In humans, RNA polymerase II is the sole source of messenger RNAs that are ultimately translated into proteins and its transcriptional activity is highly regulated. Mechanisms have evolved to control which, when, and to what degree genes are transcribed. Because most cells have the same genome, control of transcription is essential in maintaining cellular identity. Misregulation of Pol II transcription is a hallmark of both cancer and retroviral infection. This research investigates the regulation of Pol II transcription and related co-transcriptional mRNA capping. Chromatin immunoprecipitation experiments were used to characterize the composition of nucleosomes and Pol II, DSIF and NELF occupancies at bidirectional promoters and enhancers. In collaboration with Alberto Bosque and Vicente Planelles, sequencing experiments were performed in a primary T cell model of HIV latency and a role for sequence-specific recruitment of STAT5 was established in HIV reactivation. In contrast, analysis of Myc binding in vitro and in cells demonstrated that transcription machinery played a major role in recruiting Myc to genomic sites. A precise method was also developed to detect polymerase-associated nascent transcripts in nuclei. The roles of Cdk7, a subunit of TFIIH that phosphorylates Pol II during initiation, were characterized by treatment of nuclear extracts and cells with THZ1, a recently developed covalent inhibitor with anti-cancer properties. Inhibition of Cdk7 was demonstrated to cause defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. Capping of nascent RNAs was found to be spatially and temporally regulated in part by a previously undescribed THZ1-sensitive factor present in nuclear extract. THZ1 impacted pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to misloading of DSIF. In vitro and sequencing methods were used to describe an extremely rapid and global transcriptional response to hydrogen peroxide. During periods of oxidative stress, termination was likely inhibited and Pol II accumulated at promoters and enhancers after as few as two minutes, and clearance of these polymerases required P-TEFb. In the presence of flavopiridol, a potent P-TEFb inhibitor, non-productive elongation was observed and a potential role for P-TEFb in termination was proposed.

Gene therapy has the potential to treat a wide variety of diseases. Delivering nucleic acids, such as DNA and mRNA, allows for the production of an aberrant or absent protein that is causing the disease. Delivery of genes via viruses is very efficient but falls short because of other issues. Nonviral delivery, on the other hand, struggles with efficiency but has advantages in terms of lack of immunogenicity, ease in production, and carrying capacity. DNA is much more stable than mRNA, and the protein production from DNA persists for a longer time. However, DNA delivered to cells must pass through the nuclear envelope to produce protein. Nuclear penetration with nonviral DNA delivery in vivo has not yet been accomplished. mRNA only needs to be delivered to the cytoplasm. Recent interest in nonviral delivery of mRNA has surged upward because delivery of mRNA to various cells in vivo has proven successful. Yet mRNA still struggles with nuclease stability, which is a major impediment toward efficient expression. A polyacridine PEG-peptide (PEG-peptide) has been previously used to stabilize DNA against nuclease hydrolysis by binding through ionic and intercalative interactions. Binding of PEG-peptide to DNA results in a PEGylated nanoparticle, or polyplex, and which protects the DNA. The same PEG-peptide was applied to mRNA. To increase the ability of PEG-peptide to bind through intercalation, a reverse complementary strand was hybridized to the mRNA, forming double stranded mRNA (dsmRNA). In a similar manner to DNA, complexing dsmRNA or single stranded mRNA (ssmRNA) with PEG-peptide resulted in formation of PEG-peptide polyplexes. A dsmRNA polyplex was much more resistant to ribonuclease challenge in vitro than a ssmRNA polyplex. The mRNA constructs were tested in vivo by hydrodynamic dosing. dsmRNA was found to be translationally competent by producing a high level of luciferase reporter enzyme in the liver of mice. When the reverse strand length was modified such that it hybridized with only the coding region, leaving the untranslated regions (UTRs) and poly(A) tail single stranded, the in vivo translatability (level of expression) and persistence (duration of expression) of dsmRNA was equivalent to that of ssmRNA. Full hybridization of the reverse strand with the coding region, the UTRs, and poly(A) tail resulted in a decrease of in vivo translatability. However, the circulatory stability (an in vivo measure of resistance to degradation in blood) was greatly increased when the reverse strand was fully hybridized. The persistence of expression of exogenously delivered mRNA is poor in comparison to DNA. The first step in mRNA decay in the cytoplasm is predominantly poly(A) tail shortening, or deadenylation. To address the persistence issue, mRNA with nonadenosine extensions at the 3’ end of the poly(A) tail was synthesized to inhibit deadenylation-dependent mRNA decay. However, increase of the length of tail extension resulted in a concomitant overall decrease in translatability and no increase in persistence. Hybridization of a DNA oligo to the origin of the tail extension activated endogenous RNase H, cleaving the tail extension, exposing the poly(A) tail, and reactivating the mRNA for high level translation, although no increase in persistence was seen with this strategy. A structured tail extension consisting of two human β-globin 3’UTR sequences increased persistence but also decreased overall translatability. Enzymatic poly(A) tailing of this structured tail extension brought back the translatability but simultaneously lost the persistence gain. While this study on poly(A) tail extension mRNA did not produce a highly active mRNA that had increased persistence, its results may be applicable toward other gene therapy applications. Other efforts to increase the metabolic stability or persistence of mRNA were pursued. Scavenger receptors on resident liver macrophages remove polyplexes from the blood by phagocytosis. Saturation of the scavenger receptors by coadministration of a scavenger receptor inhibitor resulted in increased circulatory stability of dsmRNA. However the scavenger receptor inhibitor was toxic in mice. Another effort to increase the persistence of gene expression in vivo was utilizing an autogene. Autogenes are able to drive the expression of a DNA-based gene outside of the nucleus. In its final form, the autogene did not produce expression. It is an exciting time to be in the field of mRNA gene therapy. Hopefully the research presented in this thesis will factor in to the knowledge base that can treat and cure human diseases.

Peptides are emerging as promising therapeutics due to their inhibitory affinity towards protein-protein interactions (PPI). However, peptides have been limited mainly by their poor bio-stability and lack of cell permeability. Efforts to generate drug-like peptides have led to the development of macrocyclic peptides, which exhibit improved stability. Yet, most macrocyclic peptides still require the assistance of a cell penetrating peptide (CPP) for cellular entry. High throughput technologies have been exceptional tools for the discovery of peptides to interrupt PPIs. This work details the recent advancements we have made to improve our high throughput technique, mRNA display, to yield more therapeutically relevant peptides to inhibit PPIs. Our advancements are focused on cell permeability, protease stability, and secondary structure for enhanced affinity. Here we develop and optimize a cyclic CPP that can be included in future mRNA display libraries. We also tested the ability of our CPP to deliver an impermeable peptide cargo into cells. We rationally designed and tested linear and cyclic peptides to improve affinity to the BRCA1 protein. We used computational work to complement our experimental results for our CPPs and BRCA1 inhibitors. We examined peptides that arose from a library containing a mix of linear, monocyclic, and bicyclic peptides constructed using orthogonal cyclization chemistries. We rationally designed cyclic peptides and tested their affinity against Hsp70. We proposed a novel selection strategy to find optimal CPP motifs.

Les ARNs H/ACA remplissent des fonctions variées dans la cellule. Ils servent d'ARNs<br />guides pour les conversions d'uridines en pseudouridines des ARNs ribosomiques mais<br />également des snARNs du spliceosome. Les ARNs H/ACA nucléolaires, les snoARNs,<br />guident les modifications des ARNr alors que ce sont des ARNs H/ACA qui s'accumulent<br />dans les Cajal bodies, les scaARNs, qui guident les modifications des snARNs du<br />spliceosome transcrits par l'ARN polymérase II. Nous avons montré par une large analyse<br />mutationnelle d'un ARN chimérique artificiel que la localisation des scaARNs dans les Cajal<br />bodies ne dépendait que d'un court motif de quatre nucléotides situé au niveau des boucles<br />terminales 5' et 3' du domaine H/ACA et appelé « boîte CAB ». La boîte CAB est conservée<br />chez de nombreux organismes et est nécessaire et suffisante à la localisation des scaARNs<br />dans les Cajal bodies. De façon plus inattendue, l'ARN de la télomérase, hTR, responsable de<br />l'élongation des télomères lorsqu'il est associé à la reverse transcriptase hTERT, comporte<br />également un domaine scaARN dans sa partie 3'. Ce domaine H/ACA possède en effet une<br />boîte CAB, située au niveau de la tige boucle 3' du domaine H/ACA, qui est responsable de la<br />localisation de hTR dans les Cajal bodies. Nous avons voulu comprendre la signification<br />biologique de cette accumulation dans les Cajal bodies tout au long du cycle cellulaire par une<br />approche d'hybridation in situ et d'immunofluorescence. L'étude de la dynamique<br />intranucléaire de hTR par microscopie à fluorescence nous a permis de mettre en évidence un<br />rôle de hTR, associé aux Cajal bodies, dans la régulation de l'élongation des télomères. Les<br />Cajal bodies pourraient en effet délivrer hTR, potentiellement associé à hTERT, directement<br />au niveau des télomères.<br />Nous nous sommes intéressés dans un deuxième temps, à l'expression et à la maturation des<br />sno/scaARNs H/ACA introniques. Alors que plusieurs travaux mettent en évidence<br />l'existence d'une synergie entre la machinerie d'épissage et la machinerie d'assemblage des<br />snoRNPs C/D, nos résultats montrent qu'au contraire, l'épissage et l'assemblage de la<br />particule H/ACA chez l'homme sont deux évènements indépendants. Nous apportons<br />également l'évidence que l'expression correcte des ARNs H/ACA nécessite une transcription<br />par l'ARN polymérase II et que la particule H/ACA s'associe très précocement au niveau du<br />pré-ARNm.

<p>Chicken fetuses exposed to chronic hypoxia suffer from growth retardation and</p><p>induces an overall sympathetic activity, including elevation of the concentration</p><p>of circulating catecholamines. Simultaneously, hypoxic fetuses display a</p><p>lowered β-adrenoreceptor (βAR) density in myocardial tissue. In vertebrates,</p><p>β1AR and β2AR are the most important signalling pathways for acute elevation</p><p>of cardiac performance. The aim of this study was to see how chronic hypoxia</p><p>affects the level of messenger RNA (mRNA) for the β1AR in the fetal chicken</p><p>heart at different developmental ages. The broiler chicken is a suitable model</p><p>organism for studying the progression of heart failure because the fast growth</p><p>rate requires a large increase in blood perfusion at the end of fetal development.</p><p>The β1AR sequence of the broiler chicken is 1587 bp and located on</p><p>chromosome 6. When running a PCR for quantification of the sequence,</p><p>primers for almost the whole sequence failed (1404 bp) and so did primers of</p><p>1193 bp; instead primers of 692 bp of the sequence were used and made</p><p>quantification possible. Similar results were obtained from both the heart and</p><p>liver of day 15 fetal chickens. The PCR product was cloned into a TOPO vector</p><p>and sent for sequencing, to enable the making of a probe for a northern blot</p><p>analysis of the mRNA in the fetal chicken hearts.</p>

<p>Adenovirus infected cells provide a good model system for studying nuclear organization during RNA production and transport. This thesis is focused on the dynamic organization of splicing factors during the late phase of Adenovirus infection in HeLa cells, the nuclear localization of viral RNA, and the pathway used for viral RNA transport to the cytoplasm.</p><p>Splicing factors are relocalized from interchromatin granule clusters to sites of transcription in Adenovirus infected cells at intermediate times of infection. Later, splicing factors and viral RNA accumulate posttranscriptionally in interchromatin granule clusters. The release of the splicing factors from transcription sites was energy dependent or preceded by energy requiring mechanisms. Our data indicated that phosphorylation events inhibited by staurosporine, and 3' cleavage of the transcript are two possible mechanisms involved prior to the release of the RNP complex from transcription sites.</p><p>A viral protein derived from orf6 of early region 4, 34K, is important for the nuclear stability and transport of late viral mRNA derived from the major late transcription unit. A viral mutant lacking this region is defective for posttranscriptional accumulation of viral mRNA in interchromatin granule clusters, and for the accumulation of viral RNA in the cytoplasm. These results suggest that posttranscriptional accumulation of viral RNA in interchromatin granule clusters may contribute to the maturation of the RNP complex or sorting of RNAs and proteins, to prepare the final RNP complex for transport to the cytoplasm.</p><p>A previous model suggested that adenoviral late mRNA is transported to the cytoplasm by utilizing the CRM-1 pathway. This pathway can be blocked by the drug leptomycin B. The data presented in paper IV suggests that this model might not be applicable, since leptomycin B did not inhibit adenoviral late gene expression.</p>

<p>Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. </p><p>During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. </p><p>The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing.</p><p>We show that ICP27 inhibits pre-mRNA splicing <i>in vitro</i> in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. </p><p>In order to learn more about how splicing is organized in the cell nucleus <i>in vivo</i>, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.</p>

<p>DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. </p><p>To expand the knowledge of protein–DNA recognition, we have utilized<i> in vitro</i> evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. </p><p>Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring <i>K</i>_D values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure.</p><p>mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. </p><p>The two <i>in vitro</i> selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.</p>

This research investigates the physiological and transcriptional responses of Nitrosomonas europaea when exposed to chlorinated aromatic compounds and heavy metals under varying environmental conditions. It was found that transcriptional responses of identified sentinel genes correlate well with nitrification inhibition. Sorption of metals to biomass was also investigated and found not to correlate well with N. europaea inhibition. Whole genome microarray experiments were performed to define the transcriptional response of N. europaea when exposed to chlorobenzene. 13 out of 2460 N. europaea genes were significantly up-regulated after a 1-hour exposure to 4 μM chlorobenzene. HPLC analysis revealed that chlorobenzene was being oxidized primarily into 4-chlorophenol, and further physiological studies revealed that the presence of 4-chlorophenol could account for the inhibitory responses observed. RT-qPCR analysis of several differentially regulated genes verified that similar transcriptional responses were occurring for both chlorobenzene and 4-chlorophenol. 50% inhibitory concentrations of chlorobenzene and 4-chlorophenol resulted in moderate up-regulation of studied genes, however, increasing the concentration of 4-chlorophenol to achieve nitrification inhibition of 93% or more dramatically increased the fold regulation of several of the identified up- and down- regulated genes of interest. Increasing the 4-chlorophenol exposure time to 3 hours at the higher inhibition levels led to a general decrease in amplitude of transcriptional response for all genes tested. Cultures of N. europaea were exposed to various amounts of cadmium in aqueous solution containing EDTA, a strong metal-chelating organic, to control free ionic cadmium²⁺ (Cd²⁺) concentrations. Inhibition of ammonia oxidation as well as transcriptional up-regulation of merA, an identified sentinel gene for exposure to cadmium was found to correlate well with the concentration of Cd²⁺. The concentration of Cd²⁺ required to significantly affect N. europaea cells was found to be in the nanomolar range, which is several orders of magnitude lower than values reported in the literature for cadmium inhibition to mixed-culture activated sludge systems. The sorption of cadmium to the cells was found to be proportional to both the concentration of total cadmium and the concentration of Cd²⁺. At the concentration of metals required to cause approximately 50% nitrification inhibition, specific oxygen uptake results indicate the inhibition is specific to AMO with HAO and downstream energy-generation processes intact. To investigate more closely the inhibitory interactions between heavy metals and AMO, N. europaea inhibition by cadmium, zinc and silver was studied under substrate-limiting conditions. Unlike incubation in oxic environments, 1 hour incubations of N. europaea with cadmium and silver under anoxic conditions did not cause inhibition of nitrification activity after re-suspension in oxic media. In contrast, zinc, which is normally considered an analogue of cadmium in terms of toxic effect and transport mechanisms, was non-inhibitory to N. europaea when exposed in media lacking ammonia. Transcriptional response of merA closely followed the inhibition patterns, with samples which were inhibited after the removal of the metal having significant up-regulation of the gene, and those samples which were uninhibited showing no significant change in merA transcript levels compared to controls. Although sorbed metal concentrations were not found to be predictive of either extent of inhibition or transcriptional response, significantly more cadmium, zinc and silver were sorbed to biomass when incubated in aerobic media compared to anoxic media. Sorption in oxic media was found to be independent of AMO activity and similar results were obtained using Deinococcus radiodurans, a non-nitrifying gram-positive extremophile. The results indicate that greater heavy metal sorption to biomass in oxic environments may be due to general membrane chemistry effects. / Graduation date: 2011 / Access restricted to the OSU Community at author's request from March 31, 2011 - March 31, 2012

Ruminal acidosis is a persisting digestive disorder in modern ruminant production; however, the susceptibility of cows to ruminal acidosis differs among cows fed a common diet. The overall objective of this research was to evaluate factors affecting the susceptibility of cows to ruminal acidosis. This research demonstrated that feeding sucrose in replacement for corn grain to Holstein cows in early lactation does not increase the risk for ruminal acidosis and may actually increase ruminal pH. However, regardless of dietary treatment, cows in early lactation were at risk for ruminal acidosis. In Study 2, diets differing in the forage-to-concentrate ratio were fed to Holstein cows to evaluate changes in the in vivo rate of short-chain fatty acid absorption (SCFA) and the expression of genes coding for transporters and enzymes involved in the absorption and metabolism of SCFA in ruminal tissue. Contrary to the hypothesis, the fractional rate of absorption and expression of genes involved in SCFA absorption and metabolism were not affected by the forage-to-concentrate ratio. Considerable variation among individual cows for the severity of ruminal acidosis was detected for cows on the diet containing the low forage-to-concentrate ratio. To determine the cause of this variation, a ruminal pH measurement system was developed to accurately and precisely measure ruminal pH in non-cannulated small ruminants. Sheep were then subjected to a ruminal acidosis challenge model in vivo, and the absorption of acetate and butyrate across the isolated ruminal epithelia was measured in vitro in Ussing chambers. The results of this study demonstrated that differences in the severity of ruminal pH depression among animals could largely iv be accounted for by differences in the absorptive capability of the ruminal epithelium. In summary, although ruminal acidosis is a common digestive disorder in dairy production systems, variation in the susceptibility to ruminal acidosis is common. The cause for much of this variation is due to differences in the absorptive capacity of the ruminal epithelia. / Animal Science