4 Abstract The antral follicle provides a specialized microenvironment or niche, which is necessary for production of high quality oocyte. The developmental competence of bovine oocyte is influenced by the follicle size. Oocytes originated from medium or larger follicles (≥6 mm) have greater developmental competence (ability to develop to the blastocyst stage). The changes in cytoplasmic factors, for example mRNAs, could explain differences in oocyte developmental potential. Using Bovine Oligonucleotide microarrays the differences in gene expression profiles of oocytes at germinal vesicle and MII stages from medium (MF, 6-10 mm) or small (SF, 2-5 mm) follicles were characterized. The aim was to find differencies between oocytes diverse developmental competence. The expression fold change between the two experimental groups was in 61 genes. Subsets of 15 differentially expressed genes were validated by quantitative RT-PCR. Before maturation, significant differences were confirmed at the level of ATP5C1, MAP3K13, MTRF1L, TAF1A and UBL5. Subpopulations of oocytes were classified according to atresia of cumulus cells and follicle size. We determined the level of 12 individual transcripts after maturation. ATP5F1 remained stable in all experimental groups of oocytes. The level of BRD7 transcript remained stable...

Le 1er transfert de brin, étape cruciale de la transcription inverse impliquant la protéine de nucléocapside du VIH-1 (NC), repose sur l’appariement de la séquence r de l’ADN « strong stop » (ADNss) avec la séquence 3’ R de l’ARN viral (3’UTR) qui forme les tiges-boucles TAR et polyA. La séquence r est supposé former les tiges-boucles cTAR et cpolyA. Le transfert repose donc probablement sur l’hybridation de molécules structurées. La structure secondaire de l’ADNss n’a jamais été déterminée. L’objectif a été d’identifier les interactions et structures gouvernant l’hybridation de l’ADNss avec l’ARN 3’UTR. Les outils de la biologie moléculaire et trois sondes de structure ciblant l’ADN ont été utilisés pour atteindre cet objectif. Nos résultats sont les suivants : 1) l’ADN cTAR nu se replie sous la forme de deux conformations différentes qui sont en équilibre ; 2) la NC peut déplacer l’équilibre vers l’une des conformations et se fixer préférentiellement sur la boucle interne du cTAR ; 3) la NC est exigée pour former un hétéroduplex constitué de l’intégralité de l’ADNss et du 3’ UTR ; 4) l’hybridation ADNss-3’UTR peut être initiée à partir de plusieurs sites dans 0,2 mM MgCl2 ; 5) l’ADNss forme deux conformations en équilibre dans 0,2 mM MgCl2 et principalement une seule dans 2 mM MgCl2 ; 6) dans l’ADNss, la NC se fixe préférentiellement au niveau de la région simple-brin qui relie les tiges-boucles cTAR et cpolyA. Cette fixation joue probablement un rôle important dans l’hybridation des tiges-boucles ARN et ADN complémentaires. Notre étude permet de mieux comprendre la transcription inverse et la recombinaison qui dépend du transfert de brin interne. / The 1st strand transfer, a crucial step of reverse transcription involving the HIV-1 nucleocapsid protein (NC), relies on base pairing of the r sequence of strong-stop DNA (ssDNA) with the 3’ R sequence of viral RNA (3’ UTR) which forms the TAR and polyA stem-loops. The r sequence can form the cTAR and cpolyA stem-loops. Therefore, the transfer relies probably on annealing of folded molecules. This process is not well known at the molecular and structural level. The tools of molecular biology and three DNA-targeted probes were used to get insights into the annealing process. Our results were the following: 1) in the absence of NC, the cTAR DNA folds into two distinct conformations in equilibrium; 2) NC slightly shifts the equilibrium toward one conformation and binds tightly the internal loop of the cTAR hairpin; 3) NC is required for the formation of heteroduplex of the full-length ssDNA and 3’ UTR; 4) the annealing of ssDNA to 3’ UTR can be initiated from different sites in the presence of 0.2 mM MgCl2; 5) the full-length ssDNA folds into two conformations in equilibrium in 0.2 mM MgCl2 but mainly into one conformation in 2 mM MgCl2 ; 6) NC preferentially binds to the single-stranded region between the cTAR and cpolyA hairpins in ssDNA. This binding site probably plays an important role in the annealing of complementary DNA and RNA hairpins. This study helps us to gain insights into the reverse transcription process and the associated genetic recombination.

Les travaux présentés dans cette thèse concernent le développement d'une puce à cellules multiplexée pour l'étude de réponses cellulaires parallélisées. La lignée de cellules issues de cancer de la prostate, les cellules LNCaP, ont servi de modèle d'étude grâce à leur capacité à sécréter l'antigène prostate-spécifique (PSA) et la β-2-microglobuline (B2M) en réponse à l'induction par des hormones telles que la dihydrotestostérone (DHT). Nous avons ainsi pu détecter en temps réel et sans marquage ces molécules lors de leur sécrétion par de petites populations de cellules LNCaP adhérentes (de 1 à 100 cellules) à des positions déterminées sur une biopuce SPRi. Trois approches différentes ont été envisagées pour cette biopuce. La première consistait à microtexturer la surface d'or d'un support de SPRi afin d'obtenir des micropuits dont le fond révèle la surface d'or (région cytophile) et dont l'extérieur est composé de polystyrène (cytophobe) afin de créer un contraste d'adhésion pour la culture cellulaire. Des anticorps ont pu être immobilisés de façon contrôlée dans les micropuits grâce à un automate de micro-dépôt non contact. Dans ce système miniaturisé, différentes lignées cellulaires ont pu être co-cultivées sur une surface de 1 cm², ouvrant la voie au multiplexage. Une petite population de cellules (de 1 à 100) a été déposée de façon automatisée dans chaque micropuits. Afin de maintenir les cellules dans un milieu hydraté au cours du dépôt, un polymère d'alginate biocompatible a été utilisé. Cette méthode permet l'encapsulation de cellules dans un très petit volume (< 50 nL). La capacité de cet hydrogel à maintenir les cellules encapsulées à une position donnée sur le support a conduit à la conception d'une deuxième approche pour la fabrication de la biopuce. En effet dans cette approche la surface n'est pas modifiée et les composés biologiques (anticorps et cellules) sont directement déposés de façon automatisée sur la couche d'or. Enfin une dernière approche a été développée en immobilisant cette fois-ci les cellules sur un support microtexturé placé en face de la couche sensible de SPRi. Dans les trois approches, les cinétiques de sécrétion du PSA et de la B2M sécrétées ont pu être suivies par SPRi / The work reported in this thesis focuses on the development of a multiplexed cell chip for the study of parallelized cellular responses. The lineage of cells from Prostate cancer LNCaP cells, were used as a study model thanks to their ability to secrete prostate-specific antigen (PSA) and β-2-microglobulin (B2M) in response to induction by hormones such as dihydrotestosterone (DHT). We were able to detect in real time these label-free molecules and their secretion by small populations of adherent LNCaP cells (from 1 to 100 cells) at specified positions on a SPRi biochip. Three different approaches were considered for this biochip. The first was to pattern the gold surface of a SPRi slide to obtain microwells whose bottom reveals the gold surface (cytophilic area) and an outer shell composed of polystyrene (cytophobe) to create an adhesive/non-adhesive surface for cell culture. Antibodies were immobilized in a controlled manner in the microwells using a piezo electric spotter. In this miniaturized system, different cell lines were co-cultured on a surface of 1 cm², paving the way for multiplexing. A small population of cells (1 to 100) was deposited in an automated manner into each microwell. In order to maintain the cells in a hydrated environment during deposition, a biocompatible alginate polymer was used. This method allows the encapsulation of cells in a very small volume (<50 nL). The ability of the hydrogel to maintain the encapsulated cells in a given position on the support led to the design of a second approach for the production of the biochip. In this second approach the surface is not altered and biological compounds (antibodies and cells) are directly deposited in an automated manner on the gold layer. Finally, a last approach was developed by immobilizing the cells on a patterned substrate placed in front of the sensitive layer SPRi. In all three approaches, the kinetics of PSA secretion and secreted B2M could be followed by SPRi

Bei konventionellen Mikroarray-Experimenten zur Genexpressionsanalyse wird fluoreszenz- oder radioaktiv-markierte cDNA oder RNA mit immobilisierten Proben hybridisiert. Für ein gut detektierbares und auswertbares Ergebnis werden jedoch pro Array mindestens 15 - 20 &#181;g Hybridisierungstarget benötigt. Dazu müssen entweder 15 - 20 &#181;g RNA direkt durch Reverse Transkription in markierte cDNA umgeschrieben werden oder bei Vorhandensein von weniger Startmaterial die RNA amplifiziert werden (Standard- Affymetrix-Protokolle, Klur et al. 2004). Oft sind damit zeit- und kostenintensive Probenpräparationen verbunden und das Ergebnis ist nicht immer reproduzierbar. Obwohl es inzwischen einige Protokolle gibt, die dieses Problem zu lösen versuchen (Zhang et al. 2001, Iscove et al. 2002, McClintick et al. 2003, Stirewalt et al. 2004), eine optimale, leicht handbare und reproduzierbare Methode gibt es weiterhin nicht, weshalb in dieser Arbeit ein weiterer Lösungsansatz gesucht wurde.<br> In der vorgestellten Arbeit werden zwei einfache Methoden beschrieben, mit denen Gene aus geringen RNA-Mengen nachgewiesen werden können: erstens die On Chip- RT-PCR mit cDNA als Matrize und zweitens diese Methode als One-Step-Reaktion mit RNA als Matrize.<br><br> Beide Methoden beruhen auf dem Prinzip der PCR an immobilisierten Primern auf einer Chipoberfläche. Diese Möglichkeit der exponentiellen Amplifikation ist reproduzierbar und sensitiv.<br><br> In Experimenten zur Etablierung des On-Chip-PCR-Systems wurden für die Immobilisierung der Primer verschiedene Kopplungsmethoden verwendet. Die affine Kopplung über Biotin- Streptavidin erwies sich als geeignet. Die On-Chip-Reaktion an kovalent gebundenen Primern wurde für amino-modifizierte Primer auf Epoxy-Oberflächen sowie für EDC-Kopplung auf silanisierten Oberflächen gezeigt. Für die letztgenannte Methode wurde die On-Chip-PCR optimiert, dass Spottingkonzentrationen der Primer von 5 - 10&#181;M schon ausreichend sind. Der Einsatz von fluoreszenz-markierten Primern während der PCR ermöglicht eine unmittelbare Auswertung nach der Synthese ohne zusätzliche Detektionsschritte. In dieser Arbeit konnte außerdem mit der vorgestellten Methode der simultane Nachweis zweier Gene gezeigt werden. Die Methode kann noch als Multiplex-Analyse ausgebaut werden, um so mehrere Gene in gleichzeitig einem Ansatz nachweisen zu können.<br><br> Die Ergebnisse der Versuche mit Matrizen aus unterschiedlichen Zelltypen deuten darauf hin, dass die On-Chip-RT-PCR eine weitere optimale Methode für den Nachweis von gering exprimierten Genen bietet. / The detection of low quantities of mRNA is often difficult and methods like microarray analysis require large amount of starting material or have to be amplified before application. The reverse transcription polymerase chain reaction (RT-PCR) is often the chosen method to detect specific RNA sequences on account of its high sensitivity. The solid phase amplification technology by On-Chip-PCR provides a combination of amplification of rare target material and its on chip detection in one step.<br><br> In this report a novel application for the On-Chip-PCR technology is described. It was suitable to identify mRNA sequences and genes, respectively. For this approach we amplified cDNA sequences using immobilized specific primers and fluorescent labeled primers. They were used for genes coding subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm) and Atpase (Atp2a2). The cDNA templates were synthesized before the On-Chip application by Reverse Transcription from mRNA. For this application only at most 500 pg of total-RNA preparation was sufficient for detectable results and no pre-amplification steps were needed.<br><br> Furthermore the handling of RT-PCR could be minimized by using a One-step- RT-PCR protocol. This method used immobilized primers on glassy supports detecting specific mRNA sequences from 5 pg or less of total RNA preparations.<br> Moreover to the detection of low quantities of RNA preparation, low abundant genes could be detected by this method.

The 1st strand transfer, a crucial step of reverse transcription involving the HIV-1 nucleocapsid protein (NC), relies on base pairing of the r sequence of strong-stop DNA (ssDNA) with the 3' R sequence of viral RNA (3' UTR) which forms the TAR and polyA stem-loops. The r sequence can form the cTAR and cpolyA stem-loops. Therefore, the transfer relies probably on annealing of folded molecules. This process is not well known at the molecular and structural level. The tools of molecular biology and three DNA-targeted probes were used to get insights into the annealing process. Our results were the following: 1) in the absence of NC, the cTAR DNA folds into two distinct conformations in equilibrium; 2) NC slightly shifts the equilibrium toward one conformation and binds tightly the internal loop of the cTAR hairpin; 3) NC is required for the formation of heteroduplex of the full-length ssDNA and 3' UTR; 4) the annealing of ssDNA to 3' UTR can be initiated from different sites in the presence of 0.2 mM MgCl2; 5) the full-length ssDNA folds into two conformations in equilibrium in 0.2 mM MgCl2 but mainly into one conformation in 2 mM MgCl2 ; 6) NC preferentially binds to the single-stranded region between the cTAR and cpolyA hairpins in ssDNA. This binding site probably plays an important role in the annealing of complementary DNA and RNA hairpins. This study helps us to gain insights into the reverse transcription process and the associated genetic recombination.

La qPCR est actuellement la technique de référence en matière de quantification d'ADN. Elle peut être définie comme une amplification exponentielle, cyclique et ciblée de la séquence d'ADN cible. Le caractère exponentiel de la qPCR est à la fois à l'origine de la sensibilité de la méthode mais aussi d'une potentielle variabilité inter-échantillons. Cette variabilité est compensée par le caractère cyclique de la méthode qui entraine une synchronisation de la réaction pour tous les échantillons à chaque cycle. L'amplification ciblée de la séquence choisie traduit quand à elle la spécificité de la méthode. Néanmoins, cette dernière propriété de la PCR reste la moins vérifiée. La spécificité de la qPCR est indiscutable lorsque la cible à détecter se trouve en quantité suffisante (>100 copies environ). En revanche, pour des quantités plus faibles ou en présence d'inhibiteurs, la spécificité diminue ou disparaît (limites de détection et de quantification). Cette perte de spécificité retentit de façon significative sur la précision et la reproductibilité du dosage à réaliser. Cependant, si l'hybridation non spécifique est inéluctable dans ces conditions, l'amplification non spécifique consécutive peut être limitée, voire supprimée au moyen d'amorces de PCR soit plus spécifiques, soit moins susceptibles de générer des différences inter-échantillons. La RT-qPCR, grâce à une étape initiale de transcription inverse (conversion d'un ARN en un ADN complémentaire) permet la quantification des ARN. Cependant, la transcription inverse reste moins reproductible que la PCR, générant des différences inter-échantillons délétères en diagnostic comme en recherche. Au cours de ces travaux, je propose des méthodes originales améliorant de façon significative différentes étapes de ces techniques. Premièrement, je propose une amélioration de la standardisation de l'étape de transcription inverse capable de diminuer de façon significative la variabilité inter-échantillons ; l'utilisation d'un volume constant d'extrait d'ARN pour chaque échantillon améliore considérablement la précision de la quantification d'ARN messagers. Deuxièmement, je propose une modification des amorces par incorporation de résidus d'acides nucléiques bloqués (LNA) ; cette modification permet d'augmenter la spécificité des amorces aux limites de la détection. Enfin, je propose une méthode simple et économique permettant la mesure directe de la température de fusion (Tm) dans les conditions réelles de la PCR. Ce paramètre, considéré comme capital pour la réalisation des dosages par qPCR, est généralement obtenu par des méthodes prédictives peu précises. De plus, cette méthode doit permettre de déterminer précisément les paramètres thermodynamiques des amorces (∆G, ∆H et ∆S) et ainsi avoir accès d'une part au pourcentage réel d'amorces hybridées en fonction de la température et d'autre part d'identifier la susceptibilité des amorces aux inhibiteurs. / QPCR is currently the gold standard for DNA quantification of DNA. It can be defined as an exponential, cyclic and targeted amplification of a known DNA sequence. The exponential character of qPCR is both the cause of the sensitivity of the method but also a potential variability between samples. This variability is offset by the cyclical nature of the method that causes a synchronization of the reaction for all samples in each cycle. The targeted amplification of the selected sequence translates the specificity of the method. Nevertheless, this last property of PCR remains the least tested. The specificity of qPCR is unquestionable when the target to detect is sufficient (> 100 copies). However, for smaller quantities or in the presence of inhibitors, the specificity decreases or disappears (limits of detection and quantification). This loss of specificity will have a significant effect upon the accuracy and reproducibility of the assay to be performed. However, if non-specific hybridizations are unavoidable in these conditions, consecutive nonspecific amplification may be limited or eliminated using more specific PCR primers or less likely to produce inter-sample differences. RT-qPCR allows RNA quantification because of an initial step of reverse transcription (conversion of an RNA into a complementary DNA). However, reverse transcription is less reproducible than PCR, generating harmful inter-sample differences both in diagnosis and in the research field. In this work, I propose innovative methods improving significantly different steps of these techniques. First, I propose an improved standardization of the reverse transcription step that can significantly reduce the variability between samples; using a constant volume of RNA extract for each sample substantially improves mRNA quantification accuracy. Second, I propose a primers modification by incorporating Locked Nucleic Acid residues (LNA); this modification increases the specificity of the primers to the limits of detection. Finally, I propose a simple and economical method for direct measurement of the melting temperature (Tm) in real PCR conditions. This essential parameter in the achievement of qPCR assays is generally obtained by predictive methods with low accuracy. Furthermore, this method should determine the thermodynamic parameters of the oligonucleotide sequence (∆G, ∆H and ∆S), on the one hand allowing access to the actual percentage of annealed primers according to the temperature and on the other hand, to identify the primers susceptibility in the presence of inhibitors.

Miniaturization of biological analysis is a trend in the field of biotechnology aiming to increase resolution and sensitivity in biological assays. Decreasing the reaction volumes to analyze fewer analytes in each reaction vessel enables the detection of rare analytes in a vast background of more common variants. Droplet microfluidics is a high throughput technology for the generation, manipulation and analysis of picoliter scale water droplets an in immiscible oil. The capacity for high throughput processing of discrete reaction vessels makes droplet microfluidics a valuable tool for miniaturization of biological analysis. In the first paper, detection methods compatible with droplet microfluidics was expanded to include SiNR FET sensors. An integrated droplet microfluidics SiNR FET sensor device capable of extracting droplet contents, transferring a train of droplets to the SiNR to measure pH was implemented and tested. In paper II, a workflow was developed for scalable and target flexible multiplex droplet PCR using fluorescently color-coded beads for target detection. The workflow was verified for concurrent detection of two microorganisms infecting poultry. The detection panel was increased to multiple targets in one assay by the use of target specific capture probes on color-coded detection beads.   In paper III, droplet microfluidics has been successfully applied to single cell processing, demonstrated in paper III, where reverse transcription was performed on 65000 individually encapsulated mammalian cells. cDNA yield was approximately equivalent for reactions performed in droplets and in microliter scale. This workflow was further developed in paper IV to perform reverse transcription PCR in microfluidic droplets for detection of exosomes based on 18S RNA content. The identification of single exosomes based on RNA content can be further developed to detect specific RNA biomarkers for disease diagnostics. Droplet microfluidics has great potential for increasing resolution in biological analysis and to become a standard tool in disease diagnostics and clinical research. / <p>QC 20171024</p>

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by a virus of the family Bunyaviridae, genus Phlebovirus. It is responsible for extensive outbreaks of disease in livestock in Africa with significant mortality and economic impact. Virus neutralization is considered the gold standard for confirming Rift Valley fever virus (RVFV) infection but the procedure is time consuming and expensive. Real-time reverse transcription-polymerase chain reaction (rRT-PCR), histopathology, and immunohistochemistry (IHC) are the diagnostic methods most often used in South Africa to confirm or exclude a diagnosis of RVF in necropsied animals. Validated estimates of diagnostic accuracy of these tests, in naturally infected livestock, however, have not been published. The objective of this study was to estimate the diagnostic sensitivity and specificity of rRT-PCR, histopathology, and IHC using Bayesian latent class methods in the absence of a gold standard. A secondary objective was to estimate stratum-specific values based on species, age, degree of specimen autolysis, and the presence/absence of tissue pigments. The Sensitivity (Se) and Specificity (Sp) of qRT-PCR were 97.4% (95% credibility interval (CI): 95.2% - 98.8%) and 71.7% (95% CI: 65% - 77.9%) respectively. The extraordinary analytical sensitivity of PCR makes this test very susceptible to false positive reactions, and thus reduced specificity. This is more likely during large-scale epidemics due to crosscontamination of specimens at necropsy facilities or testing laboratories. The Se and Sp of histopathology were 94.6% (95% CI: 91% - 97.2%) and 92.3% (95% CI: 87.6% - 95.8%) respectively. Single cases of RVF could be confused with acute poisoning with plants, bacterial septicaemias, and viral diseases such as infectious bovine rhinotracheitis and Wesselsbron disease. Most of these conditions, however, can be excluded using histological examination of the liver, special stains, bacterial culture, and toxicological or serological investigations. The Se and Sp of IHC were 97.6% (95% CI: 93.9% - 99.8%) and 99.4% (95% CI: 96.9% - 100%) respectively. Immunohistochemistry is highly specific because characteristic positive immunolabelling of the cytoplasm of hepatocytes can be correlated with the presence of hepatocellular injury typical for RVFV infection. False negative results are sometimes obtained with IHC because of reader error or loss of the antigenic epitopes due to advanced autolysis. Scant positive immunolabelling might be missed or viral proteins might be absent from sections of liver with advanced hepatocellular damage. The stratified analysis suggested differences in test accuracy in foetuses and severely autolysed specimens. The Sp of histopathology in foetuses (83.0%) was 9.3% lower than the value obtained for the sample population (92.3%). Lesions in some foetuses are more subtle and the typical eosinophilic intranuclear inclusions are often difficult to detect. In severely autolysed specimens, the Se of IHC decreased by 16.1% and the Sp of rRT-PCR by 17.4%. There is no plausible biological explanation for this decrease in the Sp of rRTPCR since the RNA of RVFV is resistant to degradation in autolysed tissues. Conversely, the antibody used to detect RVFV using IHC detects epitopes raised against nucleoproteins of the virus and it is possible that viral proteins become too widely dispersed and/or degraded in autolysed tissues to detect by light microscopy. It is possible that the marked decrease in Se of histopathology and IHC in severely autolysed specimens caused an apparent decrease in Sp of rRT-PCR, due to the latent class method. In conclusion, the high estimated Sp (99.4%) of IHC and the low Sp of rRT-PCR (71.3%) suggests that the definitive diagnosis or exclusion of RVF should not rely on a single PCR test and that IHC would be an effective confirmatory test for rRT-PCR positive field cases necropsied during an epidemic. Immunohistochemistry results from severely autolysed specimens, however, should be interpreted with caution and aborted foetuses in areas endemic for RVF should be screened using a variety of tests. The diagnostic Se and Sp of histopathology was much higher than expected confirming the value of routine post mortem examinations and histopathology of liver specimens. The most feasible RVF testing option in areas that do not have suitably equipped PCR laboratories, and where disease is often not detected in livestock until after human cases have been diagnosed, would be routine histopathology screening with IHC confirmation. Key Words: Rift Valley fever; Rift Valley fever virus; Bayesian; latent-class model; real-time reverse transcription-polymerase chain reaction; immunohistochemistry; histopathology; diagnosis; sensitivity; specificity. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Paraclinical Sciences / unrestricted

Acute lower respiratory tract infection (ALRI) is a major cause of paediatric morbidity and mortality, annually accounting for approximately 2 million childhood deaths worldwide of which up to 90% resides in the developing world. In 12-39% of ALRI cases no aetiological agent is identified, despite comprehensive investigations, thus suggesting that additional unknown agents may be involved. Since 2001 a number of new viruses have been identified that may account for some of these cases including human metapneumovirus (hMPV), human bocavirus (hBoV), and two new coronaviruses (hCoV) NL63 and HKU1. The contribution of the recently identified respiratory viruses to annual seasonal lower respiratory tract disease in Sub-Saharan Africa where human immunodeficiency virus infections may exacerbate respiratory infections is not fully understood. In addition, the role and disease association of many of these viruses as primary or co-infecting pathogens, as well as the underlying factors that may determine the pathogenesis of these viruses, is not yet well defined. Quantitative multiplex real-time RT-PCR assays were developed and validated for the detection of 13 well recognized and newly identified viral causes of ALRI, including respiratory syncytial virus (RSV), influenza viruses A and B, parainfluenza virus (PIV) types 1, 2, and 3, adenovirus, hMPV, hBoV and hCoV-NL63, -HKU1, -229E, and -OC43. The newly designed assays were subsequently used to facilitate the investigation of the contribution of respiratory viruses in patients requiring hospitalisation or attending outpatient visits in public sector hospitals serving the Pretoria area, South Africa. During 2006, the prevalence of the aforementioned respiratory viruses was determined by investigating the well recognized viruses previously diagnosed by routine immunofluorescence assays (IFA) in 737 respiratory specimens as well as viruses retrospectively detected by multiplex real-time RT-PCR in a sample group of 319 specimens. The epidemiology and disease association of these respiratory viruses in children who were predominantly less than 5 years of age with acute respiratory tract infections was investigated. Specimens were received from 2 public sector hospitals in Pretoria, South Africa. In addition, the disease association of each virus as a single or co-infection in human immunodeficiency virus (HIV) infected/exposed and HIV-uninfected children as well as the role of viral load was investigated. The multiplex assays could detect 2.5-25 recombinant plasmid DNA/RNA (in vitro transcribed) copies/μl, with a co-efficient of variation of less than 3.1%. Validation on 91 known positive respiratory specimens indicated similar specificity to IFA or single-round PCRs used in the initial identification of the viruses. Application of the multiplex assays to IFA negative specimens improved the detection of respiratory viruses by up to 44%. In children less than 5 years of age RSV was identified in 35.1%, followed by PIV 3 (8.3%); adenovirus (5.6%); influenza A (4.2%); hMPV (4.2%); hBoV (3.8%); hCoV-NL63 (1.6%); influenza B (1.0%); and PIV1, PIV2, hCoV-OC43, hCoV-229E, hCoV-HKU1 in less than 1% of cases. Co-infections were more common for the new viruses ranging from 58% of hMPV cases to 84% for hCoV-NL63 relative to 27% of RSV cases. Viruses were most frequently identified in children <1-year. RSV activity peaked in autumn and winter, PIV 3 in spring, while influenza A and B were mostly detected in winter. The observed seasonal distribution of hBoV and hMPV was less defined compared to traditional viruses, with both viruses showing variability over the two years. Comparable hospitalisation rates were observed for RSV, hMPV, PIV 3 and adenovirus, where approximately 60% of infected children were hospitalised. In addition, a high frequency of hospitalisations was observed in patients for both hMPV and hBoV in HIV-infected/exposed children. Co-infections occurred at higher frequencies with the new viruses, were more frequently associated with severe disease and were frequent in HIV-infected/exposed patients. Viral load was associated with severe RSV disease (p=0.014) however no significant association was observed for the new viruses as single infections. However, where hMPV occurred as a co-infection, higher viral loads of either hMPV or co-infecting agents occurred in severe cases. This association was also observed for hBoV. Most cases of hCoV-NL63 and hCoV-OC43 were co-infections in hospitalised patients. The newly developed multiplex assays demonstrates an improved sensitivity and scope of detecting respiratory viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the role of co-infections and viral load in respiratory virus pathogenesis. RSV remains the most significant viral cause of paediatric ALRI in South Africa. Viruses not currently included in routine diagnostic assays collectively contributed to 11% of ALRI cases in children <2-years in South African hospitals. / Dissertation (MSc)--University of Pretoria, 2010. / Medical Virology / unrestricted

Recent breakthroughs in the RNA quantification of single cells are rapidly transforming the view on biology and medicine. Flexibility and sensitivity of reverse transcription quantitative PCR (RT-qPCR) make it an ideal method for quantification of single-cell material, but its limits had not been yet fully explored. In this thesis, various factors influencing RT-qPCR performance in single-cell application have been assessed, including conditions of sample collection and processing, importance of quality control, performance of reverse transcription, preamplification and role of qPCR assays. We showed that prolonged time for single cell collection as well as repeated freeze-thaw cycles had negligible effect on RT-qPCR data quality. Direct lysis routinely applied for RNA extraction from single cells may be scaled up to 256 cells. The comprehensive comparison of 11 reverse transcriptases in low RNA input conditions identified 2 best-performing enzymes. Decrease in preamplification volume as well as poor primer design resulted in the loss of sensitivity. Finally, the established workflow has been applied to profile gene expression of astrocytes in mouse model of amyotrophic lateral sclerosis (ALS) identifying important components of ALS-induced changes to astrocyte transcriptome. Altogether, the thesis...