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Bestimmung der Quantität der mRNA ausgewählter Proteine der extrazellulären Matrix des Alveolarknochens mithilfe der real-time RT-PCR / Determining the mRNA quantity of selected proteins of the extracellular matrix in the alveolar boneGroße Steffen, Christian 25 July 2017 (has links)
No description available.
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Analyse transcriptomique des cellules vasculaires isolées du tissu anévrysmal de l'aorte abdominale sous-rénale chez l'homme / Transcriptomic analysis of isolated vascular cells implicated in abdominal aortic aneuvrysm in humanSpear, Rafaëlle 10 December 2014 (has links)
L'anévrysme de l'aorte abdominale (AAA) est un problème de Santé Publique qui touche principalement les hommes de plus de 65 ans. L'AAA souvent asymptomatique évolue vers la rupture associée à un taux de mortalité élevé. Parmi les acides ribonucléiques (ARNs) non codants, les microARNs (miARNs), stables dans le tissu et les biofluides, sont des candidats intéressant dans la recherche de biomarqueurs. L'inflammation, la dégradation de la matrice extra-cellulaire (MEC) et la raréfaction de la média participent à l'AAA. De nombreuses cellules inflammatoires sont impliquées dans l'AAA. La raréfaction des cellules musculaires lisses (CML) est secondaire à l'anoïkis, apoptose par détachement cellulaire de la MEC. Une analyse protéomique différentielle de CML en culture, issues de patients porteurs d'AAA, réalisée au laboratoire a montré que la désintégrine et metalloprotéinase avec un motif de thrombospondine 5 (ADAMTS 5) est surexprimée dans les CML de patients présentant un AAA. L'isolement des cellules par la microdissection laser permet de conserver le phénotype des cellules isolées et de mettre en évidence des marqueurs potentiels de l'AAA masqués par l'analyse du tissu global. Mon travail de thèse a consisté à partir des cellules isolées de la paroi anévrysmale de l'aorte abdominale sous-rénale chez l'Homme: à effectuer une analyse globale des miARNS et une analyse ciblée de l'ADAMTS 5, métalloprotéase qui a une action enzymatique sur les protéines de la MEC. Les objectifs de ce travail sont une meilleure compréhension de l'AAA et l'identification de nouveaux biomarqueurs.La distribution des cellules dans la paroi anévrysmale est étudiée par immunohistochimie sur des biopsies anévrysmales et d'aortes saine obtenues chez l'Homme. Les cellules localisées sont isolées par microdissection laser. L'analyse par criblage de l'expression des miARNs des cellules isolées de l'AAA et des CML issues d'aorte saine est réalisée sur puce. L'expression différentielle de miARNs sélectionnés est analysée par PCR quantitative dans des cellules isolées de l'AAA et dans du tissu global. L'expression des miARNs sélectionnés est ensuite comparée dans le plasma des patients présentant un AAA et de patients athérosclérotiques sans AAA par PCR pour identifier de potentiels biomarqueurs. Dans l'AAA, les macrophages M1 proinflammatoires sont retrouvés dans l'adventice et les macrophages M2 anti inflammatoires dans le thrombus intraluminal, les lymphocytes de type B sont retrouvés organisé en organe lymphoïde tertiaire adventitielle ou ATLOs dans 11 échantillons sur 20 analysés. Les CML sont rares et strictement localises au niveau de la média. Sur 850 miARNs testés dans la puce, 205 miARNs sont exprimés dans les cellules isolées. Les miR-29b et let-7f sont augmentés dans le plasma de patients porteurs d'AAA et représentent de potentiel biomarqueurs.L'expression d'ADAMTS 5 dans les CML de la paroi anévrysmale est évaluée par immunohistochimie dans la paroi aortique saine et anévrysmale et quantifiée par Western-blot dans les CML isolées de la paroi aortique saine et anévrysmale.Deux morphotypes de CML anévrysmales ont été identifiés: un morphotype arrondi positif au marqueur de l'apoptose, caspase 3 et un morphotype allongé, similaire aux CML de l'aorte saine. Le profil d'expression des sous-unités d'ADAMTS 5 est diffèrent dans les CML arrondies et les CML allongées anévrysmales et saines. La mise en apoptose des CML a été mise au point in vitro pour étudier les mécanismes impliqués dans les modifications d’ expression d'ADAMTS 5 dans l'AAA et les conséquences sur son action enzymatique.L'approche systématique de l'expression transcriptomique des cellules anévrysmales isolées a identifié des marqueurs potentiels de l'AAA, les miR-29b et let-7f et l'analyse ciblée suggère l'implication d'ADAMTS 5 dans le profil évolutif des CML vers l'anoïkis dans l'AAA. Des études complémentaires permettront une meilleure compréhension de l'AAA. / Abdominal aortic aneurysm (AAA) is a public health problem, which mainly affects men older than 65 year. AAA are usually asymptomatic with a natural evolution towards rupture associated with a high mortality rate. Among non coding ribonucleic acids (RNAs), microRNAs are stable in tissue and biofluids and are interesting candidates for the search of biomarkers. Inflammation, extracellular matrix (ECM) degradation and media rarefaction are involved in AAA. Many inflammatory cells are involved in AAA. Anoikis is an apoptosis secondary to a cell detachment from ECM and is responsible for rarefaction of smooth muscle cells (SMC). Differential proteomic analysis of cultured SMC from AAA patients was performed in the laboratory and highlighted the overexpression of a disintegrin and metalloproteinase with thrombospondin motif of type 5 (ADAMTS 5) in SMC of AAA patients. Isolation of cells with laser microdissection allows to keep their phenotype and to find potential markers that may be masked by global tissue analysis.The aim of my PhD work was to perform a global miRNA screening of cells isolated from human aneurysmal wall and an analysis targeted on ADAMTS 5, a metalloprotease with an enzymatic activity on ECM proteins. The main objectives were a better understanding of AAA and the identification of new biomarkers.The distribution of cells in the aneurysmal wall was studied by immunohistochemistry in human aneurysmal and healthy aortic samples. Once located, the cells were isolated by laser microdissection and screened for miRNAs by microarrays. Differential expression of selected miRNAs was quantified by PCR in the cells isolated by laser microdissection and in whole aortas. They were then compared in plasma of AAA patients and atherosclerotic patients without AAA by quantitative PCR to identify potential biomarkers.In AAA, the M1 proinflammatory macrophages were located in the adventitia and the M2 antiinflammatory macrophages in the intraluminal thrombus; the type B lymphocytes were organized in tertiary lymphoid organs (ATLOs) in 11/20 of analysed samples. SMC were rare and restricted to the media. Among the 850 miRNAs tested on microarray, 205 miRNAs were detected in isolated cells. MiR-29b and let-7f were upregulated in plasma of AAA patients, and thus are potential biomarkers.The expression of ADAMTS 5 in aneurysmal SMC was evaluated by immunohistochemistry of healthy and aneurysmal aortic wall and quantified by Western blot in isolated SMC from healthy and aneurysmal wall.Two aneurysmal SMC morphotypes were identified: a rounded morphotype positive for caspase 3, an apoptotic marker, and a spindle-shaped morphotype similar to the healthy aortic SMC. The expression profile of ADAMTS 5 subunits was different in rounded SMC compared to aneurysmal and healthy spindle-shaped SMC. In vitro induction of apoptosis of SMC was established in order to study the mechanisms involved in ADAMTS 5 expression in AAA and their consequences on enzymatic actions.The global transcriptomic screening of aneurysmal cells isolated by laser microdissection has identified potential markers of AAA, miR-29b and let-7f. The targeted analysis suggested that ADAMTS 5 is involved in the evolution profile of SMC towards anoikis in AAA. Further investigations will allow a better understanding of AAA pathophysiology.
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Pathology of west nile virus lineages 1 and 2 in mice and horsesWilliams, J.H. (June Heather) January 2014 (has links)
West Nile virus (WNV) is a widespread emerging zoonotic neurotropic flavivirus cycling naturally between mosquitos and birds. WNV causes disease in 20% of infections in the most susceptible incidental hosts which are horses and humans. Up to 40% of affected horses and 1- to approximately 50% of affected humans develop neurological signs and/or flaccid paralysis, in some cases fatal or severely debilitating, due to variable encephalitis, meningitis and poliomyelitis. Two predominant genetic lineages exist, 1 and 2, with neurovirulent lineage 1 strains recorded in the northern and western hemispheres, the milder lineage 1 Kunjin strain in Australia, and the lineage 2 strain endemic to southern Africa and Madagascar and considered, until recently, to have mainly mildly pathogenic strains. Since 2002 investigations into South African lineage 2 WNV strains showed that they resulted in severe neurological disease in horses and humans. From 2004 lineage 2 strains were recorded for the first time in southern Europe as a cause of neurological signs and death in birds, and increasingly, in horses and humans. In 2011 the
mild lineage 1 Kunjin strain mutated to an equine neurovirulent strain in New South Wales, Australia, and in 2010 the first South African case of lineage 1 WNV was reported from the western Cape in a mare which showed severe neurological signs, abortion and death.
Laboratory strains of mice are extremely susceptible to WNV and have been mostly used in experimental studies since the 1937 discovery of the virus in Uganda. In the early 2000s studies in mice showed that field strains of lineage 1 and 2 WNV ranged from mildly pathogenic to highly neurovirulent, however, the associated pathology of the lineage 2 infections was not studied. In the current study, the macroscopic and microscopic pathology of a South African human-neurovirulent field strain of lineage 2 WNV (SPU93/01) and the neurovirulent lineage 1 (NY99/385) strain were investigated and compared in mice used as controls in 2 WNV vaccine studies. The clinical signs, CNS and extra-CNS pathology were indistinguishable between the lineages and some lesions were comparable to those previously reported. Lineage 1 WNV equine pathology has been well described but that of lineage 2 only briefly previously described. The pathology in 6 naturally-occurring fatal lineage 2 WNV-infected horses with severe neurological signs, was investigated and compared with that of the single South African lineage 1 WNV field infection. Diagnoses were confirmed by real-time RT-PCR. Similarities and some slight differences in lesions were found in both mouse and horse studies when compared with lineage 1 pathology cases and with previous reports, and the neurovirulence of the lineage 2 field strains was confirmed. WNV immunohistochemistry (IHC) of all mouse tissues allowed speculation as to pathogenesis of intestinal lesions, but in equine CNS lesions was mostly negative. Ultrastructure of IHC positive cells showed rare WNV particles. In the horse cases rabies, equine herpes virus, and other arboviral co-infections were excluded and similarities and implications of gross lesions of African horsesickness to those often seen in WNV infections were discussed. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Medical Virology / unrestricted
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Validación y evaluación de una prueba de RT-PCR en tiempo real in house para la detección de SARS-CoV-2 usando un gen específico RdRp y control endógeno GAPDHRojas-Serrano, Nancy, Lope-Pari, Priscila, Huaringa-Nuñez, Maribel, Marques Simas, Paulo Vitor, Palacios-Salvatierra, Rosa, Balbuena-Torres, Johanna, Caceres Rey, Omar Alberto, Padilla-Rojas, Carlos 13 December 2021 (has links)
Se validó y evaluó un método de RT-PCR en tiempo real usando cebadores y sondas específicas para los genes RdRP de SARS-CoV-2 y GAPDH de humanos; este último fue usado como control endógeno. Se evaluó la especificidad y sensibilidad; además, se evaluó otros parámetros como la robustez, la repetibilidad, reproducibilidad, comparabilidad y el límite de detección. La sensibilidad, especificidad, los valores predictivos positivo y negativo, la robustez, comparabilidad y la repetibilidad-reproducibilidad de la prueba de RT-PCR en tiempo real dúplex fue de 100%, con un límite de detección de 100 copias/μL, de acuerdo con los criterios de aceptación establecidos para validación del protocolo. Esta prueba estandarizada es una buena alternativa para el diagnóstico de COVID-19; además, la prueba fue aplicada de manera exitosa en personas sospechosas de la enfermedad permitiendo controlar el número de falsos negativos.
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Variações transcricionais dos genes AR, SRD5A2, KLK2, PCA3, KLK3 e PSMA e implicações no diagnóstico molecular do câncer de próstataNeves, Adriana Freitas 26 February 2007 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CHAPTER I -
Prostate cancer is a common disease in the world and in some countries it is one of the
main causes of male population mortality. Some molecular markers have been associated
with prostate carcinogenesis. To observe changes in transcript levels of the AR, SRD5A2,
KLK2, PSMA and PCA3 genes, the mRNA was analyzed in tissues with prostatic
adenocarcinoma (PCa, N= 48) and benign prostatic hyperplasia (BPH, N= 25), performed
through a differential multiplex RT-PCR assay. Significant differences were observed in
the relative expression of these genes between cancerous and non-cancerous tissues.
The optical density ratio of the cDNA amplicons between PCa and BPH for the AR gene
was 1.6-fold higher for the prostatic adenocarcinoma. On the other hand, the SRD5A2
mRNA levels were associated with BPH and were 1.4-fold higher than that of PCa. For
KLK2, PSMA and PCA3, the transcriptional levels were respectively, 1.9-, 1.9- and 5-fold
higher in PCa than those in BPH tissues. Of the diagnostics tests carried through
individually, the PCA3 gene was that presented higher sensitivity and accuracy, and the
inclusion of the serum PSA improved the sensitivity (of 76 to 92%), positive preditive value
(of 85 to 94%), negative preditive value (of 60 to 62%) and accuracy (of 74 to 78%). The
results suggest that the higher AR, KLK2, PSMA, and PCA3 and/or reduced SRD5A2
genes expression in prostatic tissues may indicate the occurrence of prostate
adenocarcinoma; however the PCA3 and serum PSA analysis together are highly
promising as auxiliary method in the diagnosis of this cancer.
CHAPTER II -
Purpose: Prostate cancer (PCa) is the most commonly diagnosed malignancy in men, and
it consists of multifactorial and multifocal events. Due to the complexity of the disease
process, which includes genome alterations, local invasion, micrometastatic cell
extravasations to circulation, invasion of secondary organ tissues, and resistance to
hormonal blockage, many markers must be used to represent the multiple and variable
events that lead to cancer development. The low specificity of the unique serum marker
for prostate cancer diagnostics, PSA, has leaded us to investigate four potential markers
in the peripheral blood of patients by detecting their mRNA levels and associating them to
clinical parameters. Methods: The expression levels of the KLK2, KLK3, PCA3 and PSMA
transcripts were determined by Nested RT-PCR. Patients with PCa (99) and with benign
prostatic hyperplasia (BPH, 36), and healthy volunteers (104) were investigated. Results:
Significant differences for the RNA relative levels have been found for the KLK2, PCA3
and PSMA transcripts between PCa and BPH patients or healthy volunteers. The KLK2
and PSMA levels also presented a positive association (P<0.05) with extra-prostatic
disease (pT3). The combined positive RT-PCR Nested for the KLK2, PCA3, PSMA genes
with serum PSA higher than 4ng/mL presented a 10-fold higher chance of cancer
occurrence than healthy controls, with sensitivity, specificity and positive predictive value
of 57%, 89% and 93%, respectively. Conclusions: The combined analysis of KLK2, PCA3
and PSMA transcripts may become a useful tool for the discrimination of PCa patients
from those with benign disease or healthy individuals. Additionally, the KLK2 and PSMA
transcripts may also be used as prognostic markers for the presence of extra-capsular
disease and assisting in the prediction of the post-operative outcome.
CHAPTER III -
Purpose: Transcripts of PCA3/DD3 gene are at the moment the most specific molecule
found in prostate cancer specimens. This mRNA can be detected in important sample
targets for clinical analyses, such as prostatic tissues, urine after prostatic massage, and
the peripheral blood. Methods: The present study evaluated the PCA3 gene expression in
prostatic tissues and in peripheral blood of BPH and PCa patients, by RT-PCR assays,
and based on its detection together with other clinical parameters, we proposed a model
for molecular monitoring in order to improve diagnosis as an auxiliary technology. Results:
The concomitant use of PCA3 transcript detection in the peripheral blood and in prostate
tissues has improved diagnosis, with sensitivity and an accuracy of 77%. For the
molecular staging, patients have been classified as: localized disease (PBL-; negative
PCA3) and circulating tumors cells disease (PBL+; positive PCA3). The higher
frequencies of PBL- had been observed in T1-T2 stages (75%); on the other hand, the
higher PCA3 positivity was observed for the T3-T4 staging (43%), while the T1-T2 stages
presented 25% positivity. A correlation was found between the molecular staging and
serum PSA < 10ng/mL before surgery, and approximately 60% of patients with T3-T4
stages that presented biochemical failure after radical prostatectomy presented a positive
PCA3 result (P= 0.05), with an odds ratio of 16-fold higher for the possibility of disease
recurrence in relation to the T1-T2 patients, and an accuracy of 82%. Conclusion: These
data demonstrated the importance of the PCA3 gene as an auxiliary method in prostate
cancer diagnosis, by distinguishing PCa from BPH patients, and also demonstrated its
prognostic value in recurrent disease for post-operative patients.
CHAPTER IV -
Approximately 98% of all the products transcribed in the human genome correspond to
non coding RNAs (ncRNA). Many ncRNA functions are attributed to this structural
particularity given mainly for the secondary structures formed from its linear sequence of
bases. Among the ncRNA types are tRNA, rRNA, small nuclear RNA, small nucleolar
RNA, small interference RNA (siRNA), microRNA (miRNA) and catalytic RNAs
(ribozymes). The bioinformatics has supplied useful tools in the prediction of optimal or
suboptimal secondary structures allowing the design of interference RNA as miRNAs or
siRNAs. In human, miRNAs have been associated with the development of diverse
complex diseases as cancer. The PCA3 (DD3) gene was molecularly characterized as
cancer and prostate specific, and its transcripts are non-coding, once no peptide products
have been found. Due to its structural characteristics, the PCA3 gene belongs thus to the
increasing family of ncRNA. In the present work, four new variant molecules of the PCA3
gene have been sequence characterized and their frequencies demonstrated in prostate
cancer and in benign prostatic hyperplasia patients, as well as in healthy individuals. We
have also investigated and predicted the putative secondary structures formed in order to
elucidate its role in prostate cancer biology. No association has been found between the
frequency of these molecules and prostate pathologies (PCa or BPH). On the other hand,
PCA3 variants were found in 10% (12/115) of cases in the general population. Similar
analyses of the possible polypeptides of these molecules demonstrated that it remains as
a non-coding RNA, and introns presents in the first, second and fourth variants suggesting
a possible role as a miRNA with intracellular activity to these molecules to the PCA3 gene.
In prostatic tissues, 100% of the prostate cancer cases presented the RNA molecule with
an exon 2 splicing. However, further investigation must be carried out to demonstrate the
true role of these splicing variants in prostate tumors and in other pathologies, once these
molecules have been preferentially found in the peripheral blood. / CAPÍTULO I -
O câncer de próstata é uma doença comum no mundo e já assumiu em alguns
países uma das principais causas de mortalidade da população masculina. Vários
marcadores moleculares têm sido associados à gênese do câncer de próstata. A fim de
demonstrar a expressão diferencial dos níveis transcricionais dos genes AR, SRD5A2,
KLK2, PSMA e PCA3 em doenças prostáticas, o RNAm foi analisado em tecidos com
adenocarcinoma prostático (CaP, N= 48) e hiperplasia prostática benigna (HPB, N= 25)
por meio da técnica RT-PCR multiplex semi-quantitativa. Foram observadas diferenças
significativas na expressão relativa desses genes entre os tecidos cancerosos e nãocancerosos.
A taxa de densidade ótica entre os amplicons para cDNA provenientes do
gene AR foi 1.6 vezes maior no adenocarcinoma prostático. Por outro lado, os níveis de
RNAm do gene SRD5A2 foi associado com a HPB e foi 1.4 vezes maior do que no CaP.
Para os genes KLK2, PSMA e PCA3, os níveis transcricionais foram respectivamente,
1.9, 1.9 e 5 vezes maior no câncer comparado a tecidos benignos. Dos testes
diagnósticos realizados, o gene PCA3 individualmente foi o que apresentou as melhores
sensibilidade e acurácia, sendo que a inclusão das medidas de PSA sérico melhorou a
sensibilidade (de 76 para 92%), o valor preditivo positivo (de 85 para 94%), o valor
preditivo negativo (de 60 para 62%) e a acurácia (de 74 para 78%). Os dados sugerem
que a maior expressão dos genes AR, KLK2, PSMA e PCA3 ou expressão reduzida do
gene SRD5A2 em tecidos prostáticos podem indicar a ocorrência do adenocarcinoma da
próstata, sendo que as análises do gene PCA3 juntamente aos do PSA sérico são
altamente promissores como método auxiliar no diagnóstico desse tipo de câncer.
CAPÍTULO II -
O câncer de próstata (CaP) e o mais comumente diagnosticado na população masculina
e consiste de eventos multifatoriais e multifocais. Devido a complexidade da doença, a
qual inclui alterações genômicas, invasão local, liberação de células micrometastáticas
para a circulação, invasão secundaria de tecidos de outros órgãos, e resistência ao
bloqueio hormonal, muitos marcadores podem ser usados para representar os eventos
múltiplos e variáveis que levam ao desenvolvimento do câncer. A baixa especificidade do
único marcador para diagnostico do câncer de próstata, PSA, tem nos levado a investigar
quatro potenciais marcadores no sangue periférico de pacientes pela detecção de seus
níveis de RNAm e associá-los a parâmetros clínicos. Os níveis de expressão dos
transcritos do KLK2, KLK3, PCA3 e PSMA foram avaliados pela RT-PCR Nested, em
pacientes com CaP (99), com hiperplasia prostática benigna (HPB, 36) e voluntários
saudáveis (104). Diferenças significativas foram encontradas para a expressão dos genes
KLK2, PSMA e PCA3 entre os pacientes com CaP e os pacientes com HPB ou
voluntários saudáveis. Os níveis do KLK2 e PSMA também apresentaram associação
positiva (P<0.05) com doença extra-prostática (pT3). A RT-PCR Nested positiva
combinada para os genes KLK2, PCA3 e PSMA com PSA sérico maior que 4ng/mL
apresentou uma chance 10 vezes maior de ocorrência do câncer comparado aos
controles saudáveis, com sensibilidade, especificidade e acurácia de 57%, 89% e 93%,
respectivamente. A análise combinada dos genes KLK2, PCA3 e PSMA pode ser uma
ferramenta útil na distinção de pacientes com CaP daqueles com doença benigna ou de
indivíduos saudáveis. Ainda, a analise dos transcritos KLK2 e PSMA podem ser usados
como marcadores prognósticos para a presença de doença extra-capsular e auxiliando
na predição de recidiva da doença no pós-operatório.
CAPÍTULO III -
Os transcritos do gene PCA3/DD3 são até o momento as moléculas mais específicas
encontradas em espécimes de câncer de próstata. Esses RNAm podem ser detectados
em importantes alvos para a análise clínica como tecidos prostáticos, na urina após
massagem prostática e em sangue periférico. O presente estudo avaliou a expressão do
gene PCA3 em tecidos prostáticos e em sangue periférico de pacientes com HPB e CaP,
por técnicas de RT-PCR, e baseado na sua detecção juntamente com os parâmetros
clínicos, foi proposto um modelo de estadiamento molecular como técnica assessória
para melhor o diagnóstico da doença. O uso concomitante da detecção dos transcritos do
gene PCA3 no sangue periférico e no tecido prostático melhorou o diagnóstico, com
sensibilidade e acurácia de 77%. Para o estadiamento molecular, os pacientes foram
classificados como contendo a doença localizada (PBL-) e em doença com células
tumorais circulantes (PBL +). Maiores freqüências de tumor localizado pelo estadiamento
molecular foram observadas nos estadios T1-T2 (75%), enquanto que 25 e 43% dos
cânceres T1-T2 e T3-T4, respectivamente, apresentaram PCA3 positivo (células
circulantes). Uma correlação foi encontrada para o estadiamento molecular para doença
localizada e PSA sérico pré-cirúrgico < 10ng/mL, e aproximadamente 60% dos pacientes
TNM T3-T4 que apresentaram falha bioquímica após a cirurgia radical apresentaram RTPCR
positiva do PCA3 (P= 0.05), com um Odds Ratio 16 vezes maior para a
possibilidade de recorrência da doença em relação aos pacientes T1-T2 e uma acurácia
de 82%. Esses dados demonstram a importância da detecção do gene PCA3 como
método no diagnóstico do câncer de próstata, por distinguir pacientes com CaP daqueles
com HPB, e também demonstrando seu valor prognóstico na doença recorrente no pósoperatório
dos pacientes.
CAPÍTULO IV -
Aproximadamente 98% de todos os produtos transcritos do genoma humano
correspondem a RNAs não codificantes (RNAnc). Muitas funções dos RNAnc são
atribuídas a suas particularidades estruturais dadas principalmente pelas estruturas
secundárias formadas a partir da sua sequência linear de bases. Dentre os tipos de
RNAnc estão os RNAt, RNAr, small nuclear RNA, small nucleolar RNA, small interference
RNA (siRNA), microRNA (miRNA) e RNAs catalíticos (ribozimas). A bioinformática tem
fornecido ferramentas úteis na predição de estruturas secundárias ótimas ou subótimas
permitindo o design de RNAs de interferência como os miRNAs ou siRNAs. Em humanos,
os miRNAs tem sido associados ao desenvolvimento de diversas doenças complexas
como o câncer. O gene PCA3 (DD3) foi molecularmente caracterizado como câncer- e
próstata- específico e os seus RNAs são os responsáveis por essa característica, isso
porque nenhum produto protéico tem sido encontrado para esse gene. Devido às suas
características estruturais, o gene PCA3, pertence assim à crescente família de RNAnc.
No presente trabalho foi analisado as freqüências de quatro moléculas variantes do gene
PCA3, além das anteriormente reportadas, como também foram preditas as suas
estruturas secundárias na tentativa de elucidar o seu papel na biologia do câncer de
próstata. Nenhuma associação foi encontrada entre a freqüência dessas moléculas e as
patologias da próstata como hiperplasia benigna ou câncer, sendo que na população
geral analisada essas variantes foram encontradas em apenas 10% (12/115) dos casos.
As análises de homologia de possíveis polipeptídeos para essas moléculas demonstram
que permanece o papel de RNA não-codificante para o gene PCA3. Ainda, a presença de
introns nas variantes 1, 2 e 4 podem sugerir um papel intracelular de miRNA para essas
moléculas do gene PCA3. Nos tecidos prostáticos, 100% dos casos de câncer foi
representando pela molécula com splicing do exon 2. Contudo, para as variantes de
splicing, novas pesquisas deverão ser realizadas incluindo outras patologias além das
doenças prostáticas e outros tipos tumorais para verificar o real impacto dessas
moléculas, uma vez que foram encontradas preferencialmente no sangue periférico. / Doutor em Genética e Bioquímica
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Molecular identification and characterisation of rodent- and shrew-borne HantavirusesIthete, Ndapewa Laudika 12 1900 (has links)
Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Throughout history disease entities have been described which match the
description of diseases now known to be caused by hantaviruses; however these
viruses were first identified as the aetiologic agent in 1976, the first species named
Hantaan virus after the river near which its natural host, the rodent species
Apodemus agrarius, was captured. Since then numerous species in the Hantavirus
genus, family Bunyaviridae, have been found, with today more than 30 species
worldwide being known.
Hantaviruses are hosted by rodents from the Muridae and Cricetidae families and by
shrews (insectivores) in the Soricidae family. There are two types of hantavirus
disease, Haemorrhagic fever with renal syndrome (HFRS) in the Old World and
Hantavirus cardiopulmonary syndrome (HCPS) in the New World. The first two
African hantaviruses were identified in 2006 in Guinea, West Africa; Sangassou virus
(SANGV) in a rodent, the African wood mouse (Hylomyscus simus), and Tanganya
virus (TGNV) in Therese’s shrew (Crocidura theresae).
In this study, rodents and shrews were trapped at localities in the Western Cape and
Northern Cape provinces of South Africa, and in the southern regions of Namibia.
RNA was extracted from their lungs and screened for hantavirus sequences by RTPCR,
using degenerate primers designed to detect all members of the Hantavirus
genus.
In addition, an in-house IgG ELISA assay was set up, based on recombinant N
antigen from Dobrava virus, DOB-rN, and Puumala virus, PUU-rN. The assay was
used to screen patient sera collected in an anonymous convenience serological
survey using residual serum samples left over from routine testing at NHLS
laboratories in the Western Cape for hantavirus-specific antibodies.
RNA from 576 animal specimens was screened by RT-PCR; no hantavirus genome
was detected in any of the specimens. Sera from 161 patients were screened for
hantavirus antibodies; 11.18% of the sera were reactive to DOB-rN, 4.97% against
PUU-rN and 2.48% against both antigens.
v
Though no virus was detected in the animals screened, this does not necessarily
mean that there are no hantaviruses present in Southern Africa. A previous
seroepidemiological survey conducted in South Africa reported on the presence of
hantavirus specific antibodies by IFA in two species of rodents trapped in the
Western Cape and Northern Cape Aethomys namquensis and Tatera leucogaster.
Our was the second known study in South Africa conducted that determined and
proved the presence of hantavirus specific antibodies in humans. / AFRIKAANSE OPSOMMING: Dwarsdeur die geskiedenis was daar beskrywings van siektes wat ooreenstem met
die beskrywing van hantavirus simptome, maar die eerste etiologiese oorsaak van
die siekte is eers in 1976 geïdentifiseer en Hantaan virus genoem, vernoem na die
rivier waar naby die gasheer, Apodemus agrarius, gevang is. Van daar af het die
soektog na nuwe hantavirusse intensief gevorder en vandag is daar meer as 30
spesies wêreldwyd wat aan die Hantavirus genus, ’n lid van die Bunyaviridae familie,
behoort.
Knaagdiere van die Muridae en Cricetidae families, sowel as spitsmuise (insekvreters)
in die Soricidae familie is gasheer vir hantavirusse. Twee tipes hantavirus
siekte is bekend, hemorragische koors met nier sindroom (HFRS) in die Ou Wêreld
en hantavirus kardiopulmonale sindroom in die Nuwe Wêreld. Die eerste twee Afrika
hantavirusse is in 2006 in Guinee Wes-Afrika geïdentifiseer; Sangassou virus
(SANGV) in ’n knaagdier, die Afrika hout muis (Hylomyscus simus) en Tanganya
virus (TGNV) in Therese se spitsmuis (Crocidura theresae).
In hierdie studie is knaagdiere en spitsmuise op verskeie plekke in die Wes- en
Noord-Kaap provinsies, asook die Suide van Namibië, gevang. RNS is onttrek vanuit
die longe en hantavirus volgordes is gesoek deur middel RT-PKR deur gebruik te
maak van Pan-Hanta primers wat ontwerp is om alle lede van die Hantavirus genus
op te spoor. ’n Self-ontwerpde IgG ELISA, gebasseer op rekombinante N antigeen
van Dobrava virus, DOB-rN en Puumala virus, PUU rN, is opgestel en gebruik om
pasiënt serum, verkry in ’n anonieme serologiese opname, te toets; oorblywende
serum, na toetse uitgevoer is deur NHLS laboratoriums in die Wes-Kaap, is verkry
en getoets vir hantavirus spesifieke teenliggaampies.
RNS van 576 dier monsters is getoets deur middel van RT-PKR en geen hantavirus
is in enige van die monsters geïdentifiseer nie. Serum van 161 pasiënte is getoets vir
hantavirus teenliggaampies; 11.18% van die serum was reaktief teen DOB-rN,
4.97% teen PUU-rN en 2.48% teen albei antigene.
Alhoewel geen virus in die diere geïdentifiseer is nie, beteken dit nie noodwendig dat
geen hantavirusse in Suidelike-Afrika voorkom nie. ‘n Vorige sero-epidemiologiese
opname wat in Suid-Afrika gedoen is het die teenwoordigheid van hantavirus
spesifieke teenliggaampies in twee knaagdier spesies, Aethomys namquensis en
Tatera leucogaster gevang in die Wes-en Noord-Kaap, gevind. Ons studie is die
tweede studie bekend in Suid-Afrika uitgevoer, wat die teenwoordigheid van
hantavirus spesifieke teenliggaampies bevind en bewys het.
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Evaluation of high-throughput methodology for multi-gene screening in patients with Non-Alcoholic Fatty Liver Disease (NAFLD)Fisher, Leslie Reginald 12 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent chronic liver disease in Western countries and is considered the hepatic manifestation of the Metabolic Syndrome (MetS). Its heterogeneous nature ranges from hepatic steatosis through steatohepatitis to advanced fibrosis and cirrhosis where the ingestion of significant amounts of alcohol has been excluded. The disease profile of NAFLD and its necro-inflammatory subset Nonalcoholic Steatohepatitis (NASH) were described in the parent study, which provided a clinically well-characterised patient cohort for the present investigation. South African patients with NASH had significantly higher mean serum cholesterol and triglyceride levels than those with fatty liver only.
The objective of this study was to implement a high-throughput real-time polymerase chain reaction (PCR) method in our laboratory to enable the assessment of cardiovascular genetic risk factors in NAFLD patients. The specific aims were to determine the clinical utility and perform analytical validation of each mutation included in the multi-gene cardiovascular disease (CVD) screening assay. The Pathology Supported Genetic Testing (PSGT) concept developed at our department provides a practical approach to personalized medicine. The CVD multi-gene screen analyses key metabolic pathways relating to atherogenic dyslipidaemia, chronic inflammation, hypercoagulation and iron dysregulation implicated in insulin resistance, which is known to be a universal factor in the pathogenesis of NAFLD. Deleterious low-penetrance mutations in the APOE (APOE2 and E4 alleles), MTHFR (677C>T and 1298A>C), F2 (20210G>A), FV (1691G>A, Leiden) and HFE (C282Y and H63D) genes were included for analysis due to their important role as genetic contributors to these biological processes. A total of 178 patients diagnosed with NAFLD and 75 controls were studied using direct DNA sequencing and a RT-PCR system for mutation detection. In addition, two patients with high ferritin levels were included as case studies. A significant association was found between HFE mutations and elevated Alanine Transaminase (ALT) levels in the NAFLD population (p = 0.04). This discovery is interpreted as the identification of a subset of patients at greater risk of developing progressive liver damage who would benefit most from genetic testing to direct more aggressive therapy at an earlier stage. The necessity of an integrative, systems-based network approach was demonstrated to more accurately distinguish between Hereditary Haemochromatosis (HH) and Insulin Resistance-associated Hepatic Iron Overload (IR-HIO) syndrome in obese patients. The PSGT approach to personalized medicine facilitates diagnosis of CVD subtypes, prevention of cumulative risk and the formulation of gene-based intervention programs tailored to the needs of the patient.
These findings support the clinical utility of the CVD multi-gene test to guide chronic disease risk management in patients with NAFLD. The HFE mutation detection component of this test is of particular relevance in directing an effective treatment strategy in patients with a medical history of CVD and/or high iron stores. / AFRIKAANSE OPSOMMING: Nie-Alkoholiese Vettige Lewer Siekte (NAFLD) is die mees algemene kroniese lewer siekte in Westerse lande en word bestempel as die hepatiese manifestasie van die Metaboliese Sindroom (MetS). Die heterogene natuur van NAFLD strek van hepatiese steatose deur steatohepatietis tot gevorderde fibrose en sirrose waar grootskaalse alkohol inname uitgesluit is. Die siekte-profiel van NAFLD en sy nekro-inflammatoriese subtipe Nie-Alkoholiese Steatohepatietis (NASH) is reeds beskryf in die ouer studie, wat ‗n klinies goed-gekarakteriseerde pasiënt groep vir die huidige ondersoek daar gestel het. Suid-Afrikaanse pasiënte met NASH het beduidend hoër gemiddelde serum cholesterol en trigliseried vlakke in vergelyking met slegs vettige lewer.
Die doel van hierdie studie was om ‗n hoë deurvoer rieëltyd polimerase kettingreaksie (RT-PCR) metode in ons laboratorium te implimenteer om kardiovaskulêre genetiese risiko faktore in NAFLD pasiënte te ondersoek. Die spesifieke mikpunte was om die kliniese nut en analitiese geldigheid van elke mutasie wat ingesluit is in die multi-geen kardiovaskulêre siekte (KVS) siftings toets vas te stel. Die Patologie Ondersteunde Genetiese Toetsing (PSGT) konsep wat by ons departement ontwikkel is, verskaf ‗n praktiese benadering tot persoonlike medisyne. Die KVS multi-geen toets analiseer belangrike metaboliese weë verwant aan atherogene dyslipidemie, kroniese inflammasie, oormatige bloedstolling en yster disregulering wat betrokke is by insulien weerstand wat bekend is as ‗n universele factor in the patogenese van NAFLD. Nadelige lae-penetrasie mutasies in die APOE (APOE2 en E4 allele), MTHFR (677C>T en 1298A>C) F2 (20210G>A), FV (1691G>A, Leiden) en HFE (C282Y en H63D) gene was ingesluit vir analise as gevolg van hul belangrike rol as genetiese bydraers tot die bogenoemde biologiese prosesse. ‗n Totaal van 178 pasiënte gediagnoseer met NAFLD en 75 kontroles is bestudeer deur gebruik te maak van direkte DNA volgordebepaling en ‗n RT-PCR metode vir mutasie opsporing. Twee pasiënte met verhoogde ferritien vlakke is ook as gevalle studies ingesluit.
‗n Beduidende assosiasie is gevind tussen HFE mutasies en verhoogde Alanien Transaminase (ALT) vlakke in die NAFLD studiepopulasie (p = 0.04) wat aanduidend is van ‗n subgroup van pasiënte wat die meeste baat sal vind uit genetiese toetsing om meer aggressiewe behandeling te rig op' n vroeër stadium. Die noodsaaklikheid van 'n geïntegreerde, stelsels-gebaseerde netwerk benadering is gewys om meer akkuraat te onderskei tussen Oorerflike Hemochromatose (HH) en Insulien Weerstand-geassosieerde Hepatiese Yster Oorlading (IR-HIO) sindroom in vetsugtige pasiënte. Die PSGT benadering tot persoonlike medisyne formuleer geen-gebaseerde intervensie programme aangepas tot die behoeftes van die pasiënt ek maak diagnose van KVS-subtipes en voorkoming van kumulatiewe risiko moontlik.
Hierdie bevindinge ondersteun die kliniese nut van die KVS multi-geen toets om riglyne vir die risikobestuur van kroniese siektes soos NAFLD daar te stel. Die HFE mutasie opsporings komponent van hierdie toets is van besondere belang om 'n effektiewe strategie vir die behandeling van pasiënte met 'n mediese geskiedenis van KVS en/of hoë yster vlakke daar te stel.
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The Origin of the Genus Flavivirus and the Ecology of Tick-Borne PathogensPettersson, John H.-O. January 2013 (has links)
The present thesis examines questions related to the temporal origin of the Flavivirus genus and the ecology of tick-borne pathogens. In the first study, we date the origin and divergence time of the Flavivirus genus. It has been argued that the first flaviviruses originated after the last glacial maximum. This has been contradicted by recent analyses estimating that the tick-borne flaviviruses emerged at least before 16,000 years ago. It has also been argued that the Powassan virus was introduced into North America at the time between the opening and splitting of the Beringian land bridge. Supported by tip date and biogeographical calibration, our results suggest that this genus originated circa 120,000 (156,100–322,700) years ago if the Tamana bat virus is included in the genus, or circa 85,000 (63,700–109,600) years ago excluding the Tamana bat virus. In the second study we estimate the prevalence of tick-borne encephalitis virus (TBEV) in host-seeking Ixodes ricinus from 29 localities in Sweden and compare our data with those of neighbouring countries. Nymphs and adult ticks were screened for TBEV using a real-time PCR assay. The mean TBEV prevalence for all tick stages combined was 0.26% for Sweden and 0.28% for all Scandinavian countries, excluding Iceland. The low prevalence of TBEV in nature may partly be explained by the fact that TBEV occurs in spatially small foci and that the inclusion of ticks from non-infected foci will reduce the prevalence estimate. In the third and fourth study, we conducted the first large-scale investigations to estimate the prevalence and geographical distribution of Anaplasma spp. and Rickettsia spp. in host-seeking larvae, nymphs and adults of I. ricinus ticks in Sweden. Ticks were collected from several localities in central and southern Sweden and were subsequently screened for the presence of Anaplasma spp. and Rickettsia spp. using a real-time PCR assay. For all active tick stages combined, the mean prevalence of Anaplasma spp. and Rickettsia spp. in I. ricinus in Sweden was estimated to 1.1% and 4.8%, respectively. It was also shown that A. phagocytophilum and R. helvetica are the main Anaplasma and Rickettsia species occurring in Sweden.
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Augmentation de la production d'hydrogène par l'expression hétérologue d'hydrogénase et la production d’hydrogène à partir de résidus organiquesSabourin, Guillaume P. 11 1900 (has links)
La recherche de sources d’énergie fiables ayant un faible coût environnemental est en plein essor. L’hydrogène, étant un transporteur d’énergie propre et simple, pourrait servir comme moyen de transport de l’énergie de l’avenir. Une solution idéale pour les besoins énergétiques implique une production renouvelable de l’hydrogène. Parmi les possibilités pour un tel processus, la production biologique de l’hydrogène, aussi appelée biohydrogène, est une excellente alternative. L’hydrogène est le produit de plusieurs voies métaboliques bactériennes mais le rendement de la conversion de substrat en hydrogène est généralement faible, empêchant ainsi le développement d’un processus pratique de production d’hydrogène. Par exemple, lorsque l’hydrogène est produit par la nitrogénase sous des conditions de photofermentation, chaque molécule d’hydrogène constituée requiert 4 ATP, ce qui rend le processus inefficace.
Les bactéries photosynthétiques non sulfureuses ont la capacité de croître sous différentes conditions. Selon des études génomiques, Rhodospirillum rubrum et Rhodopseudomonas palustris possèdent une hydrogénase FeFe qui leur permettrait de produire de l’hydrogène par fermentation anaérobie de manière très efficace. Il existe cependant très peu d’information sur la régulation de la synthèse de cette hydrogénase ainsi que sur les voies de fermentation dont elle fait partie. Une surexpression de cette enzyme permettrait potentiellement d’améliorer le rendement de production d’hydrogène.
Cette étude vise à en apprendre davantage sur cette enzyme en tentant la surexpression de cette dernière dans les conditions favorisant la production d’hydrogène. L’utilisation de résidus organiques comme substrat pour la production d’hydrogène sera aussi étudiée. / The search for alternative energy sources with low environmental impact is in
great expansion. Hydrogen, an elegant and simple energy transporter, could serve as
means of transporting energy in the future. An ideal solution to the increasing energy
needs would imply a renewable production of hydrogen. Out of all the existing
possibilities for such a process, the biological production of hydrogen, also called
biohydrogen, is an excellent alternative. Hydrogen is the end result or co-product of
many pathways in bacterial metabolism. However, such pathways usually show low
yields of substrate to hydrogen conversion, which prevents the development of
efficient production processes. For example, when hydrogen is produced via
nitrogenase under photofermentation conditions, each hydrogen molecule produced
requires 4 molecules of ATP, rendering the process very energetically inefficient.
Purple non-sulfur bacteria are highly adaptive organisms that can grow under
various conditions. According to recent genomic analyses, Rhodospirillum rubrum and
Rhodopseudomonas palustris possess, within their genome, an FeFe hydrogenase that
would allow them to produce hydrogen via dark fermentation quite efficiently.
Unfortunately, very little information is known on the regulation of the synthesis of
this enzyme or the various pathways that require it. An overexpression of this
hydrogenase could potentially increase the yields of substrate to hydrogen conversion.
This study aims to increase our knowledge about this FeFe hydrogenase by
overexpressing it in conditions that facilitate the production of hydrogen. The use of
organic waste as substrate for hydrogen production will also be studied.
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Srovnání vlastností buněčných linií rezistentních k ellipticinu, doxorubicinu a cisplatině / The comparison of properties of cell lines resistant to ellipticine, doxorubicin, and cisplatinČerná, Tereza January 2014 (has links)
7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....
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