Tesis sobre el tema "SsRNA"

Work presented in this thesis shows how chemical modifications of ssRNAs can increase their nuclease resistance, resulting in potent gene inhibition. The first part of thesis describes how a range of chemical modifications were introduced into the ssRNAs to test their effect on oligonucleotide stability and ability to inhibit gene expression. The ssRNAs were tested in a cancer cell line that stably expresses firefly luciferase. This work demonstrates that DNA nucleotides can be incorporated into ssRNA sequences without loss of potency in the RNAi mechanism. By using additional chemical modifications (2’-OMe and 2’-F) further improvement of ssRNA stability and a significant inhibition of firefly luciferase activity, reaching 91%, was achieved. Moreover, we show that the level of inhibition can be improved when the DNA dinucleotide linked by the (E)-vinylphosphonate is used as a protection of the 5’-end of the ASO (95% KD). The use of ssRNAs rather than dsRNAs is beneficial, since the possibility of off-target effects caused by the remaining sense strand is eliminated and the cost of production of such a medicine is reduced. The second part of this thesis focuses on development of an experimental system that will allow detection of the inhibition of a disease relevant target. Several approaches were used in order to detect KD of the target by the unmodified dsRNAs. First a western blotting technique was employed for detection. Although different ASOs and concentrations were used no KD was observed. More attempts were taken in order to generate target KD, but unfortunately they were unsuccessful so far.

Prior to the commencement of this project, few plant viruses had been identified from Vietnam despite virus-like symptoms being commonly observed on many crops and weeds. In limited surveys in the late 1990's, preliminary evidence was obtained indicating that potyviruses and geminiviruses were causing significant diseases. As a result, this study was aimed at developing generic PCR-based methods for the rapid detection of viruses belonging to viruses in the families Potyviridae and Geminiviridae in plant samples collected from Vietnam, and to characterise the viruses at the molecular level. Novel degenerate PCR primers were developed for the identification of begomoviruses. Using these primers, 17 begomoviruses species infecting seven crop and nine weed species in Vietnam were identified and characterised. Sequence analyses showed that ten of the viruses (six monopartite and four bipartite) were new species. Of the seven previously characterized begomoviruses, five were identified in Vietnam for the first time. Additionally, eight DNA-ß and three nanovirus-like DNA-1 molecules were also found associated with the monopartite viruses. Five of the DNA-β molecules were putatively novel. Two novel bipartite begomoviruses, named Corchorus yellow vein virus (CoYVV) and Corchorus golden mosaic virus (CoGMV), were isolated from jute plants. Analysis of these viruses showed that they were more similar to New World begomoviruses than to viruses from the Old World. This was based on the absence of an AV2 open reading frame, the presence of an N-terminal PWRLMAGT motif in the coat protein and phylogenetic analysis of the DNA A and DNA B nucleotide and deduced amino acid sequences. This is the first known occurrence of Old World viruses bearing features of New World viruses, and their presence in Vietnam suggests the presence of a "New World" virus in the Old World prior to Gondwana separation. Other interesting features relating to begomoviruses identified in Vietnam were; (i) the detection of several recombination events, particularly between the newly identified Tomato yellow leaf curl Vietnam virus (TYLCVNV), and the previously characterised, Tomato leaf curl Vietnam virus (ToLCVV), (ii) the identification of new natural hosts of Sida leaf curl virus (SiLCV), Papaya leaf curl China virus (PaLCuCNV) and Alternanthera yellow vein virus (AlYVV), (iii) the first report of variation in the geminivirus stem-loop nonanucleotide sequence (CoGMV sequence was TATTATTAC rather than TAATATTAC) and (iv) the first report of different stem sequences in the stem-loop structure of two genomic components from a bipartite begomovirus, Kudzu mosaic virus (KuMV). The sequence and phylogenetic analyses of the begomoviruses and begomovirus-associated DNAs identified in this study suggested that South East Asia, and Vietnam in particular, may be a centre of begomovirus diversity. Two pairs of degenerate primers, designed in the CI gene (CIFor/CIRev) and HC-Pro gene (HPFo/HPRev), were developed for the detection of viruses in the genus Potyvirus. Using these primers, three novel potyviruses from Vietnam were detected, namely Telosma mosaic virus (TelMV) infecting telosma (Telosma cordata), Peace lily mosaic virus (PeLMV) infecting peace lily (Spathiphyllum patinii) and Wild tomato mosaic virus (WTMV) infecting wild tomato (Solanum torvum). The fragments amplified by the two sets of primers enabled additional PCR and complete genomic sequencing of these three viruses and a Banana bract mosaic virus (BBrMV) isolate from the Philippines. All four viruses shared genomic features typical of potyviruses. Sequence comparisons and phylogenetic analyses indicated that WTMV was most closely related to Chilli veinal mottle virus (ChiVMV) and Pepper veinal mottle virus (PVMV) while PeLMV, TelMV were related to different extents with members of the BCMV subgroup. The incidence of potyviruses infecting plants in Vietnam was investigated using the potyvirus-specific primers. Fifty two virus isolates from 13 distinct potyvirus species infecting a broad range of crops were identified in Vietnam by PCR and sequence analysis of the 3' region of the genome. The viruses were Bean common mosaic virus (BCMV), Potato virus Y (PVY), Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Chilli veinal mottle virus (ChiVMV), Zucchini yellow mosaic virus (ZYMV), Leek yellow stripe virus (LYMV), Shallot yellow stripe virus (SYSV), Onion yellow dwarf virus (OYDV), Turnip mosaic virus (TuMV), Dasheen mosaic virus (DsMV), Sweet potato feathery mottle virus (SPFMV) and a novel potyvirus infecting chilli, which was tentatively named Chilli ringspot virus (ChiRSV). With the exception of BCMV and PVY, this is first report of these viruses in Vietnam. Further, rabbit bell (Crotalaria anagyroides) and typhonia (Typhonium trilobatum) were identified as new natural hosts of the Peanut stunt virus (PStV) strain of BCMV and of DsMV, respectively. Sequence and phylogenetic analyses, based on the nucleotide sequence of the entire CP-coding region of all 52 virus isolates, revealed considerable variability in BCMV, SCMV, PVY, ZYMV and DsMV. The phylogenetic analyses also suggested the possible presence of ancestral groups of BCMV, SCMV and ZYMV in Vietnam.

Prior to the commencement of this project, few plant viruses had been identified from Vietnam despite virus-like symptoms being commonly observed on many crops and weeds. In limited surveys in the late 1990's, preliminary evidence was obtained indicating that potyviruses and geminiviruses were causing significant diseases. As a result, this study was aimed at developing generic PCR-based methods for the rapid detection of viruses belonging to viruses in the families Potyviridae and Geminiviridae in plant samples collected from Vietnam, and to characterise the viruses at the molecular level. Novel degenerate PCR primers were developed for the identification of begomoviruses. Using these primers, 17 begomoviruses species infecting seven crop and nine weed species in Vietnam were identified and characterised. Sequence analyses showed that ten of the viruses (six monopartite and four bipartite) were new species. Of the seven previously characterized begomoviruses, five were identified in Vietnam for the first time. Additionally, eight DNA-ß and three nanovirus-like DNA-1 molecules were also found associated with the monopartite viruses. Five of the DNA-β molecules were putatively novel. Two novel bipartite begomoviruses, named Corchorus yellow vein virus (CoYVV) and Corchorus golden mosaic virus (CoGMV), were isolated from jute plants. Analysis of these viruses showed that they were more similar to New World begomoviruses than to viruses from the Old World. This was based on the absence of an AV2 open reading frame, the presence of an N-terminal PWRLMAGT motif in the coat protein and phylogenetic analysis of the DNA A and DNA B nucleotide and deduced amino acid sequences. This is the first known occurrence of Old World viruses bearing features of New World viruses, and their presence in Vietnam suggests the presence of a "New World" virus in the Old World prior to Gondwana separation. Other interesting features relating to begomoviruses identified in Vietnam were; (i) the detection of several recombination events, particularly between the newly identified Tomato yellow leaf curl Vietnam virus (TYLCVNV), and the previously characterised, Tomato leaf curl Vietnam virus (ToLCVV), (ii) the identification of new natural hosts of Sida leaf curl virus (SiLCV), Papaya leaf curl China virus (PaLCuCNV) and Alternanthera yellow vein virus (AlYVV), (iii) the first report of variation in the geminivirus stem-loop nonanucleotide sequence (CoGMV sequence was TATTATTAC rather than TAATATTAC) and (iv) the first report of different stem sequences in the stem-loop structure of two genomic components from a bipartite begomovirus, Kudzu mosaic virus (KuMV). The sequence and phylogenetic analyses of the begomoviruses and begomovirus-associated DNAs identified in this study suggested that South East Asia, and Vietnam in particular, may be a centre of begomovirus diversity. Two pairs of degenerate primers, designed in the CI gene (CIFor/CIRev) and HC-Pro gene (HPFo/HPRev), were developed for the detection of viruses in the genus Potyvirus. Using these primers, three novel potyviruses from Vietnam were detected, namely Telosma mosaic virus (TelMV) infecting telosma (Telosma cordata), Peace lily mosaic virus (PeLMV) infecting peace lily (Spathiphyllum patinii) and Wild tomato mosaic virus (WTMV) infecting wild tomato (Solanum torvum). The fragments amplified by the two sets of primers enabled additional PCR and complete genomic sequencing of these three viruses and a Banana bract mosaic virus (BBrMV) isolate from the Philippines. All four viruses shared genomic features typical of potyviruses. Sequence comparisons and phylogenetic analyses indicated that WTMV was most closely related to Chilli veinal mottle virus (ChiVMV) and Pepper veinal mottle virus (PVMV) while PeLMV, TelMV were related to different extents with members of the BCMV subgroup. The incidence of potyviruses infecting plants in Vietnam was investigated using the potyvirus-specific primers. Fifty two virus isolates from 13 distinct potyvirus species infecting a broad range of crops were identified in Vietnam by PCR and sequence analysis of the 3' region of the genome. The viruses were Bean common mosaic virus (BCMV), Potato virus Y (PVY), Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Chilli veinal mottle virus (ChiVMV), Zucchini yellow mosaic virus (ZYMV), Leek yellow stripe virus (LYMV), Shallot yellow stripe virus (SYSV), Onion yellow dwarf virus (OYDV), Turnip mosaic virus (TuMV), Dasheen mosaic virus (DsMV), Sweet potato feathery mottle virus (SPFMV) and a novel potyvirus infecting chilli, which was tentatively named Chilli ringspot virus (ChiRSV). With the exception of BCMV and PVY, this is first report of these viruses in Vietnam. Further, rabbit bell (Crotalaria anagyroides) and typhonia (Typhonium trilobatum) were identified as new natural hosts of the Peanut stunt virus (PStV) strain of BCMV and of DsMV, respectively. Sequence and phylogenetic analyses, based on the nucleotide sequence of the entire CP-coding region of all 52 virus isolates, revealed considerable variability in BCMV, SCMV, PVY, ZYMV and DsMV. The phylogenetic analyses also suggested the possible presence of ancestral groups of BCMV, SCMV and ZYMV in Vietnam.

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Associação do papaya meleira virus e de um segundo vírus de ssRNA à meleira do mamoeiro.

The ecological significance of mycoviruses is becoming increasingly recognised, not just for their potential as biocontrol agents but also as driving forces in the evolution and diversification of fungi. Therefore, it is important to understand how mycoviruses and fungi interact on the molecular and biochemical level. To this end the interaction between Botrytis cinerea and the mycoviruses Botrytis virus F and Botrytis virus X was studied. Relative and absolute real time PCR protocols were developed for monitoring the titres of BVX and BVF during transfection studies to monitor changes in virus titre in relation to phenotypic and metabolic changes in the fungal host. Phenotypic changes included severe phenotypical alterations, which were associated with extreme up regulation of carbohydrate, amino acid and lipid metabolism, and induction of stress responses (vacuolisation/cell lysis, increased pigmentation). To study the location and distribution of BVX in infected Botrytis the BVX coat protein was recombinantly expressed in E. coli, BVX specific polyclonal antibodies produced, and protocols developed for the serological detection and visualisation of BVX. Immuno-fluorescence microscopy was used to studying the distribution of BVX within growing Botrytis cultures indicated that the virus is present in aggregates located attached to the cell membrane, the septum, in spores, and in hyphal tips. A combination of light and electron microscopy showed that BVX is often closely associated with cell walls, suggesting that the virus may be moving across the cell wall by altering cell wall composition. If this is shown to be the case then it provides an alternative method to transmission via hyphal anastomosis, which is currently considered to be the only method of horizontal transfer.

The (+)strand RNA viruses include a very large group of viruses that cause epidemic diseases in humans, including dengue fever and gastroenteritis. The human (+)RNA viruses include Flaviviruses (FV) and Norovirus (NV). Both encode for proteins essential for viral replication, such as the RNA dependent RNA polymerase (RdRp). Since human cells lack RdRp, it appears as one of the most promising targets for antivirals development. I worked on the identification of new non-nucleotide inhibitors against FV and NV, using RdRp as the main target. In this context, suramin and NF023 have been identified in my lab as NV RdRp inhibitors that, however both are hampered in their application by pharmacokinetics problems. To overcome such problems, I analyzed the potential inhibitory role of Naf2, a fragment derived from these two molecules. Although Naf2 showed a low inhibitory activity, the crystal structures of NV RdRp/Naf2 complex revealed a new binding site. To further map this new site, I tested a Naf2 related molecule, PPNDS. The crystal structures of the RdRp/PPNDS complex revealed interesting features about the new binding site. I also focused on structurally related molecules synthesized following structure-driven information. NV RdRp crystal structures in complex with one of these compounds (Cpd6) were analyzed, providing new knowledge on the interactions between a small fragment and NV RdRps, establishing a platform for structure-guided drug optimization. In parallel to the NV work, I screened in silico a library of compounds against FV RdRp. One of the best compounds identified (HeE1-2Tyr) was able to inhibit the RdRp activity and several FVs in cell-based assays. Although the crystallographic analyses don't reveal clear enough electron density for the inhibitor, indirect evidence suggests that HeE1-2Tyr interferes with the RdRp priming loop that appears disordered.

Cancers that are deficient in BRCA1 or BRCA2 are thought to be hypersensitive to genotoxic agents because they cannot prevent or repair DNA double strand breaks, but observations in patients suggest this dogma may no longer agree with experiment. Here, we propose that single stranded DNA underlies the hypersensitivity of BRCA deficient cancers, and that defects in double strand break repair and prevention do not. Specifically, in BRCA deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. In addition, we observed gaps could be suppressed by either restored fork restraint or by gap filling, both of which conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored double strand break repair and prevention did not confer therapy resistance when gaps were present. Critically, double strand breaks were not detected after therapy when apoptosis was inhibited, supporting a framework in which double strand breaks are not directly induced by genotoxic agents, but instead are created by cell death nucleases and are not fundamental to genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose are fundamental to the mechanism-of-action of genotoxic chemotherapy.

ABSTRACT Regulation of gene transcription by structural interconversions of genomic DNA is an emerging biochemical and genetic paradigm that adds to the already diverse repertoire of eukaryotic gene regulatory mechanisms. The appearance of paranemic structures coincident with changes in gene activity, as well as participation of transcription factors that recognize and bind single-stranded DNA at numerous gene promoters in vivo illustrates the authenticity of this concept and its importance in cellular homeostasis. Despite its acceptance, this concept has been minimally described at the biochemical and biophysical levels, as the means by which sequence-specific single-stranded DNAbinding proteins exert transcriptional influence in double-stranded genomes remains largely undefined. Pur is a sequence-specific single-stranded DNA/RNA-binding protein that acts as a repressor of smooth muscle -actin (SM A) gene transcription, and mRNA translation. SM A is an important cytoskeletal protein that contributes contractile, antimigratory, and nonproliferative functions in smooth muscle. In concert with Pur protein family member Pur , and Y-box protein MSY1, Pur enacts repression of SM A gene expression by interacting with a cryptic cis-regulatory element in the 5’ region of the SM A promoter that has been shown to transiently adopt single-stranded conformations in vivo, and to confer transcriptional activation when trans-activator occupied while in a doublestranded conformation. Downregulation of SM A gene expression has been identified to be a contributing factor to cardiovascular disease progression; therefore a thorough understanding of SM A repression mechanisms is critical for clinical management of these conditions. Although highly homologous at the primary sequence level, Pur and Pur display significant conserved regions of sequence divergence that suggest these paralogs exert distinct cellular functions in various vertebrate classes. A goal of the studies presented herein was to delineate exhibited functional differences with respect to SM A repression in pertinent mouse cell lines. Loss-of-function and chromatin immunoprecipitation studies verified that Pur differs from Pur in that Pur is the dominant Pur protein repressor of SM A expression in embryonic fibroblasts and vascular smooth muscle cells, although by different, cell type-specific mechanisms. Biophysical assessment of Pur single-stranded DNA binding properties showed that despite the ability of Pur to self-dimerize in the absence of nucleic acid, Pur binds to the cryptic SM A enhancer by a sequential and cooperative mechanism, with remarkable affinity and a terminal stoichiometry of 2 to 1. Footprinting and in vitro binding site characterization confirms two Pur binding sites exist within this element and display slight degeneracy from a proposed Pur protein-binding consensus motif. These findings delineate binding mechanisms adopted by Pur and provide a means to identify putative Pur binding sites throughout the genome.

Since there is an urgent need for development of vaccines and antiviral agents to combat influenza pandemics, this study aimed to develop influenza virus-like particles (VLPs) and aptamers targeting the virus particles as vaccine and antiviral agent candidates. Influenza VLPs containing three structural proteins of hemagglutinin (HA), neuraminidase (NA) and matrix 1 (M1) derived from influenza A/Hong Kong/01/2009 (H1N1) virus (HK/01) were constructed using a Bac-to-Bac baculovirus expression system. The expressed VLPs were purified by sucrose density gradient ultracentrifugation and characterized by Western blotting analysis and transmission electron microscopy. The immune responses and protective efficacy induced by VLPs were compared with those elicited by the clinically used Panenza vaccine in BALB/c mouse model. The results showed that two-dose vaccination with both VLP and the Panenza vaccine could confer complete protection. Single-dose vaccination with VLP could also provide 100% protection against lethal virus challenge, whereas single dose of an equal amount (based on HA content) of the Panenza vaccination just provided incomplete protection (67% survival rate) against the lethal virus challenge. Compared to the Panenza vaccination, the VLP vaccination could induce higher and broader antibody responses and higher viral specific T help (Th) cell and cytotoxic T lymphocyte (CTL) responses. Notably, a novel finding in this study is that the VLP vaccination could induce antibodies to inhibit virus release from infected MDCK cells, although the underlined mechanism needed to be further studied. These results indicated that influenza VLP might be a more effective and safe vaccine candidate which could be developed into an alternative vaccine for the control of epidemic and pandemic influenza in the future. To develop aptamers as antiviral agents against influenza, I sought to use influenza VLPs as target for ssDNA aptamer selection. After 11 rounds of selection using the systemic evolution of ligandsby exponential enrichment (SELEX),the recovered DNA molecules were PCR-amplified, gel purified and cloned into pCR-Blunt II TOPO vector for sequencing. The sequencing results showed that one aptamer Va-1 was markedly enriched, which was accounted for 59% (13/22) of the selected aptamers. Compared to the other non-enriched aptamers, the enriched aptamer Va-1 showed the highest binding affinity to the UV inactivated influenza HK/01 virus. It was also shown that the aptamer Va-1 specifically bound to the HK/01 stain while it could not bind other respiratory viruses even the PR8 strain within the H1N1 subtype. It was further demonstrated that the aptamer Va-1 could only bind to NA protein in a dose-dependent manner but not bind to HA and M1 proteins. Unfortunately, the selected aptamer did not show any antiviral effects. However, it may be potentially developed into a diagnostic and analytic agent because its binding activity was comparable with that of the commercial anti-NA antibody. In conclusion, the influenza VLPs may be a promising vaccine candidate for the control of influenza virus infection and the selected aptamer may be potentially developed into an alternative tool for influenza virus detection.
published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy

A library of ssDNA aptamers has been screened and a number of molecules which show high binding affinity to 14-3-3 proteins have been isolated. The gene encoding mammalian 14-3-3 γ modified by (his)₆ tagging, was overexpressed in E. coli and purified to homogeneity. The recombinant 14-3-3 γ was characterised using a series of techniques ensuring that the expressed protein had similar physical and immunological characteristics to that of wild type 14-3-3 γ. An ssDNA aptamer library was synthesised and aptamers selected following an adapted SELEX (systematic evolution of ligands by exponential enrichment) protocol. After 16 selection rounds, aptamers contained within the enriched pool were cloned and sequenced for further analysis. A sample of twenty of the sequenced aptamers were synthesised and biotinylated and their interaction with (his)₆14-3-3γ was analysed by surface plasmon resonance and by enzyme linked oligonucleotide assay (ELONA). The two aptamers showing the greatest association to (his)₆14-3-3γ were further analysed against all 14-3-3 isoforms using ELONA and by dot-blot analysis. The high affinity aptamer, S16-8, was used to affinity purify recombinant 14-3-3 spiked into ovine serum albumin and wild type 14-3-3 in mouse brain homogenate and to detect and concentrate 14-3-3 protein in a scrapie positive cerebrospinal fluid (CSF) sample. The results of this work suggest that this aptamer could be used as a probe to detect isoforms of wild-type 14-3-3 proteins from a range of biological fluids.

Die vorliegenden Ergebnisse belegen eine essentielle Rolle der Beseitigung von intrazellulären DNA-Metaboliten aus der DNA-Reparatur für die Aufrechterhaltung von Immuntoleranz. So führt eine unangemessene Akkumulation körpereigener DNA im Zytosol, über die Erkennung durch Nukleinsäuresensoren, zu einer Aktivierung des angeborenen Immunsystems. Dies weist auf einen bisher unbekannten Zusammenhang zwischen DNA-Schäden, der DNA-Schadensantwort und einer Typ 1-IFN-vermittelten Aktivierung des angeborenen Immunsystems bei der Pathogenese von Autoimmunität hin.

Polychlorinated biphenyls (PCBs) are persistent environmental chemicals. Mono-hydroxylated polychlorinated biphenyls (OH-PCBs) are PCB metabolites found commonly in human blood, environmental water and sediment samples. Detection of small amounts of PCBs and their OH-PCB metabolites in biological matrices from epidemiological and laboratory studies remains a challenge. The application of aptamers is studied as a means to identify and quantify PCBs and OH-PCBs. Aptamers are single stranded short oligonucleotides that arrange into unique shape of three-dimensional structures when binding to their target. Like antibodies they have high affinity and specificity for their specific target. The hypothesis is that aptamers can identify PCBs and PCB metabolites in environmental and biological samples. To test this hypothesis, three different OH-PCBs, 4’-OH-PCB3, 4-OH-PCB72 and 2-OH-PCB106 along with 4-OH-biphenyl as a control, were covalently attached to beads with carboxylic acid groups on their surface. Several methods were explored to characterize covalent binding of OH-PCBs to the beads: FTIR-spectroscopy, Dynamic Light Scattering (DLS) and Zeta-Potential (ZP) measurements. The beads were then used in in vitro assays to test binding of two different aptamers specific to OH-PCBs. In this study, these aptamers were tested for the ability to distinguish structurally different OH-PCB congeners and other environmental pollutants. In future studies, aptamers can be selected for a PCB metabolite of interest, 4’-OH-PCB3, via a modified form of Systemic Evolution of Ligands by Exponential Enrichment (SELEX). Single stranded DNA (ssDNA) aptamers generated will be applied as a biosensor for the detection and quantification of traces of 4’-OH-PCB3.

Beak and feather disease virus (BFDV), a non-enveloped, icosahedral virus with a circular single stranded DNA (ssDNA) genome, is the causative agent behind psittacine beak and feather disease (PBFD), an often fatal disease affecting parrots. Symptoms include feathering abnormalities, loss of feathers, and occasionally beak and claw deformities. BFDV-induced immunosuppression results in an increased susceptibility to secondary microbial infections, which is often the cause of death in infected parrots. There is no cure, no effective treatment, and no protective vaccine for BFDV. The international trade in exotic parrots has facilitated the spread of BFDV, so that it now has a global presence. Given that over a quarter of the currently recognised 356 psittacine species are considered to be at risk of extinction in the wild, the worldwide presence of BFDV, coupled with its extreme environmental stability, poses serious concerns for the future of some of the worlds most endangered parrots. That genetic diversity exists among BFDV isolates has been established, yet in the 14 years since the genome was fully sequenced, very few full-length BFDV genome sequences have been deposited in GenBank, despite the technology to rapidly isolate and amplify entire circular ssDNA genomes being readily available. Most studies have sequenced just a portion of the genome, usually one of the open reading frames (ORFs) encoding the major viral proteins, to investigate phylogenetic relationships between isolates. However the two major BFDV ORFs, encoding the replication associated protein (Rep) and the capsid protein (CP), have been shown to evolve at different rates, with the functional Rep being generally more conserved while CP is more variable. When also considering the fact that ssDNA viruses are notoriously recombinant, it becomes clear that an analysis based on a portion of the genome is unlikely to accurately establish evolutionary relationships. Therefore the focus of the studies described in this thesis was on isolation and amplification of full-length BFDV genomes from avian blood and feather samples that first tested positive to a PCR-based BFDV screening method. Samples were collected by appropriately trained people in New Zealand, New Caledonia, and Poland, before being sent to the University of Canterbury for molecular and bioinformatic analysis. The sequences of the BFDV genomes from each region were compared to each other and to all other full BFDV genome sequences publically available in GenBank, to compare the genetic diversity among these isolates. Recombination analyses were also performed, to assess how recombination is impacting on the evolution of BFDV. New strains of BFDV and new subtypes of existing BFDV strains were discovered, indicating that the global genetic diversity may be greater than previously thought. Many strains also proved to be recombinants, in particular those from Poland. Europe has had a long history with importing and breeding exotic parrots, and the high degree of recombination among the Polish BFDV isolates coupled with the number of previously unsampled strains is an example of how maintaining populations of multiple species in captivity enables evolution through recombination, and emergence of novel viral strains. Full genome analyses can also enable tracking the source of an infection. A total of 78 full genome sequences from 487 samples tested were deposited into GenBank as a direct result of the work undertaken as part of this thesis, thereby adding to the existing knowledge base regarding BFDV. With continued global sampling and full genome analysis it may one day be possible to trace the history of BFDV to its original emergence.

DNA double strand breaks can occur from various sources and the timely and accurate repair of these breaks is critical to maintain the genomic integrity of the cell. Break-induced replication (BIR) is a repair pathway that has been shown to repair DSBs where only one end of the break can locate homology, similar to ends seen at collapsed replication forks or eroded telomeres. BIR progresses by an unusual bubble-like intermediate. The asynchrony between the synthesis of leading and lagging strand synthesis during BIR leads to the accumulation of long single-stranded DNA (ssDNA) behind the bubble. This mechanism leads to the conservative inheritance of newly synthesized DNA. BIR repair can lead to increased mutations, loss of heterozygosity and gross chromosomal rearrangements. In this thesis I investigated the deleterious effects of the ssDNA formed during BIR. Using yeast, Saccharomyces cerevisiae, I showed that the regulation of Rad51 that binds ssDNA during BIR is important to prevent the accumulation of toxic joint intermediates. Here, I demonstrate that a known Rad51-interacting protein, Srs2, plays two key roles in counteracting the accumulation of lethal recombination intermediates. First, Srs2 dislodges Rad51 from long ssDNA formed during DSB repair and therefore prevents promiscuous strand invasions that generate lethal joint molecules. Second, Srs2 helicase dismantles toxic intermediates that have already formed. We also demonstrate that the structure-specific endonucleases, Mus81 and Yen1, can resolve toxic joint molecules formed in the absence of Srs2, thus promoting cell survival. The other goal of this thesis was to study the effects of ssDNA accumulated during BIR in the formation of base-substitution mutagenesis. I test whether this ssDNA is mutagenic by analyzing BIR with and without the presence of DNA damaging agents, including methyl methanesulfonate (MMS) and APOBEC3A. I observed a hypermutagenic effect of BIR with respect to base- substitutions in both cases. Importantly, BIR synergizes with ssDNA damaging agents to produce mutation clusters similar to those previously observed in cancer. I also report the critical role translesion polymerase Polζ plays in the formation of base-substitutions resulting from BIR. Finally, I have discovered a completely novel, UNG1-dependent mechanism of supposed error-free bypasses of APOBEC-induced DNA lesions during BIR that promotes chromosomal rearrangements.

We analyze the cross-border effect of tax cuts on R&D activity in the context of profit shifting. A tax cut in one location of a multinational enterprise reduces the user cost of capital for the whole group if profit shifting is possible and exerts a positive cross-border effect on R&D output. Using micro-level data, we find an increase of patent output of 15% upon the implementation of a foreign tax cut for firms with cross-border links. In addition, we find that foreign tax cuts prohibiting profit shifting generate a negative cross-border effect on average patent quality.
Series: WU International Taxation Research Paper Series

Politicians and tax practitioners often claim that tax uncertainty negatively affects investment. In many countries, firms can request fee-based Advance Tax Rulings (ATRs) to mitigate tax uncertainty. We analyze theoretically the circumstances under which investors request ATRs, how tax authorities should price them and how they can affect investment. We assume that tax authorities integrate investors' reasoning into their decisions. We find that it is often optimal for tax authorities to charge prohibitively high fees to discourage firms from requesting an ATR. However, we find that revenue-maximizing tax authorities offer ATRs if the ruling enables them either to significantly reduce their tax audit costs or to increase the probability of detecting ambiguous tax issues. Under certain circumstances, ATRs may effectively foster investment and potentially benefit both the tax authorities and taxpayers. Our results provide new explanations for why taxpayers that face high levels of tax uncertainty often do not request ATRs, even when the fee is rather low. Our results also hold when the tax authority maximizes social wealth instead of its revenues. Regulatory changes in ATR requirements might serve as a natural quasi-experiment for an empirical study of our predictions regarding investment decisions. (authors' abstract)
Series: WU International Taxation Research Paper Series

Recent metagenomic studies have shown that there is a higher diversity of ssDNA viruses in the environment than previously thought. While some viral families are well characterised, novel ssDNA isolates discovered with sequence-independent molecular techniques are often too divergent to fit within the currently established viral taxonomy. Several factors have contributed to the gap in knowledge, including: the (previously) high cost of sequencing, the disproportionate amount of research that occurs after a threat is identified, and the use of sequence-based molecular techniques to isolate viral sequences. Recent studies have begun to explore viral diversity in the environment, however, most of these studies have occurred outside New Zealand. Several benefits would come from uncovering the true ssDNA viral diversity and global distribution including improving the resolution of the current taxonomic structure for identifying unknown isolates and inferring possible virus-host relationships, and providing baseline data for the development of disease prevention and monitoring strategies. Studies specific to the New Zealand environment are essential. With its geographical isolation and Gondwana ancestry, New Zealand will possess a unique viral sequence space. Studies on local viral diversity and the spread of ssDNA viruses are going to be most relevant if they are conducted within the established ecosystems in New Zealand. In this dissertation, a novel protocol was developed for exploring viral diversity in the New Zealand environment using basic molecular techniques and animal faecal samples. Design considerations included: identifying highly novel small circular viral sequences with DNA genomes without the use of specific primers, inflicting as little environmental impact as possible, and keeping the cost low. The faecal sampling approach does not require animal handling and therefore incorporates the use of viral reservoirs while remaining non-invasive. The molecular techniques in this protocol used non-specific rolling circle amplification (RCA) followed by restriction enzyme (RE) digests, cloning, and sequencing of the cloned genomes via sanger sequencing. This inexpensive exploratory method provided preliminary sequence information from which primers were designed for recovery of full viral genomes. The success of this protocol was demonstrated by the recovery and molecular characterisation of a novel ssDNA virus isolate from a pig faecal sample, which was tentatively named porcine stool-associated circular virus (PoSCV). This protocol was then applied to sample viruses in the faecal matter from variety of domesticated, wild, and farmed animals in New Zealand. The faecal samples were collected from the North and South Island of New Zealand as well as South East Island of the Chatham Islands (Rangatira). Several putative gemycircularviral isolates (novel viruses with similarities to geminiviruses and the recently discovered ssDNA virus infecting Sclerotinia sclerotiorum) were identified in the sequencing results based on BLASTx similarities to viral sequences available in public databases (GenBank). The full genomes of these isolates were recovered and characterised. Identification was based on genome organization, phylogenetic analysis of the replication associated protein (Rep), and full genome nucleotide pairwise identities. Fourteen novel ssDNA virus sequences relating to gemycircularviruses were discovered, of which ten were representative of new species (FaSCV-1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) and three were identified as strains of the same species (FasGCV-1). Two additional isolates were discovered to be distantly related to these viruses: Ostrich faecal associated ssDNA virus (OfaV) and Rabbit faecal associated ssDNA virus (RfaV). Additionally, this protocol was used to recover novel ssDNA viruses from the nesting material of a dead Yellow-crowned Parakeet chick found in the Poulter Valley in the South Island of New Zealand. The nesting material likely contained faecal matter and thus represented another approach strategy for exploring ssDNA viruses in the environment. Two novel ssDNA isolates were discovered and molecularly characterised: Cyanoramphus nest-associated circular X virus (CynNCXV), and Cyanoramphus nest-associated circular K virus CynNCKV.

Defects in BRCA1 and BRCA2 tumor suppressors predispose one to breast and ovarian cancer. The current treatment for BRCA-deficient cancers is mastectomy. Because both copies of the tumor suppressor need to be defective for cancer to occur, identifying cellular mechanisms that specifically target BRCA-deficient cells is of paramount importance. Luckily, recent experiments have shown that depletion of a protein named RAD52 in BRCA1 or BRCA2 cancer cells causes them to die. Therefore, we can use small molecules to stop the RAD52 protein from functioning. We need, however, to know which of the RAD52 activities to inhibit and how. One function of RAD52 that likely underlies all cellular activities is its ability to bind single-stranded DNA (ssDNA). To identify if small molecules could inhibit the RAD52-ssDNA complex, I screened a small library of compounds and found 13 potential inhibitors. We validated that these small molecules bind to RAD52 and inhibit RAD52 DNA binding and annealing activities. The identification of these small molecules is important because we can use them to dissect the function of RAD52 in normal and malignant cells, which to date remains elusive. In an attempt to further advance our understanding of RAD52 function and regulation we are also investigating how a novel binding partner, DSS1, interacts with RAD52 and modulates its activities. My data show that this protein enhances the way RAD52 finds separate complementary DNA templates and anneals them to make a double-stranded product. At least in part, these studies have identified some residues likely involved in the binding site of DSS1 on RAD52. In aggregate, the outcome of the two projects deepens our understanding of the complex and interconnected cellular pathways that support the integrity of genomes.

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Vírus que possuem genoma constituído por DNA fita simples (ssDNA) estão amplamente distribuídos na natureza. As constantes descobertas aliadas à grande diversidade genética desses vírus têm ampliado a compreensão sobre a trajetória evolutiva desse grupo. No Brasil, os vírus DNA fita simples circular causam grandes perdas nas culturas de importância agrícola. Um dos fatores que contribuiu fortemente para o desastre econômico é a transmissão de vírus, onde o maior hospedeiro deles são as plantas não cultivadas. Desde então, diversos trabalhos têm sido publicados tratando de assuntos como diversidade, caracterização, determinação da gama de hospedeiros, interações entre planta e patógeno assim como patógeno e vetor, evolução, predominância, epidemiologia, dentre outros. O presente trabalho teve como objetivo isolar, identificar e caracteriza um novo vírus de ssDNA associado a Melão de São Caetano (Momordica charantia), uma cucurbitácea não cultivada e potencialmente invasiva em cultivos agrícolas no Brasil. A amostra foi coletada em Coimbra, MG- Brasil, com suspeita de ter um vírus da família Geminiviridae por apresentar sintomas de mosaico amarelo. O DNA vegetal foi extraído e analisado através da técnica de amplificação por círculo rolante, os produtos da clivagem com enzimas de restrição foram clonados e completamente sequênciados. A análise das sequências indicou que o vírus tinha um tamanho de 2195 pares de bases e uma organização genômica que apresentava três proteínas: uma codificando o capsídeo do vírus (CP) e duas relac ionada com a replicação (Rep) separadas por um íntron, que a ser removido por inspeção manual codifica a proteína completa. Essa organização em conjunto com as análises do genoma completo mostraram uma identidade com vários isolados do gênero Gemycircularvirus, sendo proposto para esse novo vírus o nome de Momordica charantia associated circular DNA virus (MCasCV). Interessantemente, MCasCV tem uma característica única dentro dos novos ssDNA: um nonanucleotídeo (5'- TAATGTTAT-3') similar ao Hypericum japonicum associated circular DNA virus (HJasCV), com uma formação de um grampo ou “stem- loop” atípico pela ligação de três pares de base encontradas na estrutura. Com o objetivo de caracterizar a biologia do vírus MCasCV foram infectadas plantas de M. charantia e o fungo Sclerotinia sclerotiorum. Para isso, plantas foram bombardeadas com partículas do clone infeccioso de MCasCV e se observou após 21 dias que não apresentaram sintomas visíveis. A confirmação da presença viral foi realizada pelas técnicas de RCA e PCR obtendo um resultado negativo. Para inoculação do vírus em Sclerotinia sclerotiorum, foram transfectados protoplastos desse fungo junto ao clone infeccioso, após o crescimento foi realizada uma extração de DNA total, seguida da confirmação por PCR e RCA, que ao igual da planta os resultados foram negativos. Fatos que podem explicar esse resultado é a ocorrência de falhas no processo de transfecção, ou a não capacidade do vírus de replicar no interior do fungo utilizado. Novas análises de infectividade d everão ser realizadas para a confirmação do hospedeiro do vírus MCasCV.
Viruses that have genomes composed of single-stranded DNA (ssDNA) have been widespread in nature. Continuous discoveries combined with the high genetic diversity of these viruses have leaded our understanding of the historical evolution of this group. In Brazil, the circular single-stranded DNA viruses have caused enormous agricultural losses in staple crops. One factor that strongly contributed to the financial disaster is the transmission of the virus, where the most of the hosts are non-cultivated plants. Since then, several studies have been published, addressing issues such as diversity, characterization, determining the host range and inte ractions between plant and pathogen as well as pathogen and vector, evolution, prevalence, epidemiology, among others. This study aimed to isolate, identify and features a new virus ssDNA associated with Melon de São Caetano (Momordica charantia), one cucurbit uncultivated and potentially invasive in agricultural crops in Brazil. The sample was collected in Coimbra, MG, Brazil, suspected of having a virus Geminiviridae family for symptoms of yellow mosaic. The plant DNA was extracted and analyzed by the tec hnique of rolling circle amplification, the products of cleavage with restriction enzymes have been cloned and completely sequenced. Sequence analysis indicated that the virus had a size of 2195 base pairs and a genomic organization that showed three prote ins: one encoding the virus capsid (CP) and two related replication (Rep) separated by an intron, then it was removed by manual inspection to encoding the complete protein. This arrangement in conjunction with the analysis of complete genome showed an identity with several strains of genus Gemycircularvirus, it has been proposed to name this new virus from Momordica charantia associated circular DNA virus (MCasCV). Interestingly, MCasCV has a unique feature within the new ssDNA: A nonanucleotídeo (TAATGTTAT 5'-3 ') similar to the circular DNA Hypericum japonicum associated virus (HJasCV), with a formation of a staple or "stem- loop" atypical for binding three base pairs found in the structure. With the objective of characterizing the biology of the virus MCasCV M. charantia plants were infected and Sclerotinia sclerotiorum. For this, plants were bombarded with particles of the infectious clone MCasCV and were observed during 21 days, after that interval of time no showed visible symptoms. Confirmation of viral presence was made by RCA and PCR getting a negative result. For virus inoculation of Sclerotinia sclerotiorum, this fungus protoplasts were transfected with the infectious clone, after growing a total DNA extraction was performed, followed by PCR and confirmed by RCA, again the results were negative. Facts that can explain this result is the occurrence of failures in the transfection process, or no ability of the virus to replicate inside the fungus used. New analysis of infectivity should be performed to confirm the MCasCV in the virus host.

The introduction of next-generation sequencing (NGS) technologies has dramatically changed the field of virology, with many significant discoveries of novel circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses. Traditionally, most research into CRESS DNA viruses has often focused on investigating plant and animal pathogens that are of significant economic importance. This research has led to the discovery and establishment of three different CRESS DNA families including Geminiviridae, Nanoviridae and Circoviridae, which infect eukaryotes. CRESS DNA viruses can have single or multicomponent genomes, with the latter requiring all components for infection. CRESS DNA viruses have circular single-stranded DNA (ssDNA) genomes with at least one protein encoding a Rep which is responsible for viral replication. It has been shown that CRESS DNA viruses are able to evolve rapidly with nucleotide substitution rates that are similar to those observed in RNA viruses. The Rep gene has conserved regions known as motifs which are often used to determine relatedness between CRESS DNA virus. NGS has expanded our knowledge on the diversity of novel CRESS DNA viruses. Viral genomes are now routinely recovered from different sample types without any prior knowledge of the viral sequence. This has led to the development of the field of viral ecology. This field places an emphasis on viruses being one of the most abundant organisms on earth, and are therefore likely to play a major role in ecosystems. Environmental metagenomic studies have isolated CRESS DNA viruses from sea water, freshwater, faecal matter from various animals, soil, the atmosphere, sediments and sewage; dramatically increasing the known CRESS DNA viral genomes in the public domain. These studies are shedding light on the distribution of CRESS DNA viruses, as well as providing baseline data for future studies to examine virus-host interactions, community structure and ultimately viral evolution. Vector enable metagenomics (VEM) is another novel approach utilising NGS techniques for discovering CRESS DNA viruses. As many plant-infecting CRESS DNA viruses such as geminiviruses and nanoviruses are vectored by insects, this approach exploits this mechanism by using insect vectors as a surveillance tool to monitor and survey these viruses circulating in ecosystems. Recent studies have used these methods to identify known viral plant pathogens as well as novel viruses circulating in insect vectors such as whiteflies and other higher order insects such a mosquitoes and dragonflies. These approaches successfully demonstrated that VEM can be used as a unique method, with the first mastrevirus discovered in the new world being recovered from dragonfly species Erythrodiplax fusca using this approach. The research in this thesis uses metagenomics to survey CRESS DNA viral diversity in different organisms and environments. Two hundred and sixty eight novel CRESS DNA viruses were recovered and verified in this study from a range of sample types (adult Odonata, Odonata larvae, Mollusca, benthic sediment, water, Oligochaeta and Chironomidae) collected in the United States of America, Australia and New Zealand. All viral genomes isolated had two major proteins encoding for a putative Rep and coat protein (CP), with major Rep motifs identified in most Reps. Phylogenetic analysis of the Reps encoded by the viral genomes highlighted that most were extremely diverse falling outside of the previously described ssDNA viral families. A top-down approach was implemented to recover CRESS DNA viruses and possible viral pathogens from Odonata and their larvae. Thirty six viral genomes were recovered from terrestrial adult dragonflies as well as the twenty four from aquatic larvae. Dragonfly cycloviruses were isolated from the some adult Odonata species which were closely related to the isolates previously described by Rosario et al. (2012). The viruses isolated in the aquatic and terrestrial ecosystems differed substantially indicating that different CRESS DNA viromes exist in both land and water. The diversity of CRESS DNA viruses in seven different mollusc species (Amphibola crenata, Austrolvenus stutchburyi, Paphies subtriangulata, Musculium novazelandiae, Potamopyrgus antipodarum, Physella acuta and Echyridella menziesi) from Lake Sarah and the Avon-Heathcote estuary both in New Zealand, were also investigated. One hundred and forty nine novel viral genomes were recovered. Two CRESS DNA genomes were recovered from molluscs which have Rep-like sequences most closely related to those found in some bacterial genomes. Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) was originally isolated from fungal species Sclerotinia sclerotiorum in china and was later found in benthic sediments in New Zealand. As part of this study, SsHADV-1 was recovered from dragonflies (Erythemis simplicicollis, Ischnura ramburii and Pantala hymenaea) collected in Arizona and Oklahoma, USA suggesting a larger distribution of these viruses and not surprising given the near global distribution of S. sclerotiorum. Dragonfly larvae-associated circular DNA viruses (DflaCVs) that were originally isolated in Odonata larvae samples from three New Zealand lakes were later recovered from water, benthic sediment, worms and molluscs from one of the lakes initially sampled, suggesting that these viruses are ubiquitous in freshwater environments. This study has attempted to generate baseline data of CRESS DNA viruses in certain environments using NGS-informed approaches. This data was used to try and establish whether viral distribution in different samples types can potentially be explained by the food web interactions between different samples types. Although the analysis did not show any significant relationships between sample type interactions and viral distribution a few common associations between Odonata larvae and benthic sediment were evident. This was expected as the larvae live within the sediment so it could be assumed that they potentially have similar CRESS DNA viral distribution. Although the distribution of viruses varied across sample types, molluscs proved the best sampling tool for isolating largest numbers of CRESS DNA viruses in an ecosystem with extensive diversity. Overall, this research demonstrates the applications of NGS for investigating the diversity of CRESS DNA viruses. It demonstrates that some sample types such as Odonata in terrestrial systems and molluscs in aquatic environments, can be used as effective sampling tool to determine the diversity of CRESS DNA viruses in different environments as well as detecting previously isolated viruses. The CRESS DNA viruses isolated in this body of work provides baseline data that can potentially be used in future research to investigate hosts of these viruses and their interactions with hosts and potential flow in their environments.

Background. Viruses strongly influence microbial population dynamics and ecosystem functions However, our ability to quantitatively evaluate those viral impads is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenornes (vromes). This leaves the ecology of nondsDNA viruses nearly unlmovvn, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation). Methods. Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results. Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were 1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryoteinfecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion. Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Bacteriophages (hereafter referred as phages) can translate their mRNAs efficiently by maximizing the use of codons decoded by the most abundant tRNAs of their bacterial hosts. Translation efficiency directly influences phage fitness and evolution. Reengineered phages find application in controlling their host population in both health and industry. The objective of this thesis work is to examine the factors shaping codon choices of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) Escherichia coli phages. In chapter two, we employed two indices, rRSCU (correlation in relative synonymous codon usage between phages and their hosts) and CAI (codon adaptation index) to measure codon adaptation in phages. None of the analyzed ssDNA phages encode tRNAs while some dsDNA phages encode their own tRNAs. Both rRSCU and CAI are negatively correlated with number of tRNA genes encoded by these dsDNA phages. We observed significantly greater rRSCU for dsDNA phages (without tRNAs) than ssDNA phages. In addition, we propose that ssDNA phages have evolved a novel codon adaptation strategy to overcome the disruptive effect of their high C→T mutation rates in codon adaptation with host. In chapter three, we formulated an index phi to measure selection by host translation machinery and to present explicit linear and nonlinear models to characterize the effect of C→T mutation and host-tRNA-mediated selection on phage codon usage. The effect of selection (phi) on codon usage is detectable in most dsDNA and ssDNA phage species. C→T mutations also interfere with nonsynonymous substitutions at second codon positions, especially in ssDNA phages. Strand asymmetry along with the accompanying local variation in mutation bias can significantly affect codon adaptation in both dsDNA and ssDNA phages.

Bio-chemical sensors are an emerging and vibrant area of research. The use of micromechanical cantilevers is relatively new as biomechanical recognition detectors. Reactions on a gold coated and chemically functionalized surface produce a mechanical deflection of the cantilever which is used as the input signal of the detector. Within the area of biosensors, DNA-sensors have a wide range of applications such as DNA hybridization detectors, DNA mismatch sequence detectors and protein detectors.We designed and built a microcantilever sensor system which allows for control and characterization of surface conditions. This includes controlled functionalization which can be a dominant factor in signal generation and reproducibility in these systems. Additionally, we developed a multistep functionalization protocol which consists of a sequence of short incubations and characterizations of thiolated ssDNA on a gold-coated cantilever.Multistep functionalization is a new protocol that is used to control the ssDNA surface density on a gold-coated cantilever. Repeatable responses and feasible biosensors are obtained using this protocol.
Les capteurs biochimiques sont un domaine émergent et dynamique de la recherche. L'utilisation de cantilevers micromécaniques est relativement récente en tant que détecteur par reconnaissance biomécanique. Des réactions sur la surface recouverte d'or et fonctionnalisée chimiquement produisent une déviation mécanique du cantilever qui constitue la source de mesure du détecteur. Dans le domaine des biocapteurs, les capteurs d'ADN ont un large champ d'application tel que les détecteurs d'hybridation d'ADN, les détecteurs de mésappariement d'ADN et les aptamères (détecteurs de protéines).Nous avons conçu et construit un système de capteur microcantilever permettant le contrôle et la caractérisation des conditions de surface. Une fonctionnalisation contrôlée est un facteur dominant pour la génération de signal et de la reproductibilité dans ces systèmes. Afin d'atteindre cet objectif, nous avons développé le protocole de fonctionnalisation en plusieurs étapes qui consiste en une séquence d'incubations de courte durée (5min chacune) et de caractérisations d'ADN simple brin thiolé sur le cantilever recouvert d'or.La fonctionnalisation en plusieurs étapes est un nouveau protocole qui contrôle la densité de surface d'ADN simple brin sur cantilever recouvert d'or. Des réponses reproductibles et des biocapteurs faisables sont obtenus grâce à ce protocole.

A new detection reagent that could possibly augment or replace antibodies research and diagnosis methods are aptamers. Aptamers are ssDNA, RNA or polypeptide constructs that function like active antibodies. Antibodies and aptamers both specifically bind to selected target molecules, and as such they enable the detection or targeting of the presence or absence of a specific antigen. In order to ensure that ssDNA aptamers perform similarly to antibodies, anti-VEGF-A polyclonal antibody and anti-VEGF-A ssDNA aptamer were evaluated against vascular endothelial growth factor A (VEGF-A) using Surface Plasmon Resonance (SPR). It was hypothesized that the anti-VEGF-A aptamer had the same, if not better, binding kinetics than the anti-VEGF-A polyclonal antibody, and as such offers an ideal replacement for use in in field, real-time testing assays. SPR revealed that both the polyclonal antibody and ssDNA aptamer bound the target antigen, VEGF-A. Additionally, from the SPR kinetic analysis, the anti-VEGF-A aptamer had KD values of 20-28 nM and the anti-VEGF-A antibody had KD values of 16-127 uM. The binding efficacy of the aptamer was several orders of magnitude better than that of the antibody. The aptamer was also stable in solution for a longer amount of time than the antibody, which denatured in solution after two weeks.

Rapidly evolving RNA and ssDNA viruses represent a fascinating field characterized by a strict interconnection between the “speculative” study of viral evolution and its practical implications in everyday veterinary medicine. This thesis has been thought as a collection of manuscripts that aimed to investigate different aspects and levels of viral evolution while still maintaining a focus on practical repercussions. Even if different infectious diseases and etiological agents are considered, they are all functional to the study of different aspects and implications of rapid virus evolution. Considering the heterogeneous nature of the manuscripts, they have been organized according to a “crescent” scale, starting from the lowest scale of viral evolution and progressing to broader scales. The manuscript “Viral subpopulations in aMPV vaccines are unlikely to be responsible for reversion to virulence.” addresses a fine-level analysis of the population structure of the AMPV subtype B live attenuated vaccine and its potential role in the previously demonstrated phenomenon of reversion to virulence. The widespread administration of live attenuated vaccines, despite their obvious advantages in terms of reducing disease prevalence, clinical signs and economic losses, is associated with costs that are not limited to the risk of reversion to virulence or to direct economic costs. Based on a wide collection of Italian samples, “Continued use of IBV 793B vaccine needs reassessment after its withdrawal led to the genotype’s disappearance” reports the impact of these vaccines in complicating the diagnostic process and, as a consequence, the interpretation of the epidemiological scenario in the absence of known vaccine markers. Obviously, updated knowledge of the strains currently circulating in a particular area is of great relevance for the implementation of proper control strategies. With this aim in mind, a field survey, which is published in “Field survey of Avian Metapneumovirus in Northern Italy”, was conducted on hundreds of Italian farms to estimate and characterize the AMPV strains circulating in our country. To further support frequent and extensive surveys, an assay that is able to detect, quantify and genotype the two AMPV subtypes currently circulating in Italy was developed and validated. Because economic constraints often represent a major limit, efforts were made to reduce the assay costs compared with other real-time RT-PCR methods while still guaranteeing comparable or superior performances (“A Sensitive, Reproducible, and Economic Real-Time Reverse Transcription PCR Detecting Avian Metapneumovirus Subtypes A and B”). Unfortunately, the diagnosis of rapidly evolving RNA viruses is itself an arduous task that requires a continuous evaluation and updating of diagnostic tools, even in laboratory that receive samples from limited geographic areas. The manuscripts entitled “Observation of high recombination occurrence of Porcine reproductive and respiratory syndrome virus in field condition” and “Phylodynamic analysis of Porcine reproductive and respiratory syndrome virus (PRRSV) in Italy: action of selective pressures and interactions between different clades.” address the study of the molecular epidemiology of Porcine reproductive and respiratory syndrome virus (PRRSV) in Italy considering the evolutionary forces driving PRRSV evolution at the local scale (i.e., high substitution rate, recombination, interaction between different clades and action of selective pressures). The high heterogeneity of PRRSV in the national contest was then evaluated with respect to the challenges that it poses in the development and validation of RT-PCR- and qRT-PCR-based diagnostic methods (“Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus”) and assessing its impact on diagnostic accuracy (“The impact of porcine reproductive and respiratory syndrome virus genetic heterogeneity on molecular assay performances”). Similarly, “International trades, local spread and viral evolution: the case of Porcine circovirus type 2 (PCV2) strains heterogeneity in Italy” investigates the genetic variability of PCV2 within national borders and compares it with the knowledge of its molecular epidemiology available from other countries. This study provides evidence regarding the role of both “in loco” evolution and importation of different genotypes and strains from foreign countries in determining the Italian PCV2 genetic heterogeneity. The crescent amount of PCV2 sequences deposited in publically available databases has revealed its marked variability and challenged the current classification criteria. Nevertheless, at least a superficial knowledge of the PCV2 molecular epidemiology is mandatory for the planning and evaluation of control strategies. “Revisiting the Taxonomical classification of PCV2: still a real challenge” proposes new criteria for the classification of PCV2 into different genotypes. Our aim was to provide a scheme that both accounts for the constraint imposed by the biological proprieties of this virus and allows a rapid, practical and easy way to classify PCV2 strains even during routine diagnostic activity. Last, “Genetic characterisation of porcine circovirus type 2 (PCV2) strains from feral pigs in the Brazilian Pantanal: an opportunity to reconstruct the history of PCV2 evolution” investigates more speculative issues inherent to the PCV2 origin. The discovery of a PCV2c genotype, which, to date, was believed to be extinct, in a feral pig population characterized by a peculiar population history and by a complex, and still partially known, relationship network with other PCV2-susceptible species opens exciting scenarios concerning the history and origin of PCV2
I virus a RNA e a ssDNA rappresentano un affascinante campo di ricerca caratterizzato da una stretta interconnessione fra lo studio di aspetti più “speculativi”, inerenti all’evoluzione virale, e le implicazioni che questi comportano nella pratica veterinaria. La presente tesi è stata concepita come una raccolta di articoli che hanno esplorato diversi aspetti e livelli dell’evoluzione virale senza per questo trascurarne le ripercussioni pratiche. Diverse malattie infettive ed agenti eziologici sono stati selezionati onde investigare i diversi aspetti e implicazioni della rapida evoluzione di questi virus. In considerazione della natura eterogena dei manoscritti prodotti, questi sono stati organizzati secondo una “scala crescente”, dal livello più basso dell’evoluzione virale, procedendo verso scale via via maggiori. L’elaborato “Viral subpopulations in aMPV vaccines are unlikely to be responsible for reversion to virulence” affronta un analisi ad alta risoluzione della presenza di sottopopolazioni virali nei vaccini vivi attenuati basati sul sottotipo B del metapneumovirus aviare e del loro potenziale ruolo nel determinare fenomeni di reversione a virulenza. La somministrazione su vasta scala di vaccini vivi attenuati, nonostante gli ovvi vantaggi in termini di riduzione della prevalenza virale, dei segni clinici e delle perdite economiche, è associata a costi che non sono limitati a quelli di tipo meramente pecuniario. Sulla base di un ampia raccolta di campioni italiani, l’articolo “Continued use of IBV 793B vaccine needs reassessment after its withdrawal led to the genotype’s disappearance” descrive l’impatto di questi vaccini, in assenza di marker vaccinali noti, nel complicare il processo diagnostico e, conseguentemente, l’interpretazione dello scenario epidemiologico. Ovviamente, una conoscenza aggiornata degli stipiti virali circolanti in una particolare area è di fondamentale importanza per l’implementazione di adeguate misure di controllo. Con quest’obiettivo in mente, uno studio di campo , pubblicato in “Diffusione dell’infezione da metapneumovirus aviare in allevamenti di tacchini e broiler nel Nord Italia”, è stato condotto su centinaia di allevamenti al fine di stimare e caratterizzare i ceppi di AMPV circolanti nel nostro territorio. In aggiunta, al fine di favorire studi sempre più frequenti e estesi, è stato sviluppato un test diagnostico in grado di rilevare, quantificare e tipizzare i sottotipi di AMPV attualmente circolanti in Italia. In considerazione del fatto che le spese relative a questi test rappresentano spesso uno dei maggiori limiti all’attività diagnostica e di ricerca, si è cercato di ridurre i costi rispetto ad altre metodiche comunemente utilizzate, garantendo nel contempo le medesime o migliori performance diagnostiche(“A Sensitive, Reproducible, and Economic Real-Time Reverse Transcription PCR Detecting Avian Metapneumovirus Subtypes A and B”). Sfortunatamente la diagnosi dei virus a RNA, essendo caratterizzati da una rapida evoluzione, rappresenta di per se stessa un ardua sfida e richiede una continua dedizione alla rivalutazione e aggiornamento delle metodiche diagnostiche esistenti. Gli articoli Observation of high recombination occurrence of Porcine reproductive and respiratory syndrome virus in field condition” e “Phylodynamic analysis of Porcine reproductive and respiratory syndrome virus (PRRSV) in Italy: action of selective pressures and interactions between different clades.” sono incentrati sullo studio dell’epidemiologia molecolare del Porcine reproductive and respiratory syndrome virus (PRRSV) in Italia. In particolare sono state studiate le forze evolutive che ne modellano e condizionano l’evoluzione (i.e., alto tasso di sostituzione nucleotidica, interazione fra differenti clade e azione di diverse pressioni selettive). L’elevata eterogeneità genetica di PRRSV nel nostro territorio è stata poi analizzata alla luce delle sue ripercussioni nello sviluppo e validazione di metodiche diagnostiche basate sull’uso della RT-PCR e qRT-PCR (“Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus”) e ne è stato valutato l’impatto sull’accuratezza diagnostica (“The impact of porcine reproductive and respiratory syndrome virus genetic heterogeneity on molecular assay performances”). Similmente, “International trades, local spread and viral evolution: the case of Porcine circovirus type 2 (PCV2) strains heterogeneity in Italy”, indaga la variabilità genetica di PCV2 all’interno dei confine nazionali comparandola altresì con le informazioni di epidemiologia molecolare disponibili per altri Stati. Il presente studio ha permesso di fornire significative evidenze sul ruolo sia dei commerci internazionali che dell’evoluzione “in loco” nel determinare l’eterogeneità di PCV2 riscontrata in Italia. Il progressivo aumento delle sequenza di PCV2 depositate in database pubblici ne ha dimostrato la grande variabilità su scala mondiale e ha evidenziato i limiti dei criteri di classificazione attualmente in uso. Tuttavia, una conoscenza quantomeno superficiale dell’epidemiologia molecolare di PCV2 è di basilare importanza per la pianificazione e la valutazione delle strategie di controllo. “Revisiting the Taxonomical classification of PCV2: still a real challenge” propone dei nuovi criteri per la classificazione di questo virus in diversi genotipi. Il nostro intento è stato quello di fornire uno schema che, pur tenendo conto dei limiti imposti dalle caratteristiche biologiche del virus, permettesse una rapida, pratica e facile genotipizzazione dei ceppi di PCV2 e che potesse quindi rispondere alle esigenze imposte dalla routinaria l’attività diagnostica. Infine, “Genetic characterisation of porcine circovirus type 2 (PCV2) strains from feral pigs in the Brazilian Pantanal: an opportunity to reconstruct the history of PCV2 evolution”, si confronta con il campo, più specualtivo, dell’origine di PCV2. La scoperta di un ceppo appartenente al genotipo PCV2c, sino ad oggi ritenuto estinto, in una popolazione di suini selvatici caratterizzata da una peculiare storia e da complesse, a ancora solo parzialmente conosciute, relazioni con altre specie suscettibili a questo patogeno, apre nuovi ed eccitanti scenari concernenti la storia e l’origine di PCV2

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2016.
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Os anticorpos anti-ácidos nucleicos são importantes no desenvolvimento de doenças autoimunes como o lúpus eritematoso sistêmico (LES). Porém, muito pouco se sabe sobre a origem e manutenção desses anticorpos pelo organismo e das interações desses anticorpos com seus antígenos. Foi observado na literatura e em trabalhos anteriores do grupo de Imunologia Molecular (UnB), que as sequências anotadas em banco de dados da família VH10 formadora da região variável da cadeia pesada de anticorpos era composta em sua maioria de sequências formadoras de moléculas anti-DNA. Além disso, através de uma abordagem experimental observou-se que essa família apresenta uma certa independência da CDR H3 para esta afinidade. Baseando-se nesses achados, nós desenhamos quatro fragmentos de anticorpos, no formato scFv (single-chain fragment variable), fragmento que preserva o sítio de ligação ao antígeno, composto de uma cadeia pesada (VH) ligada a uma cadeia leve, conhecida surrogate light chain (SLC) na tentativa de simular o reconhecimento antigênico em momento específico do desenvolvimento dos linfócitos B, onde a auto-reatividade é usada em prol do desenvolvimento normal dessas células. Levando-se também em conta a independência da CDR H3 para a ligação ao antígeno, nós iremos avaliar também através dessas construções o papel da CDR H2 para a ligação. Após clonagem das sequências em plasmídeo para a expressão heteróloga, esses scFvs foram produzidos em cepas de Escherichia coli BL21(DE3) pLysS. Até o momento, após padronização das condições para a produção de scFvs solúveis, condições favoráveis de duas das quatro construções foram estabelecidas. Estas construções irão permitir a análise do papel do segmento gênico V, mais especificamente da CDR2, para a afinidade ao DNA por anticorpos originados do segmento germinal de VH10. Além disso, será possível simular a interação antigênica de cadeias pesadas auto-reativas e não auto-reativas em um momento específico do desenvolvimento dos linfócitos B, onde a auto-reatividade parece ser utilizada em prol do desenvolvimento dessas células. _____________________________________________________________________________ ABSTRACT
The anti-nucleic acids antibodies are important in the development of autoimmune diseases such as systemic lupus erythematosus (SLE). However, very little is known about the origin and maintenance of these antibodies by the body and interactions of these antibodies with their antigens. Studies in the literature and previous work of the Molecular Immunology team (UnB) show that the annotated sequences in database of the VH10 family that is part of the variable region of the heavy chain antibodies consisted mostly of sequences of anti-DNA molecules. Furthermore, through an experimental approach it was observed that this family has a certain independence from CDR H3 for this affinity. Based on these findings, we designed four antibody fragments in scFv format (single-chain fragment variable), this fragments preserves the antigen binding site that comprises a heavy chain (VH) connected to a light chain, known surrogate light chain (SLC) in an attempt to simulate the antigen recognition in a specific moment in the development of B lymphocytes, where autoreactivity is used to promote the normal development of these cells. Also taking into account the independence of the H3 CDR for binding to antigen, we will also access through such constructions the role of CDR H2 for binding. After cloning the sequences into a plasmid for heterologous expression, these four scFvs were produced in Escherichia coli BL21 (DE3) pLysS. To date, after standardization of the conditions for soluble scFv production, conditions favorable for two of the four constructs were established. These constructions will enable the analysis of the role of the V gene segment, more specifically CDR2, for affinity to DNA antibodies originating from the germline segment VH10. In addition, you can simulate the antigen interaction of autoreactive and not autoreactive heavy chains at a specific time of the development of B lymphocytes, where self-reactivity seems to be used for the development of these cells.

The gut microbiome is a complex ecosystem of bacteria, viruses, and fungi that strongly influences animal health. The bacterial component, for example, contributes orders of magnitude more gene products to host physiology than the host genome; thus, changes to the composition of these bacterial communities can have profound influences on the health of the animal. By infecting and lysing their hosts, viruses (particularly viruses infecting bacteria or phages) can affect critical functions in these environments, yet the consequences of these infections remain to be fully described. Most studies investigating gut viromes to date have focused on double-stranded DNA (dsDNA) phages and, consequently, little is known about the smaller single-stranded DNA (ssDNA) phages, which also inhabit gut environments. In this study, we investigated ssDNA phages of the Microviridae family within the gut of an invertebrate organism, Ciona robusta, used as a model system to better understand gut microbial interactions. As a filter feeder, Ciona concentrates dissolved organics and microbes as part of its diet, yet maintains a microbiome distinct from the surrounding water column. We identified 258 unique ssDNA phage genomes representing a diversity of Microviridae subgroups including novel members of the established Gokushovirinae subfamily and several proposed phylogenetic groups (Alpavirinae, Aravirinae, Group D, Parabacteroides prophages, and Pequeñovirus). Over 70% of the genomes belonged to the Gokushovirinae; however, 155 of these genomes did not group with previously described sequences. Our results highlight an unprecedented diversity of ssDNA phages from an animal gut. Furthermore, comparative analysis between samples collected from Ciona specimens with full and cleared guts as well as the surrounding water indicated that Ciona retains a unique and highly diverse community of ssDNA phages. The present study significantly expands the known diversity within the Microviridae family and suggests that Ciona is a promising system for studying the role of ssDNA phages within animal guts.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-19T07:31:49Z No. of bitstreams: 1 texto completo.pdf: 6452406 bytes, checksum: 11a9270502dc083faeefb66ee407bd7f (MD5)
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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Fruteiras de clima temperado são de grande importância econômica mundial e são suscetíveis a diversos patógenos transmissíveis por insetos ou nematoides. Seu caráter perene e a propagação vegetativa resultam no aparecimento de diversas doenças complexas, algumas das quais ainda etiologicamente desconhecidas. Em pomares e vinhedos brasileiros frequentemente são observados sintomas de possível origem viral, mas os agentes não foram identificados. Com o objetivo de prospectar agentes virais associados a estas culturas, 74 amostras de fruteiras foram coletadas em diferentes regiões produtoras e avaliadas por PCR e RCA. Um novo vírus altamente divergente, monossegmentado, com genoma de ssDNA circular de aproximadamente 3.400 nucleotídeos, apresentando características moleculares semelhantes às de vírus classificadas nas famílias Circo-, Nano- e Geminiviridae foi detectado em plantas de macieira, pereira e videira. A associação com os hospedeiros foi confirmada e sua infectividade comprovada por meio de inoculação com um clone correspondente ao genoma completo. A análise de sequências e suas características moleculares indicam que este novo vírus poderá pertencer a um novo gênero ou família viral. Os begomovírus (família Geminiviridae) são patógenos de grande importância econômica em diversas culturas, principalmente em regiões tropicais e subtropicais. Eles regulam, direta ou indiretamente, diversos mecanismos de defesa do hospedeiro, a exemplo da via dos pequenos RNAs (smRNAs), de forma a facilitar a infecção viral. O segundo objetivo deste trabalho foi avaliar o perfil de smRNAs em plantas de Nicotiana benthamiana infectadas pelo begomovírus Tomato yellow spot virus (ToYSV). Utilizando sequenciamento de nova geração, bibliotecas de smRNAs de plantas de N. benthamiana, sadias e infectadas com o ToYSV, foram sequenciadas a fim de investigar sua complexidade e compreender os mecanismos regulatórios envolvidos na patogênese. Os resultados indicam que os begomovírus são alvo das vias de silenciamento de RNA (VSR) nas etapas iniciais da infecção e que suas proteínas supressoras de silenciamento atuam in trans para interferir com as VSRs. O sucesso da infecção viral e a indução de sintomas estariam assim, ao menos em parte, correlacionados com a regulação negativa das VSRs, com a mudança do perfil de smRNAs e com a regulação da expressão de diversos genes do hospedeiro que transcrevem mRNAs e miRNAs. A proteína de movimento (MP) dos begomovírus bissegmentados é responsável pelo movimento célula-a-célula, indução de sintomas e na determinação da gama de hospedeiros. O terceiro objetivo deste trabalho foi identificar proteínas de hospedeiros do ToYSV candidatas a interagirem com a MP. Complexos proteicos provenientes de plantas expressando constitutiva- ou transientemente NTAPi-MP foram purificados, seguido de identificação por espectrometria de massas. Entretanto, não foi possível encontrar proteínas que interagissem com MP do ToYSV. Ensaios de pull-down com a proteína MP fusionada a uma etiqueta de seis histidinas (6His-MP) identificaram 64 proteínas da planta candidatas a interagirem com a MP. Destas, 25 foram selecionadas para avaliação da interação pelo sistema duplo-híbrido em levedura, contudo, os resultados foram negativos para todas as 25 proteínas.
Temperate fruit trees are of great economical importance worldwide and are susceptible to several insect or nematode-transmitted pathogens. Their perennial nature and vegetative mode of propagation result in the appearance of several complex diseases, some of which are aetiologically undetermined. In Brazilian orchards and vineyards, virus-like symptoms are often observed, but the causal agents have not been identified. With the objective of prospecting viral agents associated with these plants, 74 fruit tree samples were collected at different regions and evaluated by PCR and RCA. A novel, highly divergent virus with a monopartite circular ssDNA genome of approximately 3400 nucleotides, with molecular characteristics similar to viruses in the Circo-, Nano- and Geminiviridae families was identified in apple, pear tree and grapevine plants. Its association with the hosts was confirmed and its infectivity was proven by inoculation with a full-length genomic clone. Sequence analysis and molecular characteristics indicate that this novel virus will be classified in a new viral genus or family. Begomoviruses (Geminiviridae family) cause diseases of major economic importance in many crops, especially in tropical and subtropical regions. They regulate, directly or indirectly, several host defense mechanisms such as small RNA (smRNA) pathways so as to facilitate viral infection. The second objective of this work was to evaluate the profile of smRNAs in Nicotiana benthamiana plants infected by the begomovirus Tomato yellow spot virus (ToYSV). Using Next Generation Sequencing approaches, we sequenced smRNA libraries from healthy or ToYSV-infected N. benthamiana plants to understand their complexity and to explore the regulatory mechanisms involved in pathogenesis. The results indicate that begomoviruses are targets of RNA silencing pathways (RSP) early in the infection and that their silencing suppressor proteins act in trans to interfere with RSP. The success of viral infection and the appearance of ToYSV-induced symptoms may be correlated, at least in part, with RSP downregulation, smRNA profile change and regulation of expression of several host genes (mRNA- and miRNA-transcribing). The movement protein (MP) of bipartite begomoviruses is responsible for cell-to-cell movement, symptom induction and determination of host range. The third objective of this work was to identify host proteins that interact with the ToYSV MP. Protein complexes were purified from plants expressing NTAPi-MP either constitutively or transiently, followed by mass spectrometry-based identification. However, no candidate host proteins were identified. Pull-down assays using the MP with a six histidine tag (6His-MP) identified 64 candidate proteins, 25 of which were selected for evaluation of the interaction using the yeast two- hybrid system. However, results were negative for all 25 proteins.

Philosophiae Doctor - PhD
The plasticity of single-stranded viral genomes permits the formation of secondary structures through complementary base-pairing of their component nucleotides. Such structures have been shown to regulate a number of biological processes during the viral life-cycle including, replication, translation, transcription, post-transcriptional editing and genome packaging. However, even randomly generated single-stranded nucleotide sequences have the capacity to form stable secondary structures and therefore, amongst the numerous secondary structures formed in large viral genomes only a few of these elements will likely be biologically relevant. While it is possible to identify functional elements through series of laboratory experiments, this is both excessively resource- and time-intensive, and therefore not always feasible. A more efficient approach involves the use of computational comparative analyses methods to study the signals of molecular evolution that are consistent with selection acting to preserve particular structural elements. In this study, I systematically deploy a collection of computationally-based molecular evolution detection methods to analyse the genomes of viruses belonging to a number of ssRNA viral families (Alphaflexiviridae, Arteriviridae, Caliciviridae, Closteroviridae, Coronavirinae, Flaviviridae, Luteoviridae, Picornaviridae, Potyviridae, Togaviridae and Virgaviridae), for evidence of selectively stabilised secondary structures. To identify potentially important structural elements the approach incorporates structure prediction data with signals of natural selection, sequence co-evolution and genetic recombination. In addition, auxiliary computational tools were used to; 1) quantitatively rank the identified structures in order of their likely biological importance, 2) plot co-ordinates of structures onto viral genome maps, and 3) visualise individual structures, overlaid with estimates from the molecular evolution analyses. I show that in many of these viruses purifying selection tends to be stronger at sites that are predicted to be base-paired within secondary structures, in addition to strong associations between base-paired sites and those that are complementarily co-evolving. Lastly, I show that in recombinant genomes breakpoint locations are weakly associated with co-ordinates of secondary structures. Collectively, these findings suggest that natural selection acting to maintain potentially functional secondary structures has been a major theme during the evolution of these ssRNA viruses.

The ends of linear eukaryotic chromosomes resemble double strand breaks, susceptible to the vigilant cellular DNA damage response machinery. Telomeres are specialised nucleoprotein structures found at the ends of human chromosomes that function as a protective cap safeguarding the integrity of the genome. In normal human somatic cells, telomeres shorten with each replicative cell division. Eventually, shortened telomeres trigger cellular senescence and apoptosis. Cancer cells overcome this proliferative barrier by acquiring a telomere maintenance mechanism. Alternative Lengthening of Telomeres (ALT) is a homologous recombination (HR) mediated telomere maintenance mechanism utilised by approximately 10-15% of all cancers. ALT status in certain cancer subtypes, such as osteosarcomas and astrocytomas, often predicts aggressive nature and poor prognosis. Therefore, the ALT pathway presents an attractive avenue for developing novel cancer therapeutics. To date, ALT specific therapeutic targets are lacking. Our lab has previously identified the zinc finger protein ZNF827 as a promising molecular target in the ALT pathway. ZNF827 recruits the nucleosome remodelling and histone deacetylase (NuRD) complex to ALT telomeres, and collaboratively facilitates multifaceted functions to promote HR-mediated telomere synthesis. However, the precise molecular mechanisms underlying the important roles of ZNF827 at ALT telomeres have not been fully elucidated. Prior to the study by our lab, ZNF827 was a protein of unknown function. This thesis has focused on the functional characterisation of ZNF827, with an ultimate goal of validating it as a therapeutic target in cancers utilising the ALT pathway. We have characterised ZNF827 from several perspectives. First, we have explored the biological functions of ZNF827 as a transcription factor and discovered novel roles of ZNF827 in cellular processes including embryonic development and immune response. Second, we have gained new insights into the recruitment of ZNF827 to ALT telomeres, characterised structurally and functionally the interaction interface between ZNF827 and the NuRD complex, and implicated ZNF827 SUMOylation in promoting ALT activity. We have also discovered that ZNF827 is novel ssDNA binding protein implicated in HR-directed repair of replication-associated DNA damage at telomeres as well as genome-wide, and that its role as a DNA damage response and repair protein is likely to be mediated through the ATR-mediated DNA signalling pathway. Finally, we have provided preliminary evidence that supports synthetic lethality between ZNF827 inhibition and the topoisomerase I inhibitor topotecan, implicating ZNF827 as a potential molecular target for ATR inhibition. Taken together, our findings have substantially expanded our knowledge on the zinc finger protein ZNF827 and provided further support for its therapeutic potential in the development of novel cancer therapies.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2009.
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Os anticorpos anti-DNA são comumente encontrados nas doenças auto-imunes. Vários estudos têm sido realizados com o objetivo de se determinar as bases moleculares desse reconhecimento antigênico. Em trabalhos anteriores construíu-se uma biblioteca combinatória de fragmentos de anticorpos, a partir de cDNA de galinhas da raça Red/Black Cornish Cross, previamente imunizadas com BSA-fluoresceína. Os scFvs foram selecionados pela sua capacidade de ligação a DNA fita simples (Oligo dT-celulose) (Maranhão, 2001). A partir daí, quatro clones foram escolhidos. Esses scFvs foram novamente seqüenciados e analisados. Os fragmentos de anticorpos foram produzidos no sobrenadante de cultura de bactéria e posteriormente purificados por cromatografia de afinidade utilizando colunas de níquel. Para a confirmação da atividade ligante a ácidos nucléicos, bem como para a caracterização de sua afinidade e especificidade, essas proteínas foram submetidas à imunoensaios. A afinidade foi testada tanto por ELISA de ligação direta, onde o único antígeno utilizado era o Oligo dTbiotina, quanto por ELISA de competição. Neste último tipo de imunoensaio foram utilizados diversos ssDNA solúveis como competidores: Oligo dT, Oligo dA, Oligo dC, Oligo dG, Oligo dU. Além disso, dois desses scFvs foram testados quanto à capacidade de inibição da ligação a Oligo-dT pelo antígeno originalmente utilizado (FITC) para a imunização dos animais. As análises realizadas nesse trabalho mostram que os scFvs são capazes de se ligar ao antígeno utilizado na seleção da biblioteca. Além disso, existe uma dependência da composição das CDRs e a capacidade de reconhecimento dos Ac recombinantes de diferentes seqüências de DNA fita simples. Nesse sentido, um dos scFvs caracterizados apresentou uma grande polirreatividade, ligando-se a diferentes antígenos, enquanto que os outros três apresentaram ligação significativa apenas a Oligo-dT e a Oligo-dA, dentre os oligonucleotídeos testados. Também é de tamanha relevância o achado de que mesmo tendo sido obtidos a partir de uma seleção por afinidade a Oligo-dT, a ligação ao antígeno utilizado para a imunização dos animais (FITC) é mantida para os dois fragmentos de Ac testados. Todos esses dados sugerem que a afinidade e a especificidade desses fragmentos de anticorpos dependem não só da interação deste com o esqueleto de açúcar-fosfato, mas também com as bases nitrogenadas. _________________________________________________________________________________ ABSTRACT
Anti-DNA antibodies are commonly found in autoimmune disease patient sera. Several studies are made trying to understand their origin and the basis of their affinity. We had previously constructed a scFv library from BSA-fluorescein chicken immunized Ab repertoire and used it to select anti-ssDNA antibodies. Several clones were analyzed in terms of scFv production and VH and VL sequences, and among them, four representatives ones were chosen for further characterization. The aim of this work was to produce and characterize the selected scFvs in terms of theirs immunological features, comparing their specificities. The recombinant scFvs were produced in bacterial culture supernatants and were purified using metal affinity chromatography. The purified proteins were tested by ELISA immunoassays for ssDNA direct binding. The purified scFvs were also tested by ELISA immunoassays for ssDNA competition using soluble ssDNA: Oligo-dA, Oligo-dC, Oligo-dG, Oligo-dT, Oligo-dU. We also tested the remaining binding to FITC. The protein production and purification yielded enough recombinant products for immunological characterization. The recombinant affinity-purified products were able to bind to Oligo(dT)-biotin and this association was inhibited by Oligo-dT itself, Olig-dA and Oligo-dU. Also, for the two tested scFvs, FITC was able to compete with Oligo-dT-biotin binding. Although quite similar to the other scFvs, one of the characterized proteins showed a strong polyreactivity. These data showed that we were able to select specific antibodies fragments anti-ssDNA from a nonimmune source. The antibodies produced in bacterial cells relied their binding activity not only on sugar-phosphate backbone, but also on nitrogenous base recognition.

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Vírus que possuem genoma constituído por DNA fita simples circular são amplamente distribuídos na natureza. As constantes descobertas aliadas à grande diversidade genética desses vírus têm ampliado a compreensão sobre a trajetória evolutiva desse grupo. A partir de 2010 um grupo divergente de vírus com genoma de DNA fita simples circular, denominado genomovírus, passou a ser identificado em diversos ambientes (fezes e esgoto) e associados a diferentes organismos (plantas, fungos, humanos, mamíferos, moluscos e insetos). Neste trabalho foram caracterizados dois genomovírus denominados Momordica charantia associated gemycircularvirus (MorGemy) e Euphorbia heterophylla associated gemycircularvirus (EupGemy) encontrados associados às plantas silvestres Momordica charantia e Euphorbia heterophylla respectivamente. Ambos os vírus possuem organização genômica típica dos genomovírus. Ambos os vírus apresentam stem-loop com nanonucleotídeo conservado em todos os genomovírus, sendo a sequência TAATRTTAT (N pode ser A ou G). Entretanto, no isolado MorGemy foi observado uma estrutura em stem-loop extra na possível origem de replicação não presente em outros genomovírus já descritos. Em ambos os vírus foi observada a presença de um íntron, no interior da ORF que codifica a proteína Rep sendo que o íntron observado em EupGemy é maior que o observado em MorGemy. Na proteína Rep foram observados os motivos I, II e III, e os domínios Walker A, B, C e GRS, elementos encontrados tipicamente na proteína Rep dos geminivírus. O SDT (Sequence Demarcation Tool) revelou que MorGemy tem 72% de identidade com Pteropus associated gemycircularvirus 3 e Hypericum associated gemycircularvirus 1. Além disso, o EupGemy mostra 66% de identidade com Odonata asssociated gemycircularvirus 1. A taxa de identidade de nucleotídeos do genoma completo dos vírus isolados neste trabalho com outros genomovírus já descritos, permite classificar esses novos isolados como duas novas espécies.
Viruses that have genomes consisting of simple circular DNA are widely distributed in nature. The constant discoveries allied to the great genetic diversity of these viruses have broadened the understanding of the evolutionary trajectory of this group. In 2010 a divergent group of viruses with a circular ssDNA genome called Genomovírus was identified in several environments (feces and sewage) and associated with different organisms (plants, fungi, humans, mammals, mollusks and insects). In this work, two Genomovíruses found associated with wild plants were characterized. Both virus shows a structure Genomovírus-like. However two interesting facts were observed. The first, a presence a second structure on stem-loop in the MorGemy no related in other Genomovírus. Both virus presents stem-loop with nanonucleotide conserved in all Genomovírus. Being that the sequence TAATRTTAT (N could be A or G). We also observed the presence of a íntron in both genome, inside the ORF’s Rep. The íntron of EufGmey is greater than MorGemy. In the Rep protein were observed the motif I, II and III. Interestingly, also were observed the presence of Motifs Walker A to C and GRS domain. These elementes are found on protein Rep’s Geminivírus. The SDT releaved that MorGemy shows 72% of identity with Pteropus associated gemycircularvirus 3 and Hypericum associated gemycircularvirus 1. In addition EupGemy shows 66% of identity with Odonata associated gemycircularvirus 1. The rate of identity of MorGemy and EupGemy with Genomovírus associate with plants combined with analysis phylogeny are an indicative that these virus can have a possible host-virus relation with plants.

Escherichia coli transporter protein AcrB and its homologues are the inner membrane components of the Resistance-Nodulation-Division (RND) family efflux pumps in Gram-negative bacteria. It is well accepted that soluble proteins are only marginally stable, but such insight is missing for membrane proteins. The lack of stability data, including thermodynamic stability and oligomer association affinity is a result of intrinsic difficulties in working with membrane proteins. In addition, the degradation of soluble proteins in E. coli has been extensively studied whereas the degradation process of membrane proteins remains unclear. A focus of my thesis is the validation and development of methods used to measure the thermo- and oligomeric- stability of membrane proteins. I investigated the mechanism of a popular thermal-stability assay developed specifically for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). I found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal stability assay. The observed fluorescence increase is likely caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding. I then applied these methods in the study of three projects. In the first project, I investigated how suppressor mutations restore the function of AcrBP223G, in which the Pro223 to Gly mutation compromised the function of AcrB via disrupting AcrB trimerization. The results suggested that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding. In the second project, I created two AcrB fusion proteins, with C-terminal yellow fluorescence protein (YFP) and cyan fluorescence protein (CFP), respectively. YFP and CFP form a fluorescence resonance energy transfer (FRET) pair. Using this pair of fusion proteins, I studied AcrB assembly both in detergent micelles and in lipid bilayers. A positive cooperativity was observed in kinetic studies of association of AcrB trimer. Reconstitution experiment revealed that the association showed a higher FRET efficiency and faster association rate in liposome than in DDM. In the last project, I developed a fluorescence method to study the degradation of AcrB-ssrA by the ClpXP system. Comparing to the degradation of GFP-ssrA, degradation of AcrB-CFP-ssrA showed a lower maximum velocity and tighter binding to the enzymes with a positive cooperativity.

Replication Protein A (RPA), the major eukaryotic single-strand DNA (ssDNA) binding protein, is essential for replication, repair, recombination, and checkpoint activation. Defects in RPA-associated cellular activities lead to genomic instability, a major factor in the pathogenesis of cancer. The ssDNA-binding activity of RPA is primarily mediated by two domains in the RPA1 subunit. I characterized mutant forms of RPA to elucidate the contribution of specific residues in the high affinity DNA binding domains to the cellular function of RPA. These studies enhance the understanding of the properties of RPA that contribute to DNA repair and cellular checkpoints. Mutation of a conserved leucine residue to proline in the high-affinity DNA binding site of RPA (residue L221 in human RPA) has been shown to have a high rate of chromosomal rearrangements in yeast and mice. I characterized the equivalent mutation in human RPA. My studies show that the mutation causes a defect in ssDNA binding and a nonfunctional protein. Combined with the mice studies, the data suggest that haploinsufficiency of RPA causes an increase in DNA damage and in the incidence of cancer. The ssDNA-interactions of the high affinity binding domains in RPA1 are mediated by several residues including four highly conserved aromatic residues. Mutation of these residues had no effect on DNA replication but caused defects in DNA repair pathways. I conclude that DNA intermediates in different DNA metabolic pathways require different RPA binding functions and that the aromatic residues are indispensable for binding in DNA repair. These studies illustrate that different DNA metabolic pathways have distinct requirements for RPA function. A decrease in binding to ssDNA of any length has specific consequences in vivo. These data also demonstrate that a single mutation in RPA in a residue that does not even contact ssDNA can result in a non-functional RPA complex. I conclude that even a modest decrease in RPA protein levels is not compatible with long term cell survival. Taken together, these studies highlight the importance of proper regulation of RPA protein levels and its ssDNA binding affinity to proper maintenance of the integrity of the genome.

This study combines macro and micro data to quantify the revenue effects of double tax treaties. First, drawing on administrative information, the study estimates the tax sensitivity of income flows (dividend, interest, and royalty payments) at an aggregate level. The analysis finds important direct revenue costs linked to treaty restrictions on taxing rights, especially for flows into a few major investment hubs. However, high elasticities of income flows also suggest that increases in withholding rates at the individual treaty partner level would not necessarily result in more revenue collection. Second, the study uses firm- level information to estimate the sensitivity of reported profitability to changes in the relevant treaty network. The analysis of the reported earnings of multinational enterprise affiliates in Ukraine suggests that the ownership structure and operations with affiliates in certain jurisdictions explain reported profitability, and should thus receive increased attention in risk assessment and transfer pricing audit activities.
Series: WU International Taxation Research Paper Series

The paper analyses whether various aspects of a country's tax system have a positive or negative influence on individuals' attitudes towards the future. These attitudes are measured by an analysis of Google search queries derived from Google Trends which allow constructing an online futureorientation index for a sample of 58 countries. There results of this analysis indicate that capital gains taxes and value added taxes discourage future-oriented behaviour. Also, high personal income tax rates at the lowest income brackets discourage, whereas - surprisingly - the top marginal rates could positively influence an individual's future orientation. The paper contributes to existing research in three ways: First, it expands the existing tax literature by providing evidence that taxes can influence very fundamental personal values, such as individuals' general attitudes towards their future. Second, it contributes to a vast body of cross-cultural studies on future orientation by introducing tax law. Third, by using Internet search patterns the paper introduces these large, automatically gathered data sets into scientific tax research, thereby opening the possibility for further research opportunities. (author's abstract)
Series: WU International Taxation Research Paper Series

The Organization for Economic Cooperation and Development (OECD) under Base Erosion and Profit Shifting (BEPS) Action 2 indicated that tax arbitrage via hybrid mismatch arrangements "result in a substantial erosion of the taxable bases of the countries concerned" and "have an overall negative impact on competition, efficiency, transparency and fairness." The relevant action allowing for neutralising the effects of hybrid mismatch arrangements is therefore needed and justified. To achieve that purpose, the OECD developed different anti-hybrid rules under BEPS Action 2. In that regard, however, one may ask whether addressing tax arbitrage via hybrid mismatches as proposed by the OECD is of interest and relevance for developing countries. This paper aims to map that unexplored research area by means of a comparative analysis in four developing countries - Uruguay, Colombia, Brazil, and South Africa.
Series: WU International Taxation Research Paper Series