In the post genomic era, proteomics has enormous potential in biology and medicine. Among the various bioanalytical tools developed, protein microarray is one of the recent advancements which offer high throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. Fundamentally, the protein microarray involves the immobilization of interacting elements, proteins, on a few square microns of a solid support and in principle, it is capable of detecting analytes with a higher sensitivity than conventional macroscopic immunoassays. Here in the present report we delineates the design, fabrication and functional analysis of protein microarray using semi-synthetic ssDNA tagged-proteins as capturing moiety as well as address on a solid support. Optimization of the platform has been carried out by investigating various parameters such as surface chemistry, signal amplification, and conditions for homogenous liguid phase protein-protein interaction. / Singapore-MIT Alliance (SMA)

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2018-08-29T18:54:49Z No. of bitstreams: 1 texto completo.pdf: 947486 bytes, checksum: 6d7e934deec08032d3a902d1d879ef0a (MD5) / Made available in DSpace on 2018-08-29T18:54:49Z (GMT). No. of bitstreams: 1 texto completo.pdf: 947486 bytes, checksum: 6d7e934deec08032d3a902d1d879ef0a (MD5) Previous issue date: 2017-02-24 / 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.

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.

No description available.

No description available.

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 &quotNew 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.

Colorimetric methods combined with color-changing chemical probes are widely used as simple yet effective tools for identifying and quantifying a wide variety of molecules in solution. For nucleic acids (DNA and RNA), perhaps the most commonly used colorimetric probe is potassium permanganate, which can be used to identify single-stranded pyrimidines (thymine and cytosine) in polymers. Unfortunately, permanganate is not an effective probe for identifying purines (adenine and guanine), especially in the presence of the more reactive pyrimidines. Therefore, robust methods for discriminating between the purines remain elusive, thereby creating a barrier toward developing more complex colorimetric applications. In this dissertation, we demonstrate that chromophores such as permanganate and bicinchoninic acid (BCA) and copper, however, when combined with nucleobase-specific chemical cleavage reactions, can be a colorimetric probe for the identification and quantification of cytosines, adenines and/or guanines in single-stranded DNA oligomers, even in the presence of thymines. Furthermore, the reactions are stoichiometric, which allows for the quantification of cytosine, adenine and/or guanine frequency in these oligomers. The BCA/copper reagent detects the reducing sugar, 2-deoxyribose, resulting from the chemical cleavage of a given nucleotide’s N-glycosidic bond. Therefore, these colorimetric assays are effectively detecting abasic sites in DNA oligomers, which are known to occur in damaged DNA. Our analytic approach termed colorimetric identification of exposed nucleic acids (CIENA) combines the use of BCA/copper, permanganate, and diphenylamine chromophores along with digital image capture to identify and quantify each nucleobase within DNA. The digital image color properties are quantified in terms of the image’s hue, saturation, and lightness using the CIELAB color space and ΔE quantification of color. CIENA is a simple, low-cost tool that could be applicable in various types of nucleic acid analyses, such as the quantification of nucleobase composition and the identification of damaged DNA.

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.

DNA is the fundamental molecule in all domains of life, its role in heredity is well established. Although the famous double helical complementary form is indispensable for replication mechanism DNA can occupy wide range of conformations. In the past studies performed in the laboratory, DNA oligomers related to single stranded bacterial Repetitive Extragenic Palindromic (REP) showed spectral behavior suggesting complex equilibria including double helical, hairpin, and tetraplex conformations. The studies presented in this thesis extended the scope of analyzed sequences and employed circular dichroism spectroscopy and X-ray crystallography. We report spectral data and X-ray structures of three successfully crystalized oligonucleotides. All three structures acquire double helical architecture with two consecutive T- T mismatches in the center. To improve the convergence of the refinement process of the crystal structures we used novel dinucleotide conformational classes, NtC classes. The NtC class classification was also used to analyze geometries of selected non-canonical base pairs in all DNA crystal structures in the Protein Data Bank. We measured the fit between geometries of the dinucleotides involved in the non-canonical base pairing and the NtC classes and correlated this fit to the electron...

Glypican-3 (GPC3), the cellular membrane proteoglycan, has been established as a tumor biomarker for early diagnosis of hepatocellular carcinoma (HCC). GPC3 is highly expressed in more than 70% HCC tissues detected by antibody-based histopathological systems. Recently, aptamers, a short single-strand DNA or RNA generated from systematic evolution of ligands by exponential enrichment (SELEX), were reported as potential alternatives in tumor-targeted imaging and diagnosis. In this study, a total of 19 GPC3-bound aptamers were successfully screened by capillary electrophoresis (CE)-SELEX technology. After truncated, AP613-1 was confirmed to specifically target GPC3 with a dissociation constant (KD) of 59.85 nM. When modified with a phosphorothioate linkage, APS613-1 targeted GPC3 with a KD of 15.48 nM and could be used as a specific probe in living Huh7 and PLC/PRF/5 imaging, GPC3-positive cell lines, but not in L02 or A549, two GPC3-negative cell lines. More importantly, Alexa Fluor 750-conjugated APS613-1 could be used as a fluorescent probe to subcutaneous HCC imaging in xenograft nude mice. Our results indicated that modified AP613-1, especially APS613-1, was a potential agent in GPC3-positive tumor imaging for HCC early diagnosis.