Dissertations / Theses on the topic 'Virus-free'
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Richards, James Edward. "Engineering a helper virus-free reverse genetics system for rotavirus." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610743.
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Saxena, Pooja. "Development of RNA-free particles of Cowpea mosaic virus for applications in nanotechnology." Thesis, University of East Anglia, 2012. https://ueaeprints.uea.ac.uk/42355/.
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A method for the efficient production of RNA-free particles of Cowpea mosaic virus (CPMV) has been developed. These are generated by co-expression of the precursor of the coat proteins (VP60) and the viral proteinase (24K) using the highly-efficient plant expression system, CPMV-HT, in the model plant Nicotiana benthamiana. Particles thus produced were shown to be identical to CPMV on the outside and devoid of RNA on the inside and were hence named CPMV empty virus-like particles (eVLPs). The availability of large quantities of purified eVLPs represents a significant milestone in the development of CPMV-based particle technologies and their potential applications in nanotechnology have been investigated. eVLPs were shown be genuinely empty unlike other VLPs which package random cellular RNAs from the host. The high specificity of CPMV in packaging led to the investigation of the requirements for efficient packaging in CPMV where the functional coupling of replication and encapsidation was identified. Methods have been presented to extend this approach for packaging heterologous nucleic acids in eVLPs for their application as delivery vehicles. To obtain a continuous supply of eVLPs, methods for its stable expression were developed for which the suppressor of silencing deployed in the CPMV-HT system, P19, was modified as the use of wt P19 inhibits regeneration of leaf tissue. A mutant form of P19, R43W, with reduced but still substantial suppressor activity was shown to permit the regeneration of transgenic plants. P19/R43W was used for the stable expression of a variety of heterologous proteins showing the broad applicability of this system. To reduce the possibility of homologous recombination, an alternative to the CPMV-HT system was developed by deploying the UTRs from CPMV RNA-1. Expression with RNA-1 UTRs was rapid as compared to CPMV-HT and hence, the expression system was named Rapid-Trans.
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Cheung, Lok Man. "Investigation of virus inactivation and by-products formation under sequential disinfection using UV irradiation and free chlorine/monochloramine /." View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?CIVL%202004%20CHEUNG.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004.Includes bibliographical references (leaves 106-124). Also available in electronic version. Access restricted to campus users.
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Smith, Mark T. "Engineering Cell-Free Systems for Vaccine Development, Self-Assembling Nanoparticles and Codon Reassignment Applications." BYU ScholarsArchive, 2014. https://scholarsarchive.byu.edu/etd/4449.
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This dissertation reports on the technology of cell-free protein synthesis (CFPS) including 1) stabilized lyophilized cell-free systems and 2) enhanced heterogeneous cell extracts. This work further considers applications of CFPS systems in 1) rapid vaccine development, 2) functional virus-based nanoparticles, 3) site-specific protein immobilization, and 4) expanding the language of biology using unnatural amino acids. CFPS technology is a versatile protein production platform that has many features unavailable in in vivo expression systems. The primary benefit cell-free systems provide is the direct access to the reaction environment, which is no longer hindered by the presence of a cell-wall. The “openness” of the system makes it a compelling candidate for many technologies. One limitation of CFPS is the necessity of freezing for long-term viable storage. We demonstrate that a lyophilized CFPS system is more stable against nonideal storage than traditional CFPS reagents. The Escherichia coli-based CFPS system in this work is limited by the biocatalytic machinery found natively in E. coli. To combat these limitations, exogenous biocatalysts can be expressed during fermentation of cells prepared into extract. We demonstrate that simple adjustments in the fermentation conditions can significantly increase the activity of the heterogeneous extract. Towards virus-based particles and vaccines, we demonstrate that the open nature of CFPS can be utilized for coexpression of virus proteins and self-assembly of virus particles. This technique allows for the rapid production of potential vaccines and novel functional virus-based nanoparticles. Unnatural amino acids expand the effective language of protein biology. Utilizing CFPS as an expression system, we demonstrated that the incorporation of a single specific unnatural amino acid allows for site-specific immobilization, thus stabilizing the protein against elevated temperatures and chemical denaturants. Current unnatural amino acid incorporation technologies are limited to one or few simultaneous incorporations and suffer from low efficiency. This work proposes a system that could potentially allow for upwards of 40 unnatural amino acids to be simultaneously incorporated, effectively tripling the protein code. These projects demonstrate the power and versatility of CFPS technologies while laying the foundation for promising technologies in the field of biotechnology.
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Shahryarhesami, Soroosh [Verfasser], and Christoph [Akademischer Betreuer] Michalski. "Detection of bacteria and virus-associated Pancreatic Ductal Adenocarcinoma by cell-free protein microarray / Soroosh Shahryarhesami ; Betreuer: Christoph Michalski." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1208975218/34.
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Aljabr, Waleed A. "Using label free proteomics and RNA sequencing to investigate the human respiratory syncytial virus and the effects of the antiviral ribavirin." Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3004499/.
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Human respiratory syncytial virus (HRSV) is a known cause of severe lower respiratory tract infection (LRTI) in infants and young children worldwide. HRSV can cause illness in all ages especially those people at high risk including the immunocompromised and the elderly. Globally, HRSV infection leads to a significant healthcare and economic burden due to the lack of an approved vaccine and costly antiviral therapies that are potentially ineffective in some cases. Ribavirin is the only therapeutic licensed for the treatment of severe HRSV infection. It is a synthetic nucleoside with broad spectrum of antiviral activity encompassing both DNA and RNA viruses. The mechanism of action of ribavirin is unclear. It is thought to inhibit the replication of HRSV and lead to a reduction in viral load. How it does this is unknown and the subject of this thesis. This study focused on investigating the effect of the anti-viral ribavirin on cells in general and then infected with HRSV using both label free quantitative proteomics and transcriptomics. This allowed the investigation of the mutation frequency in HRSV and cellular protein abundance, which encompass several mechanisms by which ribavirin is postulated to work. This study was demonstrated that treatment of cells with ribavirin resulted in the increased transcription of selected cellular mRNAs including those involved in mediating anti-viral signalling. Additionally, ribavirin treatment caused a decrease in viral mRNA and proteins. In the absence of ribavirin, HRSV specific transcripts accounted for up to one third of total RNA reads from the infected cell RNA population. Ribavirin treatment resulted in a greater than 90% reduction in reads mapping to viral mRNA, while at the same time no such drastic reduction was detected for the abundance of cellular transcripts. The presented data revealed that ribavirin significantly increased the frequency of HRSV-specific RNA mutations in the viral genome, suggesting direct influence on the fidelity of the HRSV polymerase. The presented data shows transition and transversion substitutions occur during HRSV replication, and that these changes occurred in 'hot spots' along the HRSV genome. Examination of nucleotide substitution rates in the viral genome indicated an increase in the frequency of transition but not transversion mutations in the presence of ribavirin. In addition, the data indicated that in the continuous cell types used, and at the time points analyzed, the abundance of some HRSV mRNAs did not reflect the order in which the mRNAs were transcribed. Overall, the work describes a mechanism of action for ribavirin in the context of viral infection, that has not previously been elucidated for HRSV.
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Ludewig, Michael Hans. "The establishment of a virus free laboratory colony of Cryptophlebia leucotreta (False Codling Moth) and characterisation of Cryptophlebia leucotreta Granulovirus (CrleGV) genes." Thesis, Rhodes University, 2003. http://hdl.handle.net/10962/d1004016.
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Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
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Sanson, Raquel Koehler. "Development of a process of rabies virus production using BHK-21 cell line adapted to suspension in serum free media for vaccine production." reponame:Repositório Institucional da UFPR, 2013. http://hdl.handle.net/1884/44263.
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Orientador : Prof. Dr. Carlos Ricardo SoccolCoorientadora : Profa. Dra. Vanete Thomaz Soccol
Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa: Curitiba, 29/10/2012
Inclui referências : f.53-55
Área de concentração: Saúde humana e animal
Resumo: Soro animal é usado em cultivo celular por causa de seus fatores nutricionais, porém, seu uso é um risco potencial à saúde, devido a possível presença de agentes adventícios, tais como vírus e príons, e também é um dos principais responsáveis por reações alérgicas em cães. Células BHK-21 adaptadas à suspensão crescendo em meio de cultura padrão com 3% de soro fetal bovino foram submetidas à adaptação em meio de cultura livre de soro. Os meios de cultura livre de soro usados neste estudo foram VP-SFM, Ex-Cell 302 e Cellvento BHK-200. As células foram adaptadas ao VP por trocada direta do meio de cultura, ao Ex-Cell por troca direta e gradual do meio. Adaptação a Cellvento só foi possível usando células previamente adaptada a VP-SFM. As células adaptadas ao meio de cultura Ex Cell 302 apresentaram melhores resultados de crescimento. Todas as células adaptadas são capazes de produzir vírus da raiva. Porém, células adaptadas em VP-SFM apresentaram a melhor produtividade viral. Entretanto, produção de vírus utilizando Cellvento BHK-200 apresenta o melhor potencial econômico. Palavras chaves: vacina antirrábica veterinária, meio de cultura livre de soro, BHK-21, adaptação celular.
Abstract: Animal serum is used in cell culture because of its nutritional factors, however, their use not only is a potential risky for health, due to possible presence of adventitious agents, such as virus and príons, but also is the major responsible of allergic reactions in dogs. BHK-21 cells adapted to suspension growing in standard culture media with 3% of fetal bovine serum were submitted to adaptation to serum free culture media. Serum free culture media used in this study were VP-SFM, EX-CELL 302 and Cellvento BHK-200. Cells were adapted to VP by direct media change, to Ex Cell by direct and gradual media change. Adaptation to Cellvento was only possible by using cells priory adapted to VP-SFM. Cells adapted to Ex Cell culture media presented best growth results. All adapted cells have ability to produce rabies virus. Although, cells adapted to VP-SFM presented best virus productivity. However, virus production using Cellvento BHK-200 presents best economic potential. Keywords: veterinary rabies vaccine, serum free media, BHK-21, cell adaptation.
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Smiley, Jeffrey Raymond. "Characterization of the genomic stability of the VP2 hyper-variable region of infectious Bursal disease virus in the specific-pathogen-free Chick Embryo Host system /." The Ohio State University, 1999. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488190109870206.
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Nardini, João Paulo Calore [UNESP]. "Períodos de vernalização em bulbilhos semente livre de vírus de cultivares nobre de alho no cerrado brasileiro." Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/144466.
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O alho (Allium sativum L.) não possui semente verdadeiramente botânica, sendo assim, sua única via de propagação se dá vegetativamente, fato este que implica regularmente numa infecção viral mista, se tornando uma das principais causas da redução de produtividade. Alguns produtores já possuem acesso a semente de alho ‘Livre de Vírus’, no entanto, ainda utilizam de vernalização antiquada, preconizada pela pesquisa para alho semente infectada por vírus (material comum para maioria dos produtores nacionais), e quando utilizada pelos produtores em alho-semente ‘Livre de Vírus’, vem acarretando problemas principalmente para os produtores do cerrado, onde o plantio é antecipado. Neste estudo foi avaliado o efeito da temperatura de vernalização a 4ºC combinada por diferentes períodos (30, 40, 50 e 60 dias) em bulbilho-semente livre de vírus. O experimento foi conduzido de acordo coma a safra da cultura na região (março à outubro), em fazendas localizadas nos municípios de Santa Juliana (MG) e Campo Alegre de Goiás (GO): regiões de cerrado que se destacam atualmente pela produção de alho nobre no Brasil. Foram avaliadas três das principais cultivares existentes no mercado, sendo elas: Caçador, Quitéria e Ito. As maiores produtividades de bulbos comerciais para as cultivares Caçador em Campo Alegre de Goiás (GO) 2014 e Santa Juliana (MG) 2014 foram nos tratamentos de 30 e 40 dias de vernalização, respectivamente, conciliando produtividades de 11,3 t.ha-¹ e 12,4 t.ha-¹, com boa qualidade de bulbo. A cultivar Quitéria em Campo Alegre de Goiás (GO) 2014 alcançou melhor resultado com tratamento de 51 dias de vernalização, atingindo produtividade de 16,8 t.ha-¹. ‘Ito’ 2015 em Campo Alegre de Goiás (GO) e Santa Juliana (MG) atingiu produtividades interessantes do ponto de vista comercial aos 30 e 40 dias de vernalização, respectivamente, atingindo 16,7 e 17,1 t.ha-¹. Também foi avaliado que para bulbilhos livre de vírus ‘Caçador’, se demonstraram menos susceptíveis à formação de bulbos ‘charutos’ em baixos períodos de vernalização, do que bulbilhos convencionais. Em longos períodos de vernalizações, ‘Caçador’ livre de vírus é mais sensíveis ao aparecimento de brotações laterais do que o convencional. Verificou-se ainda que o efeito dos tratamentos de vernalização teve influência direta no IVD na semeadura, tempo de diferenciação (dias), número de folhas na diferenciação, ciclo da cultura (dias), incidência de bulbos charutos e brotações laterais em todas as cultivares avaliadas. Observou-se que do ponto de vista prático, que não é recomendável a utilização de extensos períodos de vernalização para produção de semente, já que esta possui notável relação com o estímulo à produção de bulbilhos com mais de uma gema.
Garlic (Allium sativum L.) does not really have truly botanic seeds; therefore, its only way of propagation is vegetatively, which regularly implies in a mixed viral infection that becomes the major cause of reduced productivity. Some producers already have access to garlic seed 'Virus Free', however, still use old-fashioned vernalization, recommended by the search for seed infected garlic virus (common material for most domestic producers), and when used by producers in garlic-seed 'Virus Free', has been causing problems especially for the cerrado producers, where planting is anticipated. This study evaluated the effect of vernalization temperature of 4 ° C combined for different periods (30, 40, 50 and 60 days) in virus free bulbil seed. The experiment was conducted in accordance eat the harvest of culture in the region (march to october), in farms located in the cities of Santa Juliana (MG) and Campo Alegre de Goiás (GO): cerrado regions which are currently out for garlic production noble in Brazil. Were evaluated three of the main existing cultivars on the market, which are: Caçador, Quiteria and Ito. The highest commercial bulbs for Caçador in Campo Alegre de Goiás (GO) 2014 and Santa Juliana (MG) in 2014 were the treatments of 30 and 40 days of vernalization, respectively, combining yields of 11.3 t ha-¹ and 12.4 t ha-¹, with good quality bulb. Quitéria in Campo Alegre de Goiás (GO) in 2014 achieved a better result with treatment of 51 days of vernalization, reaching productivity of 16.8 t ha-¹. 'Ito' 2015 Campo Alegre de Goiás (GO) and Santa Juliana (MG) reached interesting productivities commercial point of view at 30 and 40 days of vernalization, respectively, reaching 16.7 and 17.1 t ha-¹. It was also reported that for virus free bulbils of "Caçador", have proved less susceptible to the formation of bulbs 'cigar' in low periods of vernalization than conventional bulbils. Over long periods of vernalizações 'Caçador ' virus free is more sensitive to the onset of side shoots than conventional. It was also found that the effect of vernalization had a direct influence on the IVD during sowing, differentiation time (days) number of leafs in the differentiation, culture cycle (days), the incidence of ‘cigars’ bulbs and side shoots in all cultivars evaluated. It was observed that from a practical point of view, it is not recommendable to use long periods of vernalization for seed production, since it has remarkable relationship to stimulate production of bulbils over a gem.
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Bauer, Richard M. "Determination of feline interleukin 2 characteristics in specific-pathogen-free and feline leukemia virus-infected cats and the effects of 1,1-dimethylhydrazine on interleukin 1 and 2 activities in the murine system /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487330761218928.
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Kleidon, Jennifer. "Development of an excisable selectable marker system for banana." Thesis, Queensland University of Technology, 2019. https://eprints.qut.edu.au/134478/1/Jennifer_Kleidon_Thesis.pdf.
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This study describes the development of a recombinase-based platform for the removal of selectable marker genes from transgenic bananas. Using a steroid-inducible recombinase enzyme and dual selection vector containing the green fluorescent protein reporter gene, a protocol based on Agrobacterium-mediated transformation of Cavendish banana embryogenic cells was established. The system was then practically applied to generate the first ever marker-free banana plants with potential resistance to banana bunchy top virus. This platform will provide an effective means of improving this economically important crop into the future and is a major step towards public acceptance of genetically modified bananas.
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Soranzo, Thomas. "Approches Recombinantes pour l’Etude Structure/Fonction des Protéines E1, E2 et p7 du Virus de l’Hépatite C." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV056.
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Le virus de l'hépatite C (VHC) est une cause majeure d'affection hépatique chronique, notamment la cirrhose et le cancer du foie. On estime que 170 millions de personnes dans le monde sont des porteurs chroniques du VHC et que 3 à 4 millions de personnes sont infectées chaque année. Un des handicaps majeurs de la recherche sur le VHC est l'absence de systèmes de culture in vitro efficaces et de modèles animaux. Nous avons ainsi choisi une approche recombinante pour l'étude de protéines E1, E2 et p7 du VHC.Les protéines E1, E2 et p7 qui sont impliquées dans des étapes essentielles du cycle viral sont des protéines membranaires. Cependant, l'expression recombinante de cette classe de protéine est extrêmement complexe. En effet, la surexpression des protéines membranaires est souvent toxique pour les cellules hôtes. Ce phénomène est provoqué par l'agrégation ou la dégradation des protéines dans le cytoplasme dû à un manque de membrane disponible pour assurer leur intégration sur la cellule hôte. De plus, la surexpression de protéines membranaires induit la saturation de la machinerie cellulaire liée aux protéines membranaires. Ce détournement empêche le déroulement d'un cycle cellulaire normal et est ainsi fatal pour la cellule hôte. La forte concentration de protéines membranaires ou encore le fait que celles-ci soient hétérologues peut également provoquer la déstabilisation de la membrane de la cellule hôte et de son homéostasie. Afin de nous affranchir de ces limitations, nous avons utilisé une méthode de production des protéines membranaires sous forme native par un système acellulaire en présence de liposomes ; une technologie brevetée par l'université Joseph Fourier et exploitée par la société Synthelis. Dans un premier temps, nous avons procédé à la mise en place du système de production exploitant un lysat bactérien d'E. coli et d'un mélange énergétique complémentaire. Nous avons ensuite utilisé ce system pour étudier la viroporine p7. Cette protéine est essentielle pour la production de particules virales infectieuses et est impliquée dans l'assemblage viral ce qui en fait une cible thérapeutique intéressante. La production de protéoliposomes p7 en grande quantité nous a permis la caractérisation de la protéine par des techniques biochimiques et biophysiques. Nous avons mis en évidence l'inhibition de l'oligomérisation de p7 par le HMA qui ainsi inhibe sa fonction canal ionique. Grâce à la flexibilité du système d'expression acellulaire nous avons caractérisé la structure de la viroporine dans la membrane par réflectivité de neutron et avons confirmé la forme en entonnoir du complexe protéique. Des résultats préliminaires sur les proéoliposomes E1E2 quant à eux permettent d'espérer la production prochaine de particules virales mimant le VHC afin de mieux l'étudier et de lutter contre cette épidémie.L'ensemble de ces résultats confirment la pertinence de l'expression de protéines membranaires sous formes natives en système acellulaire en présence de liposomes. Les protéoliposomes produits constituent des nouveaux outils pour l'étude du VHC et permettent d'envisager de très grandes applications thérapeutiques ainsi que le développement de biomédicaments basés sur l'utilisation de protéines membranaires recombinantesThe Hepatitis C virus (HCV) is a major cause of chronic liver disease, including cirrhosis and liver cancer. An estimated 170 million people worldwide are chronically infected with HCV and 3 to 4 million people are infected each year. One of the major handicaps of the HCV research is the lack of effective in vitro culture systems and animal models. To adress this issue, we chose a recombinant approach to study the E1, E2 and p7 proteins of HCV.The E1, E2 and p7 proteins are involved in critical steps of the viral cycle. They are membrane proteins, a class of protein that is extremely complex to express. Indeed, overexpression of membrane proteins is often toxic to the host cells. This phenomenon is caused by protein aggregation or degradation in the cytoplasm due to a lack of available membrane space for their integration into the host cell. Moreover, overexpression of membrane proteins induces saturation of the cellular machinery linked to membrane proteins. This diversion prevents the flow of a normal cell cycle and is fatal to the host cell. Destabilization of the host cell's membrane and its homeostatis may also be caused by the high concentration of membrane proteins or their heterologous nature. To circumvent these limitations, we used a method for producing membrane proteins in their native form by a cell-free system in the presence of liposomes; a technology patented by the University Joseph Fourier and licenced by the startup company Synthelis. First, we have set up the cell-free production system using a bacterial lysate from E. coli and a complementary energy mix. We then used this system to study the p7 viroporine. This protein is essential for the production of infectious virus particles and is involved in viral assembly making it an attractive therapeutic target. The production of a large quantity of p7 proteoliposomes allowed us to characterize the protein by biochemical and biophysical techniques. We have demonstrated the inhibition of oligomerization of p7 by HMA, which thereby inhibits its ion channel function. Thanks to the flexibility of the cell-free expression system we have characterized the structure of the viroporine within the membrane in a neutron reflectivity assay and have confirmed the funnel shape of the protein complex. Preliminary results on proteoliposomes E1E2 offer hope for the production viral particles mimicking the hepatitis C virus in order to better study the virus and fight against this epidemic.Together, these results confirm the suitability of the expression of membrane proteins in native forms using a cell-free system in the presence of liposomes. Proteoliposomes products are a new tool for the study of HCV and consideration for very broad therapeutic applications and the development of biopharmaceuticals based on the use of recombinant membrane proteins
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Fogeron, Marie-Laure. "Development of a wheat germ cell-free expression system for the production, the purification and the structural and functional characterization of eukaryotic membrane proteins : application to the preparation of hepatitis C viral proteins." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10081/document.
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Alors que 30% du génome code pour des protéines membranaires, moins de 3% des structures protéiques dans la Protein Data Bank correspondent à ces protéines. En raison de leur nature hydrophobe, les protéines membranaires sont en effet très difficiles à produire dans des systèmes d'expression classique en cellules, notamment en bactéries. L'étude structurale des protéines membranaires du virus de l'hépatite C (VHC) sous forme entière et native a donc été pendant longtemps entravée. Le VHC est un virus à ARN positif dont le complexe de réplication est basé sur un réarrangement spécifique des membranes induit par l'action concertée de plusieurs protéines non structurales du virus dont NS2, NS4B et NS5A. La structure tridimensionnelle et le rôle de ces protéines dans la réplication virale sont encore mal connus. Pour surmonter les limitations qui empêchent leurs études structurales et fonctionnelles, un système d'expression acellulaire à base d'extrait de germe de blé a été développé avec succès, permettant la production des protéines NS2, NS4B et NS5A entières directement sous une forme solubilisée en présence de détergent. Ces protéines membranaires sont produites et purifiées par chromatographie d'affinité dans des quantités de l'ordre du milligramme. Des analyses par filtration sur gel indiquent que les échantillons obtenus sont homogènes. De plus, des analyses structurales par dichroïsme circulaire montrent que les protéines produites dans ce système sont bien repliées. Leur reconstitution dans des lipides est en cours d'optimisation. Le but ultime est en effet de déterminer leur structure par RMN du solide dans un environnement lipidique mimant l'environnement natifWhile 30% of the genome encodes for membrane proteins, less than 3% of protein structures in the Protein Data Bank correspond to such proteins. Due to their hydrophobic nature, membrane proteins are indeed notoriously difficult to express in classical cell-based protein expression systems. The structural study of the membrane proteins of hepatitis C virus (HCV) in their full-length and native form has therefore been for long time hampered. HCV is a positive-strand RNA virus building its replication complex on a specific membrane rearrangement (membranous web), which serves as a scaffold for the HCV replicase, and is induced by the concerted action of several HCV non-structural proteins including NS2, NS4B and NSSA. The knowledge of the three- dimensional structure of these proteins and their role in virus replication is still limited. To overcome the limitations that prevent the structural and functional studies of these proteins, a wheat germ cell-free protein expression system has been developed. A production protocol was designed which allows us to directly obtain membrane proteins in a soluble form by adding detergent during the in vitro protein synthesis. A large number of mainly viral proteins were successfully expressed, and full protocols were developed for the full-length NS2, NS4B and NSSA proteins. These membrane proteins were produced and purified by affinity chromatography using a Strep-tag II in the milligram range. These protein samples are homogenous, as shown by gel filtration analysis. Moreover, structural analyses by circular dichroism showed that the proteins produced in the wheat germ cell-free system are well folded. Reconstitution of these proteins in lipids is currently under optimization. The ultimate goal is to determine their structure by solid-state NMR in a native-like membrane lipids environment
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David, Guillaume. "Towards structural studies of Hepadnavirus subviral particles using wheat germ cell-free expression and solid-state NMR." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1336.
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Les études structurales des protéines membranaires eucaryotes sont importantes mais particulièrement difficiles à effectuer car elles nécessitent non seulement un système efficace et pratique pour produire la protéine dans une conformation native, d’une technique d’étude structurale compatible avec ce dernier. Du fait de leur modularité, les systèmes de production acellulaires in vitro sont adaptés à la production de protéines membranaires. De plus, l’amélioration de leur robustesse et de leur efficacité les rendent maintenant comme une alternative viable à l’expression cellulaire. Parmi ceux-ci, le Système d’Expression Acellulaire à partir de Germes de Blé (SEA-GB) est le plus efficace pour produire des protéines membranaires eucaryotes, et permet de plus un marquage isotopique efficace et spécifique. Ce dernier point est très utile pour la Résonance Magnétique Nucléaire (RMN), et plus spécifiquement la RMN du solide qui permet l’étude structure de protéines membranaires et d’assemblages macromoléculaires. Depuis récemment, la RMN du solide est compatible avec le SEA-GB, formant un outil puissant pour l’étude structurale de protéines membranaires et assemblages macromoléculaires. Dans ces travaux, les deux techniques ont été combinées pour la production et l’étude des protéines d’enveloppe du virus de l’Hépatite B du canard (VHBC), appartenant à la famille des Hepadnaviridae. Ces virus sont capables de sécréter des virions actifs, mais aussi des particules composées uniquement de protéines d’enveloppe, appelées particules sous-virales (PSV). Dans un premier temps, nous montrons que plusieurs milligrammes de petite protéine d’enveloppe (DHBs S) du VHBC sont produits sous forme soluble avec le SEA-GB. DHBs S forme des PSV durant la traduction, ce qui confirme la conformation native de la protéine. Après désassemblage des PSV, la protéine est majoritairement en hélice , synonyme d’un bon repliement. Après isolation par ultracentrifugation sur gradient de sucrose, les PSV ont été sédimentées dans un rotor de 0.7 mm et étudiées par RMN du solide. Des spectres 2D hNH très prometteurs ont été obtenus, avec un bon signal, des pics isolés et une résolution similaire à celle d’autres protéines membranaires sédimentées et étudiées par RMN du solide. De plus, la superposition du spectre de DHBs S avec des spectres simulés de protéines modèles possédant des structures secondaires caractéristiques confirme que DHBs S est principalement en hélice dans le contexte des PSV. Le signal doit cependant être amélioré pour pouvoir réaliser les expériences nécessaires à des études structurales approfondies, c’est pourquoi des tests d’optimisation de la production ont été effectués. D’une part, l’amélioration du rendement de production, via l’utilisation d’un extrait de germes de blé commercial, et de la stabilisation des PSV, par incubation avec du KSCN, ont été testés. D’autre part, différentes méthodes de purification ont été examinées: précipitation à l’ammonium sulfate ou au PEG6000, incubation à haute température, élimination de contaminants via une unité d’ultrafiltration, purification d’affinité ou d’exclusion stérique ainsi qu’un test de désassemblage des particules, suivie d’une purification puis de la reconstitution des PSVs en présence de lipides. Enfin, un marquage isotopique spécifique de certains acides aminés a été évalué. Dans la seconde partie, nous avons étendu les possibilités du SEA-GB via l’expression de la grande protéine d’enveloppe (DHBs L) du VHBC. In vivo, la protéine est phosphorylée spécifiquement et subit aussi une traduction alternative ; nous avons montré que c’était aussi le cas dans le SEA-GB. Nous avons aussi testé la coexpression de DHBs S, DHBs L ainsi que de la capside de DHBV pour inclure DHBs L dans les PSV, voire même reconstituer des virions entiers, ce qui augmenterait les possibilités du système. Enfin, nous avons aussi détaillé certains paramètres critiques pour la formation des PSV dans le systèmeStructural studies of eukaryotic membrane proteins are of prime importance but notoriously difficult as they not only necessitate an efficient and practical overexpression system that allows for membrane protein expression in a biologically relevant folding, but also a structural technique that you can easily combine with the chosen protein production system. In vitro cell-free systems, due to their modulable nature, are particularly suited for membrane protein expression. Furthermore, they now established themselves as a viable alternative to conventional cell-based expression, notably because of considerable advances in robustness and efficiency. Amongst them, the wheat germ cell-free production system (WG-CFPS) proved to be the most efficient for production of eukaryotic membrane proteins, and allows for efficient and specific isotope labeling. This makes it particularly convenient for Nuclear Magnetic Resonance (NMR), and more specifically solid-state NMR which is particularly appropriate for membrane protein studies and macromolecular assemblies. Thanks to very recent advances that lead to a drastic reduction of the quantity of protein needed, solid-state NMR is now compatible with WG-CFPS, creating a powerful tool for structural studies of macromolecular assemblies and membrane proteins. In this work, these two techniques are combined for the production and study of the envelope proteins from the duck Hepatitis B Virus (DHBV), that belongs to the Hepadnaviridae family. These viruses are able to secrete active virions, but also particles composed only of envelope proteins, which are called subviral particles (SVPs). In the first part, we show here that the DHBV small envelope protein (DHBs S) is produced as soluble in mg amounts using WG-CFPS. Even more, the protein forms SVPs upon translation, and is thus expressed in a biologically relevant form. After SVPs disassembly, the protein displays a mostly -helical folding, which is characteristic of a well-folded protein, and also very similar to the secondary structure of an assembly-incompetent mutant. After further isolation by ultracentrifugation on a sucrose gradient, the SVPs were sedimented in a 0.7 mm rotor and observed by solid-state NMR. Very promising hNH 2D spectra, with a good signal, were obtained. They display numerous isolated peaks and a resolution alike to other sedimented membrane proteins observed by solid-state NMR. Moreover, superimposition of the DHBs S spectrum with simulated spectra from proteins with extreme secondary structure content confirms that the protein is mostly -helical in the context of the SVPs. Nonetheless, the signal still needs to be improved in order to perform the experiments necessary for in-depth structural analysis. To that end, sample optimization assays were conducted. On the one hand, protein yield improvement, by the use of a commercial wheat germ extract, and SVPs stabilization, by incubation with KSCN, were tried. On the other hand, different methods for SVPs purification were tested, including PEG6000 or ammonium sulfate precipitation, incubation at high temperature, contaminant removal with an ultrafiltration device, affinity or size-exclusion purification as well as tests of particles disassembly, purification followed by SVPs reconstitution in lipids. Finally, amino-acid specific isotopic labeling of DHBs S was evaluated. In the second part, we could show extended possibilities of WG-CFPS through expression of DHBV large envelope protein (DHBs L). In vivo, the protein undergo specific phosphorylation as well as alternative translation, and we could show that it is also the case upon wheat germ cell-free expression. We also tested coexpression of DHBs S, DHBs L and of the DHBV capsid in order to assess the possibility of DHBs L inclusion in SVPs, or even complete virion reconstitution, which could even augment WG-CFPS possibilities. Ultimately, we also detail some critical parameters for SVPs formation in the WG-CFPS
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Delmas, Véronique. "Structure et proprietes biologiques du papovavirus de hamster." Paris 6, 1986. http://www.theses.fr/1986PA066550.
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Le papovavirus de hamster (hapv) possede un tropisme restreint in vivo vis a vis des keratinocytes et des lymphocytes. Il se replique dans les tumeurs cutanes qui apparaissent chez des hamsters syriens, et induit egalement des lymphomes chez le hamster. L'organisation genetique du hapv deduite de sa sequence a montre qu'il appartient a la famille des polyomavirus. Le hapv est present dans les lymphomes sous forme de multiples copies libres possedant toujours une deletion localisee dans la meme region du genome. Les signaux de transcription precoce du hapv semblent etre actives par la region precoce de ce virus
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Patiño, Sandra Fernanda Suárez. "Produção de proteínas recombinantes em células BHK-21 cultivadas em meio livre de soro fetal bovino." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-25082016-145258/.
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Células eucariotas usadas como plataforma de expressão de proteínas recombinantes são geralmente cultivadas com soro fetal bovino (SFB), porém, abordagens biotecnológicas atuais sobre cultura de células devem evitar o uso deste suplemento, devido a problemas de custo, variações entre os lotes e risco de contaminação. Assim, nosso objetivo foi expressar as proteínas recombinantes: GFP (proteína verde fluorescente), NS3 (proteína não estrutural 3 do vírus da hepatite C) e RVGP (glicoproteína do vírus da raiva) em células BHK-21 adaptadas em meios livres de soro fetal bovino (SFM) usando o sistema de expressão baseado no Semliki Forest Virus (SFV). Os resultados do presente trabalho mostraram que células adaptadas em SFM cresceram de forma eficiente, produziram mais partículas virais recombinantes de SFV do que células suplementadas com soro, sendo que estas partículas virais podem ser usadas diretamente para imunização, pois garantiram uma amplificação e expressão eficiente das diferentes proteínas dentro da célula hospedeira.Eukaryotic cells are cultured with serum, however current biotechnological approaches of cell culture need to avoid using of this supplement, due to the high costs, lot-to-lot variation and risk of contamination. Thus, our aim was to express the recombinant protein: GFP (green fluorescent protein); NS3 (Hepatitis C virus non-structural protein 3) and RVGP (rabies virus glycoprotein) in BHK-21 cells cultured in serum free culture based on Semliki Forest Virus system. The results of this work showed that cells cultured in serum-free media (SFM) were grown efficiently, they were produce more recombinant viral particles when compared with cells supplemented with SFB. These viral particles can be used directly for immunization, since generated amplification and expression efficient of different proteins within the host cell.
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Brandi, Tarrau Nuria Mercedes. "Estrés oxidativo en el neonato prematuro y en pacientes pediátricos infectados por el VIH." Doctoral thesis, Universitat de Barcelona, 2010. http://hdl.handle.net/10803/668859.
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El aumento en la generación de radicales libres en los sistemas biológicos asociada a un desequilibrio entre la producción y eliminación de los mismos da lugar al estrés oxidativo, que juega un importante papel en múltiples fisiopatologías.
Dado que se trata de un proceso que puede generar graves alteraciones, tanto celulares como tisulares, nos planteamos como objetivo general de esta tesis, estudiar el estrés oxidativo y el sistema antioxidante en dos situaciones patológicas, relativamente frecuentes en el campo pediátrico: prematuridad e infección por el VIH.
En la última fase de la gestación, el metabolismo fetal se modifica en preparación para el momento del parto. El feto adapta su sistema antioxidante para enfrentarse con éxito a la vida extrauterina en la que se produce un cambio brusco en las concentraciones de oxígeno del entorno. Cuando el neonato entra en contacto con una presión parcial de oxígeno superior a la intrauterina los componentes enzimáticos del sistema antioxidante responden aumentando su actividad. Los neonatos prematuros no se enfrentan de igual forma a la vida extrauterina que los nacidos a término, siendo por tanto más susceptibles a cualquier agresión oxidativa a la que sean expuestos. Algunas patologías, como la displasia broncopulmonar, se han relacionado directamente con su incapacidad para defenderse de los efectos tóxicos del oxígeno, por inmadurez de su sistema antioxidante. La mayoría de prematuros que precisan oxígeno en sus primeros días de vida, suman a su inmadurez pulmonar la de su aparato digestivo, lo que origina que no puedan alimentarse por vía enteral. Por ello se debe recurrir a la administración parenteral de nutrientes, como glucosa, aminoácidos y lípidos en forma de Intralípid® al 20%. Los ácidos grasos poliinsaturados constituyen el principal componente del Intralípid® y son altamente susceptibles a la peroxidación. Los hidroperóxidos formados, pueden originar graves alteraciones dependiendo de los niveles tisulares ya existentes y de la cantidad de antioxidantes presentes. Esto es crucial en los neonatos prematuros sometidos a terapia con oxígeno, el cual es un desencadenante importante de los fenómenos de oxidación y generación de radicales libres.
El estudio de antioxidantes y su relación con la prematuridad durante un largo período de tiempo permitirá conocer su etiología y su repercusión en niños prematuros con y sin oxigenoterapia; también se podría clarificar los agentes tóxicos asociados a la aparición de la displasia broncopulmonar.
Uno de los mecanismos que contribuye a la progresión del SIDA es el estrés oxidativo inducido por la producción de radicales libres, el cual puede jugar un papel importante en la estimulación de la replicación del VIH y en el desarrollo de la enfermedad. El OH, por ejemplo, activa el factor de transcripción nuclear NF-B imprescindible para la replicación viral por lo que ésta se ve potenciada en células infectadas por el VIH. Esta alteración oxidativa puede ser prevenida o atenuada por el sistema de defensa antioxidante. Esto depende de la integridad del sistema enzimático, que requiere una ingesta adecuada de elementos traza tales como Se, Zn, Cu y Mn y de una concentración adecuada de vitaminas A, C y E. La deficiencia plasmática de cisteína en pacientes VIH positivos altera la síntesis del glutatión, esencial para la detoxificación de los radicales libres y estudios in vitro sugieren que suprime la replicación viral. La enfermedad causada por el VIH repercute de manera importante en el crecimiento de la población pediátrica infectada y un 80% de los niños presentan algún déficit nutricional durante su evolución. Contribuyen a este problema la alteración de la función gastrointestinal, la mala absorción y la malnutrición siendo esta última un marcador de progresión de la enfermedad en niños. El retraso en el peso y la talla constituye una de las manifestaciones clínicas más comunes de la infección por el VIH en niños. La aparición de nuevos fármacos y de familias de antirretrovirales y su utilización como terapia combinada ha permitido controlar la replicación del VIH. Esto, a su vez, ha convertido la infección en una patología de curso crónico en aquellos pacientes que disponen de tratamiento de forma regular. La utilización de más de un fármaco permite asociar compuestos con actividad frente a líneas celulares distintas. Sin embargo, la terapia combinada es de cumplimiento difícil y no está exenta de toxicidad a corto, medio y largo plazo.
El estudio de la relación entre el estrés oxidativo, el estado nutricional y el tratamiento pautado en niños infectados por el VIH es importante desde el punto de vista clínico. Es de esperar que la suplementación con antioxidantes, a dosis inocuas, y el cambio de terapia según la respuesta del paciente, podría mejorar su estado clínico y virológico, y por tanto, aumentar su calidad de vida.
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Woodman, P. G. "The development of a cell-free assay for the insertion of a viral glycoprotein into the plasma membrane." Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.384535.
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Van, Boeckel Thomas. "Intensive poultry production and highly pathogenic avian influenza H5N1 in Thailand: statistical and process-based models." Doctoral thesis, Universite Libre de Bruxelles, 2013. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209417.
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Le virus de l’influenza aviaire hautement pathogène (IAHP) de type H5N1 apparu en Chine en 1996 constitue une menace pour la santé humaine en raison de sa circulation endémique dans les volailles domestiques et de son potentiel zoonotique. La sévérité de l'infection liée à l'IAHP H5N1 est variable selon les espèces d'oiseaux: certains anatidés sont porteurs sains et asymptomatiques du virus tandis que dans les élevages de poulets, l'IAHP est fortement contagieux et caractérisé par des taux de mortalité supérieurs à 90%. Chez les humains, l'impact de l'IAHP H5N1 reste à ce jour modéré (630 cas humains dont 375 morts, World Health Organization Juin, 2013) en raison de la faible transmission du virus des volailles aux humains et d'humain à humain. Cependant, étant donné les taux de létalité élevés (>50%), un changement des modalités de transmission pourrait mener à un impact beaucoup plus élevé.Depuis son émergence, l'IAHP H5N1 a eu un impact économique important dans de nombreux pays d’Asie du Sud-Est. La Thaïlande, pays qui fait partie des principaux exportateurs mondiaux de viande de volaille, a été sévèrement touchée par les multiples vagues épidémiques entre 2003 et 2005. Ces épisodes ont eu un impact sur les revenus des petits et moyens producteurs, mais également causé des pertes économiques importantes dans le secteur de la production intensive de volailles en raison de l'embargo imposé par les principaux marchés d'exportation.
L'objectif de ce travail est d’étudier quantitativement l'association entre la production intensive de la volaille et la distribution spatio-temporelle de l'IAHP H5N1 en Thaïlande. Deux approches ont été développées pour aborder cette étude: le développement d’une part de modèles statistiques visant à identifier les déterminants du risque d'IAHP H5N1, et d'autre part, de modèles mécanistiques visant à simuler des trajectoires épidémiques sur base de la connaissance des mécanismes de transmission de l'IAHP H5N1, de la structure du secteur de la production de volaille et des mesures d'intervention mises en place.
A l’aide de facteurs environnementaux et anthropogéniques, nous montrons que: (i) la distribution des canards domestiques en Asie peut être prédite en utilisant des modèles de régression non-linéaire, et (ii) la production de volailles peut être désagrégée entre production extensive et intensive sur base du nombre de volailles par éleveur. Enfin (iii), nous montrons en utilisant des arbres de régression boostés ("Boosted Regression Trees", BRT) que les principaux déterminants de la distribution du risque d'IAHP H5N1 sont les canards élevés en systèmes intensifs, le nombre de cycles de culture de riz et la proportion d'eau présente dans le paysage. Finalement, nous illustrons les potentialités des modèles mécanistiques pour évaluer l'efficacité des mesures d'intervention implémentées, tester des scénarios alternatifs d'intervention et identifier des stratégies optimales de prévention et d'intervention contre de futures épidémies
Doctorat en Sciences agronomiques et ingénierie biologique
info:eu-repo/semantics/nonPublished
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Paschoal, Juliana Fontes Beltran. "Expressão gênica empregando pseudopartículas em células de mamíferos (HEK 293T e Huh 7.0) cultivadas em diferentes meios de cultura livres de soro." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-24082016-113658/.
Full textAbstract:
Células HEK293T e Huh7 foram adaptadas em meios livres de soro fetal bovino (SFM). Parâmetros metabólicos e de crescimento foram avaliados, além da expressão gênica heteróloga, utilizando um sistema de expressão que produz pseudo-partículas (ppHCV), derivadas do vírus da Leucemia Murina (MLV) e da Hepatite C (HCV). A adaptação foi realizada através de diluição sequencial para SFM. A linhagem HEK293T foi adaptada em dois SFM: Hybridoma-SFM e CHO-S-SFMII, a linhagem Huh7 foi adaptada nos quatro SFM escolhidos. O consumo de substratos para cada linhagem foi diferente entre os SFM, apesar de o crescimento celular ter sido semelhante. Para a análise da expressão gênica, três vetores foram co-transfectados em células HEK293T. Foi observado que para a produção de ppHCV, o tempo de coleta foi de 48 horas. O método de co-transfecção por lipofectamina produziu mais cópias de vírus, sendo que quantificações de 5,30x103 cópias RNA/μL foram encontradas para vírus produzidos em células adaptadas no meio Hybridoma-SFM através de qRT-PCR. Estas ppHCV foram usadas para infectar células Huh7, células infectadas produziram cerca de 10 ng de proteína recombinante/106 células.HEK 293-T and Huh7 cells were adapted in serum free mediu (SFM). Metabolic and growth parameters were assessed, as well as heterologous gene expression, using an expression system that produces pseudo-particles (ppHCV), derived from the murine leukemia virus (MLV), and Hepatitis C (HCV). The adaptation was performed by sequential dilution in SFM. The HEK- 293T line was adapted in two SFM: Hybridoma-SFM and CHO-S-SFMII, the Huh7 line was adapted in four chosen SFM. The consumption of substrates were different for each line in SFM, while cell growth was similar. For the analysis of gene expression, three vectors were co-transfected into HEK-293T cells. It was observed that for the production of ppHCV, the collection time was 48 hours. The method of co-transfection with lipofectamine produced more copies of the virus into the cells, 5,30 x103 RNA copies/μL were found to virus produced in the cells adapted in Hybridoma- SFM, by qRT-PCR. These ppHCV were used to infect Huh 7, infected cells produced around 10 ng recombinant protein /106 cells.
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CHEN, CANG-HAI, and 陳滄海. "STUDIES ON BAMBOO MOSAIC DISEASE-VIRUS ISOLATION, SEROLOGY AND VIRUS-FREE SEED- ING PRODUCTION." Thesis, 1990. http://ndltd.ncl.edu.tw/handle/54220924030591584177.
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博士國立中興大學
植物病理學研究所
79
Bamboo mosaic disease occurs widely in bamboo plantation in Taiwan. The affected bamboo usually shows mosaic symptoms on leaves. On green bamboo (Bambusa oldhanii), the mosaic symptoms are also observed on young culms and brown streaks may appear on branches and older culms. Symptoms on Taiwan giant bamboo ( Dendrocalamus latiflorus ) also include brown lesions on interior tissues of shoot and brown streaks on culms and shoot sheath. from twenty counties of Taiwan, eighty three virus isolates were isolated from mosaic affected samples of thirteen bamboo species. Based on host plant reactions they were divided into two groups. There were eighty isolates in group I, while other three isolates were group II. Those isolates in group I could induce local lesions on Chenopodium spp. and Gomphrena flobosa. In group I, B-7, B-17 and B-1 isolates which isolated from Taipei, Taichung and ingtung, respectively were selected for further characterization. The thermal inactivation point of B-7 and B-17 isolates was 75-80 C, While that of B-1 isolate was 80-85 C. The dilution end point of B-7 and B-17 isolates was 10-5 - 10-6 and that of B-1 was 10-6 - 10-7. All three isolate remained infectious after stored at 24oc for one month. They were flexuous rod-shaped particles of 500*15 nm. There were three isolates in group II. They were isolated from severe necrotic shoot sheath of Taiwan giant bamboo. B-81 isolate was selected for further studies. It was flexuous rod-shaped particle of 600-630*15 It induced local lesions on Chenopodium spp. but not on Gomphrena flobosa. The local lesions formed on Chenoppdium spp. Were significantly different from those induced by those isolated in group I. Polyclonal antiserum was produced against group I B-1 isolate. In ochterlony tests, the results showed that group I B-1, B-7, B-17 isolates and group II B-81 isolates were serological identity. However, B-1 isolate was not serologically related to Cymbidium mosaic virus, Cassava common mosaic virus, cactus virus X and Potato virus X. ELISA was used to detect BoMV.A combination of 1 ug/ml of r-globulin and 10-3 dilution of enzyme-conjugated r-globulin was the optmium condition for ELISA test. Under which, it could detect purified BoMV at concentration of 100 ng/ml. Bamboo mosaic virus distributed unevenly in different parts of bamboo shoot. Concentrations of bamboo mosaic virus also differed in different leaf position or leaf parts. The contents of BoMV in leaves of green bamboo and Taiwan giant bamboo rised from July and reached a maximum on November. The virus could survive in roots of green bamboo for 6 weeks. The enzyme-conjugated immunoglobulin prepared by maleimide method was 5.5 fold more sensitive than that of the conjugated immunoglobulin prepared by glutaraldehyde method. It could detect the BoMV at the concentration of 10 ng/ml. Antiviral chemicals idoxuridine, amantading, moroxydine and ribavirin have been incorported into green bamboo shoottip culture medium to eradicate BoMV. The result showed that applying ribavirin at concentra- tion of 20-100 ug/ml could obtain 7-46% BoMV-free bud. However, applying ribavirin at 100 ug/ml appeared to be slightly phytotoxic to green bamboo culture. The growth rate of bud in culture was decreased. 竹嵌紋病普遍發生於臺灣各地竹園，除造成葉片嵌紋病徵外，尚會在綠竹上引起竹稈 嵌紋及褐條斑，以及在麻竹上造成竹稈褐色條斑，筍籜出現黑褐色條紋，筍肉內散生 黃色至褐色斑點呈縱向延伸．因而導至罹病竹出筍率降低，同時竹筍木質化，品質不 良． 由臺灣二十個縣市，十三種竹類嵌紋病害標本上分離得到83個單斑分離株，又依其在 寄主上的反應差異可歸納兩類，第一類共八十個分離株屬之，再由此選用台北（代號 B-7） ，台中（B-17），屏東（B-1 ）三分離株為代表，測試其寄主範圍及理化性質 反應，三者皆能在藜科植物及千日紅葉片形成局部斑，B-7、B-17、B-1三者之稀釋終 點分別為 （圖表省略） 而耐熱性則分別為75∼80℃，75∼80℃，及80∼85℃，24℃下三者之活性皆可維持一 個以上．病毒粒子形態三者大小皆為500×15nm， 屬相同病毒．第二類共有三分離株 屬之,主要來自麻竹以代號B-81 為代表，其寄主範圍和第一類所不同處為不感染千日 紅，但皆感染番杏，在奎藜上斑點形態也相異．病毒粒子形態雖然亦為絲狀，較第一 類為長，大小為600∼630×15nm． 選用南部分離株B-1 為代表，純化病毒為抗原製備抗血清，以此抗血清對B-1、B-7、 B-17及B-18進行瓊脂雙擴散反應測試，發現對不同分離株之抗原皆能產生相互融合而 無spur的沈降帶出現，由上述所得結果，認為B-81應為BoMV的麻竹系統．另外以同樣 方法以B-1 抗血清測試同屬PVX 群的Cymbidium mosaic virus, Cassava common mosaic virus, Cactus virus X 及Potato virus X等四種Potexvirus群病毒，並 無抗原抗體反應出現，而知本病毒異於此四種potexvirus． ELISA 試驗，以純化免疫球蛋白濃度1ug/m1連抗體稀釋 （圖表省略） 倍之反應條件最適當，有效偵測到100ug/m1濃度之嵌紋病毒，而田間竹類病葉汁液雖 稀釋至 （圖表省略） 倍在波長405nm 下尚有吸收值．綠竹、麻竹不同葉序，以及同一葉序的不同部位組織 中的嵌紋病毒含量雖然有所差異．但尚不至於影響ELISA 對病毒的偵測．罹病綠竹、 麻竹的各部位病毒含量也各不相同，用1,000 倍稀釋連抗體偵測150 支筍的結果， 病毒檢出率以筍尖之70％∼78％及筍芽56％∼65％∼較高，無病徵筍肉最低僅5 ％∼ 10％，一年中罹病綠竹、麻竹葉片中嵌紋病毒含量變化由7 月急速上升，至11月達到 最高．為了將來罹病竹園的更新所需，測試本病毒在綠竹根系中殘存時間為六週．調 查本省15屬，62（品）種竹類中，有2 屬，14種受嵌紋病毒為害．且以篷萊竹屬的13 種居大多數．為了要改進目前所採用的戊二醛標誌抗體易造成相互結合的缺失，以便 提高對莖頂組織培養所得培植體中，所含微量病毒之偵測，利用馬來西工胺法製備所 得鹼性磷酸連抗體使用雙層抗體連反應比較時，其靈敏度高出目前的戊二醛法所 製連體5.5 倍，且前者能偵測之病毒濃度下限可達10ng/m1 ，而後者僅至100ng/m1 ．不同濃度之四種抗病毒藥劑idoxuridine ，amantadine，moroxydine及ribavirin 添加於綠竹莖頂組織培養之培養基中以治療組織中之竹嵌紋病毒，結果僅ribavirin 一種從20ug/m1 有治療效果，其效果隨ribavirin 濃度之增加及培養時間之延長，其 無毒率亦可由7 ％增加到46％．但是當vibavirin 濃度達到100ug/m1，則對培植體有 輕微抑制植株伸長之毒化作用，導至芽的生長速率減緩． ///////
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Chen, Yu Chuang, and 陳宥庄. "Studies on the production and cultivation of virus free shallot(." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/35840774320471123787.
Full textAbstract:
碩士國立中興大學
園藝學系
85
Local and introduced shallot cultivars 'hia-Yi'''hwei-Chia,'' Fu-Chen'','S-28'','Philippine'' and 'Thailand'' were used to study the infuluence of shoot apex size,number of weeks in heat therapy and growth regulator for obtaining the highest propagation rate and best culture condition of producing virus- free seedings. When shoot apex with one leaf primordium (approximately 300 m)hadbetter virus-free and survival rate. If shoot apex culture with heat therapycould increase the virus- free rate. One handred percent virus-free plantlet could be obtained when shallot plant subjected to 37 for 16 hours and 32 for 8 hours daily continued for 8 weeks. The best culture medium for 'Chia-Yi'' shallot was the MS medium with Picloram 0.140 mg/l,and BA 8.6 mg/l, or with Picloram 0.39 mg/l, and TDZ 0.03 mg/l, with NAA1.1 mg/l and BA 5.3 mg/l, as well as with NAA 0.4 mg/l and TDZ 0.3 mg/l. The best culture medium for 'Thailand'' shallot shoot apex was the MS medium with Picloram 0.03 mg/l and TDZ 1.0mg/l. Yield trial in the field and storage test from virus-free plantletoriginated from shoot apex culture showed plant hight was better than ordinary plants. Number of tiller. average plant of number of bulbs per plant, and singlebulb weight of virus-free plant were higher than ordinary plant. The bulb weight decereased in ordinary bulbs was three times higher than the virus-freebulbs after 6 weeks storage was 3 times heavier of that of normal bulb.
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Lin, Ching-Yi, and 林靜宜. "Development of marker-free transgenic plants with resistance to Tomato leaf curl Taiwan virus and Tomato spotted wilt virus." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/03032368461375267573.
Full textAbstract:
博士國立中興大學
植物病理學系所
98
Abstract Whitefly-transmitted geminiviruses (Geminiviridae) and thrips-borne tospoviruses (Bunyaviridae) are two groups of extremely important plant viruses. Current transgenic approach based on genetic engineering technology provides an efficient strategy to breed plants to resist viral infection. However, public concerns about the use of antibiotic- and herbicide-resistance genes for the selection of transgenic plants during the transformation process have increased tremendously. Therefore, the objectives of this study were to develop the reliable transformation system that could remove selectable markers while generating transgenic plants that would resist the infection of geminiviruses and tospoviruses. Firstly, Tomato leaf curl Taiwan virus (ToLCTWV), a predominant tomato-infecting geminivirus in Taiwan, was subjected to investigate which viral gene fragments can confer high resistance to geminiviruses in transgenic plants. Individual transgenic constructs covering the entire ToLCTWV genome was transformed into Nicotiana benthamiana plants. Four constructs including IRC1 (intergenic region flanked with 5′ end of C1), C2 (partial C2 ORF), C2C3 (overlapping region of C2 and C3 ORFs) and Rep2 (3′ end of the C1 ORF) of high resistance for ToLCTWV have been observed. The detection of siRNA in transgenic plants confirmed that the mechanism of resistance was via gene silencing. Moreover, the middle half of the N gene of Tomato spotted wilt virus (TSWV), which is the type member of Tospovirus, was fused with the partial C2 ORF of ToLCTWV as the chimeric transgene and transformed into N. benthamiana and tomato to develop transgenic plants with multiple viral resistance. The transgenic plants remained symptomless post agro infected with ToLCTWV and exhibited high resistance to TSWV. The detectable siRNAs demonstrated that the resistance was mediated by gene silencing mechanism. The results also explained that linking multiple gene fragments of two viruses with different genomic organization was an effective strategy to engineer plants against both DNA and RNA viruses. Meanwhile, we developed three strategies of co-transformation to generate marker-free transgenic plants; they were (1) pGANP-CP1/pBin19, which comprises two individual plasmids carrying T-DNA of the target and marker genes separately; (2) pGA2T-CP1, which consists of one plasmid carrying two T-DNAs for the target and marker genes; and (3) pGA2TNH, which contains two T-DNAs in one plasmid in which one T-DNA carries the bi-selectable marker which can be used for more plant species especially those with low sensitivity to kanamycin. The co-transformation frequencies of the R0 transgenic N. benthamiana plants for both selection marker and target gene were similar. The co-transformation frequencies of three vector systems were about 50%. Segregation of transgene and selectable marker gene was revealed in the progeny of some co-transformed lines. The highest production ratio of marker-free transgenic plants was 24.1% in two plasmids system, followed by 18.6% in one plasmid system and 17.5% in bi-selectable marker system. We demonstrated that these strategies were feasible and efficient to eliminate the marker genes, and can provide a practical and simple tool for generating marker-free transgenic plants. Therefore, previously mentioned gene fragments that confer high resistance to ToLCTWV including IRC1, C2, C2C3 and Rep2 were linked together and fused with the middle half of the N gene from TSWV to make a chimeric transgene to be constructed into the binary vector, pGA2TNH, for the generation of viral resistance marker-free transgenic N. benthamiana. The transgenic R0 plants resistant to ToLCTWV were obtained and the marker-free resistant progeny plants were segregated by self-pollination. Overall, the results showed in this study have important implications for field deployment of transgenic strategies to control geminivirus and tospovirus. Moreover, the plant transformation systems that can generate marker-free plants would certainly boost the public acceptance of transgenic crops.
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Chen, Chun-Cheng, and 陳俊丞. "PCR free detection of hepatitis B virus DNA using a nanostructured impedance biosensor." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/41461326880034226870.
Full textAbstract:
碩士國立中興大學
機械工程學系所
102
Chronic hepatitis B virus (HBV) infection is an important health burden because of its worldwide prevalence and potential adverse outcomes, including liver cirrhosis and hepatocellular carcinoma. Although the DNA level of HBV in the serum is an important biomarker associated with several important outcomes of HBV patients, it is not regularly monitored in clinical practice because of its high cost. So it is necessary to develop a highly sensitive and low-cost technique for effective detection of HBV concentration in blood. In this study, a PCR free technique for effective detection of HBV DNA obtained directly from clinical samples was presented. We use a sensitive nanostructured biosensor with a sensing electrode of gold nanoparticles (GNPs) uniformly deposited on a uniform nanohemisphere array. A specially designed single-strand gene fragment of the target genomic DNA of HBV based on the genome sequences of HBV was immobilized on the nanostructured electrode as the capture probe. Target HBV DNA obtained from clinical samples were hybridized to the sensing probes. Detection results illustrate two dynamic linear ranges, 10^2-10^3 and 10^3-10^5.1 copies/mL, having R^2 values of 0.801 and 0.996 could be obtained, respectively. The detection limit of the proposed sending scheme was measured to be 111 copies/mL. The total of 45 target samples, including 20 samples with HBV concentration being lower than 102 copies/mL and 25 samples with HBV concentration being in the range of 10^3-10^5.1 copies/mL. A detection limit of 186 copies/mL could be achieved. The concentration of these 45 HBV DNA samples was measured by the COBAS Ampliprep system. Comparing the measured results of the COBAS Ampliprep and our system, it was illustrated that the HBV DNA concentrations measured by the proposed method in this study had a high linear correlation with the COBAS Ampliprep, having R^2 values of 0.983 .The proposed sensing scheme is highly feasible for future clinical applications.
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LI, CHUN-MING, and 李俊明. "A study on the Business Models of Providing Free Anti-Virus Software Services Companies." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/7426dh.
Full textAbstract:
碩士國立高雄第一科技大學
資訊管理系碩士專班
105
ABSTRACT As the rapid development and population of the Internet, The IT company supply a free software for using has become a new management model, such as software , online games and Apps. This study is going to explore how to get a benefit from free software. This studyapplied the nine elements of Osterwalder (2012) as a tool to analyze the business model of these three free anti-virus companies.The resuit of analysis shows the anti-virus software provides scan,virus cleaning and also other additional funtions.(such like the improvement of PC efficacy,cleaning trash,encryption management…and so on. )to attract clients. Taking care of Customer-centric and using Community website to understand what clients need to develop useful functions to fit uesrs. Establish membership to help marketing expansion and strategy. The anti-virusof these three free anti-virus companies are free forever,manufacturers according to customer segments (family,industry) to apply simple, updrade and industry value and get benefit from ads, computing platform income and license fee for software.The research tells free marketing mode is a trend. The advantage of company is up to the value(efficacy,funtions and operabilities) of production under the market competition. Superior quality to attract users and improve market share. Key words: Free Business Model, Business Model Canvas, Antivirus Software
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Chiou, Shyan-Song, and 邱賢松. "Molecular and epidemiologic characteristics of Japanese encephalitis virus isolated from a paddy-free islet." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/57372751325896512408.
Full textAbstract:
博士國立臺灣大學
流行病學研究所
89
The distribution of Japanese encephalitis (JE) has been widespread throughout Asia, from the southeastern ex-USSR in the North to Australia in the South. In Taiwan, the incidence of clinical JE cases significantly declined since mass vaccination was initiated in 1968, although sporadic case is reported periodically. Liu-Chiu, the islet located in the southwest coast of Taiwan, has been free of JE cases in the past decades. However, it is believed that JE virus has been circulating in Liu-Chiu because natural JE antibody has been prevalent among swine and the virus T1P1 strain was isolated from locally collected Armigeres subalbatus mosquitoes. In this study, we have analyzed molecular and epidemiologic characteristics of JE virus isolated from Liu-Chiu. The results were summarized into three parts: Seroepidemiology of Japanese encephalitis among residents in Liu-Chiu. In order to unravel this epidemiological phenomenon, we first analyzed the prevalence of both neutralizing and IgM antibodies specific to JE virus (T1P1 strain) in 219 blood samples collected from residents of Liu-Chiu islet. The overall positive rate was 48.4% for neutralizing (Nt) and 4.1% for IgM antibodies. In specific, the older the age, the higher the positive rate of Nt antibodies but the lower the IgM. Because the positives of both antibodies did not cluster around the port area, the virus circulating on the islet may have been endemic but not imported. In comparison with another strain CC-27 that was previously isolated in the main island of Taiwan, the Nt titer specific to T1P1 was higher in samples obtained from Liu-Chiu and vice versa. It indicated that the circulating JE virus in Liu-Chiu must be or rather close to T1P1, which was genetically different from CC-27. Characteristics of JE virus T1P1 strain isolated from Liu-Chiu. In mouse neuroblastoma-derived Neuro-2a cells, T1P1 appeared a significantly lower productivity than another local isolate CH1392. It implied that this new isolate possesses a characteristic viral replication pattern other than CH1392. T1P1 has also shown lower in neurovirulence which was reflected by a significantly higher LD50 (2.44 x 106 PFU) in comparison with CH1392 (2.87 x102 PFU). In comparison on the full-length RNA sequences between T1P1 and CH1392, a total of 7 nucleotides including one in preM/M and two each in NS3, NS5 and 3’-end noncoding region (NCR) appeared different. Of them, only the changes in NS3 (position 325; “U” for CH1392, “A” for T1P1 and position 364; “G” for CH1392 and “A’ for T1P1) have resulted in substitutions of deduced amino acid. There were two additional nucleotide changes appeared in the 3’-noncoding region (3’-NCR). The amino acids 109 “Phe” and 122 “Glu” in NS3 of CH1392 were substituted by “Ile” and “Lys”, respectively, in T1P1. The unique growth properties and low virulence of T1P1 presented in this report were likely related to abnormal enzymatic activity due to mutations of the NS3 gene and possibly to the mutations in the 3’-NCR. The naturally attenuation of T1P1 that has been circulating in the paddy-free Liu-Chiu islet may account for the absence of clinical JE cases in past years. Selection of JE virus variants in vitro and in vivo. Alteration of plaque size has been seen in both T1P1 and CH1392 strains of JEV after only one passage in mouse neuroblastoma-derived Neuro-2a cells. Further analysis on this phenomenon has shown several interesting results: (1) existence of different variants within each JE virus strain; (2) the selective effect differ between insect and mammalian cells; (3) selection in mosquitoes may be responsible for slow evolution of arboviruses; (4) organ-specific selection may exist for each JE virus variant; and (5) a JE virus quasispecies model has been establish to study pathogenesis of the virus. These findings have implicated that each JE virus subpopulations may play a role in viral evolution and pathogenesis. In summary, the intensive transmission of Japanese encephalitis virus may actually exist in Liu-Chiu, a region conventionally free of epidemic encephalitis. The absence of clinical cases may be associated with the circulation of the low virulent JE virus strain. In addition, the uniqueness of this virus strain obtained from paddy-free Liu-Chiu may be resulted from evolution of JE virus in such as independent and unconventional ecosystem.
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Venugopal, Vishnu. "Modelling How Refractoriness to Interferon Compromises Interferon-Free Treatment of Hepatitis C Virus Infection." Thesis, 2017. http://etd.iisc.ernet.in/2005/3612.
Full textAbstract:
Hepatitis C virus (HCV) infection globally affects 130-150 million people. It causes both acute and chronic infections. Due to the severe side effects and low success rates of interferon based treatments, which formed the standard treatment for HCV, the treatment paradigm shifted to direct acting antivirals (DAAs).
DAAs have revolutionized the treatment of hepatitis C virus infection. Clinical trials with combinations of DAAs have recorded >90% response with shorter treatment durations and fewer side effects than earlier treatments involving IFN. Outside the controlled setting of a clinical trial, however, response rates with DAA combinations are much lower (<70%). DAAs can fail if HCV accumulates mutations that confer drug resistance. Interestingly, the pre-existence of mutant frequency in the virus appears not to influence treatment outcome. A better predictor for DAA treatment outcome is yet to be unravelled. Surprisingly, individuals who respond poorly to IFN appear to be more likely to fail DAA treatment. IFN is a generic antiviral that improves immune responses and is expected not to have any bearing on DAA treatment outcomes. Why individuals with poor IFN sensitivity fail DAA treatment remains a mystery. In a recent study of the IFN signalling network, HCV has been shown to compromise IFN activity. It induces bistability in the network leading to distinct phenotypic responses of cells to IFN exposure. In particular, individuals who respond poorly to IFN tend to have a higher percentage of cells that are refractory to IFN; these cells allow viral persistence despite IFN exposure. We hypothesized here that in such individuals, greater ongoing replication would allow increased development of resistance and thus lead to the failure of DAAs. We constructed a model of viral dynamics that accounts for the distinct phenotypic responses of cells to IFN, viral replication and mutation, and the development of resistance to DAAs. Our model predicted that although the relative prevalence of pre- existing mutants is unaffected by IFN sensitivity, in agreement with observations, the growth of drug resistant mutants is accelerated in individuals with poor IFN sensitivity. Based on a distribution of IFN sensitivity across individuals, our model accurately described clinical observations of the response rates to different current treatment protocols. With this model, we predict that the common strategy of increasing the genetic barrier by adding more drugs to the combination was not necessary to avert the development of drug resistance. Instead, an optimised increase in DAA dosage alone or DAA+PR or PR dosage depending on the patient’s IFN sensitivity could help achieve success.
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Choijilsuren, Gansukh, and 札蘇光士. "Heparin at physiological concentration can enhance PEG-free in vitro infection with human hepatitis B virus." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/19861337332921895539.
Full textAbstract:
博士國立陽明大學
生化暨分子生物研究所
105
Hepatitis B virus (HBV) is a blood-borne pathogen responsible for chronic hepatitis, cirrhosis, and liver cancer. The mechanism of HBV entry into hepatocytes remains to be investigated. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was discovered as a major HBV receptor based on an in vitro infection system using NTCP-reconstituted HepG2 cells. However, this infection system relies on the compound polyethylene glycol (4% PEG), which is not physiologically relevant to human infection. High concentration of heparin has been commonly used as an inhibitor control for in vitro infection in the field. Surprisingly, we found that heparin at physiological concentration can enhance HBV infection in a PreS1-peptide sensitive, NTCP-dependent manner in both HepaRG and HepG2-NTCP-AS cells. O-sulfation of heparin is more important for the infection enhancement than N-sulfation. This system based on the HepG2-NTCP-AS cells can support in vitro infection with HBV genotypes B and C, as well as using serum samples from HBeAg positive and negative chronic carriers. In summary, our study provides a PEG-free infection system closely resembling human natural infection. In addition, it points to a future research direction for heparin and heparin-binding host factor(s) in the blood, which are potentially involved in viral entry.
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Lee, Jeng-Jie, and 李正傑. "Expressions of mdm2, p53 and Other Host Genes in Epstein-Barr Virus-Infected and -Free Nasopharyngeal Carcinoma Cells." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/99647698550575683016.
Full textAbstract:
碩士國立臺灣大學
病理學研究所
88
The purpose of this experiment was to understand whether the p53 regulated mdm2 gene expression was altered in an EBV-infected NPC cell line and biopsy specimens. Immunolocalization of MDM2 in the NPC cell line revealed that MDM2 was distributed in the cytoplasm and nuclei. When NPC-TW01 cells were infected by EBV through endocytosis of EBV-IgA-SC complex, the intensity of anti-MDM2 reaction product was moderately enhanced. The expression of mdm2 mRNA was also increased. RT-PCR analysis of 5 NPC biopsy specimens showed four positive and one negative mdm2 mRNA expression; however, in NPC biopsy specimens only a few cases appeared with negative immunostaining. Most other cases showed some cells containing reaction products and other cells revealing negative staining. Double localization of MDM2 and EBER-1 revealed that some tumor cells contained both EBV signal and MDM2 protein; other cells showed EBV signal or MDM2 protein only. Another cell fraction revealed neither EBV infection nor MDM2 protein expression. This phenomenon was also shown in an animal model in which NPC solid masses were induced by transplantation of EBV-infected and EBV-free NPC culture cells. Double localization of MDM2 and p53 protein showed that while only some MDM2 positive cells contained p53 protein, most of p53 positive cells contained MDM2 reaction product. Subcloning and sequencing of mdm2 gene from NPC-TW01 showed a heterozygous deletion in two regions (466-600 and 814-1004). Transfection of wild and mutant p53 genes separately into NPC-TW01 cells revealed up-regulation of MDM2 expression in wild-type p53 gene transfected cells only. It is concluded that EBV infection may up-regulate mdm2 mRNA and protein expression in vitro, but in vivo, MDM2 expression may not totally be up- regulated by EBV infection and p53 protein. This difference indicates that some cancer cells may increase mdm2 gene expression through other unidentified factors. The expression of MDM2 in the host cells can not be affected by either heterozygous deletion or mutation of mdm2 gene. Additionally, it was desired to understand whether EBV could regulate the expressions of the genes other than mdm2 and p53 genes in EBV-infected and -free human biopsy specimens and in tumor masses induced in the animal model which were produced by transplantation of EBV-infected and EBV-free NPC cells. The observed genes: MMP-2, MMP-9, VEGF, bFGF, and EGFR. MMP-2 and MMP-9 were involved in the metastasis of the tumors for the degradation of collagen. By immunostaining, reaction products were seen in certain NPC cytoplasm; however, a few mesenchymal cells such as fibroblast some mononuclear cells in the stroma regions also showed reaction products. Double localization of EBV and these enzymes revealed that the expressions of MMP-2 and MMP-9 were seen in EBV positive and negative cells. However, the reaction product of MMP-9 is stronger than that of MMP-2. The reaction products of VEGF were aggregated on certain NPC cellular cytoplasm and stroma regions, and in the vascular endothelial cells in some specimens. VEGF was also expressed in EBV positive and negative tumor cells. The reaction products of bFGF were also appeared in NPC cytoplasm and independent on the distribution of EBER-1 products. When EGFR was investigated, the reaction product was seen on NPC cell membranes; the expression of EGFR was consistently matched with the distributions of EBER-1 in EBV-infected mouse specimens. However, EGFR was also expressed in EBER-1 negative cells. It is concluded that EGFR expression could be up regulated by EBV infection in some NPC cells, but the overexpressed EGFR in EBV-free cells indicates that other factors in the NPC cells also play some role in regulation of its expression. The expressions of other genes were not regulated by EBV in both human biopsy specimens and mouse specimens.
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Wu, Kai-Syuan, and 吳愷軒. "A serum-free Vero cell culture process for the production of a local infectious bursal disease virus isolate." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/qnhmg2.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
107
Infectious bursal disease virus (IBDV) belongs to the genus Avibirnavirus of family Birnaviridae, and mainly infects chicken fibroblast lymphocytes resulting in a death by severe immunosuppression and secondary infection. According to the previous literature, the very virulent infectious bursal disease virus (vvIBDV) was adapted to grow in Vero-cell line after ninth serial passages. This ninth passage virus was evaluated as live attenuated Vero-cell adapted vaccine (Rasool and Hussain 2006). To produce a local infection for the establishment of serum-free Vero cell culture technology .The labor of the diseased bursal disease virus is developed for the follow-up vaccine. In this study, the Vero cells were cultured in a small amount in a serum-free medium, and the infection was continued with the IBDV P3009 native virus strain. The virus was adapted to the cell replication production. After that, it was observed that the viral titer was significantly improved. Afterwards, the cells were cultured in a Microcarrier and a roller bottle, respectively, and infected with a virus. A serum-free Vero cell suspension culture system was constructed by microcarriers combined with spinner flask .Vero cells which can be grown in serum-free state, and adjust the type and volume of the medium, stirring speed, ventilation and other factors. To establish optimal conditions for cell growth. Roller bottles are mainly used with roller mixers to allow cells to grow on a large area of the roller wall. The system culture of both systems is stable. Vero cells were infected with IBDV P3009 at different multiplicity of infection (MOI) of 10-1, 10-2 or 10-3, respectively, and the virus titer was determined with TCID50 (Median tissue culture infectious dose) assay. Analysis of progeny virus production in cell culture revealed a maximum titer of 107 pfu/ml after 96 hours post infection MOI of 10-2. The produced virus solution was subjected to immobilized-metal ion affinity chromatography (IMAC) for virion purification and the complete IBDV particles were confirmed after transmission electron microscopy examination. In the future, continuous research will be conducted to increase the value of the virus produced by spinner and to achieve the purpose of mass production with fermenter operation.
32
Burgener, Adam D. "A bioprocess to produce reovirus : serum-free media development and the relationship between intracellular nucleotide levels and virus producton." 2005. http://hdl.handle.net/1993/17970.
Full text
33
張超銘. "Isolation of plasmid-free strain in Lactococcus lactis, and Cloning Epstein-Barr virus (EBV) gp25 gene with L.lactis cloning vector." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/55214993448152301700.
Full textAbstract:
碩士國立清華大學
生物技術研究所
90
Lactic acid bacteria (LAB) are Gram-positive bacteria and generally regarded as safe (GRAS) organisms. LAB could be used for heterologous protein secretion and fused antigen to form fusion protein. They are good potential candidates as antigen delivery vehicles. We developed an efficient secretion system in the Lactococcus lactis model. Staphylococcal nuclease (Nuc protein) is a small, stable, and biochemically well-characterized enzyme secreted by Gram-positive bacteria. It was used as the reporter protein. Plasmid pNuc10 encoding LEISSTCDA-NucT is a L. lactis cloning vector. The secretion efficiency was reduced further to ~30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). A 9-residue synthetic propeptide, LEISSTCDA , was fused immediately after the signal peptide cleavage site. Secretion efficiency was increased to ~90% by LEISSTCDA insertion without altering the signal peptide cleavage site. Because the cloning vector, pNuc10, could not replicate in E.coli. We fused pBluescriptⅡSK(+) and pNuc10 to form the shutter vector pBN. We transformed DNA to E.coli by heat shock, and it would replicate in supercoil form for ColE1 origin of E.coli. Then the expression vector is transferred to the L. lactis MG1363 strain by electroporation. In this study, the gp25 glycoprotein of EB virus was fused with histidine tag as a heterologous protein, and was cloned between NucT and LEISSTCDA to form fusion protein. We hope that the L.lactis MG1363 transformed with this expression vector can be used as a live vaccine system.
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Zhang, Bingyang. "Hepatitis B core (HBc) virus like particle (VLP) as a platform for innovation of chimeric adjuvant-free VLP vaccines targeting oncoviruses." Thesis, 2021. http://hdl.handle.net/2440/130894.
Full textAbstract:
Immunotherapy is an advanced technology for treatment of oncoviruses leading cancers. However, lack of effective and safe vaccines against the oncoviruses has limited the development. This thesis aims to apply Hepatitis B core (HBc) virus like particle (VLP) as a platform for innovation of chimeric adjuvant-free VLP vaccines targeting oncoviruses. Two chimeric HBc VLP-based vaccines presenting Epstein–Barr virus nuclear antigen 1 (EBNA1) epitope (short and non-structural epitope) and Hepatitis C virus (HCV) core epitope (long and structural epitope) were successfully expressed and purified in the Escherichia coli (E. coli) expression system with high production yields, 62.1 mg/g and 40.3 mg/g of wet cell weight, respectively. To further understand and evaluate the influence of insertion of different epitopes to HBc VLP, the stability of chimeric HBc VLP vaccines under different stresses were analysed in comparison with non-chimeric HBc VLP. Computational protein modelling was employed to assist the understanding of the possible cause for the differences. Results indicate that the stability of chimeric HBc VLP vaccines was related to the hydrophobicity of chimeric HBc monomers. The stability of chimeric HBc VLP decreased with the decrease of hydrophobicity of its monomer. This finding would help and improve the efficiency in the development and design of chimeric HBc VLP-based vaccines. In the immunogenicity evaluation, both adjuvant-free EBNA1-HBc VLP and HCV core-HBc VLP induced strong epitope-specific immune response in mice compared with other reported vaccine candidates of EBV and HCV. The achieved immune responses of adjuvant-free EBNA1-HBc VLP and HCV core-HBc VLP groups were comparable to the groups with aluminium adjuvant. No side effect and death of mice were detected during the examination. This confirms that adjuvant-free HBc VLP can present either short non-structured epitope or long structured epitope and can induce strong epitope-specific immune response with low safety risks. Chimeric EBNA1-HBc VLP tended to elicit predominated humoral immune response, while chimeric HCV core-HBc VLP induced predominated cellular immune response. This indicates that the nature of antigens presented by HBc VLP has an impact on the immune response performance, which should be considered in the design of chimeric HBc VLP vaccines in the future. The thesis also found that the addition of aluminium adjuvant would improve the humoral immune response while supressing the cellular immune response of chimeric HBc VLP vaccines. EBNA1-HBc VLP was less affected by the adjuvant on the immune response tendency compared with HCV core-HBc VLP. At last, long-term immunogenicity of two chimeric HBc VLPs were examined by evaluated the epitope specific memory T cells. Both HCV core-HBc VLP and EBAN1-HBc VLP showed good potential for long-term protection. With all above findings, chimeric adjuvant-free HBc VLP-based vaccine is promising to present different types of oncoviruses epitopes with high epitope-specific immune response and low risks. More epitopes targeting different oncoviruses could be presented by chimeric adjuvant-free HBc VLP platform for cancer treatment, and further computational protein modelling is helpful in the design and investigation of these novel chimeric HBc VLP-based vaccines.Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering and Advanced Materials, 2021
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Ruttum, Joanne C. "Development of in vitro lily scale budlets as related to virus elimination." Thesis, 1991. http://hdl.handle.net/1957/36587.
Full textAbstract:
Lily hybrids vary in their ability to produce
virus-free (VF) bulblets when grown from virus-infected
scales in tissue culture. Asiatic hybrids typically
produce a higher percentage of in vitro VF scale bulblets
than do Lilium longiflorum cultivars. Three hypotheses
concerning the cause of this variation are tested on five
lily hybrids: an Asiatic hybrid, two L. longiflorum
cultivars, an Oriental hybrid and L. candidum.
The first hypothesis states that VF scale bulblets
originate from wound tissue that is naturally low in virus
concentration and blocks the passage of virus particles
from one cell to the next. The second hypothesis says that
scale-to-bulblet vascular connections, which serve as virus
pathways, occur in hybrids showing high percentages of
virus-infected scale bulblets, while connections are absent
in those hybrids with low numbers of virus-infected
bulblets. The third hypothesis concerns the virus
concentration in the scale at the site of bulblet origin:
bulblets of hybrids producing large numbers of VF bulblets
originate from scale tissues low in virus concentration;
bulblets of low percentage VF bulblet hybrids originate
from scale tissues high in virus concentration.
The first two hypotheses are not supported by the
results of this study. First, lily bulblets do not
originate from wound tissue. Second, scale-to-bulblet
vascular connections consistently occur in 'Enchantment,'
an Asiatic hybrid, and occasionally occur in L. candidum.
Vascular connections are not detected in the low VF bulblet
producers, L. longiflorum cultivars 'Ace' and 'Nellie
White,' nor are they seen in the Oriental hybrid
'Stargazer.'
Speculative support exists for the third hypothesis
concerning uneven virus concentration in the scale.
Distinct virus particles are observed with the electron
microscope in the double virus-infected L. longiflorum
cultivars and not in the other singly-infected lilies. The
doubly-infected lilies produce a continuous layer of
divided cells in the adaxial subepidermis of the scale
where bulblets originate, whereas the singly-infected
lilies produce cell division masses in the same area but
only beneath forming bulblets.
This study suggests that virus particles in
L. longiflorum cultivars are more uniformly distributed
than particles in the other lilies examined. This occurs
not only at the site of bulblet origin but also throughout
the scale mesophyll. Whether this is due to concurrent
viral infection or to hybrid variation is unknown.Graduation date: 1992
36
Hsieh, Pei-Chun, and 謝佩君. "Classical swine fever (CSF): monitoring from outbreak to field virus free at farm level and the feasibility of postcolostral intradermal vaccination of CSFV." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/whgsn5.
Full textAbstract:
碩士國立中興大學
獸醫病理生物學研究所
98
Classical swine fever (CSF) is a highly contagious and lethal disease, and along with foot and mouth disease, have been programmed for disease eradication in Taiwan. The lack of clinical CSF case and the identification of CSF virus (CSFV) free in farm are the critical steps for determination of stop vaccination. The aim of this study was to continuously monitor the progress of CSFV status in a pig farm (Farm RL) with CSF outbreak in 2003. Excretion of CSFV in saliva, vertical transmission in umbilical cord blood, and specimens from sick nursery pigs was monitored by molecular technique and pathology. The presence of CSFV antibody in sentinel pigs was also used to evaluate the horizontal transmission among weaned pigs that had been raised in the hospital pens. Results indicated that CSFV cannot be detected by RT-PCR in a total of 87 samples of saliva of sows in five collections between 2008 and 2010. However, by using nested PCR, the positive rates were found to be between 0% and 88.2%. Moreover, few field virus might persist in the pig farm after nucleic acid sequencing and utilization of differential primers. There was no CSFV nucleic acid detected in umbilical blood and no seroconversion in sentinel pigs in Farm RL. In addition, a cross-sectional study of 187 saliva of sows and 80 samples of umbilical blood from other five farms with CSF history were studied. The average of positive rate of CSFV nucleic acid was 38.0% (71/187) in saliva and 1.3% (1/80) in umbilical blood. After 4 to 5 repeats of the experiment with sentinel pigs, with the exception of two sentinel pigs in one monitor, all exhibited no seroconversion. These results indicated though wild CSFV may, in long-term, persist in sows, CSFV circulated infection in weaned pigs may be gradually controlled in farm with the implementation of CSFV vaccination and biosecurity. However, CSFV persistence is still a hamper for CSFV eradication even at farm level. Meanwhile, the major difficulty of CSF vaccination in field is the interference of maternal antibody. Though precolostral vaccination can induce good protection for CSF, more labor and mid-night delivery of sows may make the strategy not practical. Therefore, the second part of the experimental was to evaluate the feasibility of intradermal postcolostral intradermal (ID) vaccination of CSF. The first result in weaned pigs displayed that ID vaccination could induce good immune response under less maternal interference. The second trail showed that ID precolostral vaccination could induce similar CSFV antibody profile at week 6 and 12 of ages as the expression of standard intramuscular (IM) precolostral vaccination. In contrast, poor immune response was noted in ID postcolostral vaccination with 1 injection site in neonates. Moreover, good immune response was noted in these neonates receiving 2 injections of postcolostral ID vaccination. Those results suggested that the alternative postcolostral ID vaccination can avoid maternal antibody interference and may be applied on CSF prevention in some farms with special requirment.
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Chao, I. Ting, and 趙怡婷. "Evolution of the possibility of establishing a caprine arthritis encephalitis virus (CAEV) free flock and study on the mechanism of CAEV-induced chronic arthritis." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/24090969744456463098.
Full textAbstract:
碩士國立中興大學
獸醫學系
88
Abstract Caprine arthritis encephalitis is a chronic progressive debilitating disease. Taiwan has become a high prevalent area. The first aim of the experiment was to evaluate the possibility of establishing a CAEV free flock. A dairy goat farm which had been regular surveillance on CAEV infection in our laboratory was used to evaluate the possibility of establishing CAEV free flocks. Two flocks, 17 months old (F1-1) and 4 months old (F1-2) both which were born from the same dam (F0) and fed by heated pooled colostrum were used in this study. Blood were regularly sampled and tested every 3-4 month interval and CAE seropositive goats were culled or separated. After one year intensive surveillance and culling test, the accumulative seropositive rate of F1-1 and F1-2 flocks were 40.1% and 14.8%, respectively, which were compared to 67.9% of F0 dam tested before the experiment. Comparison the accumulative seroconversive rate at the age of 9 - 10 months old of F1-1 , F1-2 , and F2 (kids delivered from F1-1, F1-2) were 16.7 %, 8.3 %, and 4.4 %, respectively. Moreover, a cross-section serological surveillance on 6 dairy goat farms using heated colostrum to feed neonate kids, the average seropositive rate of 10 months old goat for CAE were 42.3+29.0 % (0 - 81.8 %), which was significant higher than the farm with intensive serological surveillance and culling system (P<0.05). In contrast to another 4 dairy goat farms using non-heated colostrum to feed neonate kids, the average seropositive rate of 10 months old goat were 74.1+27.2% (42.9 — 100 %), which was significant higher than previously heat treated colostrum groups (P<0.05). The results suggest that it is possible to establish a CAE free farm by gradually decreasing the reservoirs, feeding kids using heated colostrum, culling or separating system by intensive serological surveillance, and obtaining the full cooperation by raiser. On the other hand, the immunopathological appearance of CAEV-induced arthritis is similar to the lesion of rheumatoid arthritis (RA) in human. Thus, in order to understand the mechanism of CAEV-induced chronic progressive arthritis, the experiment was studied on the phenotype of peripheral blood mononuclear cells (PBMC) and synovial fluid (SF) using flow cytometry and the change of cellular compartment of elbow lymph nodes using computer-assisted morphometric analysis. The result showed that the relative number of CD4+ T lymphocytes in PBMC was significantly increased with the lesion of arthritis, whilst, IgG+ B cells in PBMC was significantly decreased (P<0.05) as compared to negative control. Markedly increasing CD8+ T lymphocytes in synovial fluid was noted in those CAEV- infected goats with swollen joints. There was a significant increasing IgG concentrations in synovial fluid from CAEV infection with or without swollen joints as compared to unifected controls. T cell dependent area (paracortex) in elbow lymph nodes from CAEV infected goats with marked synovial lesion were marked hyperplasia, when compared to uninfected goats without synovial lesion. Moreover, virus antigen expression in lymph nodes and synovial membrane were markedly increased with lesion of synovial membrane. Those results suggest that an abnormal regulation in controlling viral replication may be the crucial point in the formation of progressive arthritis during CAEV infection.
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Owusu, Frank K. "Mathematical modelling of low HIV viral load within Ghanaian population." Thesis, 2020. http://hdl.handle.net/10500/26903.
Full textAbstract:
Comparatively, HIV like most viruses is very minute, unadorned organism which cannot
reproduce unaided. It remains the most deadly disease which has ever hit the planet
since the last three decades. The spread of HIV has been very explosive and
mercilessly on human population. It has tainted over 60 million people, with almost half
of the human population suffering from AIDS related illnesses and death finally. Recent
theoretical and computational breakthroughs in delay differential equations declare that,
delay differential equations are proficient in yielding rich and plausible dynamics with
reasonable parametric estimates.
This paper seeks to unveil the niche of delay differential equation in harmonizing low
HIV viral haul and thereby articulating the adopted model, to delve into structured
treatment interruptions. Therefore, an ordinary differential equation is schemed to
consist of three components such as untainted CD4+ T-cells, tainted CD4+ T-cells (HIV)
and CTL. A discrete time delay is ushered to the formulated model in order to account
for vital components, such as intracellular delay and HIV latency which were missing in
previous works, but have been advocated for future research. It was divested that when
the reproductive number was less than unity, the disease free equilibrium of the model
was asymptotically stable. Hence the adopted model with or without the delay
component articulates less production of virions, as per the decline rate. Therefore
CD4+ T-cells in the blood remains constant at 𝛿1/𝛿3, hence declining the virions level in
the blood. As per the adopted model, the best STI practice is intimated for compliance.Mathematical Sciences
Ph.D. (Applied Mathematics)
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Yurdakul, Celalettin. "Interferometric reflectance microscopy for physical and chemical characterization of biological nanoparticles." Thesis, 2021. https://hdl.handle.net/2144/43096.
Full textAbstract:
Biological nanoparticles have enormous utility as well as potential adverse impacts in biotechnology, human health, and medicine. The physical and chemical properties of these nanoparticles have strong implications on their distribution, circulation, and clearance in vivo. Accurate morphological visualization and chemical characterization of nanoparticles by label-free (direct) optical microscopy would provide valuable insights into their natural and intrinsic properties. However, three major challenges related to label-free nanoparticle imaging must be overcome: (i) weak contrast due to exceptionally small size and low-refractive-index difference with the surrounding medium, (ii) inadequate spatial resolution to discern nanoscale features, and (iii) lack of chemical specificity. Advances in common-path interferometric microscopy have successfully overcome the weak contrast limitation and enabled direct detection of low-index biological nanoparticles down to single proteins. However, interferometric light microscopy does not overcome the diffraction limit, and studying the nanoparticle morphology at sub-wavelength spatial resolution remains a significant challenge. Moreover, chemical signature and composition are inaccessible in these interferometric optical measurements. This dissertation explores innovations in common-path interferometric microscopy to provide enhanced spatial resolution and chemical specificity in high-throughput imaging of individual nanoparticles.
The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system's classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts.
This dissertation makes the following four major contributions in the wide-field common-path interferometric microscopy field: (1) formulating vectorial-optics based linear forward model that describes interferometric light scattering near planar interfaces in the quasi-static limit, (2) developing computationally efficient image reconstruction methods from defocus images to detect a single 25 nm dielectric nanoparticle, (3) developing asymmetric illumination based computational microscopy methods to achieve direct morphological visualization of nanoparticles at 150 nm, and (4) developing bond-selective interferometric microscopy to enable multispectral chemical imaging of sub-wavelength nanoparticles in the vibrational fingerprint region. Collectively, through these research projects, we demonstrate significant advancement in the wide-field common-path interferometric microscopy field to achieve high-resolution and accurate visualization and chemical characterization of a broad size range of individual biological nanoparticles with high sensitivity.
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Rey, Michel Richard. "On-farm evaluation of a needle-free injection device to vaccinate beef calves under Western Canadian conditions." 2013. http://hdl.handle.net/1993/14423.
Full textAbstract:
This study was conducted to compare animal performance, presence of skin reactions and immune response following vaccination of beef calves via needle-free (NF) and needle-syringe (NS) vaccination techniques. Spring-born (Study A) and fall-born (Study B) calves were vaccinated against bovine viral diarrhea virus (BVDV) and Clostridium chauvoei (C. chauvoei) via NF and NS vaccination techniques. The parameters measured in this study included body weight (BW), skin reactions and serum antibodies. Animal performance and antibody levels against BVDV and C. chauvoei did not differ between vaccination techniques. However, NF vaccinated calves had a greater frequency of skin reactions when compared to NS vaccinated calves, except for day 42 of Study B. It can be concluded that a needle-free injection device (NFID) can be used effectively to stimulate an immune response without impacting animal performance, but may cause a greater frequency of skin reactions.
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Akomeah, Belinda. "Impact of in vitro induced epigenetic variation on the nutritional value of three Ghanaian sweet potato genotypes: implications on biofortification." Thesis, 2016. http://hdl.handle.net/2440/114123.
Full textAbstract:
Front matter only available electronically. The complete thesis in print form is available from the University of Adelaide Library.Biofortification aims to increase crop nutritional value to combat nutrient deficiency. Due to the prevalence of viruses, healthy cultivars of biofortified genotypes are produced through micropropagation techniques. However, during micropropagation, plants are exposed to conditions that could induce somaclonal variation, and result in phenotypic changes affecting the crop’s nutritional value. Currently, sweet potato (Ipomoea batata) is biofortified for enhanced beta-carotene content to alleviate Vitamin A Deficiency (VAD). Undesired somaclonal abnormalities acquired during in vitro culture could alter key nutrients such as beta-carotene, protein, or zinc. Therefore, it is important to ensure the clonal fidelity of the micropropagated biofortified lines. This study assessed the extent of in vitro induced epigenetic variation in the genome of meristem-cultured plants, and its correlation with the nutritional composition in three Ghanaian sweet potato genotypes (Bohye, Ogyefo and Otoo). Micropropagated plants presented no observable leaf and storage root abnormalities, but relatively lower levels of iron, protein, zinc, and glucose. Methylation Sensitive Amplification Polymorphism analysis showed a high level of in vitro induced molecular variation in micropropagated plants. Ogyefo showed the least viral incidence and epigenetic differentiation but the most profound nutritional changes, while Bohye showed the highest epigenetic variability. Further analysis indicated that epigenetic, rather than genetic, accounts for most of the observed variability. Taken collectively, this study offers an insight into the impact of micropropagation in methylation profiles, and its correlation to root quality in the improved sweet potato genotypes. The implications of these results to the ongoing bio-fortification projects are also discussed.
Thesis (M.Bio.(PB)) -- University of Adelaide, Masters of Biotechnology (Plant Biotechnology), School of Agriculture, Food and Wine, 2016.
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Moreku, Dikeledi Caroline. "The role of professional nurses on anti-retroviral therapy adherence among children living with HIV/AIDS in Lejweleputstwa District: Free State, South Africa." Diss., 2017. http://hdl.handle.net/11602/883.
Full textAbstract:
MCurDepartment of Advanced Nursing Science
Survival of children with HIV/AIDS has increased considerably with the use of effective antiretroviral therapy. However, the benefits of this therapy are limited by the difficulty of adherence to the treatment. This study sought to explore the role of professional nurses on anti-retroviral therapy adherence among children in Lejweleputswa district: Free State, South Africa. An exploratory descriptive qualitative research design was used to identify and describe role of professional nurses toward anti-retroviral therapy adherence among children. Population for this study included seventeen (17) professional nurses working in four purposively sampled Primary Health Care clinics invited to participate in the study. Four focus group discussions were conducted in which each group had 6 participants. The transcribed data was analysed using the framework approach of data analysis. Professional nurses in Lejweleputswa district report poor knowledge of parents/caregivers of children, perceived poverty, stigma and discrimination, inappropriate care approaches, and parental dynamics as factors influencing poor ART adherence. Recommendations for enhancing children ART adherence levels in Lejweleputswa district included: mainstreaming adherence counselling in children ART and adopting a comprehensive family centered care approach were identified as measures for improving children ART adherence. Other measures included integration of ART services into Primary Health Care (PHC) services, parental empowerment, development of a programme to reduce stigma and discrimination in the community.
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Silva, Ana Catarina Martins Mandeiro da. "Advanced cell-based biosensors for detection of label-free viral pathogens." Master's thesis, 2019. http://hdl.handle.net/10362/89378.
Full textAbstract:
The last decades have been marked by a rising number of diseases attributed to newly discovered viruses, as well as an increasing incidence of diseases caused by viral pathogens outside of their endemic areas. Simultaneously, interest on virus-based biopharmaceuticals (VBBs), for therapeutic or prophylactic ends, has increased dramatically. However, fundamental and applied research in virology depend on fast, reliable, and high-throughput approaches for detection and quantification of infectious viruses, which the most commonly used methods fail to deliver.
Herein, we developed a novel cell-based sensing system using a switch-on Cre recombinase strategy. This modular strategy consists on a protease-sensing Cre recombinase (ProCre) and a reporter module. The latter is activated by Cre-mediated DNA recombination events, resulting in a signal output. Cre always-on activity was successfully controlled by structural distortion of a truncated Cre, harbouring a viral protease specific cleavable linker. Transient transfection results showed that Cre activity was effectively constrained, delivering a “vigilant state”. As proof-of-concept, sensor activation with tobacco etch virus protease indicated that Cre activity could be efficiently recovered upon specific proteolysis at the cleavable linker. ProCre capacity to detect proteolytic activity of clinically relevant human viruses was demonstrated with the human rhinovirus protease. Moreover, ProCre was also adapted to detect adenovirus, chikungunya and Zika viruses proteolytic activity. Although no substantial increase in GFP expression was observed, sensor activation upon proteolysis was confirmed. Further optimizations are currently ongoing to allow robust detection of these viruses.
Overall, we developed a novel strategy to control the always-on nature of Cre recombinase, enabling its use as a protease-dependent sensor. The versatility of this system further allows its adaptation to detect other proteases, either viral or cellular, providing a permanent and robust output signal of interest.
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Lugongolo, Masixole Yvonne. "Optical micro-manipulation in HIV-1 infected cells for improved HIV-1 treatment and diagnosis." Thesis, 2020. http://hdl.handle.net/10500/26551.
Full textAbstract:
Laser application in the field of biological and medical sciences has significantly grown, thereby
strengthening the field of Biophotonics. Research conducted in Biophotonics focuses on the concept
of using light especially in the visible and near infrared regions of the electromagnetic radiation for
the evaluation of living systems. In this thesis new discoveries are presented about low level laser
therapy, optical trapping, transmission spectroscopy, luminescence spectroscopy and structured
illumination microscopy (SIM), displaying the impact each technique has on HIV infected cells. The
results showed that the irradiation of HIV-1 infected TZM-bl cells with low power red laser reduces
HIV-1 infection. The outcomes of this study further proved that when irradiation is used in
conjunction with efavirenz, an antiretroviral drug, HIV-1 infection could be reduced to undetectable
levels in TZM-bl cells. Through the coupling of transmission spectroscopy with optical trapping, and
separately, use of luminescence spectroscopy, label free diagnosis of HIV in infected cell samples
was achieved. This finding affirms that HIV-1 infection can be detected in a label free manner when
using laser based techniques. Furthermore, the photoluminescence spectrometer system was
employed to generate a decay curve, which was necessary so as to have some understanding on
lifetime of the luminescent signal in infected TZM-bl cells. Finally, in order to confirm that indeed
TZM-bl cells were infected, an established super-resolution microscopy system SIM was used to
detect HIV-1 infection in TZM-bl cells. Indeed in the infected cells viral molecules p24 and gp41
were detected through SIM, while they were not detected in uninfected cells. In future studies, super
resolution microscopy would be coupled to an optical trapping system in order to confirm that each
trapped cells is whether infected or uninfected so as to improve HIV diagnosis.College of Science, Engineering and Technology
Ph. D. (Science, Engineering and Technology)
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Dixit, Karuna. "NMR Solution Structures of Human γC-Crystallin & the Intrinsically Disordered Viral Genome Linked Protein in the Free & Bound Form." Thesis, 2016. http://hdl.handle.net/2005/3102.
Full textAbstract:
This thesis describes the tertiary structures and dynamic studies of two protein systems. The first is human γC -crystallin protein, which is present in the nucleus of the human eye lens and the other is the plant viral protein VPg (an intrinsically disordered protein) in its free as well as its protease bound forms. The structural studies described here have been carried out using high-resolution solution NMR spectroscopic methods.
Project I: Determination of solution structure and dynamics of Human γC-crystallin
(HGC) using NMR spectroscopy
The crystallins are the most abundant proteins in the eye lens of vertebrates. These proteins are packed in short-range spatial order to provide the transparency and appropriate refractive index gradient that are required for vision. The crystallins belong to two gene families, which are categorized as the alpha and beta/gamma crystallins respectively. The classification on the basis of molecular size and structure results in the proteins being referred to as alpha, beta and gamma crystallins. Again, each of the crystallins has two or more subtypes. The stoichiometry of the subtypes of α, β and γ crystallins varies with the age of the organism, but the order of abundance remains as β > α > γ irrespective of age. The most abundant crystallins in the nucleus (central region) of eye lens are the γ -crystallins. In the human lens, only three members of the γ− crystallin family are mainly expressed i.e. γS- (HGS), γC - (HGC) and γD - (HGD). HGS is expressed postnatally and thus is present mainly in the cortical region of the lens unlike HGC and HGD crystallins, which are present in the nucleus. It is known that aging and some cataract-associated genetic mutations alter the structure of these proteins. Other point mutations result in minimum structural perturbation but with drastically lowered solubility. Mutation in the human γC -crystallin leads to congenital cataract such as Coppock-like cataract, while structural information is available for HGD & HGS but no structure is available for HGC. However, recently a model structure has been reported for HGC based on a mouse orthologous. Based on this model structure, it was argued that HGC is an insoluble protein and was explained by lower magnitude of dipole moment and fluctuation in N-terminal domain of the model structure. However it is shown that HGC is very soluble protein.
Solution structure of human γC-crystallin has been determined from an analysis of multidimensional triple resonance NMR spectroscopy using distance restraints from unambiguously assigned 1H-1H NOE peaks and dihedral angle restraints from HNHA and HNHB spectra. 15N relaxation average T1 and T2 correspond to 0.729 ± 0.02 and 0.060 ± 0.04 second from 15N backbone relaxation study, which gives average rotational correlation time 10.87 ns that shows human γC-crystallin is monomer in solution of molecular weight 21 kDa (173 residues). The ensemble of 20 lowest energy structures shows a root mean square deviation of 0.60 ± 0.12 Å for the backbone atoms, and 1.03 ± 0.09 Å for all heavy atoms. The comparison between the calculated NMR structure with backbone chain atoms C`, Cα and NH, of the x-ray crystal structure of the mouse γC - crystallin shows that the structure determined here of human γC-crystallin is very similar with an RMSD of 1.3 Å, which is not surprising given the 84.5% amino acid sequence identity between the two proteins.
More importantly, the NMR structure reported here shows the subtle differences in the orientation of specific residues as well as the domain interface between the human and mouse orthologs. The orientation of the calculated dipole moment for this NMR structure differs from earlier reported for model structure. However it is similar to the other known soluble proteins. The determined solution structure of human γC-crystallin also enables us to estimate the effect of cataract-associative mutations on the structure and properties of the protein. Several such mutations are already known, and the work presented here could likely shed light on the molecular basis of these cataracts.
Project II: Solution structural studies of intrinsically disordered protein VPg in free and bound forms from Sesbania mosaic virus
Sesbania mosaic virus (SeMV) is a plant virus, which infects the Sesbania grandiflora tree. SeMV belongs to Sobemovirus genus, which is not defined under any family. The length of this viral genome is ~4kb. This viral genome has four open-reading frames (ORF). ORF1 and ORF2 encode movement and coat proteins, respectively. ORF2 is again split into two ORFs i.e. ORF2a and ORF2b by a -1 shift in the reading frame and encode two polypeptide chains. These polypeptide chains generate several functional proteins upon polyprotein processing. Polyprotein processing is a mechanism employed by animal and plant viruses to produce several functional proteins from a single polypeptide chain. The two polyproteins expressed are catalytically cleaved by a serine protease, thus releasing the four proteins: VPg (viral protein genome linked), RdRP (RNA dependent RNA polymerase), P10, and P8.
VPg (“Viral Protein genome linked”) as its name suggests, is covalently linked to the 5` end of the viral RNA. VPgs are generally known to be intrinsically disordered proteins and have many interacting partners. Intrinsically Disordered Proteins (IDPs) are not explained by the 3D structure–function dogma. However, they are important for biological functions such as molecular recognition, signal transduction and regulation. It is known that SeMV protease becomes inactive in the absence of the VPg domain at its C-terminal. VPgs of animal viruses are well studied as compared to VPgs of plant virues. The size of VPg varies across the Sobemovirus genus. It is important to know the structure of VPg since it is necessary for protease activity. The studies conducted here focus on the structural analysis of the VPg in its free and bound forms with protease (VPg complex) as well as some aspect of full-length ProVPg.
For structural studies, two constructs of VPg as fusion protein with Cytb5 tag, one lacking 23 residues at its C-terminal using the pET21a(+) plasmid vector have been designed. Sub-cloning was also done to add a thrombin recognition site to remove the hexa-His tag from new constructs of full-length ProVPg and protease (PRO). These proteins were highly expressed, isotopically labeled and purified for NMR study. The sample used for structural studies of the ProVPg 23 complex was prepared using selectively protonated Ile, Leu and Val; and isotopically labeled i.e. 2H, 13C, and 15N-VPg 23 protein.
VPg in its free form is an intrinsically disordered protein and this has been confirmed by its dynamic nature observed using solution NMR spectroscopy. VPg binds to its partner protease and adopts a 3D-structure, which has been shown here. The tertiary structure has been determined using distance restraints from 1HN-1HN NOEs and methyl 1HN NOEs, and dihedral angle predicted from analysis of chemical shift values. The tertiary structure of ProVPg 23 complex has one β -sheet composed of three antiparallel β-strands and an α-helix. The ensemble of 20 lowest energy structures shows a root mean square deviation of 0.42 ± 0.09 Å for the backbone atoms, and 1.09 ± 0.11 Å for all heavy atoms for residues 15 to 50 that are primarily involved in structure formation. On the other hand RMSD is 2.34 ± 0.72 Å for the backbone and 2.55 ± 0.60 Å for all heavy atoms for all residues including both termini. That the tertiary fold of VPg both in full-length ProVPg and when complexed with protease domain (PRO) are the same has been shown here. The NMR structure reported here provides a structural basis for the origin of resonances in the up-field region of one–dimensional proton spectrum of full length ProVPg. The binding surface based on the structures of ProVPg 23 complex determined here and X-ray structure of PRO; has been determined using HADDOCK. The structural model here of full length ProVPg 23 shows the presence of aromatic interaction between Trp271 of PRO and Trp46 of VPg, which is consistent with the earlier biochemical studies.