9 Abstract Enhancers are distal cis - regulatory DNA sequences that regulate (enhance) transcription of the respective gene driven by its promoter. Enhancers are found in non-coding DNA upstream or downstream of the gene coding sequence, or in introns or coding regions that are located up to hundreds kb away from the gene. Superenhancers are newly discovered clusters of multiple enhancers that play a vital role in activating tissue-specific genes, determining cell identity and regulating differentiation. PU.1 is the transcription factor (TF) that is necessary for normal haematopoiesis, specifically for the development of myeloid and lymphoid blood lineages. Distinct levels of PU.1 induce differentiation of hematopoietic cells into different cell lineages whereby disruption of PU.1 levels leads to leukemogenesis. High PU.1 levels stimulate macrophage development, while intermediate levels stimulate the development of granulocytes. This diploma thesis seeks to contribute to addressing the interesting biological question of what are the regulatory mechanisms to ensure that granulocytic genes are activated only at the intermediate concentration of PU.1, whereas macrophage genes are activated only at its high levels. The aim of this diploma thesis was to create a series of reporter vectors carrying regulatory...

The vertebrate vasculature is formed by populations of endothelial and mural cells that arrange into functionally and molecularly distinct arterial, venous and capillary beds. Although a number of signalling pathways and transcriptional regulators have been implicated in these processes of vascular differentiation, a clear picture of how arterial-specific gene regulation is achieved is yet to emerge. In this study I have investigated the transcriptional regulation of arterial identity from two different directions: characterisation of enhancers to identify the transcription factors that bind and direct arterial specification; and direct study of the function of one particular transcription factor expressed specifically in the arterial vasculature. I have identified a novel gene enhancer that directs arterial-specific expression of Flk1 (Vegfr2) in transgenic mouse and zebrafish models. Dissection of inputs from individual transcription factor binding sites within this enhancer shows a requirement for Gata factors for enhancer function in endothelial cells, whereas arterial-specification is directed by Rbpj-mediated repression of enhancer activity in veins. This work demonstrates that Flk1 expression in arterial endothelial cells is downstream of the Notch/Rbpj pathway, and also describes a novel transcriptional mechanism of arterial differentiation. In parallel, I have uncovered a novel role for the transcription factor Tbx2 in the regulation of arterial mural cell identity. Histological analysis demonstrates the previously unreported expression of Tbx2 exclusively in mural cells of peripheral arteries and microvessels, and genetic deletion experiments in mice suggest a role for Tbx2 in mural cell recruitment, survival, proliferation, and differentiation upstream of Notch3 and Pdgfrβ. Together, these results contribute valuable insights into our understanding of the establishment of vascular identity by identifying novel transcriptional regulators of arterial fate in both endothelial and mural cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22326号 / 医博第4567号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 小川 誠司, 教授 遊佐 宏介 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Drug delivery via skin is a non-invasive, patient compliant, and effective method for circulatory or skin-targeted therapeutic treatment. Based on its mechanism of action, a topical system employing cromolyn sodium (CS) poses as a cheaper, safer alternative to current treatments for atopic dermatitis, an allergic skin disease. Clinical studies have successfully treated atopic dermatitis with CS emulsions; however, semisolid CS gels have not been investigated and no commercial formula is available to date. Additionally, clinical doses of CS do not passively permeate skin, although different chemical enhancers can be incorporated into formulation to enhance cutaneous drug absorption. This study aimed to investigate salcaprozate sodium (SNAC) as a chemical enhancer for optimized drug delivery to the dermis for potential remedial effects of CS gels. Gels were prepared weight-to-weight by combining 4% CS, 1% hydroxypropyl cellulose as gelling agent, and respective amounts of propylene glycol as base. For SNAC gels, contents included 2.5%, 4.5%, and 9% SNAC, and amount of propylene glycol was adjusted accordingly. CS gel (4%) containing no SNAC was used as a control. After overnight shaking, gels were sonicated for 30 min to use in in vitro permeation studies. Porcine ear skin was mounted on Franz diffusion cells maintained at 37°C, and permeation studies were performed over 24 h for each formulated gel to determine their effect on CS permeation across skin. Donor compartment contained 100 μL gel while the receptor held phosphate buffered saline (PBS). At predetermined timepoints, 300 μL of receptor solution was sampled, replaced with fresh PBS, and analyzed using HPLC with CS detection at 236 nm. Following 24 h, remaining gel was removed, and skin surface was cleaned. Skin layers were manually separated, minced, and left to shake for 4 h to extract permeated drug using methanol. These samples were vacuum dried overnight and reconstituted with PBS to be analyzed using HPLC. Efficiency of skin extraction methods was evaluated by assessing amount of drug recovered from skin compared to amount of drug absorbed where results were plotted, and subsequent equations were used to correct skin data. Student’s T test with Welch’s correction was applied to confirm statistical significance between gels. Passive delivery of the 4% CS control gel to the dermis was 0 μg/cm2. The SNAC containing gels demonstrated significantly improved drug delivery to the dermis when compared to control for 2.5% (36.26 ± 13.05, p=0.05), 4.5% (11.64 ± 1.45, p=0.001), and 9% (35.87 ± 2.23, p=0.004) SNAC groups. No significant differences were observed between any SNAC gel group and the control gel regarding drug delivered to the epidermis or receptor over 24 h. This study observed the greatest delivery of CS to the dermis with the 2.5% SNAC gel, posing as a promising option for a commercially available topical CS gel for the skin-targeted treatment of atopic dermatitis.

Indiana University-Purdue University Indianapolis (IUPUI) / The immune system provides resistance to the myriad of pathogens in the environment, but can also respond inappropriately causing allergic inflammation and autoimmune disease. CD4+ T cells, which play a crucial role in adaptive immune system, can be divided into several subsets based on their effector functions. T helper 9 (Th9) cells, derived by the IL-4/STAT6 and TGF-β signaling pathways, produce IL-9 as a hallmark cytokine, as well as IL-10. Through IL-9 production, Th9 cells protect against parasite infection but are also involved in allergic inflammation and autoimmune diseases. Transcription factors that promote Th9 development include STATs, PU.1, BATF, and IRF4. In this study, we identify ETV5 as a factor that promotes IL-9 and IL-10 production by binding to cis-acting regulatory elements in the respective genes. At the Il9 gene, ETV5 cooperates with PU.1 in regulating gene expression. At the Il10 gene, ETV5 facilitates binding of other transcription factors to the locus. These studies and others suggested that there may be additional cis-acting regulatory elements in the Il9 gene. We demonstrate that a conserved noncoding sequence (CNS) located 25 kb upstream of the Il9 transcription start site, termed Il9 CNS-25, is critical for regulating Il9 expression in Th cell subsets. Th9 cells derived from Il9 CNS-25 mutant (Il9 ΔCNS-25) mice produce significantly less IL-9. Il9 CNS-25 promoted chromatin modifications at the promoter and accessibility of the locus. Il9 ΔCNS-25 mice showed attenuated airway inflammation compared to control mice. The Il9 CNS-25 region in mice is conserved with an IL9 CNS-18 region in the human genome. We deleted CNS-18 in primary human Th9 cells and observed diminished IL-9 production. Thus, we have identified transcription factors that regulate multiple cytokines in Th cell lineages and have demonstrated that the Il9 CNS-25/IL9 CNS-18 elements are respectively critical for Il9/IL9 gene expression.

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Humans consume cannabis for the pharmacological effects mediated by the primary psychoactive cannabinoid, delta-9-tetrahydrocannabinol (THC). However, there is little evidence to suggest that THC acts as a primary reinforcer in non-human models because the drug alone does not support robust self-administration. We hypothesized that THC may have more potent reinforcement enhancing effects – meaning that THC may enhance the reinforcing effects of other non-drug rewards in a user’s environment. In the present experiments, we explore the effects of THC on operant responding for saccharin (SACC) or a visual stimulus (VS). In all experiments rats were shaped to respond for their assigned reinforcer. Drug challenge tests were conducted every 72 hours, rats were injected with the assigned dose of THC and responding for each reinforcer was measured. Our initial findings indicated possible sex differences between male and female rats – THC injections increased lever-pressing for SACC in male rats but not female rats. However, in follow-up experiments we used a different response (nose-key press instead of lever press) that facilitated operant responding in rats that were different sizes – adult males are significantly more massive than adult females. In that experiment THC enhanced nose-key presses for SACC in both male and female rats across a range of doses. Moreover, this latter experiment confirmed that the effect of THC was motivational in nature, THC injections increased effort to obtain SACC under a progressively increasing schedule of reinforcement (progressive ratio). Finally, using a third operant response (head entry into a receptacle) we demonstrated that THC increased reinforcement by the VS across a range of doses. The present studies indicate that THC acts as a reinforcement enhancer, increasing motivation in male and female rats to obtain both SACC and VS throughout a range of doses. By demonstrating that THC enhances the reinforcing effects of both gustatory and non-gustatory reinforcers, our evidence supports the hypothesis that THC’s effect on the brain facilitates incentive motivation regardless of sensory modality of the reinforcer.

Spatial navigation in humans correlates with activity of cells in hippocampus that respond when we traverse specific locations in our environment. Hippocampal pyramidal cells in rodents called "place cells" may contribute to episodic memory by encoding location in physical space. Place cells display plasticity by "remapping" or altering their firing rates and patterns of activity in response to changes in spatial environment. Impaired remapping may underlie age-related deficits in spatial memory tasks. Using in vivo high-density electrophysiology to record place cell activity in awake, behaving rats, we tested the hypothesis that CA3 neuron hyperactivity in aged animals could be normalized by pharmacotherapy. Results show that acute, systemic administration of low dose levetiracetam and sodium valproate ameliorates deficits in the aged hippocampal network by reducing firing rates, decreasing place field area, and increasing the spatial selectivity of CA3 place cells. We then tested the hypothesis that place cell activity, field area, and spatial selectivity may be an indicator for therapeutic enhancement of spatial memory in young adult rats. The results demonstrate that α5IA enhances hippocampal-dependent spatial memory as measured by the location novelty recognition task in rats, consistent with the previously established action of α5IA as an enhancer of spatial memory in the water maze test. Electrophysiological recordings on the same animals carried out in parallel demonstrate that α5IA increases place cell firing rates, reduces field area, and increases spatial selectivity. Together, these results suggest that reducing place field area and enhancing spatial selectivity correlate with the age-independent therapeutic improvement of spatial memory. The increase in place cell firing rates by α5IA likely results from its known action as a negative allosteric modulator of α5-subunit-containing receptors (α), which are located extrasynaptically at the base of dendritic spines on CA1 and CA3 pyramidal cells. Thus, to potentially target extrasynaptic tonic inhibition in the hippocampus, we synthesized and validated two α specific miRNAs as a platform for future attempts to improve spatial memory in young adult and aging animals via molecular genetics.

High­-throughput sequencing has become pervasive in all facets of genomic analysis. I developed computational methods to analyze high­-throughput sequencing data and derive biological conclusions in two research areas -- transcriptional regulation in mammals and evolution of virus under immune pressure. To investigate transcriptional regulation, I integrated data from multiple experiments performed by the ENCODE consortium. First, my analysis revealed that Transcription Factors (TFs) prefer to bind GC-­rich, histone­-depleted regions. By comparing in vivo and in vitro nucleosome dynamics, I observed that while histones have an innate preference for binding GC-­rich DNA, TF binding overrides this preference and produces a negative correlation between GC content and histone enrichment. In the next project, I found that the binding events of multiple TFs co-­occur at genomic regions enriched in activating histone marks that are typically associated with gene enhancers and promoters, suggesting that these regions may be enhancers or have TSS-­distal transcription. Lastly, I used supervised machine ­learning techniques to train histone enrichment signals and sequence features to predict transcriptional enhancers to be validated in mouse-­transgenic assays. In a post­-clinical trial exploratory analysis of Hepatitis C Virus (HCV), I traced the evolutionary path of the envelope proteins E1 and E2 in HCV-infected liver transplant patients, in response to a novel antibody. I developed a systematic amino acid­-level analysis pipeline that quantifies differences in amino acid frequencies in each position between two time points. Upon applying this method across all positions in the E1/E2 region and comparing pre-­liver­-transplant and post­-viral­-rebound time points, mutations in two positions emerged as being key to antibody evasion. Both these mutations--N415K/D and N417S--were in the epitope targeted by the antibody, but surprisingly, did not co­-occur. In post­-rebound viral genomes that contain the N417S mutation but retain the wild-­type variant at 415, N-­linked glycosylation of 415 is another possible escape mechanism. Using the same analysis pipeline, I also identified additional candidate escape mutations outside the epitope, which could be potential therapeutic targets.