels of complexity. We uncovered five sub-circuits of EMT control, which integrated into the GRN provide a novel view of how a complex morphogenetic movement is controlled by the embryo. We also described a new functional role for alternative splicing in the GRN where the transcriptional targets for two splice variants of Erg are unique subsets of the total set of ∑Erg targets.

The identification and quantification of protein-protein interactions in large scale is critical to understanding biological processes at a systems level. Current approaches for the analysis of protein -protein interactions are generally not quantitative and largely limited to certain types of interactions such as binary and strong binding interactions. They also have high false-positive and false-negative rates. Described here is the development of and application of mass spectrometry-based proteomics metehods to detect and quantify the strength of protein-protein and protein-ligand interactions in the context of their interaction networks. Characterization of protein-protein and protein-ligand interactions can directly benefit diseased state analyses and drug discovery efforts.

The methodologies and protocols developed and applied in this work are all related to the Stability of Unpurified Proteins from Rates of amide H/D Exchange (SUPREX) and Stability of Protein from Rates of Oxidation (SPROX) techniques, which have been previously established for the thermodynamic analysis of protein folding reactions and protein-ligand binding interactions. The work in this thesis is comprised of four parts. Part I involves the development of a Histidine Slow H/D exchange protocol to facility SURPEX-like measurements on the proteomic scale. The Histidine Slow H/D exchange protocol is developed in the context of selected model protein systems and used to investigate the thermodynamic properties of proteins in a yeast cell lysate.

In Part II an isobaric mass tagging strategy is used in combination with SPROX (i.e., a so-called iTRAQ-SPROX protocol) is used to characterize the altered protein interactions networks associated with lung cancer. This work involved differential thermodynamic analyses on the proteins in two different cell lines, including ADLC-5M2 and ADLC-5M2-C2.

Parts III and IV of this thesis describe the development and application of a SPROX protocol for proteome-wide thermodynamic analyses that involves the use of Stable Isotope Labeling by Amino acid in cell Culture (SILAC) quantitation. A solution-based SILAC-SPROX protocol is described in Part III and a SILAC-SPROX protocol involving the use of cyanogen bromide and a gel-based fractionation step is described in Part IV. The SILAC-SPROX-Cyanogen bromide (SILAC-SPROX-CnBr) protocol is demonstrated to significantly improve the peptide and protein coverage in proteome-wide SPROX experiments. Both the SILAC-SPROX and SILAC-SPROX-CnBr porotocols were used to characterize the ATP binding properties of yeast proteins. Ultimately, the two protocols enabled 526 yeast proteins to be assayed for binding to AMP-PNP, an ATP mimic. A total of 140 proteins, including 37 known ATP-binding proteins, were found to have ATP binding interactions.

Edaphic factors--those pertaining to the substrate or soil--are thought to play an important role in the diversification of flowering plants. Although edaphic factors are widely interpreted as causal agents in plant diversification, little is known about the evolutionary origin of most edaphic endemic plants, preventing inference of potential mechanisms by which substrate properties may influence speciation. The North American plant genus Ceanothus (Rhamnaceae) contains 9 edaphic-endemic species, taxa restricted to soils derived from specific geological materials. The three components of my dissertation research aim to improve understanding of how edaphic ecology has influenced the diversification of Ceanothus. First, I use DNA sequence data from the low-copy nuclear gene nitrate reductase to reconstruct the phylogeny of Ceanothus and elucidate diversification of this group into the California Floristic Province (CFP) of western North America, including the evolution of edaphic endemism. This research indicates that diversification of the two Ceanothus subgenera (Cerastes and Ceanothus) is centered on the CFP and is characterized by shallow divergence and phylogenetic relationships defined predominantly by geography. Divergence time estimation suggests that diversification of both Ceanothus subgenera began approximately 6 Ma. The nine edaphic-endemic taxa are not phylogenetically clustered in my analyses, suggesting that the origin of edaphic endemism has occurred on multiple occasions, including multiple examples of serpentine endemism. The second chapter of my dissertation uses soil chemistry data in combination with a more detailed examination of genetic variation in nitrate reductase to elucidate the evolution of a single edaphic endemic species.

Bryophytes, by their haploid dominant life cycle, possess several unique qualities ideal for study of mating patterns. In particular, the possibility of intragametophytic selfing in some species, and the vegetative propagation of gametes allow for a unique window into the haploid stage that is intractable in other groups. Despite these advantages, there have been relatively few studies on mating patterns bryophytes in natural populations. Sphagnum (peatmoss) is an excellent case study in the interactions between sexual condition, ecology, and mating patterns. In the first Chapter, we use microsatellites to characterize the genetic diversity and mating patterns in fourteen species of Sphagnum, diverse in sexual condition (separate vs combined sexes in the haploid stage) and ecology (microhabitat variance along the water table). We find that genetic diversity and mating patterns are related only in species with separate sexes, that sexual condition and ecology have interacting effects on inbreeding coefficients, and that inbreeding depression is not a common phenomenon in Sphagnum. In the second Chapter, we conduct an intensive survey of one population of Sphagnum macrophyllum, to detect whether variance in haploid fecundity and mating success is related to diploid fitness. We find a relationship between mating success and fecundity (a signal of sexual selection), and fitness of the diploid generation is connected to the parentage of the haploid generation. Finally, in Chapter 3 we use phylogenetic comparative methods to track the phylogenetic signal in microhabitat preference in Sphagnum. We find extremely fast rates of evolution along the micronutrient gradient, but high phylogenetic signal along a hydrological gradient. Given that Sphagnum species living high above the water table have reduced water availability, phylogenetic signal in the hydrological gradient has macroevolutionary implications for mating systems in Sphagnum.

This dissertation investigates the processes of hybridation, polyploidy, and apomixis and their roles in the evolution of myriopterid ferns. First, I examine patterns of hybridization in members of the Cheilanthes yavapensis complex using a suite of techiniques, ranging from molecules to morphology--including isozymes, spore measurements, and molecular phylogenetics based on chloroplast and nuclear DNA markers--to elucidate relationships in this notorious group of ferns. Second, I utilize the rules of traditional taxonomy set by the International Code of Botanical Nomenclature to recircucmscribe and resurrect the genus Myriopteris from within cheilanthoid ferns. This revised classification is bolstered by results from my molecular phylogenetic analysis of DNA sequence data in the subsequent chapter. Then, using morphological and cytological analyses, I examine the evolution of indument, leaf and rachis shape, vernation, chromosome number, and reproductive mode across the myriopterid tree. In my concluding chapter I develop microsatellite markers for the apomictic triploid, M. lindheiemeri, and explore whether premeiotic chromosome duplication facilitates the production of genetically distinct offspring in this otherwise asexual lineage.

Lichens are symbioses between a fungus and a photosynthesizing partner such as a green alga or a cyanobacterium. Unlike mycorrhizal or rhizobial symbioses, the lichen symbiosis is not well understood either morphologically or molecularly. The lichen symbiosis has been somewhat neglected for several reasons. Lichens grow very slowly in nature (less than 1 cm a year), it is difficult to grow the fungus and the alga separately and, moreover, it remains difficult to resynthesize the mature symbiosis in the laboratory. It is not yet possible to delete genes, nor has any transformation method been established to introduce genes into the genomes of either the fungus or the alga. However, the lack of genetic tools for these organisms has been partially compensated for by the sequencing of the genomes of the lichenizing fungus Cladonia grayi and its green algal partner Asterochloris sp. This work uses the model lichen system Cladonia grayi and the associated genomes to explore one evolutionary and one developmental question concerning the lichen symbiosis.

Chapter One uses data from the genomes to assess whether there was evidence of horizontal gene transfer between the lichen symbionts in the evolution of this very intimate association; that is, whether genes of algal origin could be found in the fungal genome or vise versa. An initial homology search of the two genomes demonstrated that the fungus had, in addition to ammonium transporter/ammonia permease genes that were clearly fungal in origin, ammonium transporter/ammonia permease genes which appeared to be of plant origin. Using cultures of various lichenizing fungi, plant-like ammonium transporter/ammonia permease genes were identified by degenerate PCR in ten additional species of lichen in three classes of lichenizing fungi including the Lecanoromycetes, the Eurotiomycetes, and the Dothidiomycetes. Using the sequences of these transporter genes as well as data from publically available genome sequences of diverse organisms, I constructed a phylogy of 513 ammonium transporter/ammonia permease sequences from 191 genomes representing all main lineages of life to infer the evolutionary history of this family of proteins. In this phylogeny I detected several horizontal gene transfer events, including the aforementioned one which was demonstrated to be not a transfer from plants to fungi or vise versa, but a gene gain from a group of phylognetically unrelated hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina), which was subsequently lost in most lineages but retained in even distantly related lichenized fungi. Also demonstrated was the loss of the native fungal ammonium transporter and the subsequent replacement of this gene with a bacterial ammonium transporter during the early evolution of the fungi. Several additional recent horizontal gene transfers into lineages of eukaryotes were demonstrated as well. The phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). I show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: AMT family as defined here exhibits standard parent-to-offspring inheritance, whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs) into eukaryotes. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with ammonium transporters and ammonia permeases forming two distinct and possibly monophyletic groups.

In Chapter Two I address a follow-up question: in key lichenizing lineages for which ammonium transporter/ammonia permease (AMTP) genes were not found in Chapter One, were the genes lost? The only definitive infomation which can demonstrate absence of a gene from a genome is a full genome sequence. To this end, the genomes of eight additional lichenizing fungi in the key clades including the Caliciales (sensu Gaya 2011), the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales were sequenced using the Ilumina HiSeq technology and assembled with the short reads assembly software Velvet. These genomes were searched for ammonium transporter/ammonia permease sequences as well as 20 test genes to assess the completeness of each assembly. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacteria as a primary photobiont or living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the Acarosporomyetidae and Arthoniomycetes. In addition, the four AMTP genes from Cladonia grayi were shown to be functional by expression of the lichen genes in a strain of Saccharomyces cerevisiae in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source.

In Chapter Three I use genome data to address a developmental aspect of the lichen symbiosis. The finding that DNA in three genera of lichenizing fungi is methylated in symbiotic tissues and not methylated in aposymbiotic tissues or in the free-living fungus (Armaleo & Miao 1999a) suggested that epigenetic silencing may play a key role in the development of the symbiosis. Epigenetic silencing involves several steps that are conserved in many eukaryotes, including methylation of histone H3 at lysine 9 (H3K9) in nucleosomes within the silenced region, subsequent binding of heterochromatin-binding protein (HP1) over the region, and the recruitment of DNA methyltransferases to methylate the DNA, all of which causes the underlying chromatin to adopt a closed conformation, inhibiting the transcriptional machinery from binding. In this chapter I both identify the genes encoding the silencing machinery and determine the targets of the silencing machinery. I use degenerate PCR and genome sequencing to identify the genes encoding the H3K9 histone methyltransferase, the heterochromatin binding protein, and the DNA methyltransferases. I use whole genome bisulfite sequencing of DNA from the symbiotic structures of Cladonia grayi including podetia, squamules and soredia as well as DNA from cultures of the free-living fungus and free-living alga to determine which regions of the genome are methylated in the symbiotic and aposymbiotic states. In particular I examine regions of the genomes which appear to be differentially methylated in the symbiotic versus the aposymbiotic state. I show that DNA methylation is uncommon in the genome of the fungus in the symbiotic and aposymbiotic states, and that the genome of the alga is methylated in the symbiotic and aposymbiotic states.

Tuberculosis (TB) is considered the most lethal infectious disease throughout human history. Mycobacterium tuberculosis (Mtb), the causal agent of tuberculosis, has consistently infected human hosts for millennia, and today, approximately 10 million TB cases and over 1 million deaths are caused by Mtb annually. Infection with Mtb, most often in the lungs via airborne transmission, can produce a spectrum of disease outcomes. An unknown proportion of resistant hosts efficiently contain and sterilize Mtb infection. If a host cannot clear the bacterium, Mtb can lie dormant over the course of decades. From this latent state, there is a 5-15% risk that the case will progress to active disease, characterized by coughing, fever, and cachexic wasting, which can be fatal if left untreated. Although TB is treatable with courses of antibiotics that can range from 3 to 9 months in duration, the emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains increases the urgency of understanding the genetic factors within the host and in the pathogen that give rise to these variable disease outcomes.

To explore the contributions of host and bacterial genetic variation to clinically divergent TB outcomes, we combined mammalian models of natural genetic diversity with next-generation models of engineered mycobacterial diversity. Together, these systems allowed quantitative trait locus (QTL) mapping of the host genome for phenotype-to-genotype associations underlying bacterial fitness and host disease phenotypes following Mtb infection. For over a century, mice have served as tractable mammalian models for the interrogation of Mtb pathogenesis. The studies presented in this dissertation utilize modern genetic reference populations to maximize host genetic diversity for identification of TB QTL and classical mouse breeding schemata to isolate these regions of interest for functional interrogation.

In Chapter 1, we review the history of QTL mapping in Mtb-infected mice. We discuss murine models of natural genetic diversity, QTL mapping as a statistical approach, and vignettes of QTL mapping in Mtb-infected mice. The chapter concludes by contextualizing the work presented in this dissertation as it relates to previous in vivo mapping studies.

In Chapter 2, we report an infection screen of recombinant inbred BXD strains alongside the BXD panel parental strains, Mtb-resistant C57BL/6J (B6) and Mtb-susceptible DBA/2J (D2) with a comprehensive Mtb mutant library (TnSeq). We identified 140 transposon mutant fitness QTL across the host genome as well as a cluster of 4 highly significant QTL on mouse chromosome 6. The work reported in this chapter reveals early bacterial predictors of host divergence in TB susceptibility.

In Chapter 3, we report the investigation of TB susceptibility QTL on chromosomes 7, 15, and 16 (named Tip1-Tip4), which were identified between CC001 and CC042 of the octoparental Collaborative Cross (CC) recombinant inbred panel. Tip2 was caused by a private deletion in CC042, but the genetic causes of Tip1, Tip3, and Tip4 remain unknown. We report bioinformatic and classical congenic approaches that serve to narrow these QTL causal intervals and identify putative candidate genes underlying these TB susceptibility loci.

In Chapter 4, we screened multiple CC strains that exhibited similar lung Mtb burden but divergent TB outcomes to identify markers of disease resilience, independent of Mtb burden. Despite our observation that CC030 mice are highly Mtb-susceptible with high lung burden and inflammation, the CC030 haplotype within a QTL on chromosome 7 (Tip8) is associated with low lung burden and CXCL1. We generated congenic mice with CC030 Tip8 on an isogenic background to identify biomarkers of TB disease resilience in a burden-controlled context, revealing restriction of lung IL-1β and CCL3 a pathway to TB resilience.

In Chapter 5, we explore a region on mouse chromosome 15 in which genomic inheritance from wild-derived CAST/EiJ mice was associated with TB resistance QTL (Tip3, Tip7, Tip10) in several independent infection screens. We report a ~0.4Mb region within the chromosome 15 resistance locus exhibiting non-synteny between all eight of the CC founders, suggesting evolutionary selection at this resistance locus. We further leverage novel RNA-Seq-based CC founder genome annotations to identify novel strain-specific genes within this locus that were previously obscured in reference genome alignment approaches, highlighting mouse apolipoprotein L (Apol) genes as putative promoters of host TB resistance.

In Chapter 6, we explore a potent TB susceptibility locus observed on chromosome 2 (Tip5) in our previous infection screen of 52 CC lines and the eight founder lines. Within this locus, cathepsin Z (Ctsz; also, Ctsx), which has been associated with TB susceptibility in multiple independent TB patient cohorts, was identified as the lead candidate that may underlie Tip5. We report in vivo and ex vivo infection studies that highlight a protective role for Ctsz during Mtb infection for the first time, offering mechanistic insights that may assist in the development of therapeutics for TB patients harboring deleterious SNPs in CTSZ.

In Chapter 7, we summarize the work reported in this dissertation and discuss remaining questions and future directions for each chapter. We conclude the chapter with perspectives on the advances in high-throughput phenotyping, pangenomics, mouse reference populations, and bioinformatic tools and analyses that could support future work in defining the host-pathogen interactions during Mtb infection.

Collectively, the body of work presented in this dissertation represents a multipronged approach to uncovering the host and microbial genetic factors that promote unique TB disease outcomes. To date, the spread of TB remains a public health crisis, profoundly impacting communities across the globe with reduced healthcare accessibility. This work will assist in the development of therapeutics for the eradication of a pathogen that has impacted an estimated one-fourth of the global population.

Bacteria are found throughout the environment, from the air to the soil, but more importantly, they reside within the human body. Crucial to their survival in each of these environments is the constant interplay between these organisms and their surroundings. Inadvertently, the ways in which these stimuli are processed can have a profound impact on human health. With potentially negative or positive consequences, it becomes critical to understand how microorganisms encode and decode signals.

Understanding bacterial signal processing is crucial to tackling the treatment of infectious diseases, especially with the rise of antibiotic resistant organisms. Antibiotic resistance has become a global health issue as bacteria have developed or acquired genes that confer resistance to all antibiotics currently in use today. This has serious implications for the future treatment of infectious diseases, potentially limiting options to those from a pre-antibiotic era. However, as with other external factors, antibiotics are just another signal that bacteria need to decode and encode a response to. As such, it is of utmost importance to better understand how bacteria process stimuli.

In my dissertation, I analyzed the ways in which bacteria both encode and decode information. In particular, I focused on how information is processed from signals with a temporal domain. To start, I developed a computational framework to understand how organisms decode signals, specifically oscillatory signals. With this model, I examined the capability of an incoherent feedforward loop motif to exhibit temporal adaptation, in which a network becomes desensitized to sustained stimuli. I discovered that this property is crucial for networks to distinguish signals of varying temporal dynamics.

In terms of information encoding, I utilized the complexity of this process to predict bacterial characteristics of interest. The fundamental premise behind this work is to increase the information content of phenotypes for the prediction of bacterial characteristics. Specifically, I used the temporal domain of growth for the prediction of genetic identity and traits of interest. I demonstrated that temporal growth dynamics under standardized conditions can differentiate among hundreds of strains, even strains of the same species. While growth dynamics could, with high accuracy, differentiate between unique strains, it was insufficient to quantify how genetically different these strains were. This absence highlighted the challenges in using genomics to infer phenotypes and vice versa. Bypassing this complexity, I showed that growth dynamics alone could robustly predict antibiotic responses. Together, my findings demonstrate the ability to develop applications that take advantage of the complexity of bacterial information encoding.

This work highlights the importance of understanding how bacteria decode signals with temporal dynamics. Additionally, I demonstrated one application for utilizing bacterial signal encoding, the prediction of bacterial characteristics.

Bacteria are found throughout the environment, from the air to the soil, but more importantly, they reside within the human body. Crucial to their survival in each of these environments is the constant interplay between these organisms and their surroundings. Inadvertently, the ways in which these stimuli are processed can have a profound impact on human health. With potentially negative or positive consequences, it becomes critical to understand how microorganisms encode and decode signals.

Understanding bacterial signal processing is crucial to tackling the treatment of infectious diseases, especially with the rise of antibiotic resistant organisms. Antibiotic resistance has become a global health issue as bacteria have developed or acquired genes that confer resistance to all antibiotics currently in use today. This has serious implications for the future treatment of infectious diseases, potentially limiting options to those from a pre-antibiotic era. However, as with other external factors, antibiotics are just another signal that bacteria need to decode and encode a response to. As such, it is of utmost importance to better understand how bacteria process stimuli.

In my dissertation, I analyzed the ways in which bacteria both encode and decode information. In particular, I focused on how information is processed from signals with a temporal domain. To start, I developed a computational framework to understand how organisms decode signals, specifically oscillatory signals. With this model, I examined the capability of an incoherent feedforward loop motif to exhibit temporal adaptation, in which a network becomes desensitized to sustained stimuli. I discovered that this property is crucial for networks to distinguish signals of varying temporal dynamics.

In terms of information encoding, I utilized the complexity of this process to predict bacterial characteristics of interest. The fundamental premise behind this work is to increase the information content of phenotypes for the prediction of bacterial characteristics. Specifically, I used the temporal domain of growth for the prediction of genetic identity and traits of interest. I demonstrated that temporal growth dynamics under standardized conditions can differentiate among hundreds of strains, even strains of the same species. While growth dynamics could, with high accuracy, differentiate between unique strains, it was insufficient to quantify how genetically different these strains were. This absence highlighted the challenges in using genomics to infer phenotypes and vice versa. Bypassing this complexity, I showed that growth dynamics alone could robustly predict antibiotic responses. Together, my findings demonstrate the ability to develop applications that take advantage of the complexity of bacterial information encoding.

This work highlights the importance of understanding how bacteria decode signals with temporal dynamics. Additionally, I demonstrated one application for utilizing bacterial signal encoding, the prediction of bacterial characteristics.

Cancer is a disease state that arises as a result of multiple alterations in signaling pathways that are critical for making key cell fate decisions in normal cells. Understanding how these pathways operate under normal circumstances, therefore, is crucial for comprehensive understanding of tumorigenic process. With Myc and E2F pathways being central components for controlling cell proliferation, an important property that defines a cancer cell, as well as expanding roles for microRNAs(miRNA) in control of gene expression, we asked if we may better understand the underlying regulatory (transcription factor, microRNA) structure that contribute to Myc and E2F pathway activities. Through integrative analysis of mRNA and miRNA expression profile, we observe a distinct regulatory pattern in which, in the case of Myc pathway, Myc-induced miRNAs were contributing to the repression of negative regulators of cell cycle, including PTEN, while in case of E2F pathway, E2F-induced miRs were forming an incoherent Feed-Forward Loop(iFFL) with a number of E2F-induced genes including cyclin E. We further demonstrate through functional studies, as well as through single cell imaging of gene expression dynamics that miRNAs, depending on the context of either Myc or E2F pathway, play distinct roles in ensuring that cell fate decisions relevant to these pathways are properly executed.

How geographical distance and historical events affect patterns of population divergence and gene flow is an important question in evolution and biogeography. Sphagnum subgenus Acutifolia is one of the four major subgenera of Sphagnum peatmoss comprising numerous species with broad geographic ranges and diverse ecological niches within wetland habitats. One group of particular interest within the subgenus is the S. capillifolium complex which contains at least seven closely related species. Five species within the complex are circumboreal and all have overlapping geographic ranges, one species is endemic to subtropical region of eastern North America, and one species is in the tropical regions of Central and South America. The presence of both species with overlapping and disjunct distributions makes the Sphagnum capillifolium complex a natural experiment to investigate gene flow and population divergence in multiple phylogenetic scales (within and between species). Chapter 1 describes patterns of phylogenetic discordance across the genome in the Sphagnum capillifolium complex using whole genome resequencing data. The species tree phylogeny was generally well supported but phylogenetic discordance among genomic regions was prevalent, especially at nodes in the backbone. Alternative topologies for each of the backbone nodes were not random, suggesting the presence of introgression, in addition to incomplete lineage sorting (ILS). Analyses of introgression signals using ABBA/BABA tests and branch length distributions (QuIBL) showed that there were several possible introgression events within the S. capillifolium complex involving both extant and ancestral species. Most of the introgression events occurred between species that currently have overlapping geographic ranges. Further investigation of one introgression event using comparisons of terminal branch lengths showed that the biased pattern of shared derived alleles likely derives from introgression, not ancient polymorphism. These findings show that introgression played a significant role in generating phylogenetic incongruence within the S. capillifolium complex. We also show that the use of multiple phylogenomic methods and investigating localized genomic regions are essential to infer complex introgression scenarios. Chapter 2 describes phylogenetic structure of Sphagnum subgenus Acutifolia and population structure of circumboreal species within the S. capillifolium complex. Genome scale data (RAD-seq) was generated for the subgenus, with an intensive population sampling of circumboreal species within the S. capillifolium complex. Most of the species are resolved as monophyletic, although relationships among species are weakly supported in some parts of the phylogeny. Some currently recognized species are phylogenetically discernable while others are not distinguishable from the well-supported species. Within the S. capillifolium complex, five circumboreal species show similar patterns of population structure. One population system comprises plants in eastern North America and Europe, and sometimes includes plants from eastern Eurasia and the Pacific Northwest of North America. Another group comprises plants in the Pacific Northwest, or around the Beringian and Arctic regions. Our results suggest that populations of circumboreal species survived in multiple refugia during the last glacial maximum (LGM). Long-distance dispersal out of refugia, population bottlenecks, and possible adaptations to conditions unique to each refugium contribute to current geographic patterns. There are patterns of genetic admixture among distinct genotype groups within species in some restricted areas. Alaska is a hotspot for both intraspecific genetic diversity and admixture. These genetic results indicate the important role of historical events, especially Pleistocene glaciation, in shaping the complex population structure of plants with broad distribution ranges. Chapter 3 assesses the pattern of gene flow between a pair of sister Sphagnum species within the S. capillifolium complex: S. warnstorfii and S. talbotianum. The two species have different distribution ranges: S. warnstorfii is circumboreal while S. talbotianum is circumarctic, but they overlap in Alaska. Genetic data from chapter 2 were used in this chapter. Analyses of interspecific gene flow and population sizes were accomplished using coalescent simulations of site frequency spectra (SFSs), and the signature of gene flow was further corroborated by ABBA/BABA statistics. Our results indicate that S. warnstorfii and S. talbotianum were isolated after divergence. S. warnstorfii was relatively recently established in Alaska and Alaska is the only region that shows evidence of gene flow between S. talbotianum and S. warnstorfii. Gene flow occurred in only one direction from S. talbotianum into S. warnstorfii, which can possibly help S. warnstorfii survive in subarctic conditions. Molecular evidence further suggests gene flow from Alaska S. warnstorfii to other regional populations of that species. S. warnstorfii suffered a stronger population bottleneck than S. talbotianum, suggesting that Beringia could have harbored larger populations during the last glacial maximum than other, likely more southern, refugia. Although the two species are very closely related, S. talbotianum has larger pores on the convex surfaces of branch leaf apices than S. warnstorfii. Our results represent a case study of a recent gene flow between geographically sympatric peatmoss species using genomic data. Our results also support S. talbotianum as a distinct species from S. warnstorfii.

Why do species exist where they do? Understanding the forces and processes that shape species’ ranges—and that affect their dispersal and range expansion—have long fascinated biologists. In this work, I focus on understanding diversity and dispersal in Cheilanthes ferns. I first describe a species new to science, Cheilanthes ecuadorensis, from among the understudied South American members of this genus. I then turn to studying the widely distributed, asexual, Australasian species C. distans. Careful review of samples from this species allowed me to find sexual specimens previously unknown to science that exist in a narrow range, as well as to catalogue extensive spore diversity that has gone unrecorded. I find strong evidence for trade-offs related to spore size, with larger spores having higher germination while smaller spores have greater dispersal. Excitingly, I find that spores previously catalogued as abortive are in fact viable, and contribute to the spore size diversity I observe. I then place these findings into phylogenetic context by building a phylogeny for all Australasian Cheilanthes, and use it to explore the relationships of sexual and asexual lineages, of different ploidy levels, and of geographic distributions. These analyses reveal that most dispersal in C. distans occurs over shorter rather than longer distances, in contrast to previous hypotheses posited by fern biologists. I observe that lineages are not limited to particular geographic regions, as well finding that dispersal is asymmetrical and seems to be tracking trade winds. For all my work I rely heavily on herbarium specimens and use them to catalogue morphological variation as well as to obtain DNA sequences that are used for phylogenetic analysis. I implement a variety of statistical and systematic analyses to explore correlations between spore size, reproductive mode, ploidy, germination, and dispersal. While this work expands our knowledge of fern diversity and biogeography, much still remains to be understood, including cataloguing possible novel species, understanding the biology behind spore size determination, and exploring the role of niche in the dispersal and range expansion of C. distans.

The fern family Cystopteridaceae has been a thorn in the side of fern phylogeneticists, on many levels. Until this thesis, its basic existence (as a deeply isolated clade) and composition were unrecognized, hypotheses as to the relationships of its constituents within the broader fern tree-of-life were wildly inconsistent, the relationships of its genera to each other were contested, the species limits within those genera weakly understood, and the relationships among those species unknown. This thesis first establishes the broad evolutionary context for the family, which is that it is the first-diverging branch in Eupolypods II (it is sister to the rest of the eupolypod II clade). Eupolypods II is a large clade, containing nearly a third of extant fern species, making the Cystopteridaceae's position pivotal to a full understanding of fern evolution.

The evolution of the Eupolypods II is marked by an "ancient, rapid radiation" at the base of the clade, which helps to explain the difficulty that this broad group has historically posed to evolutionary biologists. Molecular data from five plastid loci show that Eupolypods II is comprised of 10 deeply divergent lineages, each worthy of recognition at the rank of family: Cystopteridaceae, Rhachidosoraceae, Diplaziopsidaceae, Hemidictyaceae, Aspleniaceae, Thelypteridaceae, Woodsiaceae, Onocleaceae, Blechnaceae, and Athyriaceae. The ancestors of Cystopteridaceae diverged from those of the rest of the clade approximately 100 million years ago, and the family is now comprised of five extant genera: Acystopteris, Cystoathyrium (the only genus for which we lack molecular data--it may be extinct), Cystopteris, Gymnocarpium, and ×Cystocarpium.

Within the family, the relationships of Cystoathyrium are unknown. Acystopteris is sister to Cystopteris, and those two genera, together, are sister to Gymnocarpium. Gymnocarpium is the maternal parent of ×Cystocarpium, so that genus falls within Gymnocarpium in phylogenetic trees based on maternally transmitted loci (i.e., plastid or mitochondrial loci). Plastid data resolve a basal trichotomy in Gymnocarpium, among the G. disjunctum clade, the G. robertianum clade, and core Gymnocarpium. The earliest diverging branch of core Gymnocarpium is the morphologically anomalous G. oyamense, followed by a split that separates G. appalachianum and G. jessoense parvulum (on one side) from G. remotepinnatum and G. jessoense jessoense, on the other. In Acystopteris, the first division surprisingly separates A. taiwaniana (which is frequently treated as a variety of A. japonica) from A. japonica + A. tenuisecta (which are morphologically very distinct from each other).

The evolution of Cystopteris is, as expected, more complex. The first lineage to diverge from the rest of the genus is the one that gave rise to C. montana. The next division, however, is unclear; molecular data infer a trichotomy among the sudetica clade (containing C. sudetica, C. moupinensis, and C. pellucida), the bulbifera clade (containing C. bulbifera and its related allopolyploids C. tennesseensis and C. utahensis), and the C. fragilis complex. Within the C. fragilis complex relationships (and species limits) get particular messy. The diploid species of eastern North America--C. protrusa--is sister to the rest of the complex, but after that point the major named species (including C. fragilis and C. tenuis) cease to be monophyletic, being found on both sides of a major split, alongside such taxa as the Australian/New Zealand C. tasmanica, the Hawaiian C. douglasii, and the Mexican C. membranifolia and C. millefolia.

In the context of the deep divergence of Gymnocarpium from Cystopteris, and the complicated species-level patterns of relationship within each genus, it is particularly surprising that molecular data confirm that ×Cystocarpium is a hybrid between Gymnocarpium dryopteris and a European tetraploid member of the Cystopteris fragilis complex. The ancestors of Cystopteris diverged from those of Gymnocarpium approximately 58 million years ago, meaning that the ×Cystocarpium hybridization event (which happened very recently) united genomes that contain, between them, over 100 million years of independent evolution. This breadth of divergence makes ×Cystocarpium the most extreme example of wide hybridization currently documented, with important implications for the pace of evolution of reproductive isolation, and thus for species formation.

This thesis ends with a tentative synopsis of the Cystopteridaceae (Appendix E). The family, as construed here, contains five genera and approximately 36 species (three in Acystopteris, one in Cystoathyrium, ~25 in Cystopteris, seven in Gymnocarpium, and one in ×Cystocarpium), plus two named subspecies (one each in Cystopteris and Gymnocarpium), and eight named sterile hybrids (three in Cystopteris and five in Gymnocarpium). Each of these tallies is highly subjective--much further research, with an emphasis on cytological and low-copy nuclear data, is necessary before we can hope to have any confidence in the species limits and finer-scale evolutionary patterns in this family.

Morphological characters from the gametophyte and sporophyte generations have been used in land plants to infer relationships and construct classifications, but sporophytes provide the vast majority of data for the systematics of vascular plants. In bryophytes both generations are well developed and characters from both are commonly used to classify these organisms. However, because morphological traits of gametophytes and sporophytes can have different genetic bases and experience different selective pressures, taxonomic emphasis on one generation or the other may yield incongruent classifications. The moss order Hookeriales has a controversial taxonomic history because previous classifications have focused almost exclusively on either gametophytes or sporophytes. The Hookeriales provide a model for comparing morphological evolution in gametophytes and sporophytes, and its impact on alternative classification systems. Sometimes, placement of certain groups within Hookeriales remains challenging even at the molecular level. That is the case of the genus Calyptrochaeta. We study diversification dynamics in this genus to elucidate possible mechanisms obscuring its placement and we address biogeographic questions using the Tropical Conservatism scenario as our null hypothesis. Furthermore, to better understand biogeographic patterns in the Southern Hemisphere, infraspecific molecular patterns are compared in two species of the genus Calyptrochaeta (i.e., C. apiculata and C. asplenioides) and vicariance and recent long distance dispersal are tested to explain the disjunct distributions observed in these species.

In this study we reconstruct relationships among pleurocarpous mosses in or associated to the Hookeriales, in Calyptrochaeta, and within Calyptrochaeta. Six molecular markers are explored in total from all three genome compartments to reconstruct the evolution of morphological characters and habitat preferences in our phylogenies. Divergence times are estimated in a Bayesian framework using a relaxed molecular clock, and diversification rates are calculated on the chronograms resulting from these estimations.

As a result, we found that the Hookeriales, as currently circumscribed, are monophyletic and that both sporophyte and gametophyte characters are labile. We documented parallel changes and reversals in traits from both generations. We show that diversification rates in Calyptrochaeta have changed through its history. Also, though we lack support to clearly reject the tropical conservatism hypothesis, our data point to a more complex scenario where both temperate and tropical species can be ancient and give rise to one another, since shifts between tropical and temperate regions seem to be possible in any direction. Finally, we have show that recent long distance dispersal best explains the distribution of both C. apiculata and C. asplenioides in the Southern Hemisphere.

Cells can sense and respond to various environmental cues. In the past 25 years, this ability has been exploited in engineering many innovative applications, ranging from bioproduction and metabolic engineering, to living therapeutics and biosensing. Despite tremendous advancements in complex genetic circuit development, the field still suffers from several limitations. For instance, evolutionary pressure can hamper the long-term genetic stability and functionality of circuits. The long incubation times required for cell growth serve as a fundamental rate limiting step for routine microbiology experiments and circuit engineering. Available biological parts, such as promoters and ribosome binding sites, often confer a limited dynamic range of gene expression levels and are incompatible, exacerbating the construction of higher order circuits.

Plasmids are extrachromosomal DNAs, usually circular, that replicate independently of the host genome. Because they are easy to manipulate and engineer, plasmids have served as a popular workhorse for programming desired functions in microbial populations. Plasmids can maintain steady average copy numbers in hosts, and a specific plasmid type can be chosen to express genes at a desired level. However, recent studies have focused on the dynamic modulation of plasmid copy number as a new engineering strategy, which is still underexplored. Understanding plasmid dynamics can provide insights to harness powerful tools for engineering microbial communities and offer a new avenue to overcome the current challenges in synthetic biology.

In this dissertation, I used mathematical modeling and synthetic biology approaches to develop methods for engineering microbial communities by exploiting and manipulating plasmid dynamics. First, I examined the sources of circuit failure and studied design strategies for enhancing synthetic gene circuits’ stability in microbial hosts for robust long-term performance. I summarized the engineering strategies into two categories: (1) to suppress the chance of mutant emergence by reducing the evolutionary pressure; and (2) to suppress the relative fitness of mutants by selecting against genetic variants.

Applying some of the identified engineering strategies, I developed synthetic gene circuits, named Red Queen circuits, that can modulate the host cell viability according to its growth rate. Using the circuit, I achieved a 250% increase in host cell growth rates at the end of a 100-day long-term adversarial laboratory evolutionary experiment, during which the circuit continuously suppressed slow-growing cells. The results suggest that the circuit can serve as an effective strain engineering strategy to accelerate biotechnology and molecular biology research.

Next, I constructed another gene circuits, named ADEPT system, to regulate the collective gene expression of an engineered microbial community by modulating plasmid dynamics. By dynamically tuning the plasmid loss rate, horizontal gene transfer rate, and plasmid-mediated fitness effects, I demonstrated that the ADEPT system can tune the total gene expression with a significantly amplified dynamic range.

Finally, in the Appendix, I engineered gene circuits for targeted conjugative plasmid elimination from microbial communities. The results illustrate the potential of plasmid dynamics modulations in engineering complex microbial communities.

Antibiotic discovery and widespread usage has revolutionized the treatment of infectious diseases. However, this golden age of modern-day medicine is threatened by the increasing prevalence of antibiotic-resistant pathogens. As the antibiotic development pipeline increasingly slows, we find ourselves falling behind in the race between innovation and evolution.

Among the various means of bacterial evolution, horizontal gene transfer (HGT) – or the non-genealogical transmission of DNA between organisms – is the dominant mode responsible for the acquisition of antibiotic resistance genes. Combined with antibiotic overuse and misuse, HGT primarily via conjugation, has compromised the efficacy of nearly every single antimicrobial available. Tight coupling between HGT and antibiotic-mediated selection, along with a lack of quantitative experiments, has led to the general belief that antibiotics themselves promote gene transfer; however, antibiotic action could modulate the rate of gene transfer (as is assumed), the resulting population dynamics, or both. Therefore, it is critical to decouple these two processes to definitively determine the influence of antibiotics on conjugation.

In my dissertation, I quantified the extent to which antibiotics influence conjugation in the presence and absence of antibiotic-mediated selection dynamics. To do so, I implemented a synthetically engineered conjugation system, which facilitates precise quantification of conjugation dynamics. Using this platform, I quantified the rate of gene transfer, or the conjugation efficiency, in the absence of confounding selection dynamics. I discovered that, in contrast to conventional wisdom, antibiotics did not significantly increase the conjugation efficiency. This finding was general to 10 antibiotics, as well as nine native and clinically relevant plasmids. Instead, antibiotic selection dynamics alone could account for conjugation dynamics.

I next investigated the potential strategies to minimize, or ideally reverse, plasmid-mediated resistance. Traditionally, reducing overall antibiotic use has been the primary approach to reversing resistance; minimizing selection takes advantage of costly resistance genes to competitively displace resistant bacteria with their sensitive counterparts. However, despite widespread antibiotic stewardship initiatives, even costly resistance persists for long periods of time. One potential explanation is that sufficiently fast conjugation enables plasmid persistence in the absence of selection. Similar challenges in quantifying conjugation have prevented general conclusions, and overall, the extent to which conjugation enables persistence is unknown.

Using the same platform, I showed that conjugation enables the persistence of costly plasmids, even in the absence of selection. Conjugation-assisted persistence was true for nine common conjugal plasmids, and in microbial populations consisting of varying degrees of complexity. Finally, I showed that by reducing the conjugation efficiency and promoting plasmid loss, it is possible to reverse resistance. Together, these findings contribute to basic understanding of the propagation and persistence plasmids, and elucidate a novel therapeutic strategy by taking advantage of ecological/evolutionary dynamics to to reduce or even reverse the spread of resistance.

The mouse gonad begins its development as a bipotential primordia, capable of developing into a testis or ovary depending on the presence of the sex-determining gene, Sry. In the XY gonad, opposing pro-testis and pro-ovary pathways compete in gonadal supporting cells. While the individual cellular decision process is well understood, the higher-level process of coordination of cell fates across the gonad remains to be explained. The testis and ovary exhibit distinct patterns of differentiation, suggesting that either development of each organ requires a particular organizing principle or bipotentiality requires regional separation for fate specification or stabilization. The overall goal of this work is to improve characterizations of the spatiotemporal features of sex determination and gonadogenesis, including cell fate organization, morphogenic processes, and system context.Though several hypotheses have connected gonad morphogenesis to sex determination, the morphogenic processes that occur in the gonad have not been sufficiently characterized for formulating testable hypotheses. To capture and analyze the complexity of genital ridge morphogenesis, we generated a 3-dimensional time course of gonad development in native form and context using whole embryo tissue clearing and light sheet microscopy. Analysis revealed that the early gonad exhibits anterior-to-posterior patterns as well as increased rates of growth, rotation, and separation in the central domain. In extending characterization to the neighboring nephric ducts, we found a close alignment of gonad and mesonephric duct movements as well as delayed duct development in Cbx2 mutants, which undergo XY sex reversal and gonad dysgenesis. These data support mechanical integration of gonad and mesonephric duct morphogenesis. In investigating the mechanisms underlying the center-to-pole pattern of testis differentiation, we performed anteroposterior axis analyses and ex vivo gonad reconstruction cultures. These experiments allowed us to rule out two commonly accepted theories in the field: paracrine relay and center-first Sry expression. After searching for patterns in other cellular processes during gonadogenesis, including cell cycle arrest and coelomic epithelium proliferation, we uncovered a center-biased pattern of supporting cell precursor ingression. The updated model indicates that differences between the patterns of differentiation in the testis and ovary are due to features of their respective regulatory networks connecting their fate dynamics to different general gonadal organizing principles acting upstream of supporting cell differentiation. Following recent work on the rete testis and rete ovarii suggesting these structures contribute to gonadal supporting cell populations, we characterized early development of the rete and adjacent tissues in both sexes. Comparison of the GATA4+/PAX8+ presumptive rete with mesonephric and gonadal cells led to the identification of undescribed patterns in mesonephros development which may play a role in sexual dimorphism of the rete. Cells of the rete may derive from mesonephric condensates in a process similar to kidney nephron development. Cell cycle analysis revealed the mesonephric tubules and early rete to be a largely non-proliferating population of cells, suggesting expansion through recruitment of new cells. These results were used to establish preliminary theories for lineage relationships in early urogenital development. Initial attempts at lineage tracing to test the theory were unsuccessful. The findings presented here contribute to a more comprehensive and systems level understanding of sex determination and gonad development. In particular, the incorporation of high-resolution spatial information into theories of sex determination serves to connect individual cell fate decisions to organ level patterns of differentiation in space and time. These results will be useful for novel hypothesis generation as well as for designing more detailed models and simulations of sex determination and gonadogenesis.

Transcription factors (TFs) control the temporal and spatial expression of target genes by interacting with DNA in a sequence-specific manner. Recent advances in high throughput experiments that measure TF-DNA interactions in vitro and in vivo have facilitated the identification of DNA binding sites for thousands of TFs. However, it remains unclear how each individual TF achieves its specificity, especially in the case of paralogous TFs that recognize distinct target genomic sites despite sharing very similar DNA binding motifs. In my work, I used a combination of high throughput in vitro protein-DNA binding assays and machine-learning algorithms to characterize and model the binding specificity of 11 paralogous TFs from 4 distinct structural families. My work proves that even very closely related paralogous TFs, with indistinguishable DNA binding motifs, oftentimes exhibit differential binding specificity for their genomic target sites, especially for sites with moderate binding affinity. Importantly, the differences I identify in vitro and through computational modeling help explain, at least in part, the differential in vivo genomic targeting by paralogous TFs. Future work will focus on in vivo factors that might also be important for specificity differences between paralogous TFs, such as DNA methylation, interactions with protein cofactors, or the chromatin environment. In this larger context, my work emphasizes the importance of intrinsic DNA binding specificity in targeting of paralogous TFs to the genome.