Generation and characterization of activation tagged potato mutants could aid in functional genomic studies. Morphological and molecular studies were conducted to compare potato cv. Bintje, its two mutants, underperformer (up), and nikku generated using the activation tagging vector pSKI074, and nikku revertant plants. Mutant up exhibited a dwarf phenotype (plant height 42 cm vs. 73 cm in cv. Bintje), abundant axillary shoot growth (3.1 shoots/plant compared to 0.7 shoots/plant in cv. Bintje; in vitro plants), greater tuber yield, altered tuber traits and early senescence compared to wild-type Bintje under in vitro conditions. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant were consistent, but the tuber yield of up was less (250 g/plant compared to 610 g/plant in wild-type Bintje) and had fewer axillary shoots compared to wild-type (1.9 shoots/plant in up vs. 4.7 shoots/plant in Bintje). Mutant nikku plants exhibited an extremely dwarf phenotype (plant height 2 cm in nikku vs. 6 cm in Bintje), had small hyponastic leaves, were rootless, and infrequently produced small tubers when compared to cv. Bintje. The overall nikku phenotype was suggestive of a constitutive stress response, which was further supported by the higher expression levels of several stress-responsive genes in nikku. The nikku revertant plants exhibited near normal stem elongation, larger leaves and consistent rooting, and it was a case of partial reversion. Southern blot analyses indicated the presence of single T-DNA insertions on chromosome 10 in the up and on chromosome 12 in the nikku mutant. The reversion in the nikku plants was not associated with the loss of enhancer copies from the original nikku mutant. Reverse transcriptase PCR analyses indicated transcriptional activation/repression of several genes in the up and nikku mutants, suggesting pleiotropic effects. In revertant, the expression levels of several genes which were differentially regulated in the nikku mutant were similar to Bintje. The gene immediately flanking the right border of the T-DNA insertion, which encoded a novel BTB/POZ (Broad complex, Tramtrac, Bric a brac; also known as Pox virus and Zinc finger) domain-containing protein, was highly up-regulated in the up mutant. This protein domain plays an important role in several important developmental, transcriptional and regulatory pathways. The mRNA-seq analyses resulted in 1,632 genes that were differentially expressed between mutant up and Bintje and the total number of up-regulated genes (661) were less than the number of genes down-regulated (971 genes) in the up mutant. Further analyses indicated that a variety of biological processes including decreased cell division, cell cycle activity, and abiotic stress responses were modified in the up mutant. In the nikku mutant, two potato genes, encoding an Acyl-CoA N-acyltransferases (NAT) superfamily protein, and a predicted major facilitator superfamily protein (MFS) were identified and overexpression lines Bintje/35S::NAT1 and Bintje/35S::PMT1 were created for recapitulation of the nikku mutant phenotype. Methylated DNA-PCR between the nikku and the revertant indicated a change in methylation status of the 35S enhancers, suggesting that the nikku revertant phenotype may be associated with some epigenetic modification. / Ph. D.

In the United States corn is one of the leading agricultural products and one of the top exports. The majority of U.S corn is grown in the Midwestern region of the U.S. known as the Corn Belt where the bacterial disease Stewart's Wilt reduces crop yield. Pantoea stewartii subsp. stewartii (Pss) is transmitted into corn via the corn flea beetle insect vector, Chaetocnema pulicaria. As the beetle feeds on the corn plant leaves, Pss deposited in beetle feces enter the leaf through lesions. The early stage of Pss infection begins in the mesophyll apoplast of the corn leaf where a type III secretion system (T3SS) and its associated effectors induce water soaking (WS) and nutrient release. Ultimately, Pss will enter the plant xylem apoplast (will be referred to as the xylem) and use quorum sensing (QS) to initiate a lifestyle shift. Within the xylem, Pss grows to high cell density and secretes exopolysaccharide (EPS), forming a biofilm which eventually obstructs water transport, leading to wilting and necrosis. Previous Tn-Seq experiments provided insights into genes that are essential for in planta survival, including the master transcriptional regulator, Leucine-responsive Regulatory Protein (Lrp). To better understand the role of Lrp when Pss inhabits the xylem, RNA-Seq experiments comparing Pss wild-type and ∆lrp strains grown in planta were conducted to ascertain differential gene expression. The RNA-Seq data was further analyzed using DESeq2 and validated using qRT-PCR methods. Following validation, the Pss genome was annotated using Blast2GO software and genes upregulated and downregulated by Lrp were linked with biological processes. Lrp was found to be involved in regulating capsule biosynthesis and nitrogen-associated assimilation and metabolism during Pss survival in the xylem. This provides further insight into how Pss contends with harmful host defense compounds and extracts scarce nutrients present in the in planta xylem environment. A corn xylem fluid extraction method was developed that has enabled more physiologically relevant growth experiments to be conducted in vitro. Extracted xylem fluid was used to grow Pss wild-type and ∆lrp mutant strains as monocultures to observe any differences in growth patterns in different growth media. When grown separately in xylem fluid or Luria-Bertani (LB) medium, the Pss wild-type and ∆lrp mutant strains grew at similar rates and to final cell densities . The Pss ∆lrp mutant strain greatly outcompeted the wild type when grown together in LB medium. However, when the two Pss strains were growth together in xylem fluid, a shift in relative competition was observed, providing evidence of the wild type slightly outcompeting the ∆lrp mutant. Analysis of the composition of extracted xylem fluid through metabolomics will help define the nutrients specifically utilized by Pss in planta. Altogether, the outcome of these research projects was to provide pertinent discoveries to contribute to understanding the mechanisms used by Pss to survive in the corn xylem environment. Broadly, increased understanding of Pss pathogenesis may translate to understanding pathogenesis mechanisms in other bacterial wilt-disease causing plant pathogens. / Master of Science / Corn is a significant agricultural product and export in the United States. This important crop is used as a food source for humans, a primary nutrient source of livestock, and a major ingredient for corn-based industries manufacturing commodities such as culinary additives, biofuels, and preservatives. Certain bacteria are greatly beneficial to plants, able to increase their overall health and growth, while other bacteria share a more insidious relationship with plants and cause disease. The research discussed in this thesis focuses on the bacterial pathogen Pantoea stewartii subspecies stewartii (Pss), the causal agent of Stewart's wilt disease in corn. Pss grows inside the plant xylem (vascular tissues which distribute water throughout the plant) and forms a biofilm that causes plant wilt leading to lower crop yield and even plant death. Previous research on Pss identified important genes for successful Pss survival inside the corn plant xylem. One of those genes codes for the Leucine-responsive Regulatory Protein (Lrp). Using a combination of experimental (RNA-Seq) and computational (bioinformatics) analyses, Lrp was found to control other genes related in biological process important for living inside the plant, necessary for the metabolism of available nutrients and production the protect slime layer within biofilm. By better understanding the key bacterial genes needed for Pss to grow inside the xylem, new disease intervention strategies can be developed to disrupt these genes and impede the ability of the bacterium to infect the plant. A second part of this research project was to develop a method for extracting corn xylem fluid from the plant. Using this extracted xylem fluid, experiments could be conducted in the laboratory to study Pss growth in more detail. The original strain of Pss (wild type) was grown separately and in combination with a Pss mutant lacking the Lrp gene in the extracted xylem fluid. Both strains grew similarly in the xylem fluid, but the wild type slightly outcompeted the mutant strain when they were grown in competition. Future work in the lab will use extracted xylem fluid to determine its precise nutrient composition and the development of synthetic xylem fluid that will enable a more detailed analysis of mechanisms used by Pss to grow in the xylem. Work on Pss serves as a model for the study of other bacterial wilt-disease causing pathogens.

Pantoea stewartii subsp. stewartii is a bacterium that causes Stewart’s wilt disease in corn plants. The bacteria are transmitted to the plants via an insect vector, the corn flea beetle Chaetocnema pulicaria. Once in the plant, the bacteria migrate to the xylem and grow to high cell densities, forming a biofilm by secreting excess capsular exopolysaccharide, which blocks water transport and causes wilting. The timing of virulence factor synthesis is regulated by the cell-density dependent quorum sensing (QS) system. Such temporal regulation is crucial in establishing infection and is orchestrated by the QS-dependent transcriptional regulator EsaR. EsaR represses expression of capsular exopolysaccharide at low cell densities. At high cell densities, an acylated homoserine lactone (AHL) molecule produced during growth by the cognate AHL-synthase EsaI accumulates. The AHL binds to and inactivates EsaR, causing derepression of capsule production. EsaR is a member of the LuxR family of QS-dependent transcriptional factors. Most LuxR homologs are unstable and/or insoluble in the absence of AHL which has hindered structural studies. Chapter Two describes the changes in the structure of EsaR due to binding of AHL ligand as determined through biochemical methods. EsaR was found to be stable and retain its multimeric state in the absence or presence of AHL, but intra- and inter-domain changes occurred that affect its DNA-binding capacity. Apart from repressing expression of capsule at low cell-densities, EsaR represses its own expression and activates production of a small RNA, EsaS, with unknown function. In Chapter Three a proteomic approach was used to identify an additional 30 QS-controlled proteins. Genes encoding three of these proteins are directly regulated by EsaR and the EsaR binding sites in the respective promoters were defined. In Chapter Four, a high-throughput RNA-Seq method identified even more genes in the QS regulon that the proteomic approach overlooked. RNA-Seq analysis of rRNA-depleted RNA from two strains of P. stewartii was used as a screen to help identify 11 promoters, subsequently shown to be directly regulated by EsaR in vitro. Most of the genes controlled by QS grouped into three major physiological responses, capsule & cell wall production, surface motility & adhesion and stress response. In Chapter Five, the role of two QS regulated genes, dkgA (encoding 2, 5-diketo-D-gluconate) and lrhA (encoding a repressor of chemotaxis, adhesion and motility), in plant virulence were examined. These studies have better characterized the QS regulator EsaR and its interaction with the AHL ligand, and shown that QS has a more global response in P. stewartii than previously recognized. Further characterization of the genes identified in this study could facilitate identification of factors crucial in plant pathogenesis or insect-vector symbiosis and aid in the development of molecular-based approaches for possible disease intervention. / Ph. D.

Although bed bug eggs are a difficult life stage to control with our currently labeled insecticides, few studies have examined how bed bug egg morphology and physiology is potentially related to pesticide resistance in bed bug eggs. Bed bug egg morphological features were examined using scanning electron microscopy (SEM) and the chorion and respiration structures were identified. Scanning electron microscopy photographs and bed bug egg measurements indicated there were no morphological differences between different bed bug egg strains (susceptible and resistant). Bed bug egg respiration rates measured by the amount of oxygen consumed (standard metabolic rate; SMR) also indicated there was no difference in SMR between different bed bug egg strains. Water conservation during respiration is vital to terrestrial insects. Therefore, similar patterns would be expected between egg water loss and respiration rates. However, susceptible strain eggs lost more water than one resistant strain of bed bug eggs, which was dissimilar from the respiration results, indicating that bed bug egg water loss and respiration are not directly related. Dose- response bioassays using two insecticide formulations (Temprid; imidacloprid/β-cyfluthrin, and Transport; acetamiprid/bifenthrin) indicated that bed bug eggs collected from pyrethroid resistant adult bed bug strains are also highly resistant. RNA sequencing of bed bug eggs from two resistant strains indicated that egg resistance may be directly related to the overexpression of multiple genes associated with insecticide resistance. / Master of Science in Life Sciences

Early growth response 1 (Egr1) is a transcription factor critical for learning and memory in the hippocampus and pituitary cell differentiation. Egr1 has been shown to extend continuation of the long-term potentiation in the hippocampus and is credited for forming long-term memories. The somatotrophs in the pituitary produce growth hormone and are found to be decreased in Egr1KO mice. These animals are also found to be sterile due to a decrease in LHB, which blocks ovulation. All previous studies have evaluated these physiological processes with complete Egr1KO research strains or antisense oligonucleotides, up until now, no data specific to individual type of cells has been generated. In an attempt to focus on the understanding of the functions of Egr1 gene in neural cell lineage, we are using an Egr1cKO Nestin-Cre model. Nestin allows for targeting neuronal lineage specific cells. In Chapter 1, we provide a systemic view of Egr1 gene and Nestin-Cre as a system for generating conditional knockout mouse strains. The Chapter begins with the identification of Egr1 gene and its protein structure, then proceeds to grasp its link to memory with behavior testing. The critical role of Egr1 in the pituitary and what cell populations are affected is also described. The same goes for Nestin-Cre, along with its limitations and understanding how to account for them in a study. The Egr1cKO Nestin-Cre system is the best form to understand neurological cell populations with Egr1 removal. In Chapter 2 and Chapter 3, we employ the Egr1cKO Nestin-Cre mouse model to understand cell-specific knockout of Egr1 in the nervous system by evaluating the hippocampus and pituitary. We explore learning and memory through behavioral tests and ribonucleic acid sequencing (RNA-seq) analysis to understand gene expression changes with Egr1 removal. Females showed higher activity during behavior tests, with more movement in the elevated plus maze and lower freezing times during the contextual fear conditioning. RNA-seq had higher changes in females than males but was not affected by the Nestin-Cre system overall. The same RNA-seq changes in the pituitary gland were present, with females having higher genomic differentiation. Females had growth-specific pathways altered by Nestin-Cre. / Doctor of Philosophy / Genetics has become a very important forerunner in scientific research. One gene that has become important in many different research arenas is Early growth response 1 (Egr1). This particular gene is critical for learning, memory, and cell changes in the pituitary. In Chapter 1, we have analyzed the current research landscape of information on Egr1 in its functions with learning and memory, as well as the pituitary. Most previous studies that have been completed only evaluate this gene by its removal from the entire body. This leaves a large gap in information about how this gene functions with specific cell types. To limit the type of cells from which Egr1 has been removed, we have selected Nestin-Cre, a tool to remove genes from neuronal stem cells. The capabilities and limitations of this tool have also been explained in this chapter, along with how the two together can accomplish a cell-specific knockout of Egr1. In Chapter 2, we have constructed an experiment with behavioral tests for mice, along with RNAseq data from the hippocampus to evaluate what changes have occurred in the Egr1cKO Nestin-Cre model. Female mice are more active in the behavioral test, including the elevated plus maze (EPM) and Contextual fear conditioning (CFC), than male mice. The same holds for differences in the RNAseq data as well. In Chapter 3, the pituitary of Egr1cKO Nestin-Cre mice is the main focus. We evaluated RNAseq data and determined growth rates of transgenic mice. The mice had different growth rates over twelve weeks between the controls and the knockout. The RNAseq data also revealed many differences between males and females. Female mice had specific growth genes effects by the knockout of Egr1

Yes / Virus-encoded miRNAs are emerging as key regulators of persistent infection and host-cell immune evasion. Merkel cell polyomavirus (MCPyV), the predominant aetiological agent of Merkel cell carcinoma (MCC), encodes a single miRNA, MCV-miR-M1, which targets the oncogenic MCPyV large T antigen (LT). MCV-miR-M1 has previously been shown to play an important role in establishment of long-term infection, however, the underlying mechanism is not fully understood. A key unanswered question is whether, in addition to auto-regulating LT, MCV-miR-M1 also targets cellular transcripts to orchestrate an environment conducive for persistent infection. To address this, we adopted an RNA-Seq-based approach to identify cellular targets of MCV-miR-M1. Intriguingly, bioinformatics analysis of transcripts that are differentially expressed in cells expressing MCV-miR-M1 revealed several genes implicated in immune evasion. Subsequent target validation led to the identification of the innate immunity protein, SP100, as a direct target of MCV-miR-M1. Moreover, MCV-miR-M1-mediated modulation of SP100 was associated with a significant decrease in CXCL8 secretion, resulting in the attenuation of neutrophil chemotaxis towards Merkel cells harbouring synthetic MCPyV. Based on these observations we propose that MCV-miR-M1 targets key immune response regulators to help facilitate persistent infection, which is a pre-requisite for cellular transformation in MCC. / Funded in part by a University of Bradford studentship to PA and a Royal Society research award to JRB.

La transcriptómica estudia el nivel de expresión de los genes en distintas condiciones experimentales para tratar de identificar los genes asociados a un fenotipo dado así como las relaciones de regulación entre distintos genes. Los datos ómicos se caracterizan por contener información de miles de variables en una muestra con pocas observaciones. Las tecnologías de alto rendimiento más comunes para medir el nivel de expresión de miles de genes simultáneamente son los microarrays y, más recientemente, la secuenciación de RNA (RNA-seq). Este trabajo de tesis versará sobre la evaluación, adaptación y desarrollo de modelos estadísticos para el análisis de datos de expresión génica, tanto si ha sido estimada mediante microarrays o bien con RNA-seq. El estudio se abordará con herramientas univariantes y multivariantes, así como con métodos tanto univariantes como multivariantes. / Tarazona Campos, S. (2014). Statistical methods for transcriptomics: From microarrays to RNA-seq [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48485 / Premios Extraordinarios de tesis doctorales

The rapid development of biotechnology has enabled researchers to collect high-throughput data for studying various biological processes at the genomic level, transcriptomic level, and proteomic level. Due to the large noise in the data and the high complexity of diseases (such as cancer), it is a challenging task for researchers to extract biologically meaningful information that can help reveal the underlying molecular mechanisms. The challenges call for more efforts in developing efficient and effective computational methods to analyze the data at different levels so as to understand the biological systems in different aspects. In this dissertation research, we have developed novel Bayesian approaches to infer alternative splicing mechanisms in biological systems using RNA sequencing data. Specifically, we focus on two research topics in this dissertation: isoform identification and phenotype-specific transcript assembly. For isoform identification, we develop a computational approach, SparseIso, to jointly model the existence and abundance of isoforms in a Bayesian framework. A spike-and-slab prior is incorporated into the model to enforce the sparsity of expressed isoforms. A Gibbs sampler is developed to sample the existence and abundance of isoforms iteratively. For transcript assembly, we develop a Bayesian approach, IntAPT, to assemble phenotype-specific transcripts from multiple RNA sequencing profiles. A two-layer Bayesian framework is used to model the existence of phenotype-specific transcripts and the transcript abundance in individual samples. Based on the hierarchical Bayesian model, a Gibbs sampling algorithm is developed to estimate the joint posterior distribution for phenotype-specific transcript assembly. The performances of our proposed methods are evaluated with simulation data, compared with existing methods and benchmarked with real cell line data. We then apply our methods on breast cancer data to identify biologically meaningful splicing mechanisms associated with breast cancer. For the further work, we will extend our methods for de novo transcript assembly to identify novel isoforms in biological systems; we will incorporate isoform-specific networks into our methods to better understand splicing mechanisms in biological systems. / Ph. D. / The next-generation sequencing technology has significantly improved the resolution of the biomedical research at the genomic level and transcriptomic level. Due to the large noise in the data and the high complexity of diseases (such as cancer), it is a challenging task for researchers to extract biologically meaningful information that can help reveal the underlying molecular mechanisms. In this dissertation, we have developed two novel Bayesian approaches to infer alternative splicing mechanisms in biological systems using RNA sequencing data. We have demonstrated the advantages of our proposed approaches over existing methods on both simulation data and real cell line data. Furthermore, the application of our methods on real breast cancer data and glioblastoma tissue data has further shown the efficacy of our methods in real biological applications.

Zebrafish thrombocytes exhibit characteristics of human platelets and megakaryocytes, making them valuable for studying megakaryopoiesis and thrombopoiesis. Using single-cell RNA sequencing, we analyzed gene expression in young and mature zebrafish thrombocytes. We identified 394 protein-coding genes unique to young thrombocytes, many corresponding with human orthologs, suggesting shared regulatory mechanisms in zebrafish and humans. We hypothesized knocking down these 394 genes should identify the novel regulatory genes that control thrombocyte maturation. To address this, we used the piggyback knockdown method to knock down these genes to study their biological functions in zebrafish thrombopoiesis. We first found the knockdown of nfe2, nfe2l1a, and nfe2l3 reduced both young and mature thrombocyte counts, confirming their role in thrombopoiesis. A comprehensive knockdown screening of the uniquely expressed genes in young thrombocytes identified 7 candidate genes associated with thrombopoiesis. We selected the spi1b gene for further mutant characterization, which revealed its critical role in young thrombocyte development, with homozygous mutations leading to embryonic lethality. Considering megakaryocyte properties in thrombocytes, we studied the potential for polyploidization in zebrafish thrombocytes. The inhibition of AURKA led to the development of polyploid thrombocytes resembling mammalian megakaryocytes, suggesting the retention of genetic programs for megakaryocyte development in zebrafish thrombocytes and providing insights into the evolutionary basis of thrombopoiesis. Thus, our study reveals critical gene expression patterns and regulatory factors in zebrafish thrombocyte development, offering insights into conserved mechanisms relevant to developmental biology and research in thrombosis and hemostasis disorder.

Cell death shapes multicellular organism development and sustains tissue and organ homeostasis. Over the past decade we have begun to understand the breadth of physiological and biochemical diversity in cell death and clearance pathways, which play vital roles in organismal development and heath. While apoptosis and necrosis have been studied extensively across many model systems and contexts, the discovery of non-apoptotic paradigms of cell death and their roles in disease has greatly expanded the field. Collectively called Regulated Cell Death (RCD), these death pathways are regulated in a tissue and context-dependent manner (e.g. disease state). This dissertation is a culmination of multiple projects investigating cell death and clearance events spanning the ovary and the brain of the model organism, Drosophila melanogaster. We undertook the first multi-modal, high-throughput survey, involving single-cell RNA-seq, TRAP-seq, and proteomics, to compare two different archetypes of germline death in the fly egg chamber - apoptosis and phagoptosis. Our analysis identified several important candidates and pathways that are either unique to or shared between the germline death modalities and affecting oogenesis upon their disruption. We also observed that V-ATPases, proton pumps required for germline phagoptosis, are differentially localized throughout oogenesis, and we identified the specific subunits upregulated in phagoptosis. Furthermore, we identified a novel exon splicing event in the ‘a’ subunit isoform of V-ATPases that may facilitate its sub-cellular localization. Using a novel image analysis method involving image segmentation and spatial statistical inference, we determined that circulating immune cells agglomerate at specific niches within the abdomen, in response to egg chamber degeneration resulting from physiological stress of protein-deprivation. We then turned our focus to phagocytosis in the fly brain, which is essential for pruning synapses and for the removal of dying neurons and misfolded proteins. Disruptions to glial phagocytosis results in a range of age-dependent neurodegenerative phenotypes, primarily exemplified by vacuolization of brain tissue. Using a pre-trained deep-learning model to perform image segmentation and 3D reconstruction of vacuoles, we characterized the severity of neurodegeneration in brains lacking the phagocytic receptor Draper in glia and further demonstrated that this phenotype is attenuated by knockdown of the NF-κB transcription factor Relish in flies lacking glial Draper. Collectively, the methods and results described herein will have applications beyond the Drosophila model and the field of cell death, with important implications in understanding fertility and the underpinnings of cognitive disorders.