Dissertations / Theses on the topic 'Molecular virology'
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Fowotade, Adeola. "Molecular virology of KSHV : elucidating vIRF2 and vIRF4 function." Thesis, University of Surrey, 2017. http://epubs.surrey.ac.uk/813233/.
Full textSanfilippo, Luiz Francisco. "Epidemiologia e caracterização molecular do vírus da Influenza em quatro espécies de pinguins na Região Antártica." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-11082011-105843/.
Full textEpidemics and pandemics of influenza usually refer to infections in human beings. The influenza virus is not, however, restricted to humans and can cause infirmity and death in other species including horses, swine, marine mammals, birds, and others. Ecological studies of viral infections have led to the hypothesis that the influenza viruses that attack mammals have their origin in the accumulation of these viruses in birds (avian flu). In some countries with influenza cases caused by the avian H5N1 virus, there was monitoring of wild birds but little had been done in Antarctica. The present work was therefore carried out during the Antarctic summer seasons of 2006, 2007, and 2008 in two Antarctic locations: The Commander Ferraz Antarctic Station, on the Keller Peninsula of King George Island, and at the Base of Advanced Studies located on Elephant Island (61°08S, 55°07W). Two hundred eighty-three (283) samples from four different penguin species Pygoscelis adeliae, Pygoscelis papua, Pygoscelis antarctica; and Aptenodytes patagonicus were collected for this study. Diagnoses of the samples were performed not only by application of direct detection and amplification according to the RT-PCR method in agar-gel, but also by Real-Time PCR (Applied Biosystems), and by RT-PCR gene scan at the Laboratory of Clinical and Molecular Virology of the Department of Microbiology of the University of Sao Paulo. Eight of the penguin samples tested positive for the Influenza-A virus. The positive samples, as determined by RT-PCR, were sent to the Influenza Laboratory of the Department of Infectious Diseases of the St. Jude Research Hospital in Memphis, Tennessee, USA, to be isolated in egg embryos where no further growth of the Influenza-A virus took place. Four of these positive samples could be sequenced and compared with those of Influenza-A on deposit at the Gene Bank and ranged from 96.85 to 100% when compared with the control samples (100% positive), thus confirming the presence of the virus in the tested birds.
Hughes, Fiona Lesley. "Molecular investigations of subgroup I geminiviruses." Doctoral thesis, University of Cape Town, 1991. http://hdl.handle.net/11427/21979.
Full textThe diversity of Subgroup I geminiviruses causing streak disease in maize, sugarcane, and indigenous wild grasses was investigated. The virus. isolates studied originated from maize (several southern African isolates), two sugarcane cultivars (from Natal province, South Africa, and from Mauritius), wheat, and three grasses (Panicum, Setaria, and Eleusine spp. from South Africa). The following methods were used: analysis of restriction fragment length polymorphisms (RFLPs) between viral genomes in individual infected plants; DNA cross-hybridization between virus isolates; restriction endonuclease mapping of whole virus genomes; and nucleic acid sequencing. The complete genome of the Natal sugarcane streak virus isolate was sequenced. Partial sequences were obtained for other isolates, either by sequencing the ends of cloned viral genomes, or by sequencing a 250 base pair fragment of a highly conserved open reading frame that had been amplified using the polymerase chain reaction technique. The viruses being studied were compared both among themselves and with other Subgroup I geminiviruses of known DNA sequence, on the basis of sequence (nucleotide and amino acid) and restriction map data. Distance matrix methods were used to infer phylogenetic relationships between Subgroup I geminiviruses from restriction map and sequence data. Phylogenies deduced from sequence data were considered to be more accurate than those deduced from map data. Regardless of the method of analysis used, however, the relationships between the Subgroup I geminiviruses studied here remained constant. Thus, three strains of MSV (maize, Setaria, and Eleusine strains) were distinguished. Streak viruses distinct from MSV were also identified: panicum streak virus (PanSV), and two distantly related strains (Natal and Mauritius) of sugarcane streak virus (SSV). Restriction mapping of different geographical isolates of the maize strain of MSV demonstrated that variation existed within a single strain of virus. RFLP analysis indicated that minor variation existed between virus genomes within single diseased plants. Methods used to. type Subgroup I geminiviruses were evaluated, and discrepancies in the serological typing of geminiviruses from Subgroups I and III were pointed out. A unified scheme was proposed for distinguishing between distinct Subgroup I geminiviruses and strains of geminiviruses. The origins of maize and sugarcane streak viruses were speculated upon.
Rizotto, Laís Santos. "Metapneumovírus aviários em aves silvestres." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-17052017-125020/.
Full textAvian metapneumovirus (aMPV), family Pneumoviridae, genus Metapneumovirus, it is the etiologic agent responsible for turkey rhinotracheitis and is also associated with swollen head syndrome in chickens, two important respiratory diseases in poultry which leads to large economic losses. The aim of this study was detect the presence of aMPV in wild birds samples, to perform the phylogenetic analysis of the isolates found with the major objective of contributing to the understanding of the epidemiology of this virus in poultry farms. In total, 448 oropharyngeal (OP) and cloacal (C) swabs from 234 wild birds collected in four different locations within the state of São Paulo. The samples were processed and tested in three different ways: 1) 266 swabs were in the form of pools of one up to five animals that were in the same enclosure and respecting the same type of swab (OP or C); 2) 188 remaining swabs were grouped into pools of up to two animals, containing the oropharyngeal and cloacal swabs of each animal; 3) tracheal and pulmonary tissue samples were also collected and tested. Purification was performed using the QIAmp RNA Mini Kit Kit (Qiagen). Viral detection was performed by conventional RT-PCR technique using the OneStep RT-PCR kit (Qiagen) with primers based on the N gene, previously described with expected fragment of 115 bp. The samples were also tested by a real time RT-PCR (RRT-PCR) with specific primers previously described, for subtypes A and B, based on the G gene with fragments of 116 and 135 bp, respectively. Of the 126 samples tested by the RT-PCR N gene based, fourteen were positive: eight samples of Anseriformes (Aix sponsa, Aix galericulata, Dendrocygna viduata), three Columbiformes (Columba livia), one Falconiformes (Falco sparverius), one Psittaciformes (Psittacara leucophtalma) and one Pelecaniformes (Egretta thula). Of the swab samples, five were derived from oropharyngeal swabs and four from cloacal swabs, the other four samples were detected in the samples processed in pools of up to two animals, which contained the oropharyngeal and cloacal swabs of each bird. The positive Egretta thula sample was from a tracheal tissue sample. Based on the RRT-PCR G gene based, none of the 184 samples tested were detected. Phylogenetic analyzes were performed on two positive samples that proved to belong to aMPV subtype A, showing high similarity with the strains derived from the vaccine and with vaccine strains.
Kapoor, Sanjay. "Molecular determinants of rotavirus virulence." Thesis, University of Warwick, 1995. http://wrap.warwick.ac.uk/4250/.
Full textJanowicz, Anna Agata. "Molecular determinants of bluetongue virus virulence." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6959/.
Full textSlack, Gillian Sinclair. "Molecular and biological characterisation of orthobunyaviruses." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7303/.
Full textFernandez, Llenalia Garcia. "Statistical modelling of performance data for molecular amplification methods in diagnostic virology." Thesis, University of Abertay Dundee, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.650529.
Full textHunt, Nicholas. "Molecular analysis of the Friend virus complex." Thesis, University of Warwick, 1989. http://wrap.warwick.ac.uk/108015/.
Full textYadav, Sarita. "Isolation and molecular characterization of bluetongue virus from Southern India." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/30807/.
Full textCoburn, Alice Miranda. "Molecular determinants of influenza A virus cross-species jumps." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8394/.
Full textHalldorsson, Steinar. "Molecular determinants of phleboviral cell entry." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:56c5ef37-b023-4a8f-bdf2-8388226dc3b3.
Full textSiliņš, Ilvars. "Molecular epidemiology of human papillomavirus and cervical cancer /." Stockholm : [Karolinska institutets bibl.], 2001. http://diss.kib.ki.se/2001/91-7349-091-1/.
Full textTerrell, Shariya Louise. "Importance of the Pre-\(NH_2\)-Terminal Domain of HSV-1 DNA Polymerase for Viral Replication." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10705.
Full textChamanian, Mastooreh. "A Novel HIV-1 Genomic RNA Packaging Element and its Role in Interplay between RNAPackaging and Gag-Pol Ribosomal Frameshifting." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1363902886.
Full textDobrowolski, Curtis Noel. "HISTONE LYSINE METHYLTRANSFERASES SELECTIVELY RESTRICT HIV-1 IN CENTRAL MEMORY T-CELLS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1522842870401743.
Full textCondjella, Rachel. "Unique properties of the canine papillomavirus type II E5 protein." Connect to Electronic Thesis (ProQuest) Connect to Electronic Thesis (CONTENTdm), 2009. http://worldcat.org/oclc/457185147/viewonline.
Full textRodrigues, Juliana Nogueira Martins. "Caracterização molecular do Epstein-Barr vírus (EBV) em pacientes portadores de HIV, em tratamento, atendidos no sistema hospitalar do sistema penitenciário do Estado de São Paulo." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-27032009-154240/.
Full textThe Epstein-Barr Virus (EBV) is the only species to the genus Lymphocriptovirus that infects humans. One of the possible route for its transmission thought by contamined saliva and usually occurs in the childhood. This study analysed 165 clinical samples from HIV infected patients, treated by HARRT, attended in the Hospitalar System in the Penitentiary System from Sao Paulo State. The aim of this study was to search EBV in peripheral blood mononuclear cells by PCR, Nested-PCR and sequencing analysis. The results showed 11,51% of the analysed samples, positive for EBV. This samples, was sequenced with specifics primers from the EBNA-1 (Epstein Barr Nuclear Antigen 1) region. The samples were aligned by DNASTAR program. The aligned sequences showed the base conversion G to A in seven samples. This conversion caused no alteration in the EBNA-1 protein conformation. In the phylogenetic analysis the studied sequences with the sequences from GenBank was possible to observe two groups represented with type 1 and type 2 from EBV. 100% the samples studied was identified with the group characterized by the type 2 to EBV. So the seven samples showed the conversion, suggesting the origin of the one new subtype.
Feng, Yuqin. "Molecular epidemiological analysis of rabies viruses associated with population structure of bat hosts." Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27243.
Full textDingle, Kate Elizabeth. "The molecular characterisation of human enteric caliciviruses." Thesis, University of Southampton, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296166.
Full textBaeshen, Naseebh. "Molecular basis of adaptation of enteroviruses to different cancer cell lines." Thesis, University of Essex, 2015. http://repository.essex.ac.uk/15694/.
Full textLogan, Grace. "The molecular and genetic evolution of foot-and-mouth disease virus." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/7877/.
Full textBagashev, Asen. "MOLECULAR MECHANISM OF HIV-1 TAT INDUCED NEURONAL DYSFUNCTION." Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/255791.
Full textPh.D.
In the early years of the AIDS epidemic, being infected with the virus that causes the disease was considered a virtual death sentence. But with the development of highly active antiretroviral therapy (HAART), many infected with HIV-1 are living much longer. In fact, it is estimated that by 2015, about half of all HIV-positive individuals will be older than 50. Yet those over 50 also progress to AIDS faster than adults in their 20s or 30s. And those in the younger age bracket, even those responding well to antiretroviral therapy, still exhibit illnesses and clinical conditions commonly associated with older people, such as HIV-associated neurocognitive disorders (HAND), certain cancers, liver and bones diseases. For the most part, the reasons for this have remained a mystery. However, one may ask, how in the absence of circulating detected virus, viral proteins could cause this kind of damage. The answer is that eradication of latent viruses still unsuccessful and studies showed the persistence of HIV-1 in brain cells as well as the presence of viral proteins in CSF. This notion was supported by the compelling neuropathological data suggesting that the loss of Synaptic Plasticity occurs with the ongoing presence of virus and despite HAART. Clinically, these neuropathological data manifest by a gradual loss of working memory and learning disability, which promote alteration of synaptic plasticity that may manifest by symptoms similar to the ones observed in aged brain or what is called PREMATURE BRAIN AGING. Anatomically, working memory and learning ability functions are assured by neurons of the hippocampus, a brain area known-to-be affected by HIV-1 proteins. Mechanistically, several laboratories, including ours, demonstrated that viral proteins perform their functions through deregulation of several molecular pathways that can cause mitochondrial damage (such as depletion of mitochondrial calcium and release of ROS), inhibition of axonal transport leading to prevent neuronal communications or loss of long-term potentiation (LTP). Interestingly, CREB and BDNF proteins have been shown to play an important role in this phenomenon directly or through its downstream target genes. In here, we examined the impact of HIV-1 Tat on CREB-BDNF pathway and whether Tat is using this pathway to cause neuronal deregulation.
Temple University--Theses
Benjeddou, Mongi. "Molecular detection and genetic manipulation of the Black Queen Cell Virus." Thesis, University of the Western Cape, 2002. http://etd.uwc.ac.za/index.php?module=etd&.
Full textA reverse transcriptase PCR (RT -PCR) assay was developed for the detection of BQCV and acute bee-paralysis virus (ABPV). Complete genomes sequences w ere used to design unique PCR primers within a l-kb region from the 3' end of both genomes to amplify a fragment of 70.0 bp from BQCV and 900 bp from ABPV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing BQCV and ABPV. Sensitivities were of the order of 130 genome equivalents of purified BQCV and 1600 genome equivalents of ABPV.
Donald, Claire Louisa. "Development of molecular tools to enhance understanding of antiviral RNAi in mosquitoes." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6207/.
Full textBos, Sandra. "Undestanding the viral molecular factors involved in Zika virus pathogenicity in humans." Thesis, La Réunion, 2019. http://www.theses.fr/2019LARE0005.
Full textZika virus (ZIKV) is an unprecedented epidemiological phenomenon which surprised the world. For many years, it was considered a trivial virus responsible for only a handful of human infections, self-limited and benign, in Africa and Southeast Asia. But then, after decades of silent spread, a first epidemic broke out in Micronesia in 2007 – like a warning signal. A few years later, a sudden Zika outbreak of larger scale occurred in the Pacific islands before reaching Brazil in 2015. During this period, Zika was associated with severe neurological complications, highlighting its serious pathogenic potential for humans. Since its emergence, more than 80 countries and territories have been affected by the ZIKV pandemic, which is now recognized as a neurotropic and teratogenic virus. The association of contemporary ZIKV strains with severe forms of disease in humans, that have never been reported before, has raised the hypothesis of newly acquired pathogenicity. In this regard, my doctoral research aimed to determine whether the scope of the current epidemic was partly facilitated by viral factors that improved ZIKV fitness. To this end, my research project focused on the identification of the viral molecular factors involved in Zika virus pathogenicity in humans based on the development of molecular clones
Lowry, Kym Sheree. "The molecular determinants and consequences of recombination in the evolution of human enteroviruses." Thesis, University of Warwick, 2011. http://wrap.warwick.ac.uk/38168/.
Full textYamamoto, Brenda Michiyo. "Molecular Analysis of Human T-cell Leukemia Virus Type 2 Accessory Protein p28." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1241708950.
Full textTopley, Elize Lindsay. "Molecular detection and characterisation of RNA viruses of honeybees." Thesis, University of the Western Cape, 2009. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_6220_1298349602.
Full textPropagation methods for honeybee viruses have not changed since these viruses were discovered. There are no suitable cell lines or cell culture techniques available for honeybee viruses. Honeybee viruses have to be manually injected with virus in order for the virus to multiply and be extracted. With the presence of inapparent viruses which could co-infect pupae, a method for pure virus propagations needs to be found. Recombinant baculovirus systems have been used extensively to produce foreign proteins from different viruses using vectors and recombinant technology. In this chapter we inserted the capsid gene from BQCV into a transfer vector under the control of the p10 promoter of Autographa californica. Fractions of the sucrose gradient containing the virus like particles (VLPs) were seen under the electron microscope. A Western blot showed the four capsid proteins at the expected sizes for BQCV capsid. This study therefore has shown that a heterologous system such as baculovirus can be used for virus like particle production. Infectious virus technology has helped gain insight into how viruses work. Using this technology altering honeybee viruses could be used to observe different functionalities of the viruses. An attempt was made to interchange the open reading frames of ABPV and BQCV to observe any changes in virus assembly and infectivity. A fusion PCR strategy was employed to interchange the 5&rsquo
and 3&rsquo
ORFs of APBV and BQCV. The strategy however was unsuccessful. Alternative strategies could improve the chances of obtaining a chimeric virus.
Friday, Dillon R. "Processing of Potato Spindle Tuber Viroids (PSTVd) RNAs in Yeast, a Nonconventional Host." Thesis, University of the Sciences in Philadelphia, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10692985.
Full textThe discovery of viroids in 1971 opened the door to a whole new field of RNA biochemistry. Viroids subsequently became the first of many facets of RNA biochemistry: the first single stranded covalently closed RNA discovered in nature, the first subviral pathogen discovered, and the first pathogen of a eukaryotic system to have its genome sequenced. Viroids are the smallest known agents of infectious disease and they represent the borders of life. They replicate autonomously within their host and since they do not code for their own proteins, they act as scavengers of the host transcriptional machinery. By doing so, viroids find ways of trafficking, localizing, and replicating within their host based on the sequence and structure of the RNA alone. Once in their hosts, viroids are incredibly resilient and can cause economic damage on several commercial crops. Apart from controlling viroids for economic reasons, the more enticing feature of viroid study is the use of viroids as model systems to study essential underlying questions about the evolution of RNA pathogens, and to use viroids as models to study non-coding RNAs. The field of non-coding RNA research has surged within the past decade and viroids are becoming important vehicles to bring insight into this field of study. The study of viroids has been extensive through the years, but several questions remain: What structural conformations do viroids employ to recruit host enzymes, and what are the enzymes that cleave and ligate viroids into mature progeny. To answer some of these questions, we have looked at processing of the potato spindle tuber viroid (PSTVd) RNA in the budding yeast Saccharomyces cerevisiae. We found that one specific construct will process into a mature viroid circle in yeast and we also found that processing in this system is distinct from other plant and non-plant based host systems. This processing is a delicate interplay of ligation and degradation by host machinery. Yeast is a great system to study viroid processing as yeast allows for use of the entire toolbox of temperature-sensitive and knockout protein mutants. By employing yeast, focus can be driven towards the mechanisms of host protein recruitment, viroid processing requirements, and degradation mechanisms from the host. We have ascertained insight into PSTVd processing using yeast. We have found methods to transform and process PSTVd, investigated enzymes that effect processing, and started to establish an in vitro yeast system. Through these studies, we have also developed a method to enrich viroid RNAs from total RNA extractives. This has been vital to assays specific around viroid transcription and cleavage. Overall, this research is further testament that viroids are minimalist scavengers of a very diverse array of cellular transcriptional machinery. They can process in higher eukaryotes (plants) and simple eukaryotes (yeast). They are shown to affect each host in distinct manners using fundamental RNA biology that all organisms share.
Kissel, Jay D. "Target specificity and structural characterization of single-stranded DNA aptamer RT1t49, a broad inhibitor of HIV-1 reverse transcriptases." [Bloomington, Ind.] : Indiana University, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3274917.
Full textSource: Dissertation Abstracts International, Volume: 68-07, Section: B, page: 4320. Adviser: Donald H. Burke-Aguero. Title from dissertation home page (viewed Apr. 22, 2008).
Bowman, Latricia. "MUTAGENESIS OF AAV CAPSID PROTEINS FOR ENHANCED TRANSDUCTION OF CARDIAC CELL TYPES." Master's thesis, Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/310822.
Full textM.S.
Background: Adeno- associated virus serotype 9 (AAV9) has a high transduction efficiency for cardiac tissues, along with liver, skeletal muscle, pancreatic tissue, and the eye, versus other AAV serotypes. Unfortunately, nonspecific targeting to the intended tissue can result in the need for use of higher particle numbers to obtain intended transduction of the target tissue, which may also increase chances of initiating innate and adaptive immune responses. Such limitations require the need to bioengineer AAV9, and other serotypes including AAV6 and AAV8, in order to develop recombinant AAV vectors with cardiac tissue specific targeting and altered surface antigenic properties that may evade phosphorylation mediated degradation, neutralization, and clearance. Methods: Random mutagenesis PCR was used to mutate the external surface domains of AAV9 and chimeric AAV9-2 capsids. Mutations in the VP3 capsid sequences, cloned into helper plasmids pXR9 and chimeric pXR9-2 (substituting the 3-fold loop region of AAV9's VP3 with AAV2's), were used to generate a small library of rAAV9 or chimeric 9-2 vectors with mutant capsids. For accurate comparison, parent plasmids AAV9-2, AAV9 and AAV2 (with no mutations) were also used to generate rAAV vectors, each packaging the luciferase transgene. Mutant or parent vectors expressing luciferase were used to transduce HEK293 cells (HEK293s), neonatal cardiomyocytes (NCMs), and neonatal or adult rat cardiac fibroblasts (RCFs), in vitro (1,000 vp/cell). Relative luciferase activities and protein concentrations were measured (RLU/ug/uL), respectively. A second approach at mutagenesis of the pXR9-2 capsid was employed. Surface-exposed T and Y residues were substituted for Valine or Phenylalanine and an ischemic myocardium targeting peptide (ICTP) motif was inserted at site 588 of the VP1 capsid protein. Results: A small library of mutant rAAV9 or 9-2 vectors was screened in HEK293s, NCMs, and RCFs in vitro. Luciferase assay results led to the selection of several mutants, believed to have enhanced levels of transgene expression. After large scale production of mutant and parent vectors, in vitro transduction assays revealed mutant vectors did not transduce NCMs or RCFs cultures better than their respective parent vectors. Recombinant AAV9-2R mutants, containing all or some point mutations and/or ICTP, produced similar results. Some mutants exhibited changes in tropism properties that were different from the parent vectors. Still, parent vector AAV9-2 displayed robust transduction efficiency, much greater than the other parent or mutant vectors. These results encourage us to continue our investigation into its unique properties. Conclusions: This study reflects the difficulties of AAV capsid mutagenesis. We hope that this study can serve as a tool to further our understanding of AAV's molecular and structural biology properties.
Temple University--Theses
Leigh, Kendra Elizabeth. "Structural Studies of a Subunit of the Murine Cytomegalovirus Nuclear Egress Complex." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226065.
Full textMa, Natalie Jing. "Altering the Genetic Code to Probe and Control the Flow of Genetic Information." Thesis, Yale University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10584955.
Full textThe genetic code is highly conserved across all domains of life, enabling horizontal gene transfer (HGT) between organisms and across ecosystems via horizontally-transferred genetic elements such as viruses and plasmids. While HGT increases genetic diversity, it poses a risk to engineered biological systems by introducing new genes that destabilize engineered functions or allowing the expression of engineered genes in wild organisms with unknown effects. A model organism engineered with an alternative genetic code may provide new insight into the origins of the genetic code while also providing a stable chassis for engineered biological systems.
The Isaacs Lab recently developed an Escherichia coli strain lacking both UAG stop codons and Release Factor 1, resulting in the first genomically recoded organism (GRO) with an unassigned codon in its genetic code. Here, we demonstrate that this alternative genetic code lacking UAG codon assignment confers resistance to multiple viruses (λ, M13, PI, MS2) at titers up to 1011 PFU/mL and impairs conjugative plasmid function (F and RK2) up to 105-fold. Propagating viruses on a mixed microbial community containing standard and alternative genetic codes also reduced viral population fitness and prompted viral adaptation to the alternative genetic code. In investigating the molecular mechanism underlying the resistance to viruses and conjugative plasmids, we found that UAG-ending genes elicit ribosomal stalling and the tmRNAmediated ribosomal rescue response, resulting in degradation of UAG-ending proteins and suggesting that genomic recoding may be a broadly applicable strategy to impair horizontal gene transfer into other organisms.
To prevent the expression of engineered genes in wild organisms, we reassigned the UAG codon in the GRO to a sense codon incorporating the non-standard amino acid 4-acetylphenylalanine (pAcF) through the introduction of an orthogonal translation system (OTS). We then created a library of UAG-containing variants and assessed escape of UAG-containing genes from the GRO into wild-type organisms for both a non-selective green fluorescent protein (GFP) and selective chloramphenicol acetyltransferase (CAT) gene. While 1 UAG codon impaired the expression of GFP in wild-type organisms, at least 2 UAG codons were required in CAT to consistently prevent escaped expression in wild-type organisms with a standard genetic code. Additionally, sequencing revealed that wild-type organisms enabled expression of CAT by mutating UAG codons to UGG coding for tryptophan or CAG coding for glutamine. By placing UAG at sites in proteins that cannot tolerate a tryptophan or glutamine substitution, we can create UAG-containing genes further isolated from expression in wild organisms.
As biotechnology increasingly targets open-environment applications such as bioremediation or disease treatment in humans, we require methods to stabilize and control the genetic information that we encode in engineered biological systems. Because alternative genetic codes can both confer resistance to horizontal gene transfer into an engineered system and restrict expression of engineered genes in wild-type organisms, genomic recoding of organisms to contain alternative genetic codes is a promising path towards increasing the stability and safety of engineered biological systems. However, open-environment applications will expose engineered biological systems to new stresses not represented in the laboratory environment, and further work is required to validate these methods will be robust in conditions of limiting nutrients or other cellular stresses. Additionally, while we have demonstrated genetic isolation of the GRO with respect to genes both entering and leaving the cell, we cannot currently have both properties simultaneously because UAG is the sole open codon. We envision that current research into further codon reassignments, including the reassignment of sense codons, will pave the way for alternate genetic codes with multiple codon reassignments. By expanding recoding efforts to multiple species, we envision the development of synthetic microbial communities with alternate genetic codes that are genetically isolated and robust to perturbation by HGT.
Ali, Ibraheem Irfan. "Role of Post-translational Protein Modifications in Regulating HIV-1 and Mammalian Transcription." Thesis, University of California, San Francisco, 2019. http://pqdtopen.proquest.com/#viewpdf?dispub=13423596.
Full textThe molecular gatekeepers of nearly all gene expression in living cells are the proteins that function in the process of transcription. Transcription occurs when a cell must respond to a signal. These signals can be in the form of metabolic responses, signals for growth or differentiation, signals to defend against stress or pathogenic invasion, to name a few. The fundamentals of transcription have been extensively studied in bacterial systems and model organisms, but technical limitations have hindered their studies in mammalian and human systems. Recent developments in mass spectrometric methodologies, next-generation sequencing and techniques to study difficult-to-detect post-translational protein modifications are extensively reviewed here to highlight an important regulatory network through which gene expression is regulated. In addition, I present two vignettes: the first, a study of the regulatory mechanisms of monomethylation of the HIV-1 Tat protein in regulating HIV-1 gene expression and latency; the second, a study investigating the role of acetylation in regulating RNA Polymerase II protein modifications and gene expression in mammalian systems. Together, these studies combine new mass spectrometric techniques, modification-specific antibodies, protein purification methods, and next generation sequencing to better understand the role of these modifications in regulating the transcriptional response in mammalian systems. These findings can be applied to better understand mechanisms that regulate HIV-1 viral latency, along with fundamentally shifting the field of mammalian transcription by pinpointing unique modes of regulation only found in higher eukaryotes relevant to HIV-1 infection and cancer.
Mingot, Martí Ares. "Identification and functional characterization of P1N-PISPO, a new gene product present in sweet potato potyviruses." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/396653.
Full textEl virus Sweet potato feathery mottle virus (SPFMV) (Gènere Potyvirus, Família Potyviridae) presenta un genoma de cadena senzilla i positiva de ARN que conté una pauta de lectura oberta (ORF), traduïda com una poliproteïna que dóna lloc a un conjunt de productes gènics madurs, i una ORF curta anomenada PIPO que es troba en el marc de lectura -1 de la regió P3 de la poliproteïna. A més a més, en el seu genoma s'ha observat una ORF addicional anomenada PISPO. PISPO es troba en el marc de lectura -1 dins de la regió gènica de la P1 en SPFMV i els potyvirus més propers, començant en el motiu conservat G 1_2A6_7 i similar al motiu d'inici de PIPO. La expressió de PISPO durant la infecció del virus donaria lloc a la producció del hipotètic producte gènic P1N-PISPO. La presència de la pauta de lectura PISPO, precedida per un motiu G2A6, ha estat confirmada en un aïllat Espanyol de SPFMV. L'anàlisi de dades obtingudes per seqüenciació massiva, ha permès identificar una proporció significativa de transcrits que contenen una A extra en el motiu al començament de PISPO, així com una proporció menor de transcrits amb aquesta A extra en el motiu de PIPO. Aquests resultats han demostrat que un mecanisme d'edició de la polimerasa podria generar transcrits amb residus A extra (G2A7), els quals permetrien la traducció dels productes gènics P1N-PISPO i P3N-PIPO. L'anàlisi del productes gènics virals presents en teixits de plantes infectades amb SPFMV s'ha dut a terme mitjançant un experiment de LC-MS/MS. Pèptids corresponents a la part N-terminal de la proteïna P1 (abans del motiu d'edició) han estat detectats, així com pèptids exclusius de la part C-terminal de la P1 i de la pauta de lectura de PISPO. Aquests resultats han confirmat que tant P1 com P1N-PISPO són expressats i co-existeixen durant la infecció de SPFMV. L'expressió transitòria de productes gènics de SPFMV coagroinfiltrats amb un gen reporter en Nicotiana benthamiana ha revelat que P1N-PISPO actua com a supressor de silenciament d'ARN, un rol associat normalment a HCPro en altres potyvirus. Per últim, la mutació de motius WG/GW presents en P1N-PISPO aboleix la seva activitat com a supressor, suggerint que la funció pot està lligada a la interacció amb les proteïnes Argonauta de la maquinària de silenciament, tal i com passa en altres supressors virals.
McGeehan, John Edward. "Molecular characterisation of Herpes simplex virus type 1 deoxyuridine triphosphatase." Thesis, University of Glasgow, 1998. http://theses.gla.ac.uk/6104/.
Full textGodec, Jernej. "Molecular Mechanisms of CD8+ T Cell Differentiation." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493424.
Full textMedical Sciences
Jiménez, Melsió Alexandra. "Description of a novel species of Torque teno sus virus (TTSuV) and first insights on immunization against TTSuVs in naturally infected pigs." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/319700.
Full textAnelloviruses are a highly diverse group of circular single-stranded DNA viruses infecting vertebrates. Torque teno sus viruses (TTSuV) are ubiquitous pig-infecting anelloviruses. Three different viral species have been described, namely TTSuV1a and 1b within the genus Iotatorquevirus and TTSuVk2a, within the genus Kappatorquevirus. These viruses are genetically very distinct (>56% sequence diversity) but share similar genome organization and expression strategy. TTSuV infection in pigs is distributed worldwide, and is characterized by a persistent viremia. TTSuV themselves are considered non-pathogenic; however, it is believed that these viruses play a role in co-infection with other economically important viral porcine infections. Apparently, TTSuV infection can influence the development or may contribute to the pathogenesis of various porcine diseases during co-infection. The real impact of TTSuV on the pig health, if any, is still under debate. The present Thesis aimed to characterize a novel TTSuV species and to explore possible ways of vaccination against TTSuVs. The first study describes the discovery, genetic characterization and epidemiology of a novel TTSuV species, named Torque teno sus virus k2b (TTSuVk2b). According to phylogenetic analysis, this new virus belongs to the Kappatorquevirus genus, belonging to the same genus as TTSuVk2a. Quantitative PCR techniques based on SybrGreen technology were developed; one for quantification of total TTSuV load (TTSuV broad-spectrum qPCR) and others for quantification of each TTSuV species separately. These techniques were used for epidemiological studies and assess the geographical distribution. Moreover, prevalence and viral DNA load were determined in porcine circovirus type 2-systemic disease (PCV2-SD)-affected animals and healthy counterparts, since previous studies have associated another kappatorquevirus species, to the disease. The epidemiological study revealed that TTSuVk2b is worldwide distributed, although less abundant and displaying lower viral DNA titres in serum than TTSuV1 and TTSuVk2a. TTSuVk2b was associated with PCV2-systemic disease (PCV2-SD), which revealed that the two kappatorquevirus species are both genetically and biologically related. The second study contained two objectives. On one hand, TTSuV proteins were expressed in a baculovirus-based platform; on the other hand, the impact of a multivalent experimental vaccine was evaluated in a natural TTSuV infection model of pigs. The ORF1, ORF1-A, ORF2 and ORF3 recombinant proteins of all four known TTSuVs were successfully expressed in a baculovirus expression system. In additional, the multivalent experimental vaccine containing ORF1 and ORF3 proteins was administered by intramuscular and intradermal routes using two different vaccination schedules (twice or three times). Seroconversion and viral titres in blood were measured from 3 weeks until 15 weeks of age, using the indirect ELISA based on baculoviruses proteins and species-specific qPCRs, respectively. This study showed that vaccination induced anti-TTSuV antibodies; however the multivalent vaccine was not able to control viremia during TTSuV natural infection. Finally, in the third study, the immunization against TTSuVk2a during natural infection was evaluated using a different approach. The immunizations consisted of a combination of DNA and protein to increase the possibilities of activating both cellular and humoral immune responses. Quantitative PCR techniques were used to detect and quantify the viremia levels of each TTSuV species, while the induction of specific antibodies was monitored by indirect ELISA. The vaccinated group showed a seroconversion and a significant reduction of the TTSuVk2a viral loads compared to the control group. This study demonstrated for the first time that TTSuV viremia can be controlled by a combined DNA and protein immunization. Overall, the present Thesis contributes to increase the knowledge on TTSuV by means of describing a novel species, which may be involved in disease progression in co-infection with other pig pathogens. Moreover, TTSuV infection can be controlled by the administration of a combined DNA/protein immunization while a multivalent protein based vaccine was not efficient.
Buitrago, Laura Yaneth Villarreal. "Coronavirus em aves: detecção, caracterização molecular e filogenética e inoculação experimental em aves SPF." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/10/10133/tde-16062004-145626/.
Full textIn a broiler breeder farm located at São Paulo State two samples of intestinal contents of 2-week old birds, four fecal samples of the floor from the same flock at 18 weeks of age and one at 30 weeks of age were surveyed to coronavirus by a nested RT-PCR assay targeted to the RNA-dependent RNA-polymerase gene (pol gene). Six out of these samples were positive, two of which from the 2-week old birds, 3 from the 18-week old birds and 1 from the 30-week old birds. Phylogenetic analysis of the c-DNA sequence from one positive sample of a 2-week old bird showed that the virus clustered among group 2 coronaviruses. Oral and ocular inoculation of White Leghorn SPF birds with this coronavirus led to mild diarrhea, depigmentation and growth delay. Gross and histopatological examinations of collected tissues showed enteritis and atrophy of the villi on the small gut and rectum with mononuclear cells infiltration and exposition of the lamina propria. SPF sentinel birds placed between inoculated groups showed the same clinical signs and gross and histological findings. The inoculated coronavirus was evidenced in all experimental groups in the small gut, rectum, pancreas and lungs and also in intestinal contents. DNA sequencing and phylogenetic analysis was carried out with one positive sample per group, confirming the identity between the recovered viruses and the close relationship between this virus and group 2 coronaviruses taking into account the studied fragment. These results show that the coronavirus here found causes enteric disease in birds, with a predominant tropism by the rectum and the small gut, being horizontally transmitted in a few hours and that the pol gene studied segment bring this virus close to group 2 coronaviruses.
Wilkinson, Nicola. "Molecular biology and in vitro pathogenesis of Amsacta moorei entomopoxvirus (AmEPV)." Thesis, Oxford Brookes University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287761.
Full textMarcello, Lucio. "A bioinformatics and molecular analysis of antigenic variation in African trypanosomes." Thesis, University of Glasgow, 2006. http://theses.gla.ac.uk/5171/.
Full textKalantari, Mina. "Human papillomaviruses : role in cervical dysplasia and carcinoma, and use as molecular risk marker for progression /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3631-5/.
Full textForton, Daniel Michael. "The cerebral manifestations of chronic hepatitis C infection : cognitive, magnetic resonance spectroscopy and molecular virology studies." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414983.
Full textHou, Yixuan. "Porcine Epidemic Diarrhea Virus: Molecular Mechanisms of Attenuation and Rational Design of Live Attenuated Vaccines." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1562349484215276.
Full textSikorski, Alyssa. "Molecular characterisation of novel single stranded DNA viruses recovered from animal faeces." Thesis, University of Canterbury. Biological Sciences, 2013. http://hdl.handle.net/10092/8018.
Full textTucker, Timothy Johan Paul. "Epidemiology, molecular characterisation and tropism of the Hepatitis G Virus / GBV-C." Doctoral thesis, University of Cape Town, 1999. http://hdl.handle.net/11427/25669.
Full textZarama, Ortiz Angela María. "Efecto de la infección del citomegalovirus sobre receptores SLAM en macrófagos murinos." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/366749.
Full textThe signalling lymphocyte-activation molecules (SLAM) family of receptors encompasses a number of proteins expressed on the surface of leukocytes that play critical roles in both innate and adaptive immunity upon engagement through homotypic or heterotypic interactions amongst them. In this study, we show that murine cytomegalovirus (MCMV) drastically downregulates several SLAM receptors during the course of the infection of macrophages, in a manner dependent on viral gene expression. By screening a battery of MCMV deletion mutants, we have identified m154 as an immunoevasin that effectively reduces the cell surface expression of the SLAM family member CD48, a high affinity ligand for natural killer (NK) and cytotoxic T cell receptor CD244. m154 is a mucin-like protein, expressed with early kinetics, that can be localized predominantly at the cell surface of the infected cell. During infection, m154 leads to proteolytic degradation of CD48, primarily via a proteasomal dependent mechanism. This viral protein interferes with the NK cell cytotoxicity triggered by MCMV infected macrophages. In addition, we demonstrate that mutant MCMVs lacking expression of m154 result in an attenuated phenotype in vivo which can be substantially restored after NK cell depletion of mice. Thus, we present here a novel tactic of cytomegalovirus to subvert detection by NK cells during acute infection, based on the modulation of a SLAM family member.
Jesus, Danila Vedovello de. "Diversidade genética dos vírus influenza A detectados em crianças de São Paulo." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-17082012-090023/.
Full textInfluenza A virus infects a wide range of hosts and cause annual outbreaks. RNA segmented virus has high genetic variability and may have rearrangements between the genes of different viruses. In 2006, 521 samples of children younger than 5 years were analyzed and 25 tested positive for Influenza A virus, of which the subtype H3N2 is the most prevalent (68%). Five genes of 18 samples were sequenced and 13 sequences of HA, 12 of NP, 14 of M and 10 of NS obtained were. The presence of this several mutations, especially in the HA and NA genes probably helped the replacement of the vaccine strain in that year. All H3N2 subtype samples showed points of resistance to M2 inhibitors. The phylogenetic analysis revealed the circulation of different lineages in the same year, for both H1 and H3, and the presence of two rearrangements involving the HA and NP genes. These results indicate the influence of rearrangements in the evolution of the virus and emphasize the need for monitoring of circulation and characterization of genes.
Comone, Priscila. "Diversidade genética da hemaglutinina (HA) de vírus influenza A, entre 1995 e 2006." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-27092011-104745/.
Full textThe Influenzavirus can be classified according to their external glycoproteins hemagglutinin (HA) and neuraminidase (NA), both showing high genetic and antigenic variability. In the present study was carried out molecular analysis of the HA gene of influenza A (IA) in samples harvested from children and infants, with respiratory symptoms attended at University Hospital, University of Sao Paulo (USP), during the years 1995 to 2006. A total of 3,009 samples were analyzed by duplex RT-PCR and 4.38% (n = 132) were positive, being 12.1% (n = 16) Influenza B and 87.9% (n = 116) IA, where which 9% (n = 9) were H1N1 and 91% (n = 91) were H3N2 and 13.8% (n = 16) did not subtyped. The HA1 region of HA gene of 39 samples were sequenced and the sequences compared with vaccine strains and circulating strains in those years. The receptor-binding region was conserved in all samples and aminoacid changes were observed mainly in the antigenic sites and surroundings. Overall, the vaccine strains were consistent with those circulating in Sao Paulo.