Дисертації з теми "Molecular diagnostic tool"
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Mohamed, Moumin Neima. "DEVELOPING A MOLECULAR TOOL KIT FOR DIAGNOSTIC PCR." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-392205.
Повний текст джерелаMenmuir, Sheena. "Visible spectroscopy as a sensitive diagnostic tool for fusion plasmas." Licentiate thesis, KTH, Physics, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-572.
Повний текст джерелаTo further the understanding and knowledge about fusion plasmas and their behaviour during different conditions, it is important to be able to collect information about the plasma and the processes occurring within it. Visible spectroscopy, or the study of the visible wavelength light emitted by the plasma, is a useful tool in this search for knowledge.
This thesis is based on experiments where visible wavelength light has been measured and analysed in order to determine quantities about the emitting source. Doppler shift measurements of spectral lines have been utilised to determine the toroidal rotation velocities of plasma impurity ions and to study the correlation with mode rotation and the effect of active feedback control of the resistive wall modes. Information on the impurities present in the plasma has been determined and the calibrated intensities of spectral lines has yielded impurity concentrations, particle fluxes and electron temperature and densities. Ion temperatures have been determined from Doppler broadening measurements.
The measured vibrational and rotational band structure of deuterium molecular spectra has been analysed in order to calculate rotational and vibrational temperatures, relative populations and molecular particle fluxes. The effect of the molecular flux on simple calculations of atomic flux has also been studied. Specific molecular states and transitions of deuterium have also been probed with synchrotron radiation to study the level and transition energies.
The measurement and analysis of visible wavelength light has been demonstrated to be a sensitive diagnostic tool in the quest for increased knowledge about fusion plasmas and molecular structure.
Christopeit, Tony. "Protein Interaction Studies with Low Molecular Weight Ligands : Applications for Drug Discovery, Basic Research and Diagnostic Tool Design." Doctoral thesis, Uppsala universitet, Institutionen för kemi - BMC, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-188328.
Повний текст джерелаFlores, Carolina. "Cassava Bacterial Blight : development of a performant molecular detection tool and diversity analysis of Xanthomonas axonopodis pv. manihotis populations in Venezuela." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT161.
Повний текст джерелаCassava (Manihot esculenta L. Crantz) belongs to the group of roots and tubers and is cultivated in the tropics worldwide. This species is a nutritional alternative in many populations where no optimal crop production, general conditions nor technological support exist. Considering its potential uses, the global development of cassava crop has increased significantly but it is still considered a subsistence crop in poor regions lead by smallholder producers. Cassava is affected by biotic and abiotic factors during its life cycle, which heavily limiting its optimal performance. A variety of pests and diseases are known to affect cassava production. Among them are those caused by Xanthomonas axonopodis pv. manihotis (Xam) and Xanthomonas cassavae (Xc), causal agents of Cassava Bacterial Blight (CBB) and Cassava Bacterial Necrosis (CBN) diseases, respectively. CBB is considered the major bacterial disease that affects cassava crop worldwide which is also the case in Venezuela where it was first reported in the 70s. Since the 90s, studies were conducted to elucidate Xam genetic variability in different regions in the country, by means of different molecular tools available at that time. A high degree of polymorphism among the isolates was reported, whether collected from the same or different fields. The Xam population was distributed into eight clusters and no correlation was observed between genetic diversity and geographic origin.Our questions deal with the situation of CBB in Venezuela 20 years later : what is the current genetic diversity of Xam populations in Venezuela? what is the genetic structure of Xam populations and how do they differ with respect to Xam strains collected in 90s. Moreover, because Xam and Xc cause similar symptoms on cassava leaves and display similar physiological and morphological characteristics, we also aimed at developing a new molecular diagnostic tool allowing for fast and reliable detection of Xam and able to discriminate with Xc.To achieve our goals, we first established a duplex-PCR as a molecular detection tool of cassava-infecting xanthomonads. Based on in silico analysis of the genome sequences of 66 Xam and 1 Xc strains, we were able to select 6 Xam and 6 Xc primers pairs candidates, of which one set of primers for each was selected for further studies. We were able to develop a duplex-PCR assay that was validated upon testing 53 Xam strains and 25 Xc strains from different countries, 18 non-target strains, and 5 epiphytic strains associated to cassava, proving this technique a useful tool to detect and differentiate Xam and Xc from in vitro cultures and in planta.Secondly we assessed the diversity of Xam populations through a variable number of tandem repeat analysis (MLVA). A field survey conducted in six states in Venezuela enabled to evaluate the occurence of the disease, its status and allowed us to establish a strain collection for detailed diversity analysis. We isolated 202 Xam strains from six localities, localized in four states. Using a MVLA14-scheme, we analyzed 12 populations highlighting a high index of genetic diversity among and within populations, mainly in the east of the country.The development of this type of research is essential in the management of crops in the world and coupled with the existing agricultural policies, it will allow us to have a deeper understanding of pathogens of agricultural importance and the mechanisms involved in their establishment over time and across regions. The long-term objective of this is to apply control measures that are effective in time, thus establishing more stringent quarantine measures to prevent the spread of the disease
CHIACCHIO, TERESA. "New molecular diagnostic and immunological tools for tuberculosis research." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/916.
Повний текст джерелаEbai, Tonge. "Development of Enhanced Molecular Diagnostic Tools for Protein Detection and Analysis." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-320380.
Повний текст джерелаFluegel, Amanda M. "Validation of diagnostic assays and development of molecular epidemiological tools for brucellosis." Laramie, Wyo. : University of Wyoming, 2008. http://proquest.umi.com/pqdweb?did=1594477821&sid=1&Fmt=2&clientId=18949&RQT=309&VName=PQD.
Повний текст джерелаHolzapfel, Marion. "De l’épidémiologie moléculaire aux analyses fonctionnelles de Brucella chez les ruminants, une approche intégrée pour l’identification et l’étude de la diversité phénotypique d’un genre génétiquement homogène." Thesis, Paris Est, 2018. http://www.theses.fr/2018PESC1141.
Повний текст джерелаBrucellosis is a zoonotic disease caused by the bacterial genus Brucella (B.), whose global incidence is estimated at 500,000 human cases per year. The reservoir is animal, affecting mainly livestock. The most important species for humans are B. melitensis, B. abortus and B. suis, which share more than 90% sequence identity. Although highly genetically related, Brucella spp. exhibit a variety of phenotypic characteristics, host preference and pathogenicity. The genetic homogeneity of these species may appear as an asset for the development of robust universal diagnostic tools. On the other hand, it is a challenge to distinguish them, making it difficult to precisely characterize isolates from the same outbreak. As part of this thesis, a real-time PCR molecular diagnostic tool targeting the genus Brucella was developed and optimized. The method has been evaluated on ruminant milk samples; these samples may be an important source of Brucella and may be useful for herd-scale disease screening. Based on the detection of the IS711 insertion element, a sequence present in several copies within the genome, this method displays sensitivity and specificity values that make it interesting for a global scheme to fight against brucellosis. On the other hand, in order to improve the understanding of the genetic stability of B. melitensis, an original panel of strains isolated in an outbreak and involving four different host species was compared. Thus, using different complementary approaches, their genomic sequences, phenotypic characteristics and their behavior in an in vitro model were compared. Our results did not highlight markers that would suggest that mutations in the genome are essential to adapt to a new host
Lopes, Jéssica Sousa. "FTIR, a potential tool to dementia diagnosis trough analysis of plasma." Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/16149.
Повний текст джерелаNowadays it is still difficult to perform an early and accurate diagnosis of dementia, therefore many research focus on the finding of new dementia biomarkers that can aid in that purpose. So scientists try to find a noninvasive, rapid, and relatively inexpensive procedures for early diagnosis purpose. Several studies demonstrated that the utilization of spectroscopic techniques, such as Fourier Transform Infrared Spectroscopy (FTIR) and Raman spectroscopy could be an useful and accurate procedure to diagnose dementia. As several biochemical mechanisms related to neurodegeneration and dementia can lead to changes in plasma components and others peripheral body fluids, blood-based samples and spectroscopic analyses can be used as a more simple and less invasive technique. This work is intended to confirm some of the hypotheses of previous studies in which FTIR was used in the study of plasma samples of possible patient with AD and respective controls and verify the reproducibility of this spectroscopic technique in the analysis of such samples. Through the spectroscopic analysis combined with multivariate analysis it is possible to discriminate controls and demented samples and identify key spectroscopic differences between these two groups of samples which allows the identification of metabolites altered in this disease. It can be concluded that there are three spectral regions, 3500-2700 cm -1, 1800-1400 cm-1 and 1200-900 cm-1 where it can be extracted relevant spectroscopic information. In the first region, the main conclusion that is possible to take is that there is an unbalance between the content of saturated and unsaturated lipids. In the 1800-1400 cm-1 region it is possible to see the presence of protein aggregates and the change in protein conformation for highly stable parallel β-sheet. The last region showed the presence of products of lipid peroxidation related to impairment of membranes, and nucleic acids oxidative damage. FTIR technique and the information gathered in this work can be used in the construction of classification models that may be used for the diagnosis of cognitive dysfunction.
Atualmente, não é possível fazer um diagnóstico precoce e diferencial da doença de Alzheimer, deste modo, é necessário encontrar biomarcadores que o permitam. Para isso, os cientistas tentam encontrar um procedimento nãoinvasivo, rápido, e relativamente barato. Os resultados de vários estudos demonstraram que a utilização de técnicas espectroscópicas, tais como a Espectroscopia de Infravermelho Transformada de Fourier (FTIR) e / ou espectroscopia de Raman, podem ser ferramentas úteis para diagnosticar a DA. Uma vez que, na DA, alguns mecanismos bioquímicos podem levar a mudanças em componentes do plasma, podem então ser utilizadas amostras de sangue nas análises espectroscópicas o que torna a técnica simples e menos invasiva. Com este trabalho pretende-se confirmar algumas das hipóteses de estudos anteriores em que o FTIR foi usado no estudo de amostras de plasma de possíveis doentes com DA e respetivos controlos e verificar a reprodutibilidade desta técnica espectroscópica na análise deste tipo de amostras. Através da análise espectroscopia combinada com análise multivariada é possível discriminar as amostras controlos e dementes e identificar as principais diferenças espectroscópicas entre estes dois grupos de amostras que permitem identificar os metabolitos alterados nesta patologia. Pode-se concluir que existem três regiões espectrais, 3500-2700 cm-1, 18001400 cm-1 e 1200-900 cm-1 onde se pode extrair informação espectroscópica relevante. Na primeira região, a principal conclusão que é possível tirar é que há um desequilíbrio entre o teor de lípidos saturados e insaturados. Na região entre 1800-1400 cm-1, é possível observar a presença de agregados de proteínas e a alteração na conformação das proteínas para folha β paralela altamente estável. A última região revelou a presença de produtos de peroxidação lipídica relacionados com a insuficiência de membranas, e danos oxidativos nos ácidos nucleicos. A técnica de FTIR e a informação reunida neste trabalho pode ser utilizada na construção de modelos de classificação que possam vir a ser utilizados para o diagnóstico de disfunções cognitivas.
Englund, Stina. "Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis /." Uppsala : Dept. of Veterinary Microbiology, Swedish Univ. of Agricultural Sciences ([Institutionen för veterinärmedicinsk mikrobiologi], Sveriges lantbruksuniv.), 2002. http://epsilon.slu.se/avh/2002/91-576-6366-1.pdf.
Повний текст джерелаDzien, Piotr. "The development of novel tools for in vivo molecular imaging using hyperpolarised ¹³C labelled molecules and ¹³C magnetic resonance spectroscopy and spectroscopic imaging." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708714.
Повний текст джерелаCraven, Thomas Henry John. "Resolving uncertainty in acute respiratory illness using optical molecular imaging." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/29507.
Повний текст джерелаGomgnimbou, Kireopori michel. "Nouvelles méthodes de diagnostic, de contrôle et de surveillance de la tuberculose à bacilles sensibles ou multirésistants dans les pays à forte co-infection au VIH : applications en Santé Publique." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA112210.
Повний текст джерелаTuberculosis (TB) remains a major public health concern worldwide despite all the efforts to fight this disease. The emergence of multi drug and extensively drug resistant TB and the pandemic of HIV/AIDS constitute major threats and challenge for the TB control and eradication. TB control requires measures in public health and in individual level as accessibility to tests for early diagnostic, effective treatment and tools for tuberculosis surveillance and control.The goals of this work were research, development and validation of new molecular multiplexed methods based on polymorphism of the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) loci and single nucleotides polymorphisms. These methods are rapid, high throughput, cheap and can be applied both for public health purposes (transmission of susceptible and multi-drug resistant tuberculosis, evaluation of national TB programs) as for interest of TB patient (drug resistance testing, infra-specific identification). Thus we developed spoligoriftyping and “TB-SPRINT” tests that allow genotyping and rifampicin or rifampicin and isoniazide resistance detection. Another test was developed for subtyping of M. africanum. All these methods had high performances (sensitivity/specificity), 99/100% for the spoligoriftyping and about 95/100% for the “TB-SPRINT” and were applied for molecular epidemiology studies of countries as Nigeria, Brazil and Pakistan. Other ongoing work and developments of genotyping methods are the spoligotyping of L. pneumophila and S. enterica and comparative genomics projects.Used in routine, our methods may play key roles in TB control and would allow important advances in Public Health, in medical and environmental Microbiology
Hernández-Neuta, Iván. "Nucleic acid analysis tools : Novel technologies and biomedical applications." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-146334.
Повний текст джерелаAt the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 1: Manuscript.
Rodríguez, Pérez Eduardo M. "Study of cow subclinical hypocalcemia and development of new tools for its diagnostic and prevention." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/323908.
Повний текст джерелаDairy cows suffer blood calcium (Ca) losses as lactation begins and might be affected by hypocalcemia in its clinical (serum Ca < 6mg/dL) or subclinical state (serum Ca < 8.5mg/dL). Since clinical incidence is only about 5%, the most relevant problem concerns subclinical cases (SCHC) because of a higher prevalence. These cases cannot be treated due to the lack of diagnostic tools. In order to study the impact, consequences and regulation of SCHC and to develop preventive and diagnostic strategies, four studies were conducted. In the first study, the association of SCHC with several periparturient diseases was evaluated. Seventy five percent of cows incurred SCHC. Displaced abomasum, ketosis, retained placenta, and metritis were more likely to happen in cows with SCHC. Affected cows had a greater milk production. Normocalcemic cows showed their first heat sooner. Also, the severity of SCHC is related to the severity of the different periparturient diseases. In order to understand the exact mechanisms involved, a second study was performed to clarify the potential roles of calcitonin in the onset of SCHC and in the prevention of hypocalcemia under metabolic acidosis. A calcitonin rise in severe SHCH cows after calving impairs the recovery of blood Ca because PTH receptor (PTHR) response is not sufficient to activate vitamin D and compensate the calcitonin effect. Metabolic acidosis prevents hypocalcemia because the expression of PTHR is up-regulated in kidney. Moreover, an impairment of calcitonin activity at low pH enhances the hypercalcemic role of PTH. Based on this calcitonin role, in the third study an approach to prevent hypocalcemia through passive immunization against calcitonin was tested. Polyclonal antibodies neutralized calcitonin in vitro and in a rat model, raising the blood Ca concentration. An affordable method of mass-production was designed from a naïve ScFv phage library. The ScFv B10 recognized and neutralized calcitonin in vitro, but it did not affect blood Ca levels when administered to cattle requiring further research to understand the main difficulties of the proposed strategy. A diagnostic system would be very useful to identify and treat hypocalcemic cows. In the fourth and last study we developed a portable semiautomatic analytical system based on ion-selective field effect transistors with Ca2+ ion selective photocurable membranes. This sensor determines bovine serum calcium concentration in a reliable and fast way and can be applied in the field in cow-side measurements.
McGowan, Michael John. "Molecular regulators of smoltification and viral infection management tools for salmon aquaculture." Thesis, University of Stirling, 2018. http://hdl.handle.net/1893/28532.
Повний текст джерелаKrzywkowski, Tomasz. "iLocks: a novel tool for RNA assays with improved specificity." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-147734.
Повний текст джерелаAt the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.
Ndlovu, Thando. "Comparison of diagnostic tools and molecular based techniques for the rapid identification of Escherichia coli and coliforms in contaminated river water." Thesis, Cape Peninsula University of Technology, 2013. http://hdl.handle.net/20.500.11838/794.
Повний текст джерелаWater is an important daily requirement and in a clean, pure form, it promotes health and well-being. In addition to South Africa being one of the driest countries in the world, water availability is also being compromised by massive pollution of remaining water sources. The Berg- and Plankenburg Rivers are two of the surface water sources in the Western Cape, South Africa, which are highly polluted by sewage, industrial and agricultural run-off. The current investigation was aimed at comparing diagnostic tools, which are employed by municipalities and food industries, and molecular based techniques to routinely monitor water for indicator organisms in time- and cost-effective manner. These rivers were sampled twice a month (July 2010 to January 2011) at the sites closest to the informal settlements of Kayamandi in Stellenbosch (Plankenburg River) and Mbekweni in Paarl (Berg River). The contamination levels of the two river systems were evaluated by the enumeration of Escherichia coli and coliforms using the Colilert 18® system, Membrane Filtration (MF) and Multiple Tube Fermentation (MTF) techniques. The highest faecal coliform count of 9.2 × 106 microorganisms/100 ml was obtained in weeks 21 and 28 from the Plankenburg River system by the MTF technique, while the lowest count of 1.1 × 103 microorganisms/100 ml was obtained in week one for both river systems by the MTF technique. The highest E. coli count of 1.7 × 106 microorganisms/100 ml was obtained from the Berg River system (week 9) using the MTF technique, while the lowest count of 3.6 × 102 microorganisms/100 ml was obtained by the MF technique from the Plankenburg River system. The coliform and E. coli counts obtained by the enumeration techniques thus significantly (p > 0.05) exceeded the guidelines of 2000 microorganisms/100 ml stipulated by the Department of Water Affairs and Forestry (DWAF, 1996) for water used in recreational purposes. Overall the results obtained in this study showed that the water in the Berg- and Plankenburg River systems is highly polluted, especially where these water sources are used for irrigational and recreational purposes. For the coliform and E. coli counts obtained using the three enumeration techniques, it was noted that the MTF technique was more sensitive and obtained higher counts for most of the sampling weeks. However, the media (Membrane lactose glucuronide agar) used in the MF technique also effectively recovered environmentally stressed microbial cells and it was also better for the routine selection and growth of coliforms and E. coli. While E. coli and total coliforms were detected utilising the Colilert 18® system, accurate enumeration values for these two indicator groups was not obtained for the entire sampling period for both river systems. It has previously been shown that dilutions (up to 10-3) of highly polluted waters increase the accuracy of the Colilert 18® system to enumerate colifoms and E. coli in marine waters. As the results obtained utilising the Colilert 18® system were also not comparable to the MF and MTF techniques it is recommended that highly polluted water samples be diluted to increase the accuracy of this system as a routine enumeration technique. Water samples were directly inoculated onto MacConkey, Vile Red Bile (VRB) agar and the Chromocult Coliform agar (CCA) and single colonies were inoculated onto nutrient agar. Chromocult coliform agar proved to be more sensitive than MacConkey and VRB agar for the culturing of E. coli and coliforms. Preliminary identification of these colonies was done using the RapID ONE and API 20 E systems. The most isolated Enterobacteriaceae species by both systems, included Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli and Enterobacter cloacae in both river systems. The API 20 E system was more sensitive in the preliminary identification of the various isolates, as greater species diversity was obtained in comparison to the RapID ONE system. The Polymerase Chain Reaction (PCR) was firstly optimised using positive Enterobacteriaceae species. The optimised method was then applied to the analysis of river water samples, which were centrifuged to harvest the bacterial cells, with DNA extracted using the boiling method. The extracted DNA was amplified using conventional PCR with the aid of species specific primers. The Enterobacteriaceae species that were detected throughout the study period in both river systems include Serratia marcescens, Escherichia coli, Klebsiella pneumoniae and Bacillus cereus. Conventional PCR was the most reliable and sensitive technique to detect Enterobacteriaceae to species level in a short period of time when compared to RapID ONE and the API 20 E systems. Multiplex PCR was optimised using the positive pathogenic E. coli strains namely, Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterohaemorrhagic E. coli (EHEC) and Enteroaggregative E. coli (EAEC). It was then employed in river water sample analysis and enabled the detection of EAEC, EHEC, and EIEC strains in Berg River system, with only the EAEC detected in the Plankenburg River system. Real-time PCR was used to optimise the multiplex PCR in the amplification of E. coli strains and successfully reduced the time to obtain final results when using control organisms. Real-time PCR was found to be more sensitive and time-effective in the identification of E. coli strains, and also more pronounced DNA bands were observed in real-time PCR products compared to conventional-multiplex PCR amplicons. To sustain the services provided by the Berg- and Plankenburg Rivers in the Western Cape (South Africa), these water sources should frequently be monitored, results assessed and reported according to the practices acknowledged by responsible bodies. It is therefore recommended that the enumeration techniques be used in conjunction with the very sensitive PCR technique for the accurate detection of coliforms and E. coli in river water samples.
Rojas, Mencias Pablo David [Verfasser]. "In situ detection, molecular epidemiology, and improvement of molecular tools for the understanding and diagnosis of infections caused by spirochetes / Pablo David Rojas Mencias." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2017. http://d-nb.info/1148425233/34.
Повний текст джерелаCadd, Verity Anne. "The study of molecular markers for the progression of Barrett's oesophagus to adenocarcinoma to identify markers that can be used as diagnostic tools." Thesis, Cranfield University, 2002. http://dspace.lib.cranfield.ac.uk/handle/1826/3689.
Повний текст джерелаCalvani, Nichola Eliza Davies. "Translocation of Fasciola hepatica via international livestock movements: development of ante-mortem molecular diagnostic tools for the identification of Fasciola spp. in livestock." Thesis, The University of Sydney, 2020. https://hdl.handle.net/2123/22464.
Повний текст джерелаNUNES, Mónica Susana Claudino. "Unraveling of Borrelia burgdorferi sensu lato genospecies diversity in Portugal towards the development of more efficient diagnostic tools for Lyme disease." Doctoral thesis, Instituto de Higiene e Medicina Tropical, 2016. http://hdl.handle.net/10362/20055.
Повний текст джерелаIxodids (hard-body ticks), are the most important vectors of pathogenic agents, responsible for emerging diseases like Lyme disease (LD). This zoonosis is caused by spirochetes of Borrelia burgdorferi sensu lato (s.l.) complex, transmitted by Ixodes ricinus tick, the main vector in Europe. LD is a multisystem disorder with different clinical presentations, and a complex diagnosis. In Portugal is still underdiagnosed and the notification, although mandatory, is scarce. The main goal of this research was to develop molecular protocols, namely a TaqMan realtime PCR algorithm and an isothermal amplification, for the identification of the most prevalent genospecies of B. burgdorferi s.l. in Portugal. The development of this goal, also allowed to evaluate the bio-ecological characteristics of the ixodofauna in nine districts of the country (north, center and south), where the presence of I. ricinus tick was previously reported, and also to determinate B. burgdorferi s.l. infection rate in the vector and hosts. The obtained results are presented in 10 scientific papers, from which seven are published, two in submission and one in preparation. Of all the achieved results, is important to highlight the variations in the distribution of ticks possible into new regions, probably related to changes in the landscape, climate and vegetation, to which ticks are very sensitive. Moreover, several B. burgdorferi s.l. species were detected in ticks, apart from those commonly recognized as vectors. B. lusitaniae species was the most prevalent species in the vector, which was collected in six of the nine selected districts. Unexpectedly, during this study, three species from Relapsing Fever Borrelia complex were identified in questing ticks, namely B. miyamotoi in an I. ricinus nymph, and two possible new Relapsing Fever like-Borrelia in Haemaphysalis punctata and Rhipicephalus sanguineus tick species. Alongside, B. burgdorferi s.l. DNA was identified in biological samples from pets (dogs and cats) and sylvatic animals (wild boars), confirming the importance of these animals, mostly pets, as sentinel for early detection of emerging LD, helping to access the risk of B. burgdorferi s.l. spirochetes transmission to humans and other animals with economic importance (e.g. cattle), in restricted geographical areas. It was also possible to optimized two molecular protocols for LD laboratory diagnosis, one of which, a TaqMan real-time PCR algorithm allowing the identification of four of the most prevalent species of B. burgdorferi s.l. in Portugal, presenting high sensitivity and specificity, and contributing for a more accurate diagnosis of LD. Some of the subjects introduced and developed in this thesis deserve more detailed investigation. However, this work alerts for the introduction of possible ‘new’ Relapsing Fever Borrelia species in questing hard-body ticks, whose pathogenicity is still unknown, but it may become a risk to public health; contributes to the spatial update of important areas for LD eco-epidemiology in Portugal; and innovates in the molecular diagnosis of this zoonosis, being a valuable tool to clinicians, allowing a more accurate therapy of the patients.
Howson, Emma Lucy Anna. "The development and application of molecular tools for the diagnosis of foot-and-mouth disease in field and low-resource laboratory settings." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8607/.
Повний текст джерелаMalan, Stefanie. "Real time PCR as a versatile tool for virus detection and transgenic plant analysis." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/1921.
Повний текст джерелаENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination.
AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.
Adamu, Robert. "Molecular and Functional Characterization of Onchocerca volvulus Gene Products (Ov58GPCR and Ov-DKR-1) in the Control of Human Onchocerciasis." Doctoral thesis, Universite Libre de Bruxelles, 2018. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/279609.
Повний текст джерелаL’onchocercose est une maladie tropicale sévèrement débilitante mais négligée qui touche actuellement environ 15,5 millions de personnes, dont 12,2 millions de souffrant de maladies de la peau et 1,025 millions de souffrant de perte de vision. La maladie provoque une stigmatisation sociale, génère et perpétue la pauvreté et finit par conduire à une cécité unilatérale ou bilatérale irréversible si elle n'est pas traitée. En conséquence, l’onchocercose est un obstacle majeur au développement socioéconomique en plus d’être une préoccupation majeure pour la santé publique. De nombreux programmes de lutte ont été lancés contre la maladie, avec quelques succès en Afrique. Ces résultats sous-optimaux (limités) sont en partie dus à l’absence d’outils fiables, non invasifs et facilement applicables pour la cartographie des régions endémiques, le suivi des succès des programmes de contrôle, la détermination des paramètres de traitement et la surveillance post-élimination. Les recommandations actuelles de l’OMS pour la certification de l’élimination incluent l’utilisation du test ELISA de détection d’anticorps Ov16, entaché d’une erreur systématique intrinsèque puisque 15 à 25% des populations infectées peuvent ne pas produire d’anticorps contre cet antigène en raison de restrictions génétiques. Avec l’évolution récente de l’objectif de santé mondial de l’onchocercose de passé de la lutte à l’élimination, il est donc nécessaire de mettre au point de nouveaux outils appropriés. Ces outils nécessaires incluent, entre autres, des médicaments, des diagnostics et des vaccins. Dans ce travail, des analyses bio-informatiques combinées à des tests immunologiques ont été appliquées dans le but de développer des outils potentiels pour les programmes d'élimination actuels. En ce qui concerne la chimiothérapie, l’ivermectine, qui est le seul médicament utilisé depuis plus de 30 ans pour le traitement de l’onchocercose, ne tue que les microfilaires (mf) laissant intacts les vers adultes qui continuent à produire le mf. A ceci joint, il y a le problème récent du développement de la résistance des parasites à ce médicament. En outre, un traitement récemment approuvé, la moxidectine, est contre-indiquée chez les femmes enceintes et les enfants de moins de 12 ans qui pourraient continuer à servir de réservoirs d’infection. Il est donc absolument nécessaire d’élaborer de nouvelles stratégies de traitement, de préférence pour les médicaments macrofilaricides. Pour une éradication totale de l’onchocercose, le développement du vaccin doit être complété par le diagnostic et le traitement. Le but de ce travail est donc de caractériser un antigène d'O. volvulus, Ov58GPCR et de concevoir un antigène chimérique à base d'épitope, que nous avons appelé Ov-DKR-1, dans le cadre du développement d'outils de contrôle de l'onchocercose.Concernant le premier objectif, des peptides synthétiques représentant des épitopes B linéaires et le domaine extracellulaire (DEC) recombinant d'un récepteur couplé à la protéine G (GPCR) présentant un potentiel diagnostique ont été testés pour déterminer leur réponse immunitaire en utilisant du sérum d'individus infectés par l'onchocercose et divers témoins. Les résultats obtenus indiquent que (i) l'antigène d'O. volvulus, Ov58GPCR est un récepteur couplé à la protéine-G (GPCR) conservé dans les nématodes apparentés (ii) les peptides synthétiques prédits comme localisés dans le domaine extracellulaire de Ov58GPCR sont bien des epitopes immunogéniques chez les individus infectés par l’onchocercose, (iii) les cocktails de peptides synthétiques établissent une distinction entre les individus activement infectés avec l’onchocercose, les individus non-infectés traités et les témoins africains en bonne santé, (iv) les anticorps polyclonaux contre un des peptides ou le domaine extracellulaire exprimé au bactéries réagisent spécifiquement avec l'antigène natif dans les extraits total et de surface d'O. volvulus, (v) Ov58GPCR est transcrit aux stades larvaire et adulte, (vi) les niveaux détectés d’IgG et IgE grâce à le DEC recombinant diminuent au cours du traitement par l'ivermectine. Toutes ces découvertes suggèrent que le domaine extracellulaire recombinant et les peptides synthétiques de Ov58GPCR, ainsi que les réponses immunitaires spécifiques générées, pourraient être exploités dans le contexte du diagnostic et de la surveillance de la maladie. Pour évaluer le rôle potentiel d’Ov58GPCR dans le développement de médicaments ou de vaccins cible, un examen préliminaire de l’indispensabilité du gène Ov58GPCR pour la survie du parasite a été évalué par interférence d’ARN. Une séquence d'ARN interférant court (ARNic) ciblant le gène conçu et testé par trempage avec des vers mâles d'O. volvulus a entraîné une réduction de la motilité. Les résultats ont indiqué que le gène pourrait être impliqué principalement dans la motilité. Des investigations complémentaires sont recommandées dans cette optique.Concernant le deuxième objectif, de nombreux indicateurs révèlent la possibilité de développer des outils de protection contre l’onchocercose. En conséquence, une approche immuno-informatique a été appliquée pour concevoir un candidat-vaccin des sous-unités de multi-épitopes conservées-filarienne consistant des épitopes de cellules B et T de protéines qui seraient de nouveaux candidats vaccins. La conservation des protéines sélectionnées chez d'autres espèces parasitaires de nématodes et d'épitopes prédits suggère que la protéine chimère générée (Ov-DKR-1) pourrait être vitale pour la protection croisée. La structure 3D a été prédite, raffinée et validée bioinformatiquement. La fixation protéine-protéine du candidat vaccin chimère au récepteur TLR4 prédit une liaison favorable efficace. La simulation immunitaire prédit des niveaux significativement élevés d'IgG1, de réponses T-helper, de cellules T-cytotoxiques, de INF-γ et d'IL-2. Globalement, le peptide chimère conçu a démontré une antigénicité supérieure aux candidats vaccins actuels.
Option Biologie moléculaire du Doctorat en Sciences
info:eu-repo/semantics/nonPublished
Guimbang, Abanda Babette [Verfasser], and Alfons [Akademischer Betreuer] Renz. "Tick-borne pathogens in African cattle – novel molecular tools for diagnostics in epizootiology and the genetics of resistance / Babette Josiane Guimbang Abanda ; Betreuer: Alfons Renz." Tübingen : Universitätsbibliothek Tübingen, 2020. http://d-nb.info/1210484455/34.
Повний текст джерелаGuimbang, Abanda Babette Josiane [Verfasser], and Alfons [Akademischer Betreuer] Renz. "Tick-borne pathogens in African cattle – novel molecular tools for diagnostics in epizootiology and the genetics of resistance / Babette Josiane Guimbang Abanda ; Betreuer: Alfons Renz." Tübingen : Universitätsbibliothek Tübingen, 2020. http://d-nb.info/1210484455/34.
Повний текст джерелаVINCI, PAOLA. "From understanding the molecular basis of Graft-versus-Host Disease (GvHD), to new diagnostic tools and innovative treatments for improving the management of patients undergoing allogeneic Hematopoietic Stem Cell Transplantation (HSCT)." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2014. http://hdl.handle.net/10281/50969.
Повний текст джерелаChang, Chia-Hao, and 張家豪. "Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/ycm2d3.
Повний текст джерела國立臺灣大學
高分子科學與工程學研究所
106
In a single-cell study, isolating and identifying single cells are essential, but these processes often require a large investment of time or money. The aim of this study was to isolate and analyse single cells using a novel platform, the PanelChip™ Analysis System, which includes 2500 microwells chip and a digital real-time polymerase chain reaction (dqPCR) assay, in comparison with a standard RT-PCR (qPCR) assay. Through the serial dilution of a known concentration standard, namely pUC19, the accuracy and sensitivity levels of two methodologies were compared. The two systems were also tested on the basis of expression levels of the genetic markers vimentin, E-cadherin, N-cadherin and GAPDH in A549 lung carcinoma cells at two known concentrations. Furthermore, the influence of a known PCR inhibitor commonly found in blood samples, heparin, was evaluated in both methodologies. Finally, mathematical models were proposed and separation method of single cells was verified; moreover, gene expression levels during epithelial–mesenchymal transition in single cells under TGFβ1 treatment were measured. The drawn conclusion is that dqPCR performed using PanelChip™ has good linearity (R^2=0.9974) at 3.4 to 3.4 x 10^8 copies/μL, and it is superior to the standard qPCR in terms of, reproducibility (at 2pg/μL, dqPCR CV:1.97 < qPCR CV:3.94 ; at 200pg/μL , dqPCR CV:1.30 < qPCR CV:8.11) and heparin tolerance (dqPCR IC50: 0.02IU/mL > qPCR IC50: 0.002IU/mL). The dqPCR assay is a potential tool for clinical diagnosis and single-cell applications.
Marques, Bruno Filipe Pinheiro. "Urine : a new tool for molecular diagnosis of chronic rejection in kidney transplantation?" Master's thesis, 2015. http://hdl.handle.net/10316/30340.
Повний текст джерелаRenal transplantation has become the treatment of choice for patients with end-stage renal disease. Recognizable improvements in early graft survival and long-term graft function have made kidney transplantation a more cost-effective alternative to dialysis. Moreover, the discovery of new immunosuppressive agents has made a significant impact on short-term graft survival. Despite these improvements a substantial portion of grafts develop progressive dysfunction and fail within a decade by a process known as chronic rejection. Ongoing monitoring of kidney transplants is crucial to avoid the development of this condition. The most common approaches to monitor renal allograft function are the measurement of the serum creatinine levels, whose variations are not specific for rejection and sometimes there is a need to perform renal biopsies, which is a risky process and only diagnoses rejection once it is installed. The main objective of this study was to create an immunological and cellular profile associated with chronic dysfunction establishment that could serve as a diagnostic tool. For this purpose, a series of different analyses were carried out on 71 patients with stable/normal renal function after the transplant and the results compared to the same tests performed on 27 patients who have been diagnosed with chronic graft rejection. The development of an antibody screening method in the urine of renal transplant patients was one of the most relevant points in the study with more than half the patients diagnosed with chronic rejection presenting anti-HLA Class I antibodies in urine. The normalized gene expression values found in the urinary sediment of patients diagnosed with chronic rejection confirm the involvement of inflammation in the development of chronic rejection. Furthermore, the increased normalized gene expression levels for B cells markers (CD19 and CD79B) in chronic rejection group suggest the existence of B cells clusters that can function as a tertiary lymphoid tissue which can harbour B cell maturation into memory B cells and antibody producing plasma cells. According to the receiving operating characteristic curves, CD19, CXCL10 and TNF3 were the genes with the highest diagnostic values for chronic rejection. This study demonstrates that analysis of the urine of renal transplant patients could give valuable information that may allow the monitoring of the transplant without resort to invasive methods. Nevertheless, only the combination of results obtained in the urine and blood samples can provide a complete and accurate assessment of the allograft condition.
A transplantação renal tornou-se a terapia de eleição para doentes com insuficiência renal crónica terminal. As melhorias, em termos de sobrevivência do enxerto e da função renal a longo prazo, fizeram com que a transplantação renal tenha uma melhor relação custo/benefício do que a diálise. A acrescentar a isto, a introdução de novos agentes imunossupressores teve um impacto significativo na sobrevivência do enxerto a curto prazo. Apesar destas melhorias, uma quantidade substancial dos enxertos desenvolvem uma disfunção progressiva com perda de função total no prazo de uma década, através de um processo denominado rejeição crónica. A monitorização funcional dos rins transplantados torna-se crucial para tentar evitar o desenvolvimento desta condição. Os métodos mais comuns para realizar a monitorização da função do enxerto são as medições dos níveis de creatinina no soro, sendo que estas variações não são específicas para a rejeição e ainda, por vezes, é necessário realizar biopsias renais, que é um processo arriscado e que só diagnostica esta rejeição depois de esta estar instalada. O principal objetivo deste estudo foi desenvolver um perfil imunológico e celular, associado ao estabelecimento da disfunção crónica, que possa servir como ferramenta de diagnóstico. Tendo isto em vista, um conjunto de análises foram efetuadas em 71 transplantados renais com uma função renal estável e os resultados, comparados com os mesmos testes efetuados em transplantados renais que tinham sido diagnosticados com rejeição crónica. O desenvolvimento de um novo método de seleção de anticorpos na urina de transplantados renais foi um dos pontos mais importantes neste estudo, sendo que mais de metade dos transplantados com rejeição crónica apresentou anticorpos anti-HLA Classe I na urina. Os valores da expressão génica normalizada encontrados para as células do sedimento urinário dos transplantados renais, confirmaram o envolvimento da inflamação no desenvolvimento da rejeição crónica. Além disso, os valores mais elevados de expressão génica normalizada para os marcadores da célula B (CD19 e CD79B), no grupo da rejeição crónica, sugerem a existência de aglomerados de células B que funcionam como um órgão linfoide terciário, capaz de albergar a maturação da célula B em células de memória e em células do plasma produtoras de anticorpos. De acordo com a análise feita das curvas características de operação do recetor, os genes CD19, CXCL10 e TNF3 foram os que obtiveram maior valor de diagnóstico para a rejeição crónica. xv Este estudo comprova que a análise da urina de transplantados renais, no futuro breve, pode fornecer informações valiosas, permitindo assim, a monitorização funcional destes doentes sem recurso a procedimentos invasivos. No entanto, só a combinação dos resultados obtidos na urina e no sangue pode oferecer uma avaliação completa e precisa da condição do enxerto
Callison, Scott Andrew. "Molecular tools for the study, diagnosis, and control of infectious bronchitis virus." 2003. http://purl.galileo.usg.edu/uga%5Fetd/callison%5Fscott%5Fa%5F200305%5Fphd.
Повний текст джерелаDirected by Mark W. Jackwood. Includes articles submitted to Avian diseases, Virus genes, and The journal of clinical microbiology. Includes bibliographical references.
DURANTI, CLAUDIA. "Novel molecular tools in cancer therapy: diagnostic and therapeutic applications of antibodies targeting ion channels and receptors." Doctoral thesis, 2017. http://hdl.handle.net/2158/1077157.
Повний текст джерелаLeão, Célia Cristina Fialho. "Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex." Doctoral thesis, 2015. http://hdl.handle.net/10362/16513.
Повний текст джерелаMIGLIORINI, DUCCIO, PAOLO CAPRETTI, and ALBERTO SANTINI. "Phytophthora in natural and anthropic environments: new molecular diagnostic tools for early detection and ecological studies." Doctoral thesis, 2016. http://hdl.handle.net/2158/1028950.
Повний текст джерелаZAMPIERI, ROBERTA. "Plant viruses: the many aspects of fascinating nano-biotechnological tool." Doctoral thesis, 2017. http://hdl.handle.net/11562/961367.
Повний текст джерелаThe capsids of most plant viruses are simple and robust structures consisting of multiple copies of one or few types of protein subunits arranged with either icosahedral or helical ordered symmetry. In many cases, capsids can be produced in large quantities either by the infection of plants or by the expression of the subunits. In view of their relative simplicity, stability and easy production, plant chimeric virus particles (CVPs) or empty virus-like particles (eVLPs) have attracted attention as potential reagents for applications in bionanotechnology. In this work CVPs and eVLPs have been exploited for the expression of functional peptides, in order to stabilize them and avoid peptide low intrinsic stability and susceptibility to degradation. In particular, the viral expression platforms chosen for the expression of target peptides are based on four plant viruses widely used as scaffold for peptide display: Potato Virus X (PVX), Cowpea Mosaic Virus (CPMV), Tomato Bushy Stunt Virus (TBSV) and Turnip Mosaic Virus (TuMV). The first application explored in this work regards the therapy of Type 1 diabetes (T1D) and Rheumatoid arthritis (RA), two autoimmune diseases that share a strong social impact. Currently, there are treatments able to manage and/or stem the effects of these disorders. In particular, plant viruses displaying peptides associated to T1D and RA have been used respectively for the development of a preventive and therapeutic drug. Virus particles displaying autoantigenic peptide specific for these diseases have been expressed and used for pre-clinical studies in T1D and RA animal models. The results observed suggest that the use of viral structure for peptide display works as an adjuvant by increasing peptide modulation capability. The second part of this work regards the use of plant viruses displaying peptide as reagents for the development of innovative kit for the Sjögren’s Syndrome (SjS) and RA diagnosis. These two autoimmune diseases are difficult to be diagnosed and either for SjS of RA there are subgroups of patient seronegative to the main diagnostic serological markers. In this work, the use of filamentous particles for the display of specific SjS peptide allowed to increase the diagnostic performances of an ELISA kit in comparison to the use of the peptide alone. Moreover, autoantigenic peptides associated to RA were successfully expressed in plants on the surface of viral particles that will be exploited in the future for the development of a kit for seronegative RA diagnosis. A third part of this PhD thesis regards another possible application of plant viruses as tools for peptide display. In particular, viral particles have been used for the expression of a antimicrobial peptide (AMP) that could be exploited as eco-friendly pesticide and for “nanoagriculture” application. Finally, the possibility of developing a biotechnological tool for peptide internalisation into the cells has been exploited by fusing on the surface of an icosahedral plant virus a cell-penetrating peptide (CPP) derive from HIV. Regarding this third part, CVPs and eVLPs displaying the selected peptides have been successfully expressed in plants; however, several drawbacks have been encountered in the purification process.
Troiano, Annaelena. "FUNCTIONAL ANALYSIS OF P63-YB1 INTERACTION IN NORMAL AND TRANSFORMED EPITHELIAL CELLS: DEVELOPMENT OF NOVEL MOLECULAR TOOLS FOR SQUAMOUS CANCER DIAGNOSIS AND THERAPY." Tesi di dottorato, 2015. http://www.fedoa.unina.it/10314/1/Troiano_Annaelena_27.pdf.
Повний текст джерела