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.

This dissertation covers two projects linked by their involvement in the modification of tRNA bases. The first project focused on an investigation of a role for the modified base Archaeosine, the ubiquitous modification in tRNA in the archaeal domain. Initial work was performed on a set of in vitro prepared tRNA modified to feature either the canonical guanine base at position 15, preQ0 (TGT product) or Archaeosine (ArcS product). There was very little difference in the thermal stability of tRNAs containing these modifications in the halophilic H. volcanii tRNASer or E. coli tRNAGln. In tRNAGln taken from M. thermautotrophicum however, there was a 2°C increase in melting point in 50 mM MgCl2 upon modification to archaeosoine. Benefitting from the development of genetic tools for the generation of specific deletion mutants of the thermophile Thermococcus kodakarensis, it was possible to start investigation of tRNAs that have been hypomodified in vivo due to the lack of arcTGT (TK0760) and ArcS (TK2156). In vitro modified equivalents of the GlnCUG isoacceptor were also prepared. Thermal stability of these tRNAs show virtually identical melting transitions, with a biphasic denaturation occurring at all magnesium concentrations tested. Isolation of the CUG isoacceptor from the in vivo maturated total tRNA pool allowed melts of specifically hypomodified tRNAs. Those containg Archaeosine (WT) and genetically encoded guanine (∆tgt) showed identical melting profiles with Tm beyond the 98°C limit of the experiment. In the preQ0 containing in vivo RNA the shows a lag in its magnesium response, and a more persistent biphasic melting profile. At 10mM Mg2+ concentration the preQ0 containing tRNA is approaching a Tm of 98°C though the turn over point in the melt is not well defined. The second project was to investigate the product of base treatment of the oxidized cofactor NAD(P)+. This cofactor is involved in the biosynthesis of preQ1 from preQ0 in bacterial systems and at low concentrations it can be difficult to quantify enzyme activity based on direct quantitation. Under these conditions a fluorescence based method where by the production of NAD(P)+ is measured rather than the consumption of NAD(P)H. Base treatment of the oxidized cofactor generates a fluorescent species with an efficiency of 95%. The assay has been used extensively by our group to track activity of various enzymes including QueF, however the identity of the fluorophore had not been established. Purification of the fluorescent product was achieved by isocratic HPLC in water using a reverse phase column. It was found that the assay conditions previously used (7.5M NaOH for 2 hours) were actually counterproductive for maximizing fluorescence yield. Incubation at 2M NaOH gave a 35% increase in product yield. The isolated product was determined to have molecular weight of 123.0318 (3.6 ppm by accurate mass ESI MS). 1H and 13C NMR were used to confirm the structure to be that of 2-hydroxynicotinaldeyde. It was also possible to determine the quantum yield for the molecule to 0.11. Work carried out previously on pyridinium based NADP analogs is consistent with the identity of the fluorophore presented here.

Le complexe de liaison de la coiffe des ARN (CBC) joue un rôle essentiel dans leur maturation et déclenche une variété de réactions biochimiques, via son interaction avec différents partenaires. Deux complexes, CBC-ARS2-PHAX (CBCAP), et CBC-ARS2-ZC3H18-NEXT (CBCN), ont récemment été montré comme important pour cibler les ARN vers l'export (CBCAP) ou la dégradation (CBCN). Cependant, les mécanismes par lesquels la sélection se fait pour l'une voie ou l'autre reste mystérieuse. Ainsi, une question majeure qui reste à résoudre est de savoir quand et comment ces complexes sont recrutés sur les ARN. Dans ce travail, j'ai utilisé la procédure du iCLIP (Cross-Linking and Immuno-Precipitation), afin d'identifier les cibles de ces complexes sur l'ensemble du transcriptome humain. J'ai réalisé un iCLIP sur cinq composants de CBCAP et CBCN, et j'ai comparé les résultats à ceux obtenus avec RBM7, un composant de NEXT précédemment étudié par iCLIP. Mes résultats indiquent que: (i) CBP20, ARS2, PHAX et ZC3H18 se lient près de la coiffe des ARN, tandis que RBM7 et MTR4 se lient partout; (ii) CBP20, ARS2, PHAX et ZC3H18 s'associent à un large ensemble d'ARN transcrits par l'ARN polymérase II et montrent une faible sélectivité; (iii) la liaison de ces protéines varie avec l'état de maturation des ARN, avec le CBC enrichi sur les ARN matures, tout comme ARS2/PHAX/ZC3H18 et MTR4 (bien que dans une moindre mesure), tandis que RBM7 est préférentiellement lié sur les pre-mRNAs non épissés; (iv) une liaison différentielle de RBM7 et MTR4 sur les ARN, avec RBM7 enrichi sur les introns et les PROMPTs, et MTR4 plus présent sur les ARN mature. Bien que des expériences additionnelles soient requises, nous proposons que le CBCAP et le CBCN se lient à un même ensemble d'ARN, ce qui indique à la fois une compétition entre ZC3H18 et PHAX pour la liaison à ces ARN, et l'absence de voies de routage bien déterminées qui ciblerait les ARN vers l'une ou l'autre de ces protéines. Le devenir des ARN pourrait ainsi être déterminé par d'autres caractéristiques des ARN, ou encore par des protéines additionnelles. Ces facteurs pourraient s'allier aux protéines liées à la coiffe afin de favoriser la formation du CBCAP ou du CBCN. Dans le but d’identifier des facteurs additionnels, j'ai réalisé un screen d'interaction par spectrométrie de masse après purification de ARS2 ou CBP80. Ceci a été fait dans des conditions natives ou après un cross-link des complexes à la formaldéhyde, afin de stabiliser les interactions transitoires. Ceci a permis d'identifier de nouveaux partenaires de ARS2 et de CBP80, dont la majorité sont impliqués dans l'épissage des ARN. Des expériences additionnelles seront nécessaires pour valider ces interactions. / The cap-binding complex (CBC) plays a pivotal role in post-transcriptional processing events and orchestrates a variety of metabolic pathways, through association with different interaction partners. Two CBC sub-complexes, the CBC-ARS2-PHAX (CBCAP) and the CBC-nuclear exosome targeting (NEXT) complex (CBCN), were recently shown to target capped RNA either toward export or degradation, but the mechanisms by which they can discriminate between different RNA families and route them toward different metabolic pathways still remain unclear. A major question to be answered is how and when the different CBC subcomplexes are recruited to the RNP. Here, we used an individual nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) approach to identify the transcriptome-wide targets for 5 different components of the CBCAP and CBCN complexes, and compared results to the previously analysed NEXT-component RBM7. We report that: (i) CBP20, ARS2, PHAX and ZC3H18 bind close to the cap, while RBM7 and MTR4 bind throughout the mRNA body; (ii) CBP20, ARS2, PHAX and ZC3H18 associate with a broad set of RNA polymerase II (PolII)-derived RNAs and have only mild species preferences; (iii) binding varies with the RNA maturation stage, with the CBC being highly enriched on mature mRNA, ARS2/PHAX/ZC3H18/MTR4 less so, and RMB7 preferentially bound to pre-mRNAs; (iv) MTR4 and RBM7 show different specificities, with RBM7 being highly enriched on introns and promoter upstream transcripts (PROMPTs), while MTR4 is additionally present on mature RNAs. Although more experimental work is needed to fully support our model, we propose that CBCAP and CBCN bind overlapping sets of RNAs, indicating a competition between the proteins ZC3H18 and PHAX, and the lack of a strict RNA sorting mechanism. RNA fate may therefore be determined by additional RNA features and/or by other RNA-binding proteins, which may synergize with the cap and drive the formation of one specific CBC subcomplex instead of another. In an attempt to identify yet unknown factors that may interact with cap-bound CBCAP and CBCN, we performed a protein interaction screen leveraging affinity capture-mass spectrometry (ACMS), using ARS2 and CBP80 as bait proteins. As a complementary approach, we also employed a formaldehyde-based chemical cross-linking strategy, aimed at stabilizing weak/transient interactions. Although we failed to detect any transient interactions involving the CBC, we identified several potential CBC80 and ARS2 interactors, the majority of which are involved in pre-mRNA splicing. Additional quantitative experiments are required to validate our ACMS results and confirm the existence of such protein interactions in vivo.