It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...

A new series of fluorescent prorin (promazine-coumarin hybrid) derivatives were synthesized via piperidine catalyzed cyclization with 3-formyl-2-hydroxypromazine and ethyl acetoacetate or Meldrum’s acid. The synthesis of these new compounds focused on the introduction of electron withdrawing carbonyl groups on the 3-position of the coumarin lactone ring in order to increase polarization of the molecule which results in a longer absorbance wavelength by stabilizing intramolecular charge transfer that occurs in the excited state. The 3-acetyl derivative was further subjected to an aldol condensation with either benzaldehyde or cinnamaldehyde in order to further extend the conjugation of the molecule. The four new prorin compounds exhibited excellent photophysical properties and had Stokes shifts ranging from 160-190 nm. These properties are ideal for use as fluorescent probes to detect RNA binding ligands. The optical properties of the new prorin compounds were compared with a previous series of prorins as well as theoretical calculations in order to gain a more comprehensive understanding of how the photophysical properties of the molecule can be tuned by introducing different substituents. Although increasing polarization of the molecule with electron withdrawing substituents does red-shift the absorbance wavelength, it has little effect on the emission wavelength and can actually cause a blue-shift in some cases. It was observed that introducing aromatic substituents has the greatest effect on red-shifting the emission wavelength and increasing Stokes shift.

It is estimated that fungal infections kill greater than 1.6 million people annually, a number that is comparable to the number of deaths associated with tuberculosis. Candida species are the fourth leading cause of hospital-acquired blood infections and Candida albicans is the most common cause of these fungal blood infections (known as candidemia). Immunocompromised individuals, such as those who have HIV/AIDS, those undergoing chemotherapy treatments, or those on broad-spectrum antibiotics, are most likely to develop candidemia. Candidemia is associated with a 20-40% mortality rate. However, when patient treatment for candidemia is delayed for over 48 hours, associated mortality rates increase to 78%. Blood infections can disseminate Candida albicans throughout the body, eventually leading to infection in vital organs like the liver, kidney and brain. Optimal patient outcomes are achieved if antifungal therapy is given within 12 hours after a blood sample is obtained for culture and testing. However, current blood tests cannot reliably detect Candida this early and thus antifungals are not routinely given to patients in this time frame. Counterintuitively, it is believed that some fungi, like many bacteria, are non-harmful residents in small intestines of most adults and this hypothesis is supported by the fact that the most common fungal species in the human gut is Candida albicans. However, intestinal overgrowth of C. albicans is linked to Crohn's disease, and disease-causing forms of C. albicans can arise from commensal strains that once resided in the patient’s gastrointestinal tract. The specific molecular mechanisms by which C. albicans interacts with host immune cells versus intestinal cells, and those that trigger Candida pathogenicity remain unknown. Many strains of Candida albicans have developed resistance to azoles, the major class of drugs used to treat both superficial and systemic infections. In order to develop new treatments, we must better understand host-fungal pathogen biology to determine novel antifungal targets or therapeutics to fight fungal infections at the early stages of infection. In this work, we developed a novel tool that allows us to measure which genes are important to both the host and Candida albicans simultaneously, in specific infection states. We have applied this tool to measure gene expression in Candida albicans interacting with mammalian macrophages and small intestine epithelial cells– at both the population and single cell levels. When examining populations of sorted infection samples, we found that host immune cells both exposed to and infected with fungal cells exhibit similar expression patterns. In contrast, phagocytosed C. albicans exhibit unique expression patterns compared to those merely exposed to macrophages. We found that immune response genes in single, Candida infected macrophages exhibited bimodal expression patterns for some immune response genes. We also observed examples of expression bimodality in live Candida inside of single macrophages. Both Candida albicans and host small intestine epithelial cells demonstrate distinct patterns of expression when exposed to each other at the population level, compared to unexposed controls. However, the magnitude of these differences is dependent on the multiplicity of infection. Some expression programs overlapped with those observed in populations of Candida cells interacting with macrophages, with key differences. We also observed expression bimodality among epithelial cells infected with C. albicans. We believe the information obtained using this technique could be used when considering new antifungal or therapeutics targets; if uniform and high expression of particular in genes in Candida populations phagocytosed by macrophages or invading epithelial cells leads to high and early protein production, these proteins may be effective antifungal targets. Similarly, if some host immune response genes are not expressed in a population of Candida infected macrophages as uniformly and highly as expected, these genes or proteins could be target of a therapeutic for patients with Candida infections that are resistant to azoles.

A dissertation submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree Master of Science in Medicine / Ferritin is an iron storage protein, made up of heavy (H) and light (L) subunits. Ferritin synthesis is regulated at a post transcriptional level by iron, which induces a redistribution of ferritin mRNA from a free cytoplasmic pool to polyribosomes. Inflammatory states influence iron metabolism, causing a decrease in serum iron levels associated with an increase in reticuloendothelial ferritin synthesis and iron storage. / IT2018

Thesis advisor: Larry W. McLaughlin / Thesis advisor: Mary F. Roberts / Nucleic acids are essential biological molecules for life. For example, deoxyribonucleic acid (DNA) is the main genetic information carrier; ribonucleic acid (RNA) plays a critical role in translation and transcription. These characteristics place nucleic acids as the fundamental genetic materials of a living system. Since over a century ago, intensive attempts have been made by researchers to study the nucleic acid properties. For chemists, it is particularly interesting and important to understand the relationship between structures and properties of nucleic acids. For instance chemical modifications can alter stability of nucleic acids, and consequently influence their biochemical behaviors. In this work, we began by investigation of a 5',6-cyclo-modified nucleic acid resembling the product of DNA oxidation, and then developed a library of cyclomodifications. Our research on their structures and properties indicated that by installing cyclo-modifications we might be able to add some properties, that were not observed in nature to nucleic acids. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

在不同的模型體系和疾病microRNA-199a (miR-199a)的功能和基因表達都不盡相同而且相當複雜。它在人基因組中有個表達位點，分別是位於19 號染色體的miR-199a-1 和1 號染色體的miR-199a-2。這個位點可以產生相同的miRNA (miR-199a-3p 和miR-199a-5p)。造成miR-199a不同表達和功能的原因尚未清楚。但是，很有可能是由於個位點不同的調節機制和miR-199a 在不同的細胞和組織中可變的作用对象。在睾丸生殖細胞腫瘤(簡稱睾丸癌)和膠質母細胞瘤(簡稱膠質瘤)中研究miR-199a 的啟動子甲基化發現，在睾丸癌中miR-199a-1 和-2 共同的啟動子過甲基化導致miR-199a 表達低。而在膠質瘤中，只有miR-199a-2，而不是miR-199a-1 的啟動子低甲基化與miR-199a 的高表達相關。除啟動子甲基化，在幹細胞分化過程中升高表達的miR-199a 被證實是通過轉因子Twist1 的調控；循环通Twist1-miR-199a-5p-HIF1α控制miR-199a-5p 促進成骨分化的作用。miR-199a 的功能是通過其下游目標 (靶基因) 而成的。因此, 目標分子的功能控制miR-199a 在同的模型體系和疾病中的作用。我應用基因組學和蛋白組學的方法尋找miR-199a-5p 在睾丸癌中的下游目標(靶基因)。MAFB 被指出和證明是miR-199a-5p 在睾丸癌中的靶基因，而miR-199a 抗腫瘤細胞增殖活性的作用是通過MAFB 發揮的。通過研究miR-199a 在用系統中的調控機制和作用目標，我希望用miR-199a 作為模型明miRNA 生物學的複雜多樣性。 / microRNA-199a (miR-199a) has been shown to have diverse biological functions and behave quite differently in different physiological systems and diseases. It is encoded by two loci in the human genome, miR-199a-1 in chromosome 19 and miR-199a-2 in chromosome 1. Both loci give rise to the same miRNAs (miR-199a-5p and miR-199a-3p). The underlying mechanism responsible for the diverse action of the miRNA is not clear. However, it is likely contributed by differential regulation of the two genomic loci and variable targets of the miRNA in different cells and tissues. Studies on the promoter methylation of miR-199a in testicular germ cell tumors (TGCTs) and glioblastomas (gliomas) demonstrated that hypermethylation of both loci of miR- 199a resulted in its reduced expression in TGCTs, while hypomethylation of miR-199a-2 but not -1 in gliomas might lead to its elevated expression. In addition to DNA methylation, the functions of transcription factors in controlling the expression of miR-199a through a Twist1- miR-199a-5p-HIF1α cyclic pathway were demonstrated to play significant roles during me nchymal stem cell differentiation. The functions of miR-199a are mediated by the actions of its downstream targets. Both genomic and proteomic approaches were applied to study the targets of miR-199a-5p in TGCTs. A putative target of miRNA-199a-5p, MAFB, was identified and confirmed to be responsible for the anti-tumor proliferation activity of the microRNA. By studying the mechanisms that control the expressions of miR-199a and its various downstream targets in different systems, it is hoped that miR-199a could be used as a model to illustrate the complexity of miRNA biology. / Detailed summary in vernacular field only. / Gu, Shen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 144-163). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract --- p.I / 摘要 --- p.II / Acknowledgements --- p.III / Abbreviations --- p.VIII / Chapter 1 --- p.1 / Chapter 1.1 --- miRNAs - small yet powerful molecules --- p.2 / Chapter 1.1.1 --- Biogenesis and processing of miRNA --- p.2 / Chapter 1.1.2 --- Detection of miRNA expression --- p.7 / Chapter 1.1.3 --- Identification of putative miRNA targets --- p.11 / Chapter 1.2 --- miR-199a - an example revealing the complexity of the miRNA world --- p.19 / Chapter 1.2.1 --- miR-199a - standing out among thousands --- p.19 / Chapter 1.2.2 --- Regulation of miR-199a expression --- p.20 / Chapter 1.2.3 --- miR-199a in carcinogenesis --- p.24 / Chapter 1.2.4 --- miR-199a in cardiogenesis --- p.33 / Chapter 1.2.5 --- miR-199a in stem cell differentiation and embryo development --- p.34 / Chapter 1.2.6 --- Other functions of miR-199a --- p.35 / Chapter 1.3 --- Project overview --- p.38 / Chapter 2 --- p.39 / Chapter 2.1 --- Introduction --- p.40 / Chapter 2.2 --- Materials and methods --- p.42 / Chapter 2.3 --- Results --- p.49 / Chapter 2.3.1 --- Dysregulation of miR-199a in tumors were controlled by DNA methylation --- p.49 / Chapter 2.3.2 --- Transcriptome changes upon miR-199a-5p in TGCTs --- p.58 / Chapter 2.3.3 --- Identification of MAFB as a putative target of miR-199a-5p in TGCTs --- p.63 / Chapter 2.3.4 --- MAFB is highly expressed in malignant testicular tumor and negatively correlated with miR-199a-5p --- p.67 / Chapter 2.3.5 --- MAFB knockdown suppresses tumor cell growth in vitro --- p.69 / Chapter 2.4 --- Discussion --- p.70 / Chapter 3 --- p.75 / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Materials and methods --- p.81 / Chapter 3.3 --- Results --- p.86 / Chapter 3.3.1 --- Expression of miR-199a during MSC osteogenesis --- p.86 / Chapter 3.3.2 --- Functions of miR-199a on osteogenesis of hMSCs --- p.90 / Chapter 3.3.3 --- Involvement of HIF1α, Twist1 and miR-199a-5p during osteogenesis of hMSC at early stage and late stage of differentiation --- p.94 / Chapter 3.3.4 --- Up-regulation of miR-199a-5p was related to hypoxia enhanced osteogenesis at early stage --- p.99 / Chapter 3.3.5 --- miR-199a-5p inhibited HIF1α-Twist1 pathway to increase osteogenesis at late stage --- p.103 / Chapter 3.4 --- Discussion --- p.107 / Chapter 4 --- p.111 / Chapter 4.1 --- Overview of the project --- p.112 / Chapter 4.2 --- Summary and conclusion --- p.115 / Chapter 4.3 --- Future work --- p.116 / Chapter Supplementary Materials --- p.117 / Chapter Supplementary Table 2.1 --- p.118 / Chapter Supplementary Table 2.2 --- p.129 / Chapter Supplementary Table 2.3 --- p.142 / References --- p.144

Typescript. / Digitized by Kansas Correctional Industries

The Integrator complex (INT) is a metazoan-specific group of proteins associated with RNA polymerase II (Pol II) that has important functions in the 3'-end processing of noncodng RNAs, including uridine-rich small nuclear RNA (UsnRNA) and enhancer RNA (eRNA). Recently, INT has also been reported to be involved in Pol II transcriptional regulation of protein-encoding genes. INT contains at least 14 subunits, but the function of each subunit is difficult to predicted, because most subunits lack identifiable domains and display little similarity with other proteins. The endonuclease activity of INT is carried out by its subunit 11 (IntS11), which belongs to the metallo--lactamase superfamily and is a paralog of CPSF-73, the endonuclease for pre-mRNA 3'-end processing. IntS11 forms a stable complex with INT subunit 9 (IntS9) through their C-terminal domains (CTDs). This dissertation describes the crystal structure of the IntS9-IntS11 CTD complex at 2.1-Å resolution and summaries the structure-based biochemical and functional studies. The complex is composed of a continuous nine-stranded -sheet with four strands from IntS9 CTD and five from IntS11 CTD. Highly conserved residues are located in the interface between the two CTDs. The structural observations on the complex are confirmed by yeast two-hybrid assays and coimmunoprecipitation experiments. Functional studies demonstrate that the Int9-IntS11 interaction is crucial for proper INT function in snRNA 3'-end processing. The dissertation also presents the structural studies of a newly found mammalian mRNA deNADding enzyme, Nudt12. We determined the crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6-Å resolution. The structure provides exquisite insights into the molecular basis of the deNADding activity within the NAD pyrophosphate. Previous studies have reported that NAD-capped mRNAs in mammalian cells are hydrolyzed by the DXO deNADding enzyme. Together with biochemical and functional studies, we demonstrate that Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targets different RNAs.

No description available.

Every cell in the human body contains almost the same genetic material, therefore, cellular identity is derived from the selection of genes transcribed into RNA and the mRNAs that are made into proteins. To achieve precise control of gene expression, the transcription of messenger RNAs by RNA polymerase II is regulated at multiple checkpoints. A major control point within this system is the P-TEFb dependent transition from paused to productive elongating polymerase complexes. Reversible inhibition of P-TEFb by the 7SK small nuclear ribonucleoprotein (snRNP) is the key step in the control of transcription elongation. Due to the importance of the regulation of P-TEFb, this research investigates the structure of 7SK RNA and the interactions within the 7SK snRNP. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) was used to demonstrate a magnesium-dependent conformational change of in vitro transcribed 7SK RNA folding including a switch in the pairing of the 7SK motif, which is required for P-TEFb regulation. SHAPE was also used to determine that the 5′ end of 7SK pairs alternatively with two different regions within the RNA resulting in open and closed conformations. Moreover, SHAPE was used to show a similar conformational change in cellular 7SK snRNP complexes after the loss of P-TEFb. Assembly of the 7SK snRNP in vitro, using recombinant HEXIM1, P-TEFb, LARP7, MEPCE, and in vitro transcribed 7SK RNA were combined under optimized conditions, resulted in a complete and functional complex. These complexes demonstrated a reversible inhibition of the activity of P-TEFb as well as a similar structure to cellular complexes. LARP7 was found to contain a C-terminal MEPCE interaction domain (MID) that associates with and inhibits MEPCE after binding to the 3′ stem loop of 7SK. The inhibition of MEPCE was determined to be dependent on the overall conformation of 7SK and structural elements of the 3′ stem loop. Use of a highly selective degrader of Brd4, dBET6, also revealed a possible alternative mechanism for P-TEFb sequestration into the 7SK snRNP. Collectively, these findings aid in the understanding of gene regulation through the control of P-TEFb by the 7SK snRNP.