Dissertations / Theses on the topic 'Autoimmune diseases Molecular diagnosis'
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Narayanan, Harish Anandha. "Molecular Understanding of Selected Autoimmune Diseases." Thesis, The University of Arizona, 2010. http://hdl.handle.net/10150/146614.
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Pathogens that affect the immune system, or defects in the immune system contribute to numerous autoimmune diseases. In this paper, we are going to review, analyze and understand recent findings in literature. The papers that are to be reviewed explore the observation of loss of regulatory T cells in individuals with multiple sclerosis, the importance of CD4+ during HIV infection, the role of CCR6 in the immune system and the importance of Pan-DR-Binding Hsp60 self-epitopes in rheumatoid arthritis. So this literary review is limited to understanding major defects in specific parts of the immune system and its role in causing the specific autoimmune disease. The reason for this focus is to highlight the importance of the immune system in the functioning of many processes in our body and more specifically the importance of T cells and its regulators in maintaining the immune system.
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Halonen, Maria. "Monogenic model for autoimmune diseases : molecular basis of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED)." Helsinki : University of Helsinki, 2003. http://ethesis.helsinki.fi/julkaisut/laa/haart/vk/halonen/.
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Hall, Richard James, and n/a. "Chromosome 18 and autoimmune disease." University of Otago. Department of Biochemistry, 2005. http://adt.otago.ac.nz./public/adt-NZDU20070221.141018.
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The autoimmune diseases embody a diverse range of common human conditions that are caused by a loss of self-tolerance in the host immune system to a specific organ or tissue type. Approximately 5% of the general population are affected by autoimmune diseases which include type 1 diabetes (T1D), rheumatoid arthritis (RA) and Graves disease (GD). The majority of the autoimmune diseases are multifactorial in origin, brought about by a combination of both environmental and genetic factors. Numerous susceptibility loci have been identified for each autoimmune disease and a number of these loci have been shown to be shared amongst the autoimmune diseases. The fine-mapping of susceptibility loci to the underlying disease genes remains the current challenge facing complex disease genetics. This project aimed to further characterise the autoimmune disease susceptibility locus IDDM6 on chromosome 18q12-21. This was achieved by using a comparative mapping approach that incorporates the study of genetic association in human autoimmune disease alongside the consomic mapping of the orthologous region in the non-obese diabetic (NOD) mouse model of autoimmunity.
Deleted in colorectal carcinomas (DCC) provided a strong candidate gene at IDDM6 and the resident R201G polymorphism was identified as a functional candidate A potential mechanism for the R201G polymorphism involvement in T1D aetiology was identified where the polymorphism may affect the ability of DCC to induce apoptosis in vitro. However, no evidence for R201G association could be detected in autoimmune disease case-control datasets from the New Zealand (NZ) population (T1D n = 428, RA n = 730, autoimmune thyroid disease n = 192 (AITD); versus n = 1246 healthy controls). In addition, no evidence for R201G involvement in T1D could be provided in a transmission disequilibrium test (TDT) incorporating 382 affected sib-pair families (54.2% transmission; P = 0.15). Significant association of R201G with GD was detected in a United Kingdom (UK) dataset (P = 0.002) from the Newcastle population (423 cases vs. 393 controls) but this was not replicated in an additional dataset from the UK Birmingham population (731 cases vs 668 controls; P = 0.81). It was concluded that the R201G polymorphism may encode susceptibility to GD but is unlikely to be the sole aetiological variant that accounts for the linkage previously observed at IDDM6 in autoimmune disease. To further investigate DCC as a positional candidate at IDDM6, five SNPs were selected from a 100 kb window surrounding a DCC-resident microsatellite that had previously been associated with T1D, called "88,21". The five SNPs were genotyped in the NZ T1D dataset, and the ascertainment of estimated haplotypes in this dataset revealed association of a rare haplotype with T1D, called haplotype H (3.31% cases vs 1.17% controls; P = 0.0044), in addition to global association of all haplotypes (P = 0.018). Haplotype H was also associated in an independent case-control dataset from the UK comprised of 400 T1D subjects and 443 healthy controls (P = 0.038). Maximum support for association of haplotype H was extended when both the UK and NZ T1D datasets were combined (P = 0.0017). Association of haplotype H could not be verified in a family-based test for association using the 382 UK T1D families (P = 0.40). However, the inclusion of the DCC SNPs in a TDT analysis of the published DCC-resident microsatellites "88,21" and "55,26", that had been used to identify IDDM6, extends support for the previously-associated 2-10 haplotype (2-10 refers to the published allele nomenclature at "88,21" and "55,26" respectively; 2-10-haplotype A; 59.6% T; P = 0.0058). There was no evidence for association of the five SNPs with RA or AITD when using either individual SNP analyses or estimated haplotypes in the NZ datasets. A similar lack of association was reported for the UK Newcastle GD dataset. Taken together, these data further support DCC, or a nearby gene, as conferring susceptibility to T1D.
The human genetic data that supports IDDM6 involvement in autoimmune disease is further strengthened by consomic mapping of the orthologous region in mouse, using the non-obese diabetic mouse (NOD) model of autoimmune disease. In this thesis, the first evidence for a diabetes and thyroiditis susceptibility locus on mouse chromosome 18 is presented, which have been designated Idd21 and Sat1 respectively. This was achieved by using a chromosome-replacement strain with chromosome 18 derived from the diabetes-resistant Biozzi ABH strain on a diabetes-susceptible NOD genome, called NOD.ABH[Chr�⁸]. Mouse chromosome 18 contains orthology to both IDDM6 and the rat diabetes-susceptibility locus Iddm3. The NOD.ABH[Chr�⁸] mice showed a dramatic and significant reduction in diabetes incidence (30% of females were affected by 7 months of age versus 85% in NOD; P <0.0001) and that of thyroiditis (15.5% at 12 months compared to 37.4% in NOD; P <0.002).
The comparative mapping of the chromosome 18 autoimmune susceptibility locus IDDM6 in human and mouse presented in this thesis provides further support for this locus. This research also clearly defines the next steps required to fine-map IDDM6 to the underlying disease genes, especially in regard to the DCC gene.
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Braida, Claudia. "Molecular analysis of myotonic dystrophy type 1 patients with an unusual molecular diagnosis." Thesis restricted. Connect to e-thesis to view abstract, 2008. http://theses.gla.ac.uk/359/.
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Thesis (Ph.D.) - University of Glasgow, 2008.Ph.D. thesis submitted to the Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.
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Yang, Min, and 杨敏. "Role of regulatory B cells in autoimmune disease." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48079832.
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Although B cells are well-known for their functions in antibody production and antigen presentation, certain B cell subsets have been recently identified as regulatory B cells to modulate immune responses through cytokine production. However, the microenvironmental factors involved in the induction of regulatory B cells remain largely uncharacterized. B cell-activating factor (BAFF), a member of TNF family cytokines produced by myeloid cells, is a key regulator for B cell maturation and function. However, it remains unknown whether BAFF plays a role in modulating the generation of regulatory B cells and how regulatory B cells suppress autoimmune pathogenesis. In this study, treatment with BAFF significantly increased IL-10-producing B cells in culture of mouse splenic B cells, an effect specifically abrogated by neutralization with TACI-Fc. BAFF-induced IL-10-producing B cells showed a distinct CD1dhiCD5+(B10) phenotype. Phenotypic analysis further indicated that these BAFF-induced B10 cells were marginal-zone (MZ)-like B cells. Interestingly, BAFF treatment in vivo also increased the number of IL-10-producingB cells in splenic MZ regions. Moreover, chromatin immunoprecipitation analysis revealed that BAFF activated the transcription factor AP-1 for binding to IL-10 promoter, demonstrating a novel function for BAFF in inducing IL-10 production. Furthermore, those BAFF-induced B10 cells exhibited significant suppressive effects on CD4+T cell proliferation and Th1 cytokine production in culture.
To explore whether these BAFF-induced B10 cells possess a regulatory function in suppressing autoimmune progression in vivo, collagen-induced arthritis (CIA) mouse model was employed. In vitro-expanded B10 cells and other control B cells were intravenously transferred into DBA/1J mice on the day of 2ndcollagen II (CII)-immunization. After adoptive transfer of BAFF-induced B10 cells, CII-immunized mice exhibited a delayed onset of arthritis and substantially reduced severity of clinical symptoms. The pathogenesis of IL-17-producing CD4+T cells (Th17) in the development of arthritis has been well-recognized, which has led me to test the hypothesis whether B10 cells ameliorate the development of arthritis via modulating Th17 cells. During the progression of CIA, IL-10-producing B cells were decreasedwhereasTh17 cells were significantly increased at the acute phase of CIA. Upon transfer of BAFF-induced B10 cells, a substantially reduction ofTh17 cells in both lymphoid organs and inflamed joints were detected. To verify whether B10 cells inhibit Th17 cell generation in culture, CFSE-labeled na?ve CD4+T cells were cocultured with B10 cells in Th17 cell polarization medium. It was found that B10 cells suppressed Th17 cell differentiation via reducing STAT3 phosphorylation and RORt expression. Although adoptive transfer of Th17 cells triggered the development of CIA in IL-17-/-DBA mice, cotransfer of B10 cells with Th17 cells profoundly delayed the onset of delayed the onset of arthritisand remarkably reduced the infiltration of Th17 cells in synovial fluid.
Taken together, I have identified a novel function of BAFF in the induction of IL-10-producing regulatory B cells. My findings that adoptive transfer of BAFF-expanded B10 cells can effectively suppress the development of experimental arthritisin mice via the inhibition of Th17 cell generation may contribute to the development of new therapeutic strategies in treating human rheumatoid arthritis.published_or_final_version
Pathology
Doctoral
Doctor of Philosophy
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Mulcahy, Anthony Francis. "The molecular cloning and characterisation of autoantigens." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242453.
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Hudspith, Karl Alexander Zhivojin. "Application of genomic technologies for molecular diagnosis of genetic diseases." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:b4addaed-e9f9-4762-846a-87eb73f77235.
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Methods for sequencing deoxyribonucleic acid (DNA) have improved rapidly in the past decade. Recent methods, termed "next-generation sequencing" (NGS), have made sequencing large quantities of DNA economically viable in molecular diagnostics of genetic diseases. This thesis describes some of the first investigations to use NGS for such a purpose. Several different NGS platforms, each of which differs substantially in terms of sample preparation, chemistry, and sequencing methodology, were tested, in addition to several sequence capture and enrichment technologies that allow sequencing to be targeted, and these combinations were compared to Sanger sequencing. They were each found to have different strengths and weaknesses, which affect their accuracy, reliability, time taken to results, financial cost, and ease of use, but all showed high accuracy and dramatically increased throughput over Sanger sequencing. NGS was used to identify pathogenic mutations in two groups of patients who had either inherited retinal dystrophies (IRD), or severe early onset epilepsies. NGS was able to identify pathogenic variants, demonstrating the ability of the technology to provide medically useful information for genetically heterogeneous conditions. NGS combined with a novel data analysis pipeline was able to make a secure molecular diagnosis in 25% of a cohort of IRD patients who previously did not have a genetic diagnosis. The greatest challenge presented by NGS was found to be filtering the vast amounts of data produced to identify potential pathogenic variants. In silico pathogenicity prediction programs were used, but none were 100% accurate. Other methods were also employed to provide further evidence of pathogenicity. These included family based DNA testing for cosegregation of variants with phenotype, and transcript based analysis. In some patients, despite extensive genetic testing, a secure molecular diagnosis could not be made using DNA sequencing technologies, illustrating that challenges still remain in the field of genetic diagnostics.
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Mohamed, Nahla. "Molecular Diagnosis of Common Viral Infectious Diseases Based on Real-Time PCR." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7118.
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Xu, Jiru. "Application of PCR and DNA sequencing based molecular diagnosis in infectious diseases." Thesis, University of Ulster, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399727.
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Abdelrahman, Wael Hosny Abdellatif. "Avian intestinal spirochaetosis in British egg laying flocks : molecular diagnosis, epidemiology and economic impact." Thesis, Royal Veterinary College (University of London), 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.559017.
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Mesa, Annia. "Auto-antigenic Properties of the Spliceosome as a Molecular Tool for Diagnosing Systemic Lupus Erythematosus and Mixed Connective Tissue Disease Patients." FIU Digital Commons, 2014. http://digitalcommons.fiu.edu/etd/1126.
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Systemic Lupus Erythematosus (SLE) and Mixed Connective Tissue Disease (MCTD) are chronic, autoimmune disorders that target overlapping autoantigens and exhibit similar clinical manifestations. Despite 40 years of research, a reliable biomarker capable of diagnosing these syndromes has yet to be identified. Previous studies have confirmed that components of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) such as U1A are 1000 fold more autoantigenic than any other nuclear component in SLE patients. Based on these findings, I hypothesize that models derived from the U1 snRNP autoantigenic properties could distinguish SLE from MCTD patients. To test this hypothesis, 30 peptides corresponding to protein regions of the U1 snRNP were tested in triplicates by indirect ELISA in sera from SLE or MCTD subjects. In addition laboratory tests and clinical manifestations data from these patients were included and analyzed in this investigation. Statistical classification methods as well as bioinformatics pattern recognition strategy were employed to determine which combination, if any, of all the variables included in this study provide the best segregation power for SLE and MCTD. The results confirmed that the IgM reactivity for U1 snRNP and U1A have the power to significantly distinguish SLE from MTCD patients as well as identify kidney and lung malfunctions for these subjects (p ≤ 0.05). Furthermore, the data analysis revealed eight novel classification rules for the segregation of SLE and MCTD which are a better classification tool than any of the currently available methods (p ≤ 0.05). Consequently, the results derived from this study support that SLE and MCTD are indeed separate disorders and pioneer the description of eight novel classification criteria capable of significantly discerning between SLE and MCTD patients (p ≤ 0.05).
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Ferraz, Colomina Rosa María. "Development of allosteric biosensors for the diagnosis of infectious diseases." Doctoral thesis, Universitat Autònoma de Barcelona, 2008. http://hdl.handle.net/10803/3922.
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Los biosensores representan actualmente herramientas importantes para la detección de moléculas de interés. Los biosensores proteicos destacan por su fácil producción y uso, permitiendo un desarrollo económico de biosensores que pueden ser utilizados en todo el mundo y sobretodo, útiles en países con falta de recursos y tecnología. Un diagnóstico rápido y eficaz de enfermedades infecciosas permitiría un tratamiento más rápido y un mejor aprovechamiento de los recursos disponibles.En este estudio, el biosensor proteico NF795gpC, útil para la detección de anticuerpos anti-virus de la inmunodeficiencia humana (VIH), ha sido caracterizado. La proteína NF795gpC es una beta-galactosidasa modificada previamente sintetizada en nuestro laboratorio, que contiene el péptido antigénico P1 de la proteína gp41 de VIH insertado entre los residuos 795 y 796. La inserción de péptidos en lugares permisivos expuestos del enzima, permite la síntesis de proteínas que responden a la presencia de anticuerpos específicos. El hecho de que contengan inserciones, hace que estas proteínas tengan una actividad enzimática reducida. Sin embargo, en presencia de anticuerpos específicos para el péptido insertado, estos enzimas híbridos traducen la interacción antígeno-anticuerpo en un incremento de actividad enzimática fácil de medir. Finalmente, y teniendo un ejemplo previo con el virus de la fiebre aftosa, este principio puede extenderse a la detección de otras enfermedades infecciosas que producen una respuesta immune mediada por anticuerpos.
Primero se optimizó el ensayo buscando las concentraciones óptimas de proteína y sustrato y también analizando el efecto sobre sensibilidad, ratio señal-fondo y rango de respuesta, de seis sustratos diferentes. La proteína NF795gpC fue también inmovilizada en un soporte para analizar su capacidad de respuesta en estas condiciones, siendo el primer paso para un futuro desarrollo de un sistema de detección útil para hacer medidas de campo.
El análisis de los diferentes tipos de anticuerpos específicos presentes en muestras de suero con el sensor mostró una correlación entre la cantidad de inmunoglobulinas IgG4 y la respuesta, considerándose esta inmunoglobulina como la mayormente responsable de la activación del sensor.
Finalmente, nuevos enzimas alostéricos fueron sintetizados para mejorar la sensibilidad del sistema previamente descrito. El hecho de que la proteína NF795gpC contiene solamente un único inserto representa una limitación a la hora de diagnosticar la enfermedad, llegando solo a un 94% de coincidencia entre este y el método estándar normalmente utilizado para diagnosticar VIH. Por tanto, la inserción de otros fragmentos antigénicos del virus en la proteína permitiría aumentar el número de anticuerpos VIH específicos y, por tanto, también aumentar la sensibilidad del sistema. El desarrollo de un sistema mucho más completo representaría un gran avance en la salud pública y también sería de gran importancia para entender mejor el mecanismo de regulación enzimática en biosensores alostéricos.
Biosensors have become important tools in the detection of different types of molecules of analytical interest. Among them, protein-only biosensors stand out due to their ease production and use, what allow a cheaper development and applicability with low costs, especially important in low resource areas. A better and quicker diagnosis of illnesses would allow a faster treatment and an effective use of the available resources.
In this study, the protein-only biosensor NF795gpC, suitable for the detection of anti-human immunodeficiency virus (HIV) antibodies, has been characterized. NF795gpC protein is an engineered beta-galactosidase previously developed in our laboratory, containing the antigenic P1 peptide of gp41 protein of HIV in between the residues 795 and 796. The insertion of small peptides in permissive solvent exposed sites of the enzyme produces hybrid beta-galactosidases with a reduced enzymatic activity but allows the protein to respond enzymatically to peptide-specific antibodies. In presence of anti peptide monoclonal antibodies or polyclonal sera, these hybrid enzymes translate the antigen-antibody interaction into an easily measurable increase of the enzymatic activity. Furthermore, and having a previous example with FMDV, this principle can be extended to other infectious diseases producing an antibody response.
First, the sensing assay was optimized looking for the optimal concentrations of NF795gpC protein and substrate generating a higher sensing response, and also analyzing the sensor response regarding sensitivity, signal-background ratio and range of response with six different substrates. Protein NF795gpC was also immobilized into a support to analyze the sensor response in these conditions, being the first step to the further development of a sensor device useful for field measurements.
The analysis of the different types of antibodies present in sera samples through the sensor showed a correlation between the amount of IgG4 subpopulation of antibodies of a group of sera samples and the sensing signal, considering this immunoglobulin as the most important for sensor activation.
Finally, new allosteric enzymes were constructed in order to improve the sensitivity of the test. The fact that the NF795gpC contains only one type of HIV peptide represents a limitation to the proper diagnosis of HIV infection, arriving only to a 94% of agreement between this test and the standard method normally used for HIV detection. Hence, the insertion of other antigenic fragments of HIV into the beta-galactosidase could allow the detection of other specific antibodies. The development of a more complete device could represent big advances in public health and also be helpful in a better understanding of the mechanism of enzymatic regulation in allosteric biosensors.
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Xavier, Maria José Pinto Barreira Rego de Sousa. "Rastreio combinado do 1º trimestre e doenças autoimunes : Impacto das variaveis pré-analíticas na avaliação do risco." Doctoral thesis, Faculdade de Ciências Médicas, 2014. http://hdl.handle.net/10362/12156.
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RESUMO: Contexto: As anomalias congénitas, com particular destaque para as neuploidias , afectam aproximadamente 2% dos recém-nascidos, constituindo causas frequentes de morbilidade e mortalidade. Actualmente, a avaliação do grau de risco para as aneuploidias mais prevalentes
(T21, T13, T18) é efectuada através do “Rastreio Combinado do 1º Trimestre”, devendo as grávidas com risco acrescido ser sujeitas a exames invasivos (ex.biópsia das vilosidades coriónicas,amniocentese). Quanto mais qualidade existir num rastreio, menos falsos positivos existirão e menor o número de exames diagnósticos invasivos desnecessários.
As doenças autoimunes são doenças inflamatórias crónicas em cuja fisiopatologia se encontram distúrbios da imunidade humoral e celular, dependentes de factores genéticos, hormonais,psicológicos e ambientais. Atingem mais o sexo feminino e durante a idade fértil,podendo influenciar o outcome da gravidez e a saúde neonatal causando significativa morbilidade
e mortalidade. O lúpus eritematoso sistémico para além de potencialmente afectado
pelas alterações imunoendócrinas fisiológicas da gravidez, associa-se frequentemente a problemas de fertilidade. Recentemente, foi sugerido que as anormalidades ocorridas durante a invasão precoce do sinciciotrofoblasto, resultando em deficiente diferenciação, deficiente
maturação e diminuição na produção de hCG, poderão ser o mecanismo fisiopatológico
primário para as perdas fetais no primeiro trimestre, nos doentes com SLE.
A ocorrência de níveis elevados de hCG total e ß-hCG livre no rastreio para despiste de síndrome de Down do segundo e do primeiro trimestre foi assinalada em grávidas portadoras de lúpus, mas a escassez de estudos comprovativos e a pequena dimensão das amostras estudadas constituiu uma limitação significativa na fidedignidade dos resultados obtidos.
Objectivos: O estudo teve como objectivos i. estabelecer valores normativos Portugueses e
de distribuição para as MoM’s dos parâmetros séricos do primeiro trimestre, por semana de
gestação:(PAPP-A e ß-hCG livre), ii. avaliar a influência que as doenças autoimunes têm sobre as MoM’s individuais dos parâmetros bioquímicos PAPP-A e/ou ß-hCG livre, utilizados no rastreio pré-natal combinado do 1º trimestre, e iii. saber se as doenças autoimunes podem condicionar um aumento da taxa de resultados falsos positivos, com consequente aumento do número de amniocenteses. Metodologia: Estudo longitudinal prospectivo, consistindo num rastreio pré-natal combinado de 1º trimestre para pesquisa de aneuploidias, em duas amostras provenientes do Reino Unido (n= 45,854) e de Portugal (n=3122). Foram avaliados parâmetros socio-demográficos,
ecográficos, laboratoriais, e calculados os indicadores de desempenho do rastreio combinado.
A execução analítica dos testes bioquímicos séricos (PAPP-A e ß-hCG livre) foi realizada no autoanalisador Brahms Kryptor e no autoanalizador 6000 Delfia Xpress. Compararam-se os grupos autoimune e não autoimune das amostras.
Resultados: Relativamente às características populacionais, o grupo auto imune tinha valores significativamente superiores nas variáveis idade materna e idade gestacional. Comparando os grupos com e sem doença autoimune, constatou-se a existência de uma elevação das MoM’s da ß-hCG livre nas grávidas com doença autoimune, nomeadamente nos casos de lúpus eritematoso sistémico.
Conclusões: os resultado obtidos reforçam a indicação do rastreio combinado do 1º trimestre certificado pela FMF nas grávidas com doenças autoimunes, nomeadamente para
as doentes com LES; no entanto, devem ser calculados e introduzidos factores de correcção no algoritmo de risco, de modo a evitar a subida no número de resultados falso-positivos, e consequentemente a sobre- utilização de métodos invasivos.------------ ABSTRACT: Context: Congenital anomalies, with particular reference to aneuploidias, affect approximately 2% of newborns, and are frequent causes of morbidity and mortality. Currently, the risk evaluation for the most prevalent aneuploidias (T21, T13, T18) is carried out through the “combined first trimester screening”, and pregnant women with increased risk are subjected
to invasive tests (e.g. villus biopsy done, amniocentesis). The more quality exists in a screening, less false positives exists and fewer unnecessary invasive diagnostic exams. Autoimmune diseases are chronic inflammatory diseases in whose pathophysiology are immune humoral and cellular disorders, dependent on genetic factors, hormonal, psychological and environmental factors. The disease is more prevalent among females, during the child-bearing age, and may influence the outcome of pregnancy and neonatal health causing significant morbidity and mortality. Lupus Erythematosus in addition to potentially affected by immunoendocrine physiological changes of pregnancy, is often associated with fertility problems.
Recently, it has been suggested that the abnormalities that occurred during the early invasion of the syncytiotrophoblast, resulting in insufficient differentiation, deficient maturation and decrease in production of hCG may be the primary pathophysiological mechanism for fetal losses in the first quarter, in patients with SLE. The occurrence of elevated levels of total hCG and free ß-hCG in screening for Down’s syndrome of the second and first trimester was reported in pregnant women with lupus, but the paucity of supporting studies and the small size of the samples studied constituted a significant limitation on the trustworthiness of the results obtained.
Objectives: this study aims to i. establish normative values for the serum parameters MoM’s (PAPP-A and free β-hCG) and it’s distribution, in the first trimester, by week of pregnancy; ii. assess the influence that the autoimmune diseases have on the MoM’s of individual biochemical
PAPP-A and/or β-hCG, used in antenatal screening combined for the first trimester,
and iii. whether the autoimmune diseases may make an increased rate of false positives, with consequent increase in the number of amniocenteses.Methodology: Prospective longitudinal study, consisting of a combined first trimester antenatal screening for aneuploidies lookup in two samples from the United Kingdom (n=45.854) and Portugal (n= 3.122). Socio-demographic, echographic and laboratory parameters were evaluated, and combined screening performance indicators were calculated. The analytical run of serum biochemical tests (PAPP-A and ß-hCG) was held at the Brahms Kryptor and in Delfia Xpress 6000. Comparisons between autoimmune group and non-autoimmune group were made.
Results: Relating to population characteristics, the autoimmune group had significantly diferente values in the variables maternal age and gestational age. Comparing the groups with and without autoimmune disease, it was noted that there is an elevation of the MoM’s of free
ß-hCG levels in pregnant women with autoimmune disease, particularly in cases of systemic lupus erythematosus.
Conclusions: The results obtained reinforce the indication of FMF certified combined
screening in pregnant women with autoimmune diseases, notably to the patients with SLE;
However, correction factors should be calculated and entered in the risk algorithm, in order to avoid the rise in the number of false positive results, and consequently the over-use of invasive methods.
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Hollis-Moffatt, Jade Elissa, and n/a. "Fine mapping and characterisation of an autoimmune diabetes locus, insulin dependent diabetes 21, (Idd21) on mouse chromosome-18." University of Otago. Department of Biochemistry, 2006. http://adt.otago.ac.nz./public/adt-NZDU20070130.151657.
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Autoimmune disease is comprised of a wide variety of disorders characterised by a loss of self-tolerance towards a target organ or systemic region leading to its eventual destruction. Type 1 diabetes (T1D), autoimmune thyroid disease (AITD) and inflammatory bowel disease (IBD) are debilitating organ-specific disorders. These disorders arise from a combination of genetic factors and environmental triggers. A greater level of basic understanding of these disorders is required to delay and/or prevent their effects. Numerous autoimmune susceptibility loci have been implicated in the development of these disorders, but only a few causative genes have been identified. The aim of this project was to use comparative mapping between the human and mouse genomes to provide a greater understanding of the human autoimmune susceptibility locus, IDDM6, shown to be involved in a number of autoimmune disease conditions.
Hall et al., (2003) previously demonstrated that the mouse autoimmune diabetes locus, Idd21, on distal mouse chr18 contains orthology to human IDDM6, IDDM10, IDDM18 and rat Iddm3. As part of this project the Idd21 locus was fine mapped using the congenic mapping technique. Beginning with the consomic mouse strain, NOD.ABHChr18 (90Mb of Biozzi/ABH-derived diabetes-resistant chr18 introgressed onto a non-obese diabetic (NOD) genetic background), 13 NOD.ABHIdd21 congenic mouse strains were established. The diabetes incidences of these congenic mouse strains were statistically compared stepwise along mouse chr18 and Idd21 was fine mapped to at least four independent autoimmune diabetes loci. Idd21.1 and Idd21.2 were located on distal mouse chr18 in regions orthologous to human IDDM6 and rat Iddm3 and Idd21.3a/b and Idd21.4 were located on proximal mouse chr18 in regions orthologous to human IDDM18 and IDDM10 respectively. Candidate genes of notable interest include Map3k8, Spink5, Cd14, Dcc, Smad4 and 7, Miz1, Nfatc1 and Cd226.
Idd21.1 was further fine mapped. Beginning with the NOD.ABHD18Mit8-D18Mit214[(75-85.1Mb)] (Idd21.1) congenic strain (containing at least 10.1Mb of distal chr18 Biozzi/ABH diabetes-resistant DNA introgressed onto a NOD genetic background), seven subcongenic mouse strains were created. The diabetes incidence of these subcongenic mouse strains were statistically compared stepwise along mouse chr18 and Idd21.1 was fine mapped to at least three independent autoimmune diabetes loci; Idd21.11 (72.6-76.1Mb), Idd21.12a/b (75-76.1Mb and 80.6-81.4Mb) and Idd21.13 (84.8-85.1Mb). Candidate genes of interest in these regions include Dcc, Smad4 and 7, Miz1, Nfatc1, and Cd226.
Functional characterisation of the Idd21.1 locus was performed by adoptively transferring splenocytes from female NOD or NOD.ABHIdd21.1 mice into cohorts of severe combined immune deficient (scid) female mice, NOD/LtSz.Prkdc[scid] and NOD/LtSz.Prkdc[scid].ABHIdd21.1. There were two notable findings from this work. Firstly, NOD.ABHIdd21.1 splenocytes are not as effective as NOD at transferring diabetes to either NOD/LtSz.Prkdc[scid] (P = 0.0004) or NOD/LtSz.Prkdc[scid].ABHIdd21.1 (P = 0.0178), suggesting that Idd21.1 acquired immune cells are not as diabetogenic as NOD. Secondly, NOD/LtSz.Prkdc[scid].ABHIdd21.1 mice are more resistant to autoimmune attack than NOD/LtSz.Prkdc[scid] when injected with either NOD (P = 0.0015) or NOD.ABHIdd21.1 splenocytes (P = 0.0014), suggesting that Idd21.1 either acts by altering the intrinsic resistance of beta-cells to autoimmune attack or due to changes in the innate immune system.
Other NOD-based models of autoimmune disease, spontaneous and experimental autoimmune thyroiditis and spontaneous colitis, were also investigated to determine whether Idd21.1 is a common autoimmune disease locus. When bred onto the NOD.Cg-H2[h4] (thyroiditis model) genetic background Idd21.1 was demonstrated to increase the development of thyroiditis and reduce the incidence of insulitis in spontaneous (untreated) but not experimental (NaI-induced) NOD.Cg-H2[h4] mice. When bred onto the NOD.Cg-Il10[tm1Cgn] (colitis) genetic background Idd21.1 was demonstrated to inhibit the development of rectal prolapse in breeding female NOD.Cg-Il10[tm1Cgn] mice.
Data from this thesis demonstrate that the IDDM6 orthologous region in mouse, Idd21.1, contains several loci that influence autoimmune diabetes, thyroiditis and colitis in NOD-based mouse models. These findings are consistent with previous knowledge that IDDM6 is a common autoimmune susceptibility locus.
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Sigurdsson, Snaevar. "Large-Scale Genotyping for Analysis of the Type I Interferon System in Autoimmune Diseases." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6792.
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De, Aguiar Saldanha Pinheiro Ana Cristina <1978>. "Development of new molecular methods for the diagnosis and the study of viral diseases of fish." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6939/.
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The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.
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Breve, André Luis da Silva. "Predição In Silico de Epítopos de Microrganismos com Identidade a Autoantígenos Humanos." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-26052011-113627/.
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A origem das doenças autoimunes é multifatorial, sendo que envolve condições ambientais e predisposição genética, dificultando sua identificação. Muitos pesquisadores têm estudado a associação entre agentes infecciosos e autoimunidade, a qual pode ser disparada pelo processo conhecido por mimetismo molecular. Neste caso, respostas imunes cruzadas envolvendo antígenos próprios têm sido documentadas. O presente projeto tem como objetivo a busca in silico por associações entre epítopos de microrganismos e autoantígenos humanos. Iniciaram-se as análises pela identificação de semelhanças de sequências de aminoácidos entre epítopos de microrganismos e autoantígenos humanos por meio do alinhamento local de sequências efetuado pelo programa BLASTP. As sequências de epítopos dos microrganismos e autoantígenos humanos foram previamente adquiridas nos bancos de dados Immune Epitope Database and Analysis Resource (IEDB) e no Genbank, respectivamente. Foram também realizadas modelagens de estruturas proteicas para o antígeno e o autoantígeno que obtiveram melhores valores de alinhamento, com base no valor do E-value, por meio dos programas Modeller e Rosetta. Por fim, a predição de epítopos foi executada, pelo uso dos softwares NetMHC e NetMHCII, para avaliar a possibilidade de epítopos de microrganismos e de autoantígenos humanos se associarem aos mesmos alelos de HLA. Como resultado, foram encontradas similaridades tanto de sequências proteicas quanto de afinidade a 4 tipos de alelos de HLA entre um epítopo do antígeno LSA-1 de Plasmodium falciparum e o autoantígeno de miosina, o que sugere uma associação entre eles, atingindo o objetivo deste trabalho.The origin of autoimmune diseases is multifactorial. It involves environmental conditions and genetic predisposition that difficulties its identification. Several researchers have studied the association between infectious agents and autoimmunity, which can be initiated by a process named molecular mimicry. In this case, cross immune responses involving self antigens have been documented. This project aims to search in silico for associations between microorganisms epitopes and human autoantigens. The first step was the identification of similarities in amino acid sequences between microorganisms epitopes and human autoantigens by use of sequence local alignment performed by the program blastp. The sequences of the microorganisms epitope and the human autoantigens had been previously acquired in the Immune Epitope Database and Analysis Resource (IEDB) and Genbank, respectively. The modeling of protein structures for the antigen and autoantigen was also carried out to show the best alignment values, based on the E-value, using the programs Modeller and Rosetta. Finally, the prediction of epitopes was performed by use of NetMHC and NetMHCII softwares to evaluate the possibility of microorganisms epitopes and human autoantigens join the same HLA alleles. Similarities of protein sequences was found for both. It was possible to observe affinity of 4 HLA alleles between an epitope from LSA-1 Plasmodium falciparum antigen and the myosin, suggesting an association between them, reaching the goal of this work.
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Persson, Anja M. "Molecular characterisation of Mycoplasma mycoides subsp. mycoides SC /." Uppsala : Dept. of Veterinary Microbiology, Swedish Univ. of Agricultural Sciences ([Institutionen för veterinärmedicinsk mikrobiologi], Sveriges lantbruksuniv.), 2002. http://epsilon.slu.se/avh/2002/91-576-6364-5.pdf.
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Slager-Bastos, Armanda Duarte. "Molecular epidemiology and diagnosis of SAT-type foot-and-mouth disease in southern Africa." Thesis, University of Pretoria, 2001. http://hdl.handle.net/2263/22866.
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Foot-and-mouth disease (FMD) is an economically devastating picornaviral disease affecting over 40 species of cloven-hoofed animals. The virus occurs as seven immunologically distinct serotypes which are characterized by high levels of intra- and intertypic variation. The three South African Territories (SAT) serotypes 1-3 are endemic to sub-Saharan Africa, a region where the epidemiology of the disease is particularly complex due to the presence of six of the seven serotypes, the role of wildlife in virus maintenance and the apparently higher levels of variation in the endemic serotypes. These factors make it imperative to establish methods suited to elucidating the regional epidemiology. One of the integral parts of this process is the genetic characterization of regionally representative viruses in order to assess the variation in the field and to clarify the role of wildlife. Nucleotide sequence data and methods suited to studying the SAT-types are however limited. A first priority was therefore to establish a PCR-based nucleotide sequencing technique targeting the highly immunogenic and phylogenetically informative 1D genome region encoding the VP1 protein. The screening of multiple serotypes and subtypes prevalent on the African continent confirmed that this method was robust and well-suited to molecular epidemiological studies in the southern Africa region. The method was first applied in the characterization of FMD virus recovered from the reproductive tract of free-living African buffalo in the Kruger National Park. Nucleotide sequencing assisted in authentication of the results and indicated that carrier status was likely, but it was not possible to unequivocally demonstrate persistent infection of FMDV. In a separate study, the role of impala antelope (Aepyceros melampus) in the epidemiology of the disease in South Africa was assessed. Genetic characterization of impala and African buffalo (Syncerus caffer) viruses collected over an eleven year period confirmed that inter-species transmission occurred on several occasions and that virus can persist in impala populations for more than 12 months. Inter-species transmission and investigation of the possible mechanisms facilitating virus transmission from persistently infected buffalo focussed on the Kruger National Park in South Africa. In order to ensure regional relevance the study was broadened to incorporate buffalo populations throughout southern Africa. Viruses of the three SAT-types recovered from diverse African buffalo populations were therefore characterized. The results reveal that independently evolving viral lineages occur in distinct geographical regions for each of the SAT-types examined and that the levels of intratypic variation are in the order of 52 - 55 % on nucleotide level across the genome region characterized. Given the strict locality-specific grouping of buffalo viruses the likely usefulness of this database for tracing the origin and course of contemporary and historical SAT-type outbreaks was investigated. Molecular epidemiological studies conclusively show that buffalo are indeed the ultimate source of infection for susceptible cloven-hoofed animals occurring in close proximity, that interspecies transmission occurs between cattle and antelope and that trans-boundary transmission of virus remains a threat to disease security in southern African countries.Thesis (PhD (Microbiology))--University of Pretoria, 2007.
Microbiology and Plant Pathology
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Leite, Rodrigo Souza. "Avaliação do SWAB conjuntival para o diagnóstico molecular da Leishmaniose visceral em inquéritos caninos rotineiros." CNEN - Centro de Desenvolvimento da Tecnologia Nuclear, Belo Horizonte, 2015. http://www.bdtd.cdtn.br//tde_busca/arquivo.php?codArquivo=342.
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NenhumaO diagnóstico da leishmaniose visceral canina (LVC) é uma etapa importante do programa de controle da leishmaniose visceral no Brasil uma vez que o cão é o principal reservatório de Leishmania (Leishmania) infantum, agente etiológico da doença. O objetivo deste estudo foi avaliar o swab conjuntival (SC) como uma ferramenta de triagem em massa para o diagnóstico molecular da LVC em uma área endêmica classificada como prioritária para as ações de vigilância do Ministério da Saúde. Um total de 1.350 cães domiciliados foi envolvido neste estudo. Os animais foram avaliados por testes sorológicos (ELISA como triagem e IFI para confirmação) e moleculares. A PCR em tempo real, tendo como alvo os minicírculos do kDNA, foi o principal teste molecular utilizado para análise das amostras de SC. Dos 1.350 cães triados 369 (27,3%) foram positivos no SC-PCR em tempo real e 126 (9,3%) obtiveram resultado positivo por ELISA. Trinta e um por cento (39/126) dos cães positivos no teste de ELISA foram confirmados pelo IFI, enquanto que comparativamente 83,3% (105/126) obtiveram resultados positivos no método SC-PCR em tempo real. Entre os cães avaliados, um subgrupo de 484 animais foi adicionalmente submetido à avaliação clínica e diagnóstico molecular através do SC associado ao ITS-1 nested PCR. Neste subgrupo o número de animais positivos por SC-PCR em tempo real foi 156/484 (32,2%), por SC-ITS-1 nested PCR 44/484 (9,0%) e por ELISA 52/484 (10,7%). SC-PCR em tempo real foi capaz de detectar infecção em cães, independentemente do grau de sintomatologia (p> 0,05), enquanto ELISA foi mais sensível no grupo de cães que apresentavam três ou mais sinais clínicos relacionados à LVC (SII). Cães infectados com Leishmania (Viannia) braziliensis e L. (L.) amazonensis foram também identificados neste estudo através de RFLP (Restriction Fragment Lenght Polymorphism) dos amplicons obtidos por ITS-1 nested PCR. Os resultados do presente estudo demonstraram que o SC-PCR em tempo real foi capaz de detectar um maior número de cães infectados que o ELISA e que a prevalência de infecções caninas tem sido subestimada pelos testes sorológicos. A utilização de métodos diagnósticos altamente sensíveis como SC-PCR em tempo real, principalmente em áreas endêmicas, poderia dar uma contribuição significativa para o controle da enfermidade.
The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniais control program in Brazil once the dog is the main animal reservoir host of Leishmania (Leishmania) infantum, the etiologic agent of the disease. The aim of this study was to evaluate conjunctival swab (CS) as a mass-screening tool for CVL molecular diagnosis in an endemic area classified as priority for the Brazilian Ministry of Healthy for surveillance action. A total of 1,350 domiciled dogs were screened. The animals were evaluated by the serological tests (ELISA as screening and IFI for confirmation) and by CS associated to real-time PCR using kDNA minicircles as targets. Of the 1,350 dogs screened 369 (27.3%) were positive by CS real-time PCR and 126 (9.3%) tested positive by ELISA. Thirty one percent (39/126) of the ELISA positive dogs were confirmed by IFI, while 83.3% (105/126) tested positive in the CS real-time PCR assay. Among the dogs screened, a sub-group of 484 animals were also submitted to clinical evaluation and molecular diagnosis using CS associate to ITS-1 nested PCR. In this sub-group the number of animais positive by CS real-time PCR was 156/484 (32.2%), by CS ITS-1 nested PCR 44/484 (9.0%) and by ELISA 52/484 (10.7%). The CS real-time PCR was able to detect infection in dogs independently of the symptomatology degree (p > 0.05), while ELISA was more sensitive in the group of dogs that present three or more clinical signs related to CVL (SII). Dogs infected by L. (Viannia) braziliensis and L. (L.) amazonensis were also found in this study by means of RFLP (Restriction Fragment Length Polymorphism) analysis of ITS-1 nested PCR amplicons. The results of present study demonstrated that CS real-time PCR was able to detected a higher number of infected dogs than ELISA and that the prevalence of canine infections has been underestimated by the serological assays. The use of sensitive molecular diagnostic methods like CS real-time PCR, mainly in endemic areas, could greatly contribute to disease control.
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Hashmi, Sumaiya F. "A DNA Computer for Glioblastoma Multiforme Diagnosis and Drug Delivery." Scholarship @ Claremont, 2013. http://scholarship.claremont.edu/cmc_theses/799.
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Glioblastoma multiforme (GBM) is a debilitating malignant brain tumor with expected patient survival of less than a year and limited responsiveness to most treatments, often requiring biopsy for diagnosis and invasive surgery for treatment. We propose a DNA computer system, consisting of input, computation, and output components, for diagnosis and treatment. The input component will detect the presence of three GBM biomarkers: vascular endothelial growth factor (VEGF), caveolin-1α (CAV), and B2 receptors. The computation component will include indicator segments for each of these genes, and ensure that output is only released if all the biomarkers are present. The output component will consist of the therapeutic agent interleukin-12 (IL-12).
This study will designate four groups of animals: untreated tumor-free (control), tumor-inoculated (RG2), treated and tumor-free (DNA), and treated and tumor-inoculated (RG2/DNA). In the RG2 and RG2/DNA groups, we will inoculate adult male Fischer rats with RG2 cells into the striatum to induce tumor growth. Rats in the DNA and RG2/DNA groups will be implanted with the DNA system at the same location via recombinant adeno- associated viral vectors. The effectiveness of the DNA system will be evaluated through tumor size measurements, collected from brain slices stained with hematoxylin and eosin, and survival curve. Additionally, IL-12 localization will confirm the release of the output component.
We anticipate that the DNA treatment will result in a decrease in tumor size, leading to smaller tumor size in the RG2/DNA group versus the RG2 group. The control group is expected to survive the longest, followed by the DNA group, then the RG2/DNA group, and finally the RG2 group. In the DNA group, IL-12 is expected to stay localized to the implantation site, remaining in its unreleased stem-loop form. On the other hand, it is expected to be released and active in the RG2/DNA group.
This study provides a proof of concept to demonstrate the viability and effectiveness of a DNA system using VEGF, CAV, and B2 receptors as biomarkers and IL-12 as a therapeutic output component in the RG2 model. Further research may include varying several of the parameters used in this study, including amount of RG2 administered, choice of biomarkers, quantity and choice of output component, and choice of animal model. This system provides a promising and innovative new approach that is less invasive than surgery yet is still effective in diagnosing, targeting, and treating GBM.
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劉國培 and Kwok-pui Lau. "Clinicopathological roles of transforming growth factor alpha (TGFα) in papillary thyroid carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39558733.
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Leite, Rodrigo Souza. "Diagnostico molecular da Leishmaniose visceral canina: avaliação do swab conjuntival em cães assintomáticos e em cães vacinados." CNEN - Centro de Desenvolvimento da Tecnologia Nuclear, Belo Horizonte, 2010. http://www.bdtd.cdtn.br//tde_busca/arquivo.php?codArquivo=122.
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NenhumaO controle epidemiológico da leishmaniose visceral (LV) no Brasil envolve a eliminação de cães infectados, portanto, métodos diagnósticos confiáveis são essenciais para evitar a transmissão da doença ou o sacrifício desnecessário de animais. O programa de controle da LV no Brasil é baseado em inquéritos sorológicos. Os métodos sorológicos, no entanto, apresentam problemas de sensibilidade e especificidade. A Reação em Cadeia da Polimerase (PCR) demonstrou ser uma técnica rápida e sensível para a detecção de Leishmania, no entanto amostras não invasivas representam um instrumento essencial, neste contexto, uma vez que elas são mais simples, indolores e mais facilmente aceitas pelos proprietários de cães. Um método útil é o swab conjuntival (SC) que utiliza um swab estéril para a realização de um esfregaço das conjuntivas do cão. O SC mostrou-se altamente sensível para o diagnostico da LV por PCR. O objetivo deste estudo foi avaliar através do SC a prevalência de animais infectados em dois grupos de cães: assintomáticos e vacinados contra a LV. O primeiro grupo foi composto por 30 animais assintomáticos, positivos nos diagnósticos sorológico e parasitológico. Os resultados do SC foram comparados com os obtidos através de duas amostras pouco invasivas, sangue (S) e biopsia de pele (BP). As amostras foram analisadas por dois métodos diferentes de PCR: kDNA PCR-hibridização e ITS-1 nPCR. O volume de amostra de DNA utilizada para o kDNA PCR-hibridização foi de 1,0 L e para o ITS-1 nPCR o volume foi de 10,0 L. Utilizando-se o SC o método kDNA PCR- hybridização detectou DNA de Leishmania em 24/30 cães (80%) através da conjuntiva direita (CD) e 23/30 cães (76,6%) utlizando-se a conjuntiva esquerda (CE), 17/30 cães (56,7%) por meio de BP e 4/30 cães (13.3%) com S. A positividade do SC obtida combinando-se ambas as conjuntivas foi de 90% (27/30 cães). A análise das amostras de SC pelo método ITS-1 nPCR revelou que 25/30 cães (83,3%) foram positivos utilizando-se a CD e 20/30 cães (66,6%) foram positivos via a CE. Pelo mesmo método 15/30 cães (50,0%) foram positivos através de BP e 17/30 cães (56,7%) com S. A positividade obtida combinando-se ambas as oculares foi de 83,3%. Para o método kDNA PCR- hybridização as positividades do SC para a CD e CE foram significantemente superiores (p<0.05) as obtidas com BP e S. Diferença estatística em relação a BP e S foi verificada pelo método ITS-1 nPCR apenas para CD. Os métodos kDNA PCR-hibridação e ITS-1 nPCR apresentaram sensibilidade semelhante para SC e amostras BP. Por outro lado, a positividade do ITS-1 nPCR para amostras de S, foi significativamente maior que a obtida pelo kDNA PCR-hibridização, indicando que a sensibilidade dos métodos de PCR pode variar de acordo com o tecido examinado. A melhor sensibilidade neste estudo (90%) foi obtido através de amostras SC (combinando-se as duas conjuntivas) associada ao kDNA PCR-hibridização. Este nível de sensibilidade foi semelhante ao obtido em outros estudos utilizando SC para o diagnóstico da LV em cães sintomáticos. O segundo grupo foi de 42 cães militares, todos vacinados contra a LV. Neste grupo os resultados do SC foram comparados aos obtidos por testes sorológicos. Os testes sorológicos foram realizados de forma independente por três laboratórios. Os laboratórios 1 e 2 (Lab1 e Lab2) foram laboratórios comerciais. O laboratório 3 (Lab3) foi o Laboratório de Referência Nacional. A primeira triagem sorológica realizada pelo Lab1 mostrou 15 cães reativos e 4 cães foram classificados como indeterminados. Devido à alta positividade encontrada neste ensaio, animais com sorologia reativa e indeterminada, diagnosticados pelo Lab1, foram submetidos a novos testes sorológicos pelos Lab2 e Lab3. O Lab2 confirmou apenas 3 cães reativos e 2 animais foram indeterminados. O Lab3 confirmou 7 cães reativos e 3 cães foram classificados como indeterminados. Os cães que foram confirmados como reativos no diagnóstico sorológico do Lab3 foram submetidos à eutanásia. O diagnóstico molecular por PCR, utilizando o SC em todos os 42 animais foi capaz de detectar o DNA de Leishmania em 17 cães. Comparando os resultados da PCR com os obtidos através do teste sorológico do Lab1, a PCR foi positiva para 10 casos reativos e um caso indeterminado, mas foi negativo para 5 reativos e 3 casos indeterminados. Além disso, a PCR foi positiva em 5 casos não reativos. Os casos reativos e indeterminados, de acordo com o Lab1 que foram PCR negativos, apresentaram resultados negativos nos testes sorológicos dos Lab2 e Lab3. Para o Lab2, a PCR confirmou os 3 casos reativos e foi positiva para os 2 casos indeterminados. Em relação ao Lab3, a PCR confirmou todos os 7 casos reativos e foi positiva para os 3 casos indeterminados. O ensaio de PCR confirmou todos os casos, simultaneamente reativos nos testes sorológicos de dois laboratórios. Nossos resultados mostraram que o SC é um método sensível e prático para coleta de amostra permitindo diagnósticos confiáveis por PCR, além de apresentar sensibilidade superior a outras amostras pouco invasivas. Concluímos que o uso do SC deve ser considerado para os inquéritos caninos rotineiros para a LV
The epidemiological control of visceral Leishmaniasis (VL) in Brazil involves the elimination of infected dogs. Therefore, reliable diagnostic tests are essential to prevent the transmission of the disease or unnecessary culling of dogs. The VL control in Brazil is based on serological surveys, nevertheless serologic assays present problems related to sensitivity and specificity. Polymerase chain reaction (PCR) proved to be a rapid and sensitive technique for detection of Leishmania. However, non-invasive samples are an essential tool in this context, since they are simpler, painless and more easily accepted by dog owners. A useful method is the conjunctival swab (CS) that uses a sterile swab to sample the dog conjunctiva. The SC was highly sensitive for the VL diagnosis by PCR. The objective of this study was to evaluate by CS the prevalence of infected animals in two groups of dogs: asymptomatic and vaccinated against VL. The first group had 30 asymptomatic dogs with positive serological and parasitological tests. The SC samples were compared with two non-invasive samples: blood (B) and skin biopsy (SB). The samples were analyzed by two different PCR methods: kDNA PCR-hybridization and ITS-1 nPCR. The volume of DNA sample used for kDNA PCR-hybridization was 1.0 L and ITS-1 nPCR volume was 10.0 L. The DNA sample volume used was of 1.0 L and 10.0 L respectively. Using CS samples the kDNA PCR- hybridization was able to detected parasite DNA in 24/30 dogs (80%) using the right conjunctiva (RC) and 23/30 dogs (76.6%) with the left conjunctiva (LC), 17/30 dogs (56.7%) by means of SB and 4/30 dogs (13.3%) with B. The CS positivity obtained combining RC and LC results was of 90% (27/30 dogs). The assay of CS samples by ITS-1 nPCR revealed that 25/30 dogs (83.3%) were positive when using RC and 20/30 dogs (66.6%) were positive when using LC. Via the same method 15/30 dogs (50.0%) were positive by SB and 17/30 dogs (56.7%) with B. The CS positivity obtained by ITS-1 nPCR combining RC and LC was of 83.3%. The CS positivities for RC and LC were significantly higher (p<0.05) than SB and B for kDNA PCR- hybridization method. Statistical difference in relation to SB and B was verified by ITS-1 nPCR only for RC. Methods kDNA PCR-hybridization and ITS-1 nPCR showed similar sensitivities to CS and SB samples. Moreover, the positivity of ITS-1 nPCR 11 for B samples, was significantly higher than that obtained by kDNA PCR-hybridization, indicating that the sensitivity of the PCR may vary with the tissue examined. The best sensitivity in this study (90%) was obtained from CS samples (by combining both conjunctivas) associated with kDNA PCR-hybridization. This level of sensitivity was similar to that obtained in other studies using CS for the VL diagnosis in symptomatic dogs. The second group was of 42 military dogs, all of them vaccinated against VL. In this group CS and compared with the results obtained by serological tests. The serological tests were carried out independently by three laboratories. Laboratories 1 and 2 (Lab1 and Lab2) were private laboratories. Laboratory 3 (Lab3) was the National Reference Laboratory. The first serological screening performed by the Lab 1 showed 15 reactive dogs and 4 dogs were classified as indeterminate. Due to the high positivity found in this test, animals with reactive and indeterminate serology according Lab 1 were subjected to new serological tests by Lab 2 and Lab 3. Lab 2 confirmed only 3 reactive dogs and 2 animals were undetermined. The Lab 3 found 7 reactive dogs and 3 dogs were classified as indeterminate. Dogs that were confirmed as reactive by Lab 3 were euthanized. The molecular diagnosis by PCR, using CS, was performed in all 42 animals and was able to detect Leishmanial DNA in 17 dogs. Comparing the PCR results with those obtained by serological test of Lab 1, PCR was positive for 10 reactive and one indeterminate case, but was negative for 5 reactive and 3 indeterminate cases. In addition, the PCR was positive in 5 non-reactive cases. The reactive and indeterminate cases according to Lab 1 that were PCR negatives, tested negative in the serologic assays of Lab 2 and Lab 3. For the Lab 2, the PCR confirmed the 3 reactive cases and was positive for the 2 indeterminate cases. In relation to Lab 3, the PCR confirmed all 7 reactive cases and test positive for the 3 indeterminate cases. The PCR assay confirmed all cases simultaneously reactive in the serologic tests of two laboratories. Our results showed that the SC is a sensitive and practical method for collecting samples, allowing reliable diagnostic tests by PCR and showed higher sensitivity than other low invasive samples. We conclude that the use of CS for the regular screenings of dogs by PCR should be considered.
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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.
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Kathiravel, Ushanthine [Verfasser]. "Molecular diagnosis and pathogenesis of Marfan syndrome and related heritable diseases associated with thoracic aortic aneurysms and dissections / Ushanthine Kathiravel." Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2014. http://d-nb.info/1058241354/34.
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Tairo, Fred. "Molecular resolution of genetic variability of major sweetpotato viruses and improved diagnosis of potyviruses co-infecting sweetpotato /." Uppsala : Dept. of Plant Biology and Foresty Genetics, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/200605.pdf.
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Abrahams-Salaam, Fatima. "A molecular investigation of a mixed ancestry family displaying dementia and movement disorders." Thesis, Stellenbosch : Stellenbosch University, 2008. http://hdl.handle.net/10019.1/2432.
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Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008.A South African family of Mixed Ancestry presented with a rapidly progressive dementia and a movement disorder which affected a number of individuals across three generations. The initial symptoms included personality changes and tremors that escalated to severe dementia and eventually a completely bedridden state. It was determined that the mean age at onset was in the third decade of life and affected individuals died within 10-15 years after the onset of symptoms. The aim of the present study was to elucidate the genetic cause of the disorder in this family and to further investigate the patho-biology of the disease. Mutations that could possibly cause the observed phenotype in this family were screened for. These included loci implicated in Huntington’s disease, Parkinson’s disease, Dentatorubral-Pallidoluysian Atrophy, Spinocerebellar ataxias (types 1, 2, 3, 6, and 7), Huntington’s disease-like 2 (HDL2) and several mitochondrial disorders. Single-strand Conformation Polymorphism (SSCP) analysis and direct sequencing were used to detect possible mutations while genotyping on an ABI genetic analyser was used to detect disorders caused by repeat expansions. Haplogroup and Short Tandem Repeats (STRs) analyses of the Y-chromosome and mitochondrial DNA of one affected family member was used to determine the family’s genetic ancestry. Reverse transcriptase polymerase chain reaction (RT- PCR) and complementary DNA (cDNA) analyses of the Junctophlin-3 (JPH3) gene was performed to provide information on the expression profile of this gene. After the exclusion of several genetic loci it was shown that this family had HDL2. This is a rare disease caused by a CAG/CTG repeat expansion in an alternatively spliced version of the JPH3 gene. HDL2 occurs almost exclusively in individuals of Black African ancestry. The genetic ancestry data suggested that the family member was most likely of South African Mixed Ancestry making this the first reported family of South African Mixed Ancestry with HDL2. A pilot study investigated the repeat distribution amongst three South African sub-populations in order to determine whether there was a bias in the repeat distribution that possibly predisposes Black Africans to develop the disease. The results showed a statistically significant difference (P= 0.0014) in the distribution of the repeats between the Black African and Caucasian cohorts. However, no conclusions could be drawn as to whether Black Africans harboured larger repeats that predisposes them to developing HDL2. The expanded repeat is located in an alternatively spliced version of the JPH3 mRNA. Interestingly, this repeat is not present in the mouse homologue of the gene although the rest of the genomic sequence is highly conserved across the human, mouse and chimpanzee genomes. Using foetal brain cDNA and PCR primers designed to be specific for different JPH3 isoforms, independent confirmation of the presence of two JPH3 mRNA transcripts (the full length and a shorter alternatively spliced version) was provided. In the absence of brain tissue from an HDL2-affected individual, it was investigated whether both JPH3 mRNA transcripts could be detected in lymphocytes. Using RNA isolated from the transformed lymphocytes of two HDL2-affected family members, real-time PCR was attempted. These experiments produced inconclusive results and required further optimisation. Further RT-PCR experiments for JHP3 expression in different tissues (brain and other) obtained from HDL2-affected individuals would be of interest. The present study identified the first Mixed Ancestry family with HDL2. This family will now be able to request genetic counselling and pre-symptomatic testing for all at-risk family members. Aspects of this study provided independent confirmation of characteristics of the mutated gene. More research on HDL2 will be crucial in understanding the pathogenesis of this disease.
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Ockert, Candice. "Identification of molecular markers for the diagnostic identification of the intracellular prokaryote associated with the appearance of withering syndrome in the abalone Haliotis midae." Thesis, Link to the online version, 2005. http://hdl.handle.net/10019/1148.
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Silva, Ryan Emiliano da. "Genes de cisteíno proteases (catepsina L-like) de Leishmania infantum chagasi: caracterização, relações filogenéticas e diagnóstico molecular." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-12072018-145115/.
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Os parasitas pertencentes ao gênero Leishmania têm distribuição ubíqua. Este táxon inclui Leishmania infantum chagasi, agente etiológico da leishmaniose visceral nas Américas, uma zoonose negligenciada cujas metodologias diagnósticas acumulam uma série de limitações, requerendo a validação e padronização de metodologias diagnósticas satisfatórias. Vários fatores estão relacionados à patogênese causada por este protozoário, entre eles a catepsina L-like, uma cisteíno protease envolvida em processos regulatórios metabólicos e infecciosos. Portanto, este trabalho teve como objetivo avaliar a eficácia do gene de catepsina L-like isoforma CPA como alvo de diagnóstico molecular e como marcador filogenético que permita a compreensão das variações intraespecíficas e elucidem a história evolutiva de L. infantum chagasi no Brasil. Foram utilizados 44 isolados de L. infantum chagasi de diferentes estados brasileiros. Os fragmentos do gene de catepsina L-like foram amplificados, purificados, sequenciados, alinhados manualmente e analisados por métodos filogenéticos de máxima parcimônia e inferência bayesiana. As sequências geradas foram usadas para pesquisar e sintetizar iniciadores a serem usados em reações específicas para o parasita alvo. O gene de catepsina L-like não mostrou variabilidade intraespecífica entre os isolados analisados, sugerindo um evento recente de introdução do mesmo nas Américas. O par de iniciadores propostos amplificou o DNA alvo de isolados de L. infantum chagasi, sendo efetivo na amplificação de DNA em concentrações de até 10-11g / µl. O marcador proposto não apresentou reações cruzadas com outros hemoparasitas de importância clínica. Quando utilizado para o diagnóstico em um painel de amostras clínicas de cães, obteve-se uma frequência de positividade de 49,03% (102/208), contrastando com o valor de 14,42% (30/208) obtido com o marcador para o gene do espaçador ribossomal interno ITS. Quando testado em amostras de flebotomíneos se obteve um valor de 6,25% e em amostras de pacientes humanos o valor foi de 14,28%. Os marcadores também foram eficazes em amplificar DNA extraído de amostras de urina, de sangue fixado em papel filtro e mesmo em amostras de swab de lesões conjuntivas. Este conjunto de parâmetros permite inferir que o CatLeish- PCR é sensível e específico para o diagnóstico de L. infantum chagasi podendo ser aplicado tanto em pesquisas clínicas quanto em inquéritos epidemiológicos de vigilância.The parasites belonging to the Leishmania genus have a wide distribution. This taxon includes Leishmania infantum chagasi, the etiologic agent of Visceral Leishmaniasis in the Americas, a neglected zoonosis that requires the validation and standardization of satisfactory diagnostic methodologies. Several factors are related to the pathogenesis caused by this protozoan, as Catepsin L-like, a cysteine protease involved in regulatory and infectious processes. Given this information this work aimed to evaluate the effectiveness of Cathepsin L-like isoform CPA as a target for molecular diagnosis and as a phylogenetic marker that allows understanding the intraspecific variations and the evolutionary history of L. infantum chagasi in Brazil. We used 44 isolates of L. infantum chagasi from different Brazilian states. The cathepsin L-like gene fragments were amplified, purified, sequenced, manually aligned and analyzed by maximum parsimony and Bayesian inference methods. The sequences generated were researched to construction of oligonucleotide primers to be used in reactions specific to the target parasite. The Cathepsin L-like gene did not show intraspecific variability among the isolates analyzed, suggesting a recent event of introduction of the same in the Americas. The pair of proposed primers amplified the target DNA of L. infantum chagasi isolates, being effective in DNA amplification at concentrations of up to 10-11g/µl. The proposed marker did not present cross-reactions with other hemoparasites of clinical importance. When used for the diagnosis in a panel of clinical samples of dogs obtained a positive frequency of 49.03% (102/208), against the 14.42% (30/208) to ribosomal ITS marker. Samples of sandflies obtained a value of 6.25% and in humans the value was 14.28%. The markers were also effective in blood samples fixed on filter paper and even in samples from conjunctival lesion swabs. This set of parameters allows to infer that CatLeish-PCR is a sensitive and specific marker for the diagnosis of L. infantum chagasi in clinical and epidemiological surveys.
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Miranda, de Sousa Dias Miguel. "Novel Approaches for Molecular Diagnosis of Genetic Diseases by Next Generation Sequencing: Application to Breast Cancer and Retinitis Pigmentosa in the Clinical Practice." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/133297.
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En la pràctica clínica actual existeix una demanda creixent d’estudis moleculars de malalties genètiques. Generalment, la detecció de mutacions patològiques en gens candidats consisteix en la seqüenciació directa dels exons i les zones intròniques flanquejants mitjançant el mètode de Sanger, el qual es basa en una electroforesi capil·lar i requereix l’amplificació prèvia de cada fragment d’ADN en una reacció de PCR individual. Així, l’anàlisi de gens formats per molts exons com BRCA1 i BRCA2, associats a càncer de mama i ovari, implica un elevat nombre de reaccions de PCR i seqüenciació, i esdevé una tasca molt costosa en termes de temps i diners. D’altra banda, l’estudi de malalties monogèniques genèticament heterogènies, com és el cas de la Retinosi Pigmentària autosòmica dominant (RPad), pot requerir l’anàlisi de més de 20 gens per tal d’identificar la causa molecular de la malaltia. Tot i així, només un 20-30% dels pacients amb RPad són diagnosticats molecularment ja molts gens i mutacions associades a la malaltia són encara desconeguts.
D’aquí que l’ús de les tècniques de seqüenciació massiva d’ADN o seqüenciació de nova generació (NGS) sigui una pràctica cada vegada més habitual en els laboratoris de genètica molecular. Per a l’estudi de diversos pacients en paral·lel o per a analitzar exomes complets amb la finalitat de trobar nous gens associats a RPad, existeixen grans plataformes de seqüenciació massiva. No obstant, l’elevat cost i la gran capacitat d’aquestes plataformes es tradueix en una pèrdua de flexibilitat a l’hora de satisfer la necessitat de molts laboratoris de genètica, que sovint han d’analitzar la mostra d’un o pocs individus en un temps i amb un cost raonablement reduïts. Com a conseqüència, les empreses tecnològiques que treballen amb equips de NGS han introduït al mercat petites plataformes adaptades a l’ús clínic. Una d’aquestes plataformes, el GS Junior, ha demostrat amb èxit el seu potencial per al diagnòstic en laboratoris de genètica molecular. Amb la mateixa tecnologia bàsica que el GS 20 i GS FLX, la plataforma GS Junior utilitza les tècniques de PCR en emulsió i piroseqüenciació, però suposa una menor despesa tant de posada en marxa com de funcionament.
Aquest treball de recerca té com a objectiu provar i posar a punt tecnologies de NGS per tal d’obtenir diagnòstics moleculars a uns costos assumibles. Amb aquesta finalitat i per a l’estudi dels gens BRCA1 i BRCA2, es va idear un protocol de PCR llarga associat a dos mètodes enzimàtics de fragmentació d'ADN per tal d’obtenir biblioteques d’ADN preparades per a ser analitzades amb NGS. A més, es van assajar diferents mètodes basats en PCR llarga, multiplex o en emulsió, i en captura d’ADN per a detectar mutacions causants d’RPad.
A més, aprofitant la reducció del preu per megabase seqüenciada, també es va fer seqüenciació massiva de l’exoma. L'eficiència de les diferents metodologies avaluades ha permès crear nous protocols de treball que ja han estat implementats en la rutina del laboratori. Per tal de caracteritzar nous gens associats a RPad, es va utilitzar la NGS per analitzar un array de captura de 448 gens candidats en diverses famílies i l’exoma complet d’una d’elles. Es va observar que els pacients analitzats presentaven un gran nombre de variants. Per a poder establir el paper d’aquestes variants com mutacions causants de la malaltia, és imprescindible efectuar la segregació familiar; per tant, la validació dels resultats només es podia aconseguir en famílies amb almenys dos individus afectats i un membre no afectat. D’aquesta manera, en la seqüenciació de l’exoma, es van trobar més de 150 variants genètiques que podrien ser les causants de la RPad en la família.Molecular testing of genetic diseases is in increasing demand in routine clinical practice. Medical analysis of candidate genes to characterize the mutation that causes a disease currently requires amplification of the exonic and flanking sequences by PCR as a previous step to individual PCR fragment capillary electrophoresis sequencing (Sanger sequencing). BRCA1 and BRCA2, which are associated with the risk of breast cancer, are large genes with a high number of exons, and therefore involve a considerable number of individual PCRs and sequencing reactions to cover the coding and flanking sequences of both genes, which is a very costly and time consuming task. On the other hand, in genetically heterogeneous monogenic diseases such as autosomal dominant Retinitis Pigmentosa (adRP), mutation screening may be required in more than 20 genes in order to establish the molecular cause of the disease. Even so, using these expensive approaches, just 20-30% of adRP patients can be molecular diagnosed due to the fact that the mutation associated with the disease is yet unknown in more than 60% of all adRP cases Hence the use of massive DNA sequencing or next generation sequencing (NGS) technologies is a vital practice within any clinical genetic laboratory. Large next-generation platforms are indicated for this task. Likewise, for the analysis of the whole exome to characterise new genes associated with adRP and/or for extensive patient surveys, these large platforms are also required. However, the cost and extremely large capacity of these platforms results in a loss of flexibility regarding the needs of many genetics laboratories where it is sometimes necessary to analyse samples from only one or just a few individuals in a reasonably short time. In consequence, technologic firms participating in the NGS marketspace have introduced smaller NGS platforms adapted for clinical use. One such platform, the GS Junior, has successfully proven its potential for molecular diagnosis in molecular genetics laboratories. Sharing the same core technology as the GS 20 and the GS FLX, the GS Junior platform exploits similar emulsion PCR and pyrosequencing approaches, but with lower set-up and running costs. Accordingly, this research work aims for the successful management of NGS technologies in order to offer advanced molecular diagnosis services at assumable costs. As a result, a Long-Range (LR) -PCR approach associated with two distinct enzymatic DNA shearing methods was devised in order to prepare DNA libraries for NGS to achieve molecular testing of the large BRCA1 and BRCA2 genes. Furthermore, different methods based on LR, multiplex, emulsion PCR, or targeting DNA gene capture were assayed to detect mutation-causing adRP. In addition, and taking the advantage of the significant price reduction per Megabase sequenced, the whole exome analysis method was also put to the test. The efficiency of the distinct methodologies used for NGS in routine clinical practice was evaluated resulting in new diagnostic protocols based on this research work, which are already introduced at a clinical routine level. In order to characterize novel genes associated with adRP, NGS was used. 448 candidate genes array and the whole exome analysis approach were carried out. It was demonstrated that the analysed patients showed a large number of variants. To characterize these variants as a disease-causing mutation, segregation in the family is mandatory. Thus, validation of results only can be achieved in families with at least two affected and one unaffected member. In this case, more than 150 genetic variants putative causing of adRP were obtained in one family.
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Zhian, Samaneh. "Molecular Genetic Analysis of CRELD1 in Patients with Heterotaxy Disorder." PDXScholar, 2011. https://pdxscholar.library.pdx.edu/open_access_etds/410.
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Heterotaxy refers to the abnormal arrangement of internal organs in relation to each other. Model organism studies have shown that functions of more than eighty genes are required for normal asymmetric left-right organ development. CRELD1 has been shown to be necessary for proper heart development and mutations in CRELD1 are known to increase risk of cardiac atrioventricular septal defects (AVSD). AVSD is the most common form of heart defect associated with heterotaxy, and we have previously shown that some individuals with heterotaxy-related AVSD have mutations in CRELD1. Therefore, we propose to examine the CRELD1 gene in a large sample of patients with heterotaxy syndrome. Our goal was to determine if mutations in CRELD1 are associated with other manifestations of heterotaxy or if they only coincide with AVSD. To achieve this aim, a sample size of 126 patients with heterotaxy collected by Dr. Belmont, Baylor college of Medicine, Texas, with approximately 66% of the heterotaxy population with different types of heart defects, were used for this study. Ten exons, promoter regions, and regulatory elements in the introns of CRELD1 gene were sequenced and analyzed. In this study three different heterozygous missense mutations in CRELD1 were identified in three unrelated individuals. These three individuals were diagnosed with different forms of heart defects in addition to AVSD. All three mutations were identified in highly conserved regions of CRELD1 possibly altering the CRELD1 properties. This demonstrates that mutations in CRELD1 may increase the susceptibility of AVSD in heterotaxy population. This information can help us to find factors effecting disease susceptibility in heterotaxy patients since the heart defects are a complex trait with incomplete penetrance.
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Rudd, Matthew Francis, and mikewood@deakin edu au. "Virulence determinants of infectious bursal disease virus." Deakin University. School of Biological and Chemical Sciences, 2003. http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20050825.103742.
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The very virulent (vv) pathotype of infectious bursal disease virus (IBDV) has spread rapidly throughout Europe, Asia, and the Middle East. Although Australia is currently unaffected, there remains the potential for incursion of an exotic isolate. The aim of this study was to identify putative virulence determinants of IBDV to facilitate the development of improved diagnostic assays for detection and characterisation of vvIBDV isolates.
Sequencing of Indonesian vvIBDV Tasik94 revealed a unique substitution
[ A¨S222] in the hypervariable region (HVR) of viral protein (VP) VP2, which did not appear to impinge on virulence or antigenicity. Phylogenetic analyses indicated that Tasik94 was closely related to Asian and European vvIBDV strains. Extensive alignment of deduced protein sequences across the HVR of VP2 identified residuesI242 I256 and I294 as putative markers of the vv phcnotype.
Comparison of the pathology induced by mildly-virulent Australian IBDV 002/73 and Indonesian vvIBDV Tasik94, revealed that histological lesions in the spleen, thymus and bone marrow were restricted to Tasik94-infected birds, suggesting the enhanced pathogenicity of vvIBDV might be attributed to replication in non-bursal lymphoid organs.
The biological significance of the VP2 HVR in virulence was assessed using recombinant viruses generated by reverse genetics. Both genomic segments of Australian IBDV 002/73, and recombinant segment A constructs in which the HVR of 002/73 was replaced with the corresponding region of either tissue culture-adapted virus or vvIBDV (Tasik94), were cloned behind T7 RNA polymerase promoter sequences. In vitro transcription/translation of each construct resulted in expression of viral proteins. Co-transfection of synthetic RNA transcripts initiated replication of both tissue culture-adapted parental and recombinant viruses, however attempts to rescue non-adapted viruses in specific-pathogen-free (SPF) chickens were unsuccessful.
Nucleotide sequence variation in the HVR of VP2 was exploited for the development of a new diagnostic assay to rapidly detect exotic IBDV isolates, including vvIBDV, using reverse transcription polymerase chain reaction (RT-PCR) amplification and Bmrl restriction enzyme digestion. The assay was capable of differentiating between endemic and exotic IBDV in 96% of 105 isolates sequenced to date.
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Jew, Kara Lynn. "Development of a Prolyl Endopeptidase Expression System In Lactobacillus reuteri to Reduce the Clinical Manifestation of Celiac Disease." DigitalCommons@CalPoly, 2019. https://digitalcommons.calpoly.edu/theses/2065.
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Celiac Disease (CD) is an autoimmune disorder that emerges due to the ingestion of gluten, a protein found in a variety of common grains such as wheat, rye, and barley. Approximately 1 in 100 individuals in the US suffer from CD, making it the most commonly diagnosed gastrointestinal disorder (Ciclitira et. al., 2005). These proline-rich gluten peptides are resistant to proteolysis and accumulate in the duodenum of the small intestine. Once in the duodenum, these peptides illicit an autoimmune response resulting in villous atrophy. Current treatment for CD requires a rigorous adherence to a gluten-free diet. Nevertheless, gluten-containing grains are ubiquitous in the western diet, so accidental
exposure to gluten remains as a persistent threat.
The approach of this project centers on genetically engineering a strain Lactobacillus reuteri to secrete a Myxococcus xanthus prolyl endopeptidase (PEP), an enzyme that hydrolyzes a peptide bond adjacent to an internal proline residue. The data from this study revealed that recombinant M. xanthus PEP purified from E. coli was effective in degrading Suc-Ala-Pro-pNA, a chromogenic substrate containing an internal proline residue. When introduced into a L. reuteri expression vector, mutations accumulated in the vector over the course of 5 days. These data suggested that toxicity was possibly associated with M. xanthus PEP and the amyl signal peptide.
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Rosa-Lago, Natássia Carolina Esposito. "Investigação molecular, caracterização genotípica de parasitas patogênicos e distribuição espacial por geoprocessamento de amostras humanas e ambientais do município de Bauru-SP." Botucatu, 2020. http://hdl.handle.net/11449/192596.
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Orientador: Virgínia Bodelão Richini PereiraResumo: As infecções intestinais parasitárias são um problema mundial. Parasitas veiculados em água e alimentos como podem ser provenientes da falta de higiene durante o manuseio dos alimentos, contaminação ambiental por material fecal, irrigação de cultivos agrícolas com águas poluídas ou fossas sépticas precárias, situações comuns em países como o Brasil. Tais questões, colaboram para ocorrência de surtos por água e alimentos na população. Assim, o objetivo deste trabalho foi investigar a presença de parasitas importantes em saúde pública em hortaliças e água de irrigação de propriedades do município de Bauru, São Paulo; bem como, nas fezes e nas mãos de manipuladores dos cultivos. As amostras foram coletadas de cinco propriedades do município de Bauru, sendo uma localizada em área urbana e quatro em área rural. Foram obtidas 33 amostras de água de irrigação, 62 de hortaliças, 31 das mãos e dez fecais dos manipuladores. Todas as amostras foram submetidas a análise molecular e as águas de irrigação submetidas ainda a análise microbiológica. Na análise microbiológica foi detectado coliformes totais e E. coli em três propriedades. Na análise molecular, o parasita mais prevalente em água de irrigação e hortaliça foi Cyclospora cayetanensis. Taenia spp. foi detectada em uma hortaliça e Giardia spp. foi mais prevalente nas amostras humanas. Não foi detectado Toxoplasma gondii. As amostras de água de irrigação apresentaram maior quantidade de amostras positivas. Atividades de educação em ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Parasitic intestinal infections are a worldwide problem. Parasites carried in water and food as they may come from the lack of hygiene during food handling, environmental contamination by fecal material, irrigation of agricultural crops with polluted water or precarious septic tanks, common situations in countries like Brazil. Such issues contribute to the occurrence of outbreaks by water and food in the population. Thus, the objective of this work was to investigate the presence of important parasites in public health in vegetables and irrigation water on rural properties in the municipality of Bauru, São Paulo; as well as in the feces and in the hands of crop handlers. The samples were collected from five rural properties in the municipality of Bauru, one of them located in an urban area and another four in a rural area. Were collected 33 samples of irrigation water, 62 of vegetables, 31 of hands and 10 fecal samples from handlers. All samples were subjected to molecular analysis and irrigation water was also subjected to microbiological analysis. In this microbiological analysis, total coliforms were detected at high rates and E. coli in three of the properties. In molecular analysis, the parasite most prevalent in the analysis and most common in irrigation water and vegetables was Cyclospora cayetanensis. Taenia spp. was detected in one greenery. Giardia spp. was most detected in human samples. Toxoplasma gondii was not detected. The samples of irrigation water had a grea... (Complete abstract click electronic access below)
Mestre
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Van, der Merwe Ruben Gerhard. "The development of a novel fluorescentmarker phage technology system for the early diagnosis of tuberculosis disease." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/71908.
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Thesis (PhD)--Stellenbosch University, 2012.Includes bibliography
ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative organism of tuberculosis (TB), is a major cause for mortality and morbidity world-wide with a death toll only second to HIV among infectious diseases. Drug resistance is widespread and cases of multiple drug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) have emerged in several countries. Drug treatment is problematic and new drugs are not developed rapidly enough to offset the rapid drug resistance mutation rate of M. tuberculosis. Simple and effective diagnostics are required to contain the spread of the disease as current routine diagnostics are not fulfilling this role. Additionally, current rapid TB diagnostics are out of reach to resource poor settings due to infrastructure, cost and skill requirements. Novel TB diagnostics are thus required that meet these requirements. Mycobacteriophages are phages that infect mycobacteria and could offer a viable and cost effective alternative rapid TB diagnostics. In this study, an affinity-tagged fluorescent reporter mycobacteriophage is described, which was engineered to act as a TB diagnostic. Its performance proved favourable and superior to current existing mycobacteriophage-based TB diagnostics.
AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme verantwoordelik vir tuberkulose (TB), is `n groot bron van mortaliteit en morbiditeit wêreldwyd en slegs HIV is verantwoordelik vir groter getalle sterftes as gevolg van n aansteeklike siekte. Middelweerstandigheid is algemeen en gevalle van meervoudigemiddelweerstandige tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) kom in verskeie lande voor. Antibiotika behandeling is problematies en nuwe anti-TB middels word nie vinnig genoeg ontwikkel om die antibiotika weerstandigheid mutasie spoed van M. tuberculosis te bekamp nie. Doeltreffende diagnostiese toetse word benodig om die verspreiding van die siekte te beheer en bestaande roetine diagnostiese toetse voldoen tans nie aan hierdie vereiste nie. Behalwe hiervoor, is huidige vinnige TB diagnostiese toetse buite bereik van arm instansies weens vereistes aan infrastruktuur, meegaande kostes en werknemervaardigheid. Nuwe TB diagnostiese toetse is dus nodig om aan hierdie vereistes te voldoen. Mikobacteriofaage is fage wat mikobacteria infekteer en kan moontlik 'n lewensvatbare en koste-effektiewe alternatief bied vir vinnige TB diagnostiese toetse. In hierdie studie word 'n affiniteitgekoppelde fluoreserende rapporteringsmikobakteriofaag beskryf wat ontwerp is om op te tree as `n nuwe vinnige TB diagnostiese toets. Die werking hiervan vertoon gunstige en beter resultate as die huidige, mikobacteriofaaggebaseerde TB-diagnostiese toetse.
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Arcos, Machancoses José Vicente. "Exactitud de los criterios simplificados para el diagnóstico de la hepatitis autoinmune en población pediátrica. Una nueva propuesta basada en la clasificación ESPGHAN/NASPGHAN 2009." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/666561.
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INTRODUCCIÓN: Para facilitar la reproductibilidad de los estudios clínicos sobre hepatitis autoinmune (HAI), la International Autoimmune Hepatitis Group (IAIHG) propuso unos criterios diagnósticos que continúan vigentes desde 1999. Su aplicación práctica tiene un alcance limitado debido a su complejidad. Además, existe desde 2008 una versión simplificada que ha demostrado ser válida en adultos. Sin embargo, es posible que su exactitud diagnóstica no sea adecuada para las formas juveniles de HAI. En consecuencia, las sociedades Europea y Norteamericana de Hepatología Pediátrica (ESPGHAN/NASPGHAN 2009) han propuesto unos nuevos criterios adaptados al fenotipo clínico de niños y adolescentes.
OBJETIVO: Estudiar la exactitud diagnóstica de las versiones alternativas a los criterios clásicos AIHG, tanto desde el punto de vista de la validez como de la fiabilidad, la discrepancia de sus clasificaciones y su utilidad para la toma de decisiones clínicas.
MÉTODOS: El diseño es el de un estudio transversal de exactitud de pruebas diagnósticas, que se aplicó por separado a los criterios simplificados IAIHG 2008 y a un nuevo sistema de puntos basado en los criterios ESPGHAN/NASPGHAN 2009. Para ello se empleó una única cohorte de pacientes con disfunción hepática adquirida. Parte de esta muestra sirvió para desarrollar un modelo predictivo del diagnóstico de HAI basado en los criterios pediátricos de 2009. Los indicadores de exactitud se calcularon por intención de diagnosticar. Paralelamente, se llevó a cabo una revisión sistemática de la validez de los criterios simplificados IAIHG 2008, con el fin de obtener, mediante meta-análisis, unos indicadores agregados que incluyeran nuestros resultados. Finalmente, con la información generada en los pasos anteriores se llevó a cabo un análisis de decisiones clínicas. Complementariamente, se estudió la estabilidad diagnóstica de los modelos basados en ambos criterios mediante el índice de reclasificación neta y por curvas de decisión.
RESULTADOS: Se incluyeron 212 pacientes, entre los que 47% fueron HAI. El grupo de no-casos estuvo representado por niños con enfermedad de Wilson (EW) y colangitis esclerosante primaria, entre otras. Los criterios simplificados IAIHG 2008 obtuvieron una sensibilidad/especificidad (Se/Sp) de 72%/96%. La capacidad discriminante global de los criterios simplificados, medida como área bajo la curva de características operativas del receptor (ABCCOR), fue 0,943. La revisión sistemática demostró la existencia de otros tres trabajos similares. Sumando los datos de los estudios primarios, la Se/Sp agregada fue 77%/95%. Con los criterios ESPGHAN/NASPGHAN 2009 fue posible desarrollar un sistema diagnóstico sencillo: autoanticuerpos (0-2 puntos), hipergammaglobulinemia (1 punto), histología (3 puntos), exclusión de hepatitis viral y de EW (1 punto cada una). Se consideró obligatorio disponer de una colangiografía normal para excluir colangitis esclerosante autoinmune (CEAI). El corte óptimo se estableció en 6 puntos. En la submuestra de validación externa, la Se/Sp del nuevo score fue 96%/100%. Los valores predictivos positivo y negativo fueron, respectivamente, 100% y 96%. El ABCCOR fue 0,971. Las principales fuentes de error fueron las HAI seronegativas, causa de falsos negativos. Respecto a la utilidad clínica, la posición del umbral terapéutico permite tomar la decisión de tratar o no en base al resultado de los criterios simplificados y de la propuesta ESPGHAN/NASPGHAN 2009. El ancho de prevalencias en las que se observa este fenómeno fue de mayor magnitud en la última (5-97%). Por último, las curvas de decisión del nuevo score pediátrico trascurrieron casi de forma solapada a la de los criterios IAIHG 1999.
CONCLUSIONES: El nuevo sistema diagnóstico ESPGHAN/NASPGHAN 2009 arroja unos indicadores de validez superiores a los de los criterios simplificados IAIHG 2008. Son clínicamente útiles en una mayor diversidad de escenarios clínicos y su capacidad de acierto es igualmente excelente tanto con un resultado positivo como negativo. Además, debería de permitir diferenciar entre HAI y CEAI.BACKGROUND: To facilitate the reproducibility of clinical studies on autoimmune hepatitis (AIH), the International Autoimmune Hepatitis Group (IAIHG) proposed some diagnostic criteria that are still valid after being reviewed in 1999. Nevertheless, they have a limited scope in real practice due to its complexity. Since 2008, there is a simplified version that has proven to be valid in adults. However, it is possible that its diagnostic accuracy is not adequate for the juvenile form of the disease. Consequently, the European and North American Paediatric Hepatology Societies (ESPGHAN/NASPGHAN 2009) have proposed a new set of criteria adapted to the clinical phenotype of AIH in children and adolescents. OBJECTIVE: To study the diagnostic accuracy of the alternative versions to the classic AIHG criteria, both from the point of view of validity and reliability, the discrepancy of their classifications and their usefulness for clinical decision-making. METHODS: The design is that of a cross-sectional study of diagnostic tests, which was applied separately to the IAIHG 2008 simplified criteria and to a score based on the ESPGHAN/NASPGHAN 2009 criteria. A single cohort was used for this purpose, including patients with recent-onset hepatic dysfunction. Part of this sample was used to build a predictive model for AIH diagnosis, based on the already proposed paediatric criteria in 2009. Validity indicators were obtained by intention to diagnose. In parallel, a systematic review of the accuracy of IAIHG 2008 simplified criteria was carried out, in order to obtain, through meta-analysis, some pooled indicators including our own results. Finally, with the information generated in previous steps, a clinical decision analysis was conducted. In addition, the diagnostic stability of the models based on both alternative criteria was studied through net reclassification improvement and through decision curves. RESULTS: A total of 212 patients were included, resulting in an AIH prevalence of 47%. The group of non-cases was composed by children with Wilson's disease (WD), primary sclerosing cholangitis and others. The IAIHG 2008 simplified criteria obtained a sensitivity/specificity (Se/Sp) of 72%/96%. Their overall discriminant capacity, measured by the area under the receiver operating characteristics curve (AUROC), was 0.943. Three other studies with the same objective were identified after the systematic review. Adding up data from all primary sources, the pooled Se/Sp was 77%/95%. It was possible to develop a simple diagnostic score with the ESPGHAN/NASPGHAN 2009 criteria: autoantibodies (0-2 points), hypergammaglobulinemia (1 point), histology (3 points), exclusion of viral hepatitis and WD (1 point each). A normal cholangiography was considered mandatory to exclude autoimmune sclerosing cholangitis (AISC). The optimal cut-off was established at 6 points. Within the external validation subsample, the new score obtained a Se/Sp of 96%/100%. Positive and negative predictive values were, respectively, 100% and 96%. The AUROC was 0.971. A major source of error was seronegative AIH, leading to false negatives. Regarding clinical utility, the therapeutic threshold’s position makes it possible to decide whether to treat or not depending on the result of both simplified criteria and the ESPGHAN/NASPGHAN 2009 score. The prevalence width in which this phenomenon is observed was of greater magnitude in the latter (5-97%). Finally, the decision curves of the new paediatric score almost overlapped with the IAIHG 1999 criteria. CONCLUSIONS: The new ESPGHAN/NASPGHAN 2009 diagnostic score yields superior accuracy to that of the IAIHG 2008 simplified criteria. It is clinically useful in a greater diversity of clinical scenarios and their ability to succeed is equally excellent with both a positive and a negative result. In addition, it may allow to differentiate between AIH and AISC.
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Melki, Isabelle. "Clinical and molecular characterisation of type I interferonopathies." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB122/document.
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Les interférons de type I (IFN I) sont des cytokines antivirales aux propriétés puissantes. L’induction, la transmission et la résolution de la réponse immunitaire engendrée par les IFN I est minutieusement régulée. Le concept d’interféronopathie de type I, récemment individualisé par notre équipe, repose sur l’hypothèse que certaines pathologies seraient secondaires au déséquilibre de ces voies de signalisation complexes et à la sécrétion excessive et inappropriée d’IFN I. L’inhibition de celle-ci par des thérapeutiques ciblées permettrait de valider cette hypothèse, si les symptômes allégués s’amélioraient, voire disparaissaient. Ce travail de thèse s’est initialement concentré sur la caractérisation clinique et biologique des interféronopathies monogéniques et polygéniques, et secondairement sur l’identification moléculaire de nouvelles mutations du gène TMEM173 à l’origine de l’interféronopathie liée à STING, également appelée SAVI (STING associated vasculopathy with onset in infancy), syndrome auto-inflammatoire associant une atteinte sévère cutanée et pulmonaire. De nouvelles techniques ont permis la sélection de patients présentant une augmentation de l’IFN I en comparaison à des contrôles sains : la signature IFN I, qPCR de 6 gènes stimulés par l’IFN (IFN stimulated genes – ISGs) et le dosage d’IFN alpha sérique ou plasmatique par méthode du SIMOA (single molecule array) permettant la détection de molécules d’IFN de l’ordre du femtogramme (10-18g). Ces méthodes nous ont ainsi permis d’élargir le spectre clinique phénotypique des interféronopathies de type I, initialement considéré comme essentiellement neurologique. Les patients atteints du syndrome d’Aicardi-Goutières, première interféronopathie monogénique décrite, présentaient les signes suivants : dystonie, spasticité, décalage des acquisitions, calcifications intra-cérébrales et anomalies de la substance blanche. Cependant, l’utilisation systématique de nos méthodes de criblage associée à l’avènement des technologies de séquençage à haut débit (next generation sequencing – NGS) a permis de révéler un phénotype plus large, caractéristique des interféronopathies de type I : sur le plan cutané (engelures, vascularite nécrosante des extrémités, sclérodermie), pulmonaire (pneumopathie interstitielle isolée ou non), musculo-squelettique (arthralgies, arthrites, arthropathie de Jaccoud, myalgies et myosites), ophtalmologique (glaucome), néphrologique (néphropathies lupiques), gastro-entérologique (maladies inflammatoires chroniques intestinales précoces), associées à de l’auto-immunité ou un déficit immunitaire inconstants. Notre méthode de sélection nous a notamment permis d’identifier des patients présentant de manière variable des signes cardinaux de SAVI et une de trois nouvelles mutations activatrices dans une région spécifique du gène TMEM173 (codant pour STING). Ces mutations circonscrivent une région de la protéine à ce jour encore jamais impliquée dans le contrôle de la voie de l’IFN I. STING est une protéine du réticulum endoplasmique qui agit comme adaptateur cytosolique de senseurs intracellulaires d’ADN viral dans une voie de signalisation de l’IFN I. STING active TBK1 (TANK-binding kinase) et permet la transcription des IFN I par la phosphorylation d’IRF3. La Janus Kinase 1 (JAK1) et la tyrosine kinase 2 (TYK2) sont activées suite à la stimulation des récepteurs de l’IFN I et phosphorylent les facteurs de transcription STAT1 et STAT2, conduisant à l’expression de nombreux ISGs. Les analyses génétiques, de conformation tridimensionnelle, sur un modèle cellulaire in vitro (HEK293T) et ex vivo sur cellules mononuclées périphériques des patients nous ont ainsi permis de mettre en évidence pour ces mutations un caractère constitutionnellement activé, indépendant de la liaison au ligand cGAMP, mais transmettant ce signal à travers la voie d’aval par TBK1. (...)Type I interferons (IFN I) are antiviral cytokines with potent properties. Hence, the induction, transmission and resolution of the immune response generated by IFN I is tightly regulated. The concept of the type I interferonopathies, recently formulated by our team, rests on the assumption that some diseases arise from a disturbance of this complex signalling pathway, leading to excessive and inappropriate IFN I secretion. On this basis, targeted therapeutics should improve or cure features of such type I interferonopathies, thereby providing a validation of the underlying hypothesis. This PhD project initially focused on the clinical and biological characterisation of monogenic and polygenic interferonopathies, and secondarily on the molecular identification of novel mutations in the gene TMEM173 causing the interferonopathy called STING associated vasculopathy with onset in infancy (SAVI), an auto-inflammatory syndrome with severe cutaneous and pulmonary features. Our selection of patients in comparison to healthy controls was made possible through the use of novel screening tools: IFN signature (qPCR of 6 IFN stimulated genes – ISGs), and measurement of IFN alpha protein levels in serum or plasma (SIMOA-single molecule array - enabling the detection of molecules of IFN in the femtogram [10-18g]) range. In this way, we have been able to expand the phenotypic spectrum of the interferonopathies, which was initially considered as primarily neurological. Patients with Aicardi-Goutières syndrome (AGS), the first described of the monogenic interferonopathies, exhibit dystonia, spasticity, developmental delay, intra-cranial calcifications and white matter abnormalities. However, the systematic use of our interferon screening assays, plus the advent of next-generation sequencing technology, has revealed a much broader set of features relevant to this novel disease grouping – involving the skin (chilblains, necrotising vasculitis, scleroderma), lungs (isolated lung interstitial disease or associated with other signs), musculoskeletal system (joint pain, arthritis, Jaccoud’s arthropathy, muscle pain and myositis), eyes (glaucoma), kidneys (lupus nephritis) and gastro-intestinal tract (early inflammatory bowel disease), as well features of autoimmunity and immunodeficiency. Using our screening assays enabled us to identify three patients variably exhibiting the core features of SAVI, all of whom were found to harbour distinct novel activating mutations in STING. These mutations highlight a protein domain not previously implicated in the control of IFN I signalling. STING is an endoplasmic reticulum protein, acting as a cytosolic adaptor of intracellular sensors of viral DNA in the type I IFN signalling pathway. STING activates TANK-binding kinase (TBK1), allowing transcription of IFN I through phosphorylation of IRF3. Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) are activated following stimulation of the IFN I receptor, leading to phosphorylation of the transcription factors STAT1 and STAT2 and the subsequent induction of a large number of ISGs. Genetic analysis, conformational studies, an in vitro cellular model (HEK293T) and ex vivo experimental data (using patient peripheral blood mononuclear cells - PBMCs) enabled us to confirm the constitutive activating nature of these variants, and show that this activation did not require binding with cGAMP, but was dependent on signalling through TBK1. Ruxolitinib, a JAK1/2 inhibitor, could antagonise this constitutive activation ex vivo. These results indicate a promising therapeutic approach in such patients, and more widely in the monogenic, and perhaps even, polygenic, interferonopathy context
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Lintner, Katherine E. "The Roles of Complement C4A and C4B Genetic Diversity and HLA DRB1 Variants on Disease Associations with Juvenile Dermatomyositis and Systemic Lupus Erythematosus." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1460986052.
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Lukanji, Zinathi, and R. N. Ndip. "Isolation and molecular characterization of Bacillus cereus from cow’s raw milk." Thesis, University of Fort Hare, 2015. http://hdl.handle.net/10353/d1021284.
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Bacillus cereus is a group of ubiquitous facultative anaerobic spore forming Gram-positive rods commonly found in soil. It has been detected and implicated in several contaminated food products and raw milk in dairy farms and it causes foodborne gastroenteritis by producing several toxins. This study is aimed at characterizing virulence determinants of B. cereus from cow‟s raw milk. A total of 400 raw milk samples were collected in Fort Hare Dairy Trust and Middledrift Dairy Farm; and cultured on Polymyxin pyruvate Egg-Yolk Mannitol Bromothymol Agar (PEMBA) for 48 hours at 37°C. DNA was isolated from the isolates and 16S rDNA was amplified and sent to Central Analytical Laboratory for sequencing. The gyrB gene of B. cereus was also used to confirm the identity of the isolates. Antibiotic susceptibility profiles of the isolates together with virulence genes were investigated. Multilocus Sequence typing was used to investigate the genetic relatedness of some selected isolates. Furthermore, spores of the isolates were produced, harvested and their concentrations determined. All (100%) of the isolates were identified as having a 96-99% similarity to B. cereus, B. thuringiensis and B. anthracis using sequencing; while gyrB gene was observed in all (100%) of the isolates. Three virulence genes nheA, nheB, nheC encoding for non haemolysin enterotoxin were amplified in all (100%) the isolates. All (100%) of the isolates were susceptible to doxycycline, gentamycin, tetracycline, ciprofloxacin, chloramphenicol and streptomycin. Resistance to rifampicin and penicillin G was predominant with equal rate of 100%, while susceptibility to erythromycin, clindamycin and doxycycline ranged from 60% to 100%. The selected isolates were related and are descendants of the same ancestor. All (100%) the isolates produced spores. The B. cereus isolates contain virulence genes, has multiple antibiotic drug resistance and produce spores, which poses a health risk to the public and cannot be used as probiotics.
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Malan, 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.
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Thesis (MSc (Genetics))--University of Stellenbosch, 2009.ENGLISH 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.
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Prudent, Elsa. "Applications de l'hybridation in situ en fluorescence et stratégies moléculaires pour le diagnostic des infections bactériennes." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0253/document.
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Une partie de ce travail de thèse a consisté à appliquer les méthodes de FISH pour l’étude de trois bactéries pathogènes intracellulaires. La viabilité de Bartonella henselae a été évaluée à partir de ganglions de patients atteints de la maladie des griffes du chat (CSD). Le faible taux d’ARN détecté par biologie moléculaire, la stérilité des cultures, l'absence de détection par analyses histologiques et FISH confirment que B. henselae n'est pas ou rarement viable dans les ganglions de patients atteints de CSD. Tropheryma whipplei, l’agent de la maladie de Whipple, a été identifié et localisé par FISH, dans les macrophages d’un ganglion et d’une biopsie pulmonaire, confirmant le diagnostic infectieux. Deux méthodes de FISH ont été testées pour détecter Coxiella burnetii dans des cas d’endocardites et d’infections vasculaires en utilisant des sondes oligonucléotidiques et des sondes PNA. Les résultats ont confirmé une meilleure efficacité des sondes PNA et démontré que les techniques de FISH sont plus sensibles que l’immunohistochimie pour le diagnostic des endocardites et des infections vasculaires à C. burnetii. Nous avons également évalué les stratégies moléculaires mises en place pour le diagnostic syndromique. Bien que la PCR conventionnelle à large spectre permette l'identification de micro-organismes fastidieux et anaérobies, la PCR spécifique en temps réel révèle une supériorité significative dans le diagnostic syndromique. En conclusion, ce travail a permis de démontrer l’efficacité et l’applicabilité de la FISH pour la détection bactérienne. Cette méthode peut être utilisée comme un outil complémentaire afin d'améliorer le diagnostic de microbiologie cliniqueWe applied FISH methods to the study of three intracellular pathogenic bacteria. The viability of Bartonella henselae was evaluated in a large series of lymph nodes from patients with cat scratch disease (CSD). The results obtained, associated with sterile cultures and negative histological analyzes and FISH, as well as the low level of RNA detected by molecular biology, provide evidence that B. henselae are not or are rarely viable in the lymph nodes of patients with CSD. Tropheryma whipplei has been identified by FISH in macrophages from one lymph node and for the first time in a pulmonary biopsy, confirming the diagnosis of infection. Two methods of FISH have been tested to detect Coxiella burnetii in cases of endocarditis and vascular infections using oligonucleotide and PNA probes. The results attested to the greater efficiency of PNA probes, and demonstrated that FISH were applicable for the diagnosis of C. burnetii endocarditis. We also evaluated the molecular strategies used for syndrome-driven diagnosis of infectious diseases. Although conventional broad-spectrum PCR allows for the identification of fastidious and anaerobic microorganisms, real-time specific PCR reveals a significant superiority in syndrome-driven diagnosis. The addition of specific PCRs in real time PCR would improve our molecular strategies, for example, in the case of the detection of Staphylococcus aureus for the diagnosis of lymphadenopathy. In conclusion, this work demonstrates the effectiveness and applicability of FISH for the identification of intracellular bacteria. This method can be used as an important complementary tool to the improvement of clinical microbiological diagnosis
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Parsons, Sven David Charles. "Natural animal model systems to study tuberculosis." Thesis, Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/4505.
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Thesis (PhD (Molecular Biology and Human Genetics))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: The growing global epidemic of human tuberculosis (TB) results in 8 million new cases of this disease and 2 million deaths annually. Control thereof will require greater insight into the biology of the causative organism, Mycobacterium tuberculosis, and into the pathogenesis of the disease. This will benefit the design of new vaccines and diagnostic assays which may reduce the degree of both disease transmission and progression. Animal models have played a vital role in the understanding of the aetiology, pathogenesis, and treatment of TB. Much of such insight has been obtained from experimental infection models, and the development of new vaccines, for example, is dependant on these. Nonetheless, studies utilising naturally occurring TB in animals, such as those which have investigated the use of interferon-gamma release assays (IGRA) for its diagnosis, have contributed substantially to the body of knowledge in this field. However, there are few such examples, and this study sought to identify and investigate naturally occuring animal TB in South Africa as an opportunity to gain further insight into this disease. During the course of this study, the dassie bacillus, a distinctly less virulent variant of M. tuberculosis, was isolated from a rock hyrax from the Western Cape Province of South Africa. This has provided new insight into the widespread occurrence of this organism in rock hyrax populations, and has given impetus to further exploring the nature of the difference in virulence between these pathogens. Also investigated was M. tuberculosis infection in dogs in contact with human TB patients. In so doing, the first reported case of canine TB in South Africa was described, v a novel canine IGRA was developed, and a high level of M. tuberculosis infection in these animals was identified. This supports human data reflecting high levels of transmission of this pathogen during the course of human disease. Additionally, the fact that infected companion animals may progress to disease and potentially act as a source of human infection was highlighted. However, an attempt to adapt a flow cytometric assay to study cell-mediated immune responses during canine TB revealed the limitations of such studies in species in which the immune system remains poorly characterised. The use of IGRAs to diagnose TB was further explored by adapting a human assay, the QuantiFERON-TB Gold (In-Tube Method), for use in non-human primates. These studies have shown that such an adaption allows for the sensitive detection of TB in baboons (Papio ursinus) and rhesus macaques (Macaca mulatta) and may be suitable for adaption for use in other species. However, they have also evidenced the limitation of this assay to specifically detect infection by M. tuberculosis. Finally, to contextualise the occurrence of the mycobacterial infections described above, and other similar examples, these have been reviewed as an opinion piece. Together, these investigations confirm that animal models will continue to make important contributions to the study of TB. More specifically, they highlight the opportunities that naturally occuring animal TB provides for the discovery of novel insights into this disease.
AFRIKAANSE OPSOMMING: Wêreldwye tuberkulose (TB) epidemie veroorsaak agt miljoen nuwe gevalle en twee miljoen sterftes jaarliks. Ingryping by die beheer hiervan vereis begrip van die biologie van die mikroörganisme Mycobacterium tuberculosis, die oorsaak van TB, asook van die patogenese van die siekte self. Hierdie kennis kan lei tot ontwerp van nuwe entstowwe en diagnostiese toetse wat gevolglik beide die oordrag- en vordering van die siekte mag bekamp. Dieremodelle speel lankal 'n rol in ons begrip van die etiologie-, patogenese- en behandeling van TB. Insig is grotendeels verkry vanaf eksperimentele infeksiemodelle, en ontwikkeling van entstowwe, onder andere, is afhanklik van soortgelyke modelle. Desnieteenstaande, studies wat natuurlike TB voorkoms in diere ondersoek, byvoorbeeld dié wat op die ontwikkeling van interferon-gamma vrystellingstoetse (IGVT) fokus, het merkwaardige bydrae gemaak tot kennis en begrip in hierdie studieveld. Daar is slegs enkele soortgelyke voorbeelde. Om hierdie rede is die huidige studie uitgevoer waarbinne natuulike diere-TB geïdentifiseer en ondersoek is in Suid-Afrika om verdere kennis en insig te win aangaande TB. Die "dassie bacillus", bekend om beduidend minder virulent te wees as M. tuberculosis, is tydens hierdie studie geïsoleer vanuit 'n klipdassie (Procavia capensis) in die Wes-Kaapse provinsie, Suid-Afrika. Insig in die wydverspreide voorkoms van hierdie organisme in klipdassie bevolkings is gevolglik verkry en verskaf momentum om die aard van verskil in virulensie tussen dié patogene te bestudeer. vii Voorts is M. tuberculosis infeksie bestudeer in honde wat in kontak is met menslike TB pasiënte en word die eerste geval van honde TB dus in Suid-Afrika beskryf. In hierdie groep diere, is 'n hoë vlak van M. tuberculosis infeksie geïdentifiseer deur gebruik te maak van 'n nuut ontwikkelde IGVT vir die diagnose van honde TB. Gevolglik ondersteun dié studie bevindinge van menslike studies wat toon dat besondere hoë vlakke van M. tuberculosis oordrag voorkom gedurende die verloop van die siekte. Verder toon die studie dat geïnfekteerde troeteldiere 'n bron van menslike infeksie kan wees. 'n Poging om 'n vloeisitometriese toets te ontwikkel om die aard van selgefundeerde immuunreaksies te bestudeer in honde met TB toon die beperkings van dergelike studies in spesies waarin die immuunsisteem gebrekkig gekarakteriseer is. Die gebruik van IGVT'e in die diagnose van TB is verder ondersoek deur 'n menslike toets (QuantiFERON-TB Gold, In-Tube Method) aan te pas vir die gebruik van nie-menslike primaat gevalle. Hierdie studies toon gevolglik dat so 'n aanpassing toepaslik is vir hoogs sensitiewe deteksie van TB in chacma bobbejane (Papio ursinus) en rhesus ape (Macaca mulatta), en mag ook aangepas word vir gebruik in ander spesies. Tog word die beperkings van hierdie toets om infeksie wat spesifiek deur M. tuberculosis veroorsaak uitgelig. Ter afsluiting word hierdie studie in konteks geplaas deur 'n oorsig te gee van bogenoemde- en soortgelyke gevalle van dierlike infeksie deur mikobakterieë in Suid-Afrika. Hierdie studies bevestig dat dieremodelle steeds belangrike toevoegings maak tydens die bestudering van TB en lig veral die moontlikhede uit dat bestudering van natuulike TB in diere kan lei tot die ontdekking van nuwe insigte ten opsigte van die siekte self.
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Daniel, Rhonda W. "Dysregulation of microRNAs in Blood as Biomarkers for Diagnosing Prostate Cancer." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3975.
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Prostate cancer is the most common noncutaneous cancer among men, yet current diagnostic methods are insufficient and more reliable diagnostic markers need to be developed. The answer that can bridge this gap and enable more efficient diagnoses may lie in microRNAs. These small, single stranded RNA molecules impact protein expression at the translational level and regulate important cellular pathways. Dysregulation of these small RNA molecules can have tumorigenic effects on cells and lead to many types of cancers.
Currently the Prostate-Stimulating Antigen (PSA) is used as a diagnostic marker for prostate cancer. However, many factors can elevate PSA levels such as infections and certain medications, consequently leading to false positive diagnoses and unnecessary concern and over treatment with dire outcomes for the patient. Even worse, are the chances of false negative diagnoses, which result in prostate cancer not being diagnosed until its later stages. Therefore, although the use of the PSA level has had its uses in the clinic, it has failed to sufficiently bridge the gap or to distinguish indolent from aggressive disease.
It has long been suggested in the literature that microRNAs are drastically altered throughout the course of cancer progression. Here, RNA sequencing was used to identify changes in miR expression profiles diagnostic for prostate cancer patients compared to non-patient controls. The RNA sequencing results were also used to identify normalization miRs to be used as endogenous controls. Confirmatory qRT-PCR was then used to corroborate these results for the top seven dysregulated miRs found from the RNA sequencing data. Data analysis of the Area Under the Curve (AUC) of the Receiver Operating Curves (ROC) of the selected miRs exhibited a better correlation with prostate cancer (AUC Range= 0.819- 0.950) than PSA (AUC of PSA=0.667). In summary, a panel of seven miRs are proposed, many of which have prostate specific targets, which would represent a significant improvement over current testing methods.
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Moreno, Cristiane de Araujo Martins. "Estudo clínico, histológico e molecular na miopatia congênita nemalínica e na miopatia congênita com alterações mínimas." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/5/5138/tde-06022017-150033/.
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Introdução: As miopatias congênitas são doenças musculares genéticas caracterizadas por hipotonia e fraqueza muscular de início precoce na infância. Histologicamente são caracterizadas por alterações estruturais no músculo esquelético (corpos nemalínicos, cores ou centralização nuclear), no entanto, existem casos com alterações leves e inespecíficas, alterações mínimas, tais como, desproporção no tamanho das fibras e desarranjo na arquitetura interna das fibras (falhas focais na atividade oxidativa). Quanto ao aspecto molecular, vários genes já foram identificados em associação com os diversos subtipos, porém com grande sobreposição de achados histológicos e clínicos. Objetivo: Caracterização clínica, histológica e molecular de pacientes Brasileiros com miopatia nemalínica e com miopatia congênita com achados histológicos mínimos. Métodos: Avaliação clínica e histológica (revisão dos achados das biopsias musculares) de pacientes com diagnóstico de miopatia congênita nemalínica e com alterações mínimas, provenientes de dois centros de investigação em doenças neuromusculares da cidade de São Paulo (HC-FMUSP e UNIFESP). O estudo molecular foi realizado através de sequenciamento Sanger para os genes ACTA1, TPM3, MYH7 e SEPN1 e/ou sequenciamento de nova geração para painel de genes musculares e/ou exoma. Resultados: Foram avaliados 23 pacientes com miopatia nemalínica (20 famílias) e 22 pacientes com alterações mínimas (20 famílias). O diagnóstico molecular foi concluído em sete famílias com miopatia nemalínica, sendo quatro com variantes missense, em heterozigose, no gene ACTA1 já associadas previamente a miopatia nemalínica, e três famílias, com variantes não conhecidas, em heterozigose, no gene NEB com alta predição de patogenicidade. Na coorte de miopatias congênitas com alterações mínimas o diagnóstico molecular foi concluído em nove famílias, sendo uma com variante conhecida no gene CHRNE, descrita em miastenia congênita; duas famílias com variantes no gene TPM3, sendo uma inédita, em homozigose, e outra, em heterozigose, já conhecida; duas famílias com variantes novas, em heterozigose, no RYR1, uma no gene TTN e três famílias com variantes já conhecidas no gene no MYH7 com fenótipo de miopatia distal de Laing. A despeito da realização de sequenciamento de exoma, sete famílias ainda permanecem sem gene candidato. Conclusões: Os achados clínicos, histológicos e moleculares dos pacientes da coorte de miopatia nemalínica seguem aos padrões descritos da literatura. O estudo dos pacientes com miopatia congênita com alterações mínimas se revelou complexo e variável, tanto no fenótipo quanto no genótipo. As mutações novas no gene NEB, RYR1, TTN, TPM3 e MYH7 confirmam a importância e patogenicidade destes genes nas miopatias congênitas e ampliam o seu espectro de alterações. Diante da quantidade de genes candidatos e do tamanho de alguns genes envolvidos com essas miopatias, técnicas de sequenciamento de nova geração são de grande valorIntroduction: Congenital myopathy are a group of genetic muscle diseases characterized by hypotonia and weakness in early childhood. They are characterized by structural abnormalities in muscle biopsy (nemaline bodies, central-cores or nuclear centralization). However, it can present within mild and unspecific findings like fiber type disproportion and abnormalities on oxidative staining (minimal changes). Regarding the molecular aspects, there are many genes associated with the congenital myopathies with an important overlapping between the histological and phenotypical findings. Objectives: Clinical, histological and molecular characterization of Brazilian patients with nemaline myopathy and congenital myopathy with minimal changes. Methods: Clinical and histological evaluation (review of muscle biopsy) of patients with nemaline myopathy and congenital myopathy with minimal changes from two centers of neuromuscular diseases (HC-FMUSP e UNIFESP). The molecular study was performed using Sanger sequencing for ACTA1, TPM3, SEPN1 and MYH7 genes and/or neuromuscular panel and/or exome. Results: Twenty-three patients with nemaline myopathy (20 families) and 22 patients with congenital myopathy with minimal changes (20 families) were evaluated. The molecular diagnose were concluded in seven families with nemaline myopathy, with four families having missense, heterozygous and pathogenic ACTA1 variants and three families having unknown heterozygous and pathogenic variants in NEB gene. In the congenital myopathy with minimal findings group, the diagnose was concluded in 9 families. One presenting with a pathogenic variant in CHRNE gene previously described in congenital myasthenia, two families with pathogenic variants in TPM3, one novel homozygous and one heterozygous previously reported. Two families presented with novel and pathogenic RYR1 variants, one with novel and pathogenic TTN variants and 3 families presented with heterozygous variants in MYH7 myopathy with Laing distal myopathy phenotype. Despite the NGS realization, 7 families remain without a gene candidate. Conclusions: The clinical, histological and molecular findings of nemaline myopathy cohort follow the literature pattern. In contrast, the study for minimal change patients appear complex and variable, either on phenotype or on genotype. The new gene mutations for NEB, RYR1, TTN, TPM3 and MYH7 reinforce relevance and pathogenicity of these genes in the congenital myopathies and expand the mutation spectrum. In light of diversity of candidate genes and the size of some genes involved with these myopathies, next generation sequencing techniques have been proved essential
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Gómez, Morago Alba. "Estudio de los mecanismos terapéuticos en la inducción de tolerancia inmunológica en un modelo animal de esclerosis múltiple." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/386466.
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La terapia génica puede ser una herramienta para inducir tolerancia inmunológica de forma experimental. Nuestro trabajo anterior mostró que la transferencia de células de médula ósea que habían sido transducidas con un autoantígeno (MOG40-55) en ratones con encefalomielitis autoinmune experimental (EAE) mejoró el pronóstico de los animales. En este trabajo, hemos tratado de identificar la subpoblación de células de médula ósea transducidas que fue la principal responsable del efecto terapéutico observado. Se encontró que en los cultivos de transducción de células de médula ósea se generaban células mieloides supresoras de los dos subtipos descritos, tanto granulocíticos (CD11b+ Gr-1high) como monocíticos (CD11b+ Gr-1low), y que las células transducidas con mayor eficacia consistieron en gran medida de estos tipos de células. Tanto las células de médula ósea como las dos subpoblaciones de células mieloides supresoras presentaron una capacidad de inhibir la proliferación de células T inducidas por un antígeno in vitro. Curiosamente, a pesar de la constatación de que células CD11b+ Gr-1low transducidas fueron inmunosupresora in vitro y tenían actividades de la arginasa y de la óxido nítrico sintasa, sólo la subpoblación de células CD11b+ Gr-1high transducidas con el antígeno tenía un efecto terapéutico en ratones con EAE establecida. Por último, se observó un mayor porcentaje de células T reguladoras en los bazos de ratones tratados con la población de médula ósea total transducida con MOG y las células CD11b+ Gr-1high lo que sugiere un papel para las células T reguladoras en la mediación del efecto terapéutico.Gene therapy can be used to experimentally induce immune tolerance. Our previous work showed that transferring bone marrow cells that had been transduced with an autoantigen (MOG40-55) into mice with experimental autoinmune encephalomyelitis improved the animals’ prognosis. In this work, we sought to identify the cell subpopulation in the transduced bone marrow that was primarily responsible for the observed therapeutic effect. We found that both granulocyte-like (CD11b+ Gr-1hi) and monocyte-like (CD11b+ Gr-1lo) myeloid-derived suppressor cells were generated during the standard 4-day retroviral transduction of bone marrow cultures and that the effectively transduced cells largely consisted of these cell types. Both of the myeloid cell subtypes inhibited antigen-induced T cell proliferation in vitro, and they were significantly more suppressive than their sham-transduced controls. Interestingly, despite the finding that transduced CD11b+ Gr-1lo cells were immunosuppressive in vitro and exhibited arginase and nitric oxide synthase activities, only the antigen-transduced CD11b+ Gr-1hi cell subpopulation had a therapeutic effect in mice with the experimentally induced disease. Finally, we observed higher percentages of T regulatory cells in the spleens of mice treated with MOG-expressing total bone marrow population and CD11b+ Gr-1hi cells, which suggests a role for these T regulatory cells in mediating the therapeutic effect.
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Empereur-Mot, Charly. "Développement d’outils statistiques d’évaluation de méthodes de criblage virtuel : courbes de prédictivité & Screening Explorer." Thesis, Paris, CNAM, 2017. http://www.theses.fr/2017CNAM1126/document.
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Les méthodes de criblage virtuel sont largement utilisées dans le processus de conception de médicaments afin de réduire le nombre de composés à tester expérimentalement. Cependant, les résultats obtenus par criblage virtuel ne sont que des prédictions et leur fiabilité n'est pas garantie. L'évaluation de ces méthodes est donc essentielle pour guider le bioinformaticien dans le choix de l'outil et du protocol adaptés dans les conditions de son expérience. Dans une première étude, nous avons développé une nouvelle métrique pour l'analyse des résultats de criblage : la Courbe de Prédictivité. Cette métrique permet une analyse fine de la pertinence des scores d'affinité pour la détection de composés actifs et complète les métriques existantes, permettant une meilleure compréhension des résultats de criblage. Lors de notre projet suivant, nous avons souhaité faciliter ce processus d'analyse en intégrant l'ensemble des métriques de criblage virtuel dans un outil web interactif : Screening Explorer. Une seconde partie de ma thèse a consisté en la recherche de nouveaux inhibiteurs du VIH (Virus de l’Immunodéficience Humaine). L'équipe génomique de notre laboratoire a identifié plusieurs gènes dont l'expression influence le développement du SIDA, révèlant ainsi de potentielles cibles thérapeutiques. Une étude bibliographique a permis d'identifier plusieurs composés inhibiteurs de ces cibles. La société Peptinov, associée à notre laboratoire, va prochainement estimer le potentiel thérapeutique de ces composés dans des essais in vitro (i) d'infection par le VIH, (ii) de prolifération virale et (iii) de réactivation viraleVirtual screening methods are widely used in drug discovery processes in order to reduce the number of compounds to test experimentally. However, virtual screening results are only predictions and their reliability is not guaranteed. Evaluating these methods is crucial to guide the bioinformatician in the choice of the right tool and protocol according to the conditions of his experiment. In a first study, we developed a new metric to analyze the results of virtual screening: the Predictiveness Curve. This metric allows to finely analyze the relevance of binding scores for the detection of active compounds and complete existing metrics, allowing a better comprehension of screening results. In a following project, we facilitated the analysis process by integrating all of the virtuel screening metrics in an interactive tool: Screening Explorer. The second part of my thesis consisted in the research of novel HIV inhibitors. The genomic team of our laboratory identified several genes whose expression influence the development of AIDS, therefore revealing potential therapeutic targets. A bibliographic study allowed to identify compounds that can inhibit those targets. The company Peptinov, associated to our laboratory, is currently estimating the therapeutic potential of the compounds in vitro in assays of (i) HIV infection, (ii) viral proliferation and (iii) viral reactivation
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Giabicani, Eloïse. "Croissance et système des IGFs (insulin-like growth factors) : l’apport physiopathologique des maladies soumises à empreinte parentale New clinical and molecular insights into Silver–Russell syndrome Roles of Type 1 Insulin-Like Growth Factor (IGF) Receptor and IGF-II in Growth Regulation: Evidence From a Patient Carrying Both an 11p Paternal Duplication and 15q Deletion Diagnosis and Management of Postnatal Fetal Growth Restriction Chromosome 14q32.2 imprinted region disruption as an alternative molecular diagnosis of Silver-Russell Syndrome." Thesis, Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=http://theses-intra.upmc.fr/modules/resources/download/theses/2019SORUS304.pdf.
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La croissance fœtale est sous la dépendance de nombreux facteurs environnementaux, génétiques et hormonaux dont les interactions vont en conditionner le bon déroulement. Le système des insulin-like growth factors (IGFs) joue un rôle prépondérant, à l’interface de ces différents facteurs, pour assurer une bonne croissance fœtale. Dans ce travail, nous nous sommes intéressés aux différents acteurs du système des IGFs dans des pathologies de la croissance fœtale. Dans une approche clinique et expérimentale, nous avons décrit les conséquences fonctionnelles d’anomalies génétiques ou épigénétiques intéressant IGF-I, IGF-II et leur récepteur commun IGF1R. Ainsi, nous avons mis au point un test fonctionnel permettant d’apprécier l’activité in vitro d’IGF1R chez les patients présentant une restriction de croissance fœtale et postnatale. Nous avons également documenté la biodisponibilité d’IGF-I chez des patients présentant un syndrome de Silver-Russell, qui est une pathologie liée à l’empreinte parentale responsable d’une restriction de croissance à début ante-natal. Enfin, nous avons caractérisé le chevauchement clinique et moléculaire entre les patients présentant un SRS ou un syndrome de Temple (autre pathologie liée à l’empreinte parentale), confirmant le rôle prépondérant du défaut d’expression d’IGF2 dans ces deux syndromes. Ces résultats confirment un fonctionnement des gènes soumis à empreinte en réseau et le rôle majeur du système des IGFs dans la croissance fœtale, particulièrement altérée en cas de pathologie intéressant ces gènes soumis à empreinte parentaleFetal growth is dependant of environemental, genetic and hormonal factors which interact to ensure a proper development. Insulin-like growth factors (IGF) system plays a key role in fetal growth by interactions with these differents systems. In this work, we studied the roles of the IGF system in fetal growth restriction diseases. We used both clinical and experimental approaches to enhance knowledge on functional consequences of genetic ou epigenetic defects of IGF system actors. We set-up a functional test to assess IGF1R activity in vitro in patients with restricted fetal and postnatal growth. We also documented the IGF-I bioavailability in patients with Silver-Russell syndrome, which is an imprinting disorder responsible for fetal and postnatal growth restriction. We characterized the clinical and molecular overlap of Silver-Russell and Temple syndrome (another imprinting disease affecting growth and metabolism) and confirmed the central role of IGF2 in the physiopathology of these disorders. These results confirmed the integration of imprinted genes in a large co-regulation network and the major role of IGF system actors in fetal growth which is usually impaired when these imprinted genes are affected
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Keller, Emma Jean. "The Contribution of IFNα-Stimulated Immune Cell Populations to B6.NbA2 Lupus-likeDisease." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1625138193480211.
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ER, TZE-KIONG, and 余志強. "Molecular Diagnosis of Genetic Diseases by High-Resolution Melting Analysis." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/49092182181899780900.
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博士高雄醫學大學
醫學研究所
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Identifying and understanding genetic variation between populations and individuals is an important aim in the field of genomics. The unique genetic profile of an individual confers susceptibility to a given trait or disease. Therefore, there is a rapidly growing interest in feasible methods for mutation screening in life science research. High-resolution melting (HRM) analysis represents the next generation of mutation scanning technology and offers considerable time and cost savings prior to other screening method. HRM is a novel, homogeneous, close-tube, post-PCR method, enabling researchers to analyze genetic variations in PCR amplicons. Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Based on its ease of use, simplicity, flexibility, low cost, nondestructive nature, high sensitivity, and specificity, HRM analysis is quickly becoming the tool of choice to screen patients for pathogenic variants. Here we briefly discuss the establishment of HRM analysis for mutation screening including JAK2 V617 missense mutation and ETFDH gene mutation. Taken together, HRM analysis can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes. Janus kinase 2 (JAK2) is a tyrosine kinase involved in the cytokine signaling of several growth factors such as erythropoietin and thrombopoietin in normal and neoplastic cells. The G to T exchange at nucleotide 1849 in exon 14 of the JAK2 gene leads to a substitution of valine with phenylalanine at the amino acid position 617 (V617F) of the JAK2 protein. Currently, the occurrence of the JAK2 V617F mutation is well recognized in myeloproliferative disorders (MPDs), such as essential thrombocytosis, polycythemia vera, and primary myelofibrosis. The identification of molecular lesions specific to the myeloproliferative neoplasms, in particular JAK2 V617F, has broadened understanding of the common features within these disorders and has advanced diagnostic, prognostic, and therapeutic tools. The aim of our study was to assess the value of the HRM analysis using real-time polymerase chain reaction (PCR) (Lightcycler® 480; Roche Applied Science) for identifying the JAK2 V617F missense mutation. Our results showed that up to 5% of the JAK2 V617F mutation was successfully detected in patients with MPD using HRM analysis. The results proved 100% comparable to those obtained by ARMS assay. In conclusion, the HRM analysis is a rapid and effective technique for the detection of JAK2 V617F missense mutation. Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II is an autosomal recessive disease caused by defects in mitochondrial electron transfer system and metabolism of fatty acid. Recently, ETFDH mutations were reported to be major causes of riboflavin-responsive MADD. The present study is aimed at screening ETFDH mutations by HRM analysis. Our results showed that HRM analysis proved to be feasible in detecting 3 known (c.250G>A, c.380T>A, c.524G>T) and 1 novel (c.1831G>A) ETFDH mutations. Each mutation could be readily and accurately identified in the difference plot curves. We estimated the carrier frequency of the hotspot mutation, c.250G>A, in the Taiwanese population to be 1:125 (0.8%). In summary, HRM analysis can be successfully applied to screen ETFDH mutations. Since riboflavin-responsive MADD is often treatable, especially with mutations in ETFDH, identifying ETFDH mutations is crucial for these patients. Interestingly, two of the mutations (p.Ala84Thr and p.Phr128Ser) are located in the FAD-binding domain; however, the two amino acids do not have direct interactions with FAD according to the predicted 3D structure of ETF:QO. Therefore, to explore the effects of the mutations on ETF:QO dynamics, molecular dynamics (MD) simulations of the wild type (WT) and mutant type (MT) ETF:QO in the same model environment were compared. Besides the MD simulations, an alternative method, normal mode analysis (NMA), for studying protein motions was used to analyze the dynamic correlations between the mutation sites and the FAD-binding motif. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.
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"Steroid metabolism and pathology: biochemical and molecular diagnosis." 2014. http://library.cuhk.edu.hk/record=b6115511.
Full textAbstract:
This thesis describes biochemical and molecular methods diagnostic for a spectrum of steroid metabolic diseases. Deficiency of any enzyme in the steroid hormone biosynthetic pathways leads to disorders including congenital adrenal hyperplasia, while some cause disorders of sex development (DSD). A gas chromatography mass spectrometry-based analytical technique called urinary steroid profiling (USP) has been shown to be a useful diagnostic test for these diseases and for steroid-secreting adrenocortical tumours. To test the hypothesis that this approach would be effective in our local population, we interpreted 482 USP results using reference intervals set up from 371 local healthy subjects. Characteristic steroid metabolite excretion patterns were found in 39 patients, including 21 patients with 21-hydroxylase deficiency (21OHD) where there were grossly increased 17-hydroxyprogesterone metabolites, 12 patients with 5α-reductase 2 deficiency (5ARD) with extremely low 5α- to 5β-reduced steroid metabolite ratios, and five patients with adrenocortical carcinoma with markedly raised tetrahydro-11-deoxycortisol and 3,16,20-pregnenetriols levels.The genetic basis of 21OHD in various populations is mainly due to conversion between the CYP21A2 and the CYP21A1P genes but this has not yet been explored in our population. By using DNA sequencing and multiplex ligation-dependent probe amplification, 74 mutations were found in 35 patients with 21OHD. Gross deletion/conversion of the CYP21A2 gene accounted for 27%. c.290-13A/C>G was the most common point mutation (27%), followed by p.Ile172Asn (17.6%). One novel mutation c.1367delA was also detected. Their prevalence in our patients differs from those in other populations.
The most common cause of 46,XY DSD in Western populations is androgen insensitivity syndrome (AIS) but this has not been verified locally. A prospective study was conducted where 64 patients were recruited for comprehensive hormonal profiling and targeted molecular analysis. In this study, a genetic diagnosis was established in 22 patients, with 5ARD being the most common disease, followed by AIS. Traditionally the diagnosis of 5ARD relies on measuring dihydrotestosterone. However, with our experience in diagnosing this condition based on USP and mutational analysis of the SRD5A2 gene, two new diagnostic algorithms for 46,XY DSD were proposed where dihydrotestosterone is not required.
In vitro study is the preferred method for characterising the function of novel genetic variants. However, clinical laboratories rarely have the facilities and resources for it. In silico prediction programmes appear to be practical alternatives but their performance on testing non-synonymous variants in genes related to steroid metabolism has not been verified. Three web-based in silico prediction programmes, namely Sorting Intolerant From Tolerant, PolyPhen-2 and Pathogenic-Or-Not-Pipeline, were tested by analysing 797 published non-synonymous genetic variants in 12 genes related to steroid metabolism. The results of in vitro functional study and/or clinical phenotype were used as gold standards. The performance of these three programmes were: sensitivity (76.6%, 84.1%, 70.0%), specificity (56.6%, 56.3%, 89.4%) and accuracy (70.1%, 75.2%, 76.8%), respectively.
In conclusion, USP is a valuable biochemical phenotyping technique that helps to select patients for subsequent genetic confirmation. Since the mutation spectrum of 21OHD and the aetiological basis of 46,XY DSD in our population differ from the others, laboratory diagnostic algorithms and molecular analytical strategies must be adjusted accordingly.
Chan, On Kei Angel.
Thesis (M.D.) Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 250-269).
Appendixes includes Chinese.