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Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by arterial pruning, occlusive vascular remodeling, and inflammation contributing to increased pulmonary vascular resistance with resultant right heart failure. Endothelial cell (EC) injury and apoptosis are commonly considered triggers for PAH, the mechanisms leading from injury to complex arterial remodeling are incompletely understood. While current therapies can improving symptoms, with the exception of parenteral prostacyclin, they do not significantly prolong transplant free survival. As well, there are no therapies that can regenerate the damaged lung short of transplantation. In this project, I sought to both advance the understanding of disease pathogenesis and explore regenerative therapeutic options for PAH. To this end, I first employed single cell RNA sequencing (scRNA-seq) at multiple time points during the Sugen 5416 (SU) – chronic hypoxia (CH) model of PAH, to provide new insights into PAH pathogenesis both during onset and progression of disease. We also employed microCT analysis to visualize and quantify the arterial pruning associated with PH and found significant loss up to 65% of the healthy arteriolar volume in this model. Through scRNA-seq analysis performed at four timepoints spanning the onset and progression of disease, two disease-specific EC cell types emerged as key drivers of PAH pathogenesis. The first was the emergence of capillary ECs with a de-differentiated gene expression profile, which we termed dedifferentiated capillary (dCap) ECs, with enrichment for the Cd74 gene. Interestingly, RNA velocity analysis suggested that these cells may be undergoing endothelial to mesenchymal transition during PAH development. At later times, a second arterial EC population became apparent, which we termed activated arterial ECs (aAECs), since it uniquely exhibited persistently elevated levels of differential gene expression consistent with a migratory, invasive and proliferative state. Interestingly, the aAECs together with the smooth muscle (SM)-like pericytes, a population which was also greatly expanded in PAH, expressed Tm4sf1, a gene previously associated with a number of cancers and abnormal cell growth. Furthermore, by immunostaining, TM4SF1 was found to be spatially localized to sites of complex and occlusive arterial remodeling, associated with both endothelial cells and pericytes in these lesions, suggesting an important role for the aAECs and SM-like pericytes in arterial remodeling and PH progression. Together, these findings suggest that aAECs, dCap ECs, and SM-like pericytes are emerging cell populations responsible for lung arterial remodeling in PAH, which drives disease progression, and that TM4SF1 may be a novel therapeutic target for this disease. As a first step in trying to develop approaches to regenerate lung arterial bed that is lost in PAH, we investigated the potential role of endothelial colony forming cells (ECFCs) and mesenchymal stromal cell (MSC) derived extracellular vesicles (EVs) as novel therapeutics, on the premise that these stem/progenitor cells would stimulate lung regeneration by mainly paracrine mechanisms. Additionally, we used biomaterials to microencapsulate cells and EVs to improve their local delivery and retention. While ECFCs were found to be ineffective in treating the monocrotaline model on their own, they were poorly retained in the lung and microencapsulation of ECFCs led to enhanced lung delivery within the first 72 hours, with resultant hemodynamic improvements in this model of PAH. MSCs are well known to be immunomodulatory and proangiogenic, largely acting through paracrine mechanisms, including by the release of EVs. Yet, following intravenous administration, nano sized EVs are rapidly cleared from circulation, potentially limiting their therapeutic potential. I adapted our microencapsulation strategy for EVs, and demonstrated significantly greater retention of microgel-loaded EVs were within the lung, resulting in enhanced local cell uptake. Interestingly, the hydrogel used for microencapsulation induced a local immune response which made it unsuitable for testing any potential therapeutic benefits of MSC-EVs in this study. Nonetheless, this work demonstrated proof-of-principle for the utility of microencapsulation as a strategy to enhance EV lung delivery. Overall, this work has identified novel lung cell populations (aAECs, dCap ECs, SM-like pericytes) driving arterial remodeling associated with PH progression, demonstrated the potential of ECFCs as a regenerative cell for the treatment of PAH, and illustrated the utility of microencapsulation as a tool to enhance lung targeting of both cells and EVs.

The purpose of this thesis project was to elucidate the immune transcriptomic changes in the diaphragm of mdx mice treated with microdystrophin gene therapy with and without running wheel activity. Mdx mice are a model of Duchenne Muscular Dystrophy (DMD). Similar to DMD, mdx pathophysiology is associated with chronic inflammation due to sarcolemma fragility and cellular membrane leakage. Immune modulation has not yet been described when endurance exercise and AAV-microdystrophin gene therapy have been combined in mdx mice. An increase of physical activity in DMD individuals is a potential outcome of current clinical studies investigating microdystrophin treatment; therefore, understanding the impacts of physical activity on the immune system, particularly for the diaphragm, may be important to minimize risk. Recently, the Grange lab published the endurance and contractile property outcomes of combined microdystrophin gene therapy and running wheel activity in mdx mice.1 Diaphragm RNA-seq transcriptomic data were also collected from this study for gene expression analysis. Using this dataset, I tested the hypothesis that relative to mdxGT (mdx mice treated with gene therapy), transcripts related to the immune response such as immune cell recruitment, activation, and downstream signals that promote fibrosis deposition were unchanged or downregulated in mdxRGT (mdx mice treated with gene therapy and access to running wheel). DEGs (differentially expressed genes) were analyzed with Microsoft Excel, R, and bioinformatic tools such as KEGG and DAVID to explain immune system adaptations in response to combined microdystrophin treatment and running in mdx mice. Two major inflammatory signaling pathways, the IL-6/JAK/STAT and NF-kB signaling pathways translationally relevant to DMD patients were rescued by gene therapy towards WT expression levels. Although running maintained the majority of the rescued transcriptome profile (691 of 724 genes), some immune response-related gene expressions (33 of 724 genes) were modulated including genes related to chemotaxis and cellular migration. These changes suggested potential signaling for angiogenesis and a fast to slow fiber type shift; however, unbiased analysis with bioinformatic tools did not confirm either of these possibilities. The data from this study revealed inflammatory and fibrotic signaling pathways commonly observed in DMD patients and mdx mice were rescued by the AAV microdystrophin gene therapy and were maintained by voluntary wheel running / Master of Science / Duchenne Muscular Dystrophy (DMD) is an X chromosome-linked muscular dystrophy, a genetic disease that affects around 1 in 14,000 boys globally. DMD is lethal and currently there is no cure. Mutations in the DMD gene results in the absence of the protein dystrophin. The dystrophin protein and other proteins associated with it provide structural support to the skeletal muscle membrane. Without it, muscles are more easily damaged during contraction. This damage promotes recruitment of immune cells which initiates the first stage of muscle repair. Under normal circumstances, this inflammatory reaction caused by immune cells restores the skeletal muscles. However, in DMD patients, repeated breakdown and regeneration of skeletal muscles leads to abnormal inflammation which promotes negative outcomes such as increased fibrosis. Fibrosis impairs muscle function, especially the diaphragm . Hamm et al., 2021 from the Grange lab investigated the effects of microdystrophin gene therapy and increased physical activity in mdx mice, a mouse model of DMD, with the idea that some of the negative changes with muscular dystrophy could be improved. The results showed a positive increase of endurance capacity in mdx mice treated with gene therapy alone (mdxGT group) and a greater increase if the mice also used a running wheel (mdxRGT group) compared to untreated mdx mice (mdx group). These findings suggested that gene therapy can increase a DMD patient's ability to become more physically active. However, the effects of running and microdystrophin gene therapy on the damaging inflammatory response in the diaphragm were not reported. To address this question, gene expression data from diaphragm muscles of all treatment groups were collected in the Hamm et al., 2021 study for later analysis. In my study, these diaphragm gene expression data were used to compare inflammatory signals between the various treatment groups. Indicators of skeletal muscle damage, immune cell accumulation and fibrosis deposition were rescued (i.e., returned to healthy mice levels) by microdystrophin gene therapy (mdxGT group). Running did not exert any negative effects on the majority of genes rescued by the microdystrophin therapy (mdxRGT group). These results indicated that voluntary wheel running could maintain the reduced inflammatory signals due to the microdystrophin gene therapy in mdx mice. If the function of the skeletal muscle of dystrophic boys was similarly improved by microdystrophin gene therapy and exercise did not interfere with its positive effects, DMD boys could potentially be physically active similar to normal boys of their age.

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.