Plants have developed intricate mechanisms to control growth in response to a variety of environmental cues, to compensate its immobility and to survive in both normal and adverse conditions. The TCP proteins are a family of plant-specific, basic helix-loop-helix (bHLH) transcription factors that involve in different aspects in plant growth and developmental control. The Arabidopsis TCP20 has been shown to involve in coordinating cell growth and proliferation, and in growth arrest in response to DNA double-stranded breaks (DSB). In this thesis, the main interest is to examine the function of Arabidopsis TCP6, which shares the highest homology with TCP20, and like TCP20, contains a putative ATM phosphorylation motif that suggests potential involvement in the ATM/ATR-mediated DSB responses. Expressional analysis including transcript measurement and reporter gene tagging demonstrated that TCP6 is expressed in flowers, in particular in the first mitotic event of pollen and ovule/embryo sac development, indicating that TCP6 potentially involves in regulating the mitotic cell cycle during gametophyte development. Yet no gametophytic or fertility-affecting mutant phenotype was observed in the tcp6 single and tcp6/tcp20 double mutants, which may be due to high functional redundancy. The tcp6/tcp20 double mutant seedlings exhibited significantly higher growth performances in true leaf growth compared to wild type when treated with gamma radiation, implying that both functional TCP6 and TCP20 are involved in response to gamma radiation-generated DSBs. The work of this thesis provides the first expressional and functional characterizations of TCP6, with the results suggesting that TCP6 and other class I TCPs play a role in regulating growth under both normal and stress conditions.

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.

<p>This thesis is based on nine research publications (<b>I – IX</b>) on structure and reactivity of RNA vis-à-vis DNA. The DNA and RNA are made of flexible pentose sugar units, polyelectrolytic phosphodiester backbone, and heterocyclic nucleobases. DNA stores our genetic code, whereas RNA is involved both in protein biosynthesis and catalysis. Various ligand-binding and recognition properties of DNA/RNA are mediated through inter- and intra-molecular H-bonding and stacking interactions, beside hydration, van der Waal and London dispersion forces. In this work the pH dependant chemical shift, p<i>K</i>_a values of 2'-OH group as well as those the nucleobases in different sequence context, alkaline hydrolysis of the internucleotidic phosphodiester bonds and analysis of NOESY footprints along with NMR constrained molecular dynamics simulation were used as tools to explore and understand the physico-chemical behavior of various nucleic acid sequences, and the forces involved in their self-assembly process. <b>Papers I – II</b> showed that the ionization of 2'-OH group is nucleobase-dependant. <b>Paper III</b> showed that the chemical characters of internucleotidic phosphate are non-identical in RNA compared to that of DNA. <b>Papers IV – VI</b> show that variable intramolecular electrostatic interactions between electronically coupled nearest neighbor nucleobases in a ssRNA can modulate their respective pseudoaromatic character, and result in creation of a unique set of aglycons with unique properties depending on propensity and geometry of nearest neighbor interaction. <b>Paper VII</b> showed that the cross-modulation of the pseudoaromatic character of nucleobases by the nearest neighbor is sequence-dependant in nature in oligonucleotides. <b>Paper VIII</b> showed that the purine-rich hexameric ssDNA and ssRNA retain the right-handed helical structure (B-type in ssDNA and A-type in ssRNA) in the single-stranded form even in absence of intermolecular hydrogen bonding. The directionality of stacking geometry however differs in ssDNA compared to ssRNA. In ssDNA the relatively electron-rich imidazole stacks above the electron-deficient pyrimidine in the 5' to 3' direction, in contradistinction, the pyrimidine stacks above the imidazole in the 5' to 3' direction in ssRNA. <b>Paper IX</b> showed that the p<i>K</i>_a values of the nucleobases in monomeric nucleotides can be used to show that a RNA-RNA duplex is more stable than a DNA-DNA duplex. The dissection of the relative strength of base-pairing and stacking showed that the relative contribution of former compared to that of the latter in an RNA-RNA over the corresponding DNA-DNA duplexes decreases with the increasing content of A-T/U base pairs in a sequence.</p>

<p>Bacterial resistance to antibiotics has become a serious problem. This is due to the remarkable ability of bacteria to respond and rapidly adapt to environmental changes. Integrons are elements with the capacity for gene capture by an integron-encoded site-specific recombinase called IntI. IntI binds and acts at the recombination sites, <i>attI </i>and<i> attC</i> resulting in excision and integration of short DNA elements called gene cassettes carrying an <i>attC</i> site in the 3’ end. Several families of antibiotic resistance genes are borne on gene cassettes in integrons connected to mobile elements. Other cassettes reside in the larger and ancestral superintegrons located on chromosomes in both pathogenic and environmental bacteria. Due to their close connection with lateral gene transfer systems, it is possible that integrons are functionally dependent on those networks. This work presents arguments for such connections. The<i> attC</i> of the <i>aadA1-qacE</i> cassette junction in Tn<i>21</i> was characterized in detail. Like other <i>attC</i> sites, it contains two pairs of inverted repeats and is almost palindromic. By using electrophoretic mobility shift assays, this study showed that IntI1 binds only to the bottom strand of <i>attC</i>. Upon folding the strand into a hairpin, a few chiral hairpin distortions define both the strand choice and also the appropriate orientation of the highly symmetrical site. Structural recognition also explains the wide sequence variation among <i>attC</i> sites. We have documented the initial cleavage step in recombination in IntI extracts and integrase levels in extracts were evaluated by a new method. Mutagenesis and homology modelling were performed to find amino acid residues in IntI1 that are important for recognition of <i>attC</i> hairpin-DNA. Comparisons were made with other tyrosine family members to explain how integron integrases differ in site-recognition and also in their mechanism of strand exchange.</p>

Bacterial resistance to antibiotics has become a serious problem. This is due to the remarkable ability of bacteria to respond and rapidly adapt to environmental changes. Integrons are elements with the capacity for gene capture by an integron-encoded site-specific recombinase called IntI. IntI binds and acts at the recombination sites, attI and attC resulting in excision and integration of short DNA elements called gene cassettes carrying an attC site in the 3’ end. Several families of antibiotic resistance genes are borne on gene cassettes in integrons connected to mobile elements. Other cassettes reside in the larger and ancestral superintegrons located on chromosomes in both pathogenic and environmental bacteria. Due to their close connection with lateral gene transfer systems, it is possible that integrons are functionally dependent on those networks. This work presents arguments for such connections. The attC of the aadA1-qacE cassette junction in Tn21 was characterized in detail. Like other attC sites, it contains two pairs of inverted repeats and is almost palindromic. By using electrophoretic mobility shift assays, this study showed that IntI1 binds only to the bottom strand of attC. Upon folding the strand into a hairpin, a few chiral hairpin distortions define both the strand choice and also the appropriate orientation of the highly symmetrical site. Structural recognition also explains the wide sequence variation among attC sites. We have documented the initial cleavage step in recombination in IntI extracts and integrase levels in extracts were evaluated by a new method. Mutagenesis and homology modelling were performed to find amino acid residues in IntI1 that are important for recognition of attC hairpin-DNA. Comparisons were made with other tyrosine family members to explain how integron integrases differ in site-recognition and also in their mechanism of strand exchange.

<p>In Drosophila melanogaster, where males are XY and females are XX, dosage compensation is acheived by approximately two-fold upregulation of transcription of the single male X chromosome. This upregulation is mediated by the dosage compensation complex (DCC), which is assembled in a sequential manner on the male X chromosome and is composed of the two noncoding roX (RNA on the X) RNAs and at least five proteins, including the RNA helicase Maleless (MLE). MLE contains two highly conserved double stranded RNA binding domains (DRBDs) at the N terminus. We investigated the roles of the roX RNAs and MLE helicase through experiments using classical genetic methods to generate and analyze the effects of mutants and mutant transgenes, immunolocalization experiments to study MSL protein and roX RNA to chromosomes. For the first time in vivo, we demonstrate that MLE associates with double stranded RNA in a sequence non-specific manner that is independent of other DCC components. Importantly, we find that the DSRBDs of MLE are essential for dosage compensation but are not required for MLE localization to the male X chromosome. We propose that although the DSRBDs are not essential for ds RNA binding, they may act synergistically with other domains of MLE or other MSLs to associate with RNA in vivo. We propose that a MLE/ roX RNA association involving secondary structure formed by the roX RNAs may be involved in the assembly, stabilization, or function of the DCC.</p> / Dissertation

The study of the DNA has found application beyond our understanding of its cellular function and into a variety of materials assembly and nucleic acid detection systems. The current research investigates double-stranded DNA probes in both a colloidal particle assembly and fluorescent assay format utilizing competitive hybridization events. In both contexts, the affinity of the dsProbes is tuned by the sequence design parameters of duplex length and complementarity. These systems were incubated with nucleic acid targets of interest and, based on the mechanism of competitive hybridization, were responsive to the presence of a high affinity competitive target. In the case of the particle assemblies, incubation with the competitive target resulted in observable disassembly of particle structures. In the case of fluorescently labeled dsProbes, incubation with competitive targets resulted in a quantifiable loss of fluorescence as determined by flow cytometry. Utilizing the fluorescently labeled dsProbe system, the kinetics of competitive hybridization was characterized for nucleic acid targets of varying specificity and strand context. The results indicate promise for the development of the competitive hybridization approach in nucleic acid detection systems providing advantages over current single-stranded probe designs. By utilizing a fluorescently labeled dsProbe approach, it is unnecessary to chemically modify the target of interest to impart a signaling mechanism. Additionally, as the process of competitive hybridization of dsProbes with targets of interest is an affinity driven process, discrimination of targets based on specificity is decoupled from standard measures such as elevated temperature protocols, an important step in translating nucleic acid technologies from the controlled laboratory environment to field applications.

Monitoring viruses circulating in the human population and the environment is critical for protecting public and ecosystem health. The goal of this dissertation was to incorporate a viral metagenomic approach into virus surveillance efforts (both clinical and water quality control programs) to enhance traditional virus detection methods. Clinical surveillance programs are designed to identify and monitor etiological agents that cause disease. However, the ability to identify viruses may be compromised when novel or unsuspected viruses are causing infection since traditional virus detection methods target specific known pathogens. Here we describe the successful application of viral metagenomics in a clinical setting using samples from symptomatic patients collected through the Enterovirus Surveillance (EVS) program in the Netherlands (Appendix A). Despite extensive PCR-based testing, the viruses in a small percentage of these samples (n = 7) remained unidentified for more than 10 years after collection. Viral metagenomics allowed the identification of viruses in all seven samples within a week using minimal sequencing, thus rapidly filling the diagnostic gap. The unexplained samples contained BK polyomavirus, Herpes simplex virus, Newcastle disease virus and the recently discovered Saffold viruses (SAFV) which dominated the unexplained samples (n = 4). This study demonstrated that metagenomic analyses can be added as a routine tool to investigate unidentified viruses in clinical samples in a public-health setting. In addition, metagenomic data gathered for SAFV was used to complete four genotype 3 SAFV (SAFV-3) genomes through primer walking, doubling the number of SAFV-3 full genomic sequences in public databases. In addition to monitoring viruses in symptomatic patients, it is also important to monitor viruses in wastewater (raw and treated) to protect the environment from biological contamination and prevent further spread of pathogens. To gain a comprehensive understanding of viruses that endure wastewater treatment, viral metagenomics was used to survey the total DNA and RNA viral community in reclaimed water (the reusable end-product of wastewater treatment) (Appendix B). Phages (viruses that infect bacteria) dominated the DNA viral community while eukaryotic viruses similar to known plant and insect viruses dominated RNA metagenomic libraries suggesting that highly stable viruses may be disseminated through this alternative water supply. A plant virus, the Pepper mild mottle virus (PMMoV), was identified as a potential indicator of wastewater contamination based on metagenomic data and quantitative PCR assays (Appendix C). The metagenomic analysis also revealed a wealth of novel single-stranded DNA (ssDNA) viruses in reclaimed water. Further investigation of sequences with low-level similarities to known ssDNA viruses led to the completion of ten novel ssDNA genomes from reclaimed water and marine environments (Appendix D). Unique genome architectures and phylogenetic analysis suggest that these ssDNA viruses belong to new viral genera and/or families. To further explore the ecology of the novel ssDNA viruses, a strategy was developed to take metagenomic analysis to the next level by combining expression analysis and immunotechnology (Appendix E). This dissertation made a significant contribution to current microbiological data regarding wastewater by uncovering viruses that endure the wastewater treatment and identifying a new viral bioindicator.

DNA double strand breaks (DSB) are the most toxic of all types of DNA lesions. In Saccharomyces cerevisiae, DNA DSBs are predominantly repaired by the homologous recombination repair (HRR) pathway. The initial step of HRR requires extensive processing of DNA ends from the 5' to 3' direction by specific endo-exonuclease(s) (EE) at the DSB sites, but no endo-exonuclease(s) has yet been conclusively determined for such processing of DSBs. S. cerevisiae TRM2/RNC1 is a candidate endo-exonuclease that was previously implicated for its role in the HRR pathway and was also shown to have methyl transferase activity primarily located at its c-terminus. / In this dissertation, we provided compelling biochemical and genetic evidence that linked TRM2/RNC1 to the DNA end processing role in HRR. Trm2/Rnc1p purified with a small calmodulin binding peptide (CBP) tag displayed single strand (ss) specific endonuclease and double strand (ds) specific 5' to 3' exonuclease activity characteristic of endo-exonucleases involved in HRR. Intriguingly, purified Trm2/Rnc1p deleted for its C-terminal methyl transferase domain retained its nuclease activity but not the methyl transferase activity indicating that the C-terminal part responsible for its methyl transferase function is not required for its nuclease activity. / Our genetic and functional studies with S. cerevisiae trm2/rnc1 single mutants alone or in combination with other DNA DSB repair mutants after treatment with the DNA damaging drug methyl methane sulfonate (MMS) or IR that is believed to produce DSBs, or with specific induction of DNA DSBs at the MAT locus by HO-endonuclease demonstrated an epistatic relationship of TRM2/RNC1 with the major recombination factor RAD52. These studies suggested that TRM2/RNC1 probably acts at an earlier step than RAD52 in the HRR pathway. The genetic evidence also indicated a possible functional redundancy with the bona fide endo-exonuclease EXO1 in the processing of DNA ends at the DSB sites. / In a recent report, the immuno-purified mouse homologue of TRM2/RNC1 exhibited similar enzymatic properties as the endo-exonucleases involved in HRR. A small molecular inhibitor pentamidine specifically inhibited the nuclease activity of the mouse EE and sensitized various cancer cells to DNA damaging agents commonly used in cancer chemotherapy. We specifically suppressed expression of the mouse EE using small interfering RNA (siRNA) that conferred sensitivity of B16F10 melanoma cells to a variety of DNA damaging drugs often used in cancer treatment. This further validated our earlier claim of the endo-exonuclease as a potential therapeutic target in treating cancer.

This thesis is based on nine research publications (I – IX) on structure and reactivity of RNA vis-à-vis DNA. The DNA and RNA are made of flexible pentose sugar units, polyelectrolytic phosphodiester backbone, and heterocyclic nucleobases. DNA stores our genetic code, whereas RNA is involved both in protein biosynthesis and catalysis. Various ligand-binding and recognition properties of DNA/RNA are mediated through inter- and intra-molecular H-bonding and stacking interactions, beside hydration, van der Waal and London dispersion forces. In this work the pH dependant chemical shift, pKa values of 2'-OH group as well as those the nucleobases in different sequence context, alkaline hydrolysis of the internucleotidic phosphodiester bonds and analysis of NOESY footprints along with NMR constrained molecular dynamics simulation were used as tools to explore and understand the physico-chemical behavior of various nucleic acid sequences, and the forces involved in their self-assembly process. Papers I – II showed that the ionization of 2'-OH group is nucleobase-dependant. Paper III showed that the chemical characters of internucleotidic phosphate are non-identical in RNA compared to that of DNA. Papers IV – VI show that variable intramolecular electrostatic interactions between electronically coupled nearest neighbor nucleobases in a ssRNA can modulate their respective pseudoaromatic character, and result in creation of a unique set of aglycons with unique properties depending on propensity and geometry of nearest neighbor interaction. Paper VII showed that the cross-modulation of the pseudoaromatic character of nucleobases by the nearest neighbor is sequence-dependant in nature in oligonucleotides. Paper VIII showed that the purine-rich hexameric ssDNA and ssRNA retain the right-handed helical structure (B-type in ssDNA and A-type in ssRNA) in the single-stranded form even in absence of intermolecular hydrogen bonding. The directionality of stacking geometry however differs in ssDNA compared to ssRNA. In ssDNA the relatively electron-rich imidazole stacks above the electron-deficient pyrimidine in the 5' to 3' direction, in contradistinction, the pyrimidine stacks above the imidazole in the 5' to 3' direction in ssRNA. Paper IX showed that the pKa values of the nucleobases in monomeric nucleotides can be used to show that a RNA-RNA duplex is more stable than a DNA-DNA duplex. The dissection of the relative strength of base-pairing and stacking showed that the relative contribution of former compared to that of the latter in an RNA-RNA over the corresponding DNA-DNA duplexes decreases with the increasing content of A-T/U base pairs in a sequence.