Made available in DSpace on 2015-03-26T13:42:37Z (GMT). No. of bitstreams: 1 texto completo.pdf: 790827 bytes, checksum: 31229e606f6ff3a38ad36a37dcb0cbb9 (MD5) Previous issue date: 2007-07-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Soybean Asian Rust, a disease caused by the fungi Phakopsora pachyrhizi, has caused serious economic damages to the Brazilian and world-wide soybean crop. The development of soybean lines resistant to this fungi has been a challenge, and currently the measures of control this disease are based on the handling of the crop and application of fungicides. With the objective to advance the knowledge on the molecular mechanism of defense of the plant to the Soybean Asian Rust, it was evaluated, by PCR in real time, the expression of genes that supposedly are involved in the defense mechanism. Two genotypes had been selected for the conduction of the experiment, PI 230970, that it possess allele Rpp2 and develops type RB type injuries, when in presence of P. pachyrhizi, and Embrapa-48, susceptible, which develops injuries of Tan type. The experiment was carried out in Fitotron and for the analysis of the gene expression was done using RNAs from leaves of the soybean inoculated either with pathogen or mock-inoculated with water. Amongst the evaluated genes, chitinase presented an expression 117 times higher in the resistant genotype (PI 230970), when compared with the calibrator (PI 230970 mock-inoculated) possibly acting as a barrier of defense of the plant. In the susceptible genotype the expression of this gene was 1.75 time higher that your calibrator (Embrapa-48 mock-inoculated). It was observed a differential expression of related genes with reactive species oxygen, production of fitoalexinas as well as involved genes in the release of elicitors. These genes can be acting in the mechanism of defense against P. pachyrhizi in the resistant genotype; however its expression in the susceptible genotype can be related to a bigger settling of the pathogens in the tissues of the host or to a delayed response the infection. The identification of a defense route should be farther investigated, with the objective to identify more specific genes in the process of cellular defense and to point out new alternatives for the development of resistant cultivars. / Ferrugem asiática da soja, uma doença causada pelo fungo Phakopsora pachyrhizi, tem provocado sérios danos econômicos à sojicultura brasileira e mundial. A obtenção de linhagens de soja resistentes ao fungo tem sido um desafio, e atualmente as medidas de controle da doença são baseadas no manejo da cultura e aplicação de fungicidas. Com o objetivo de avançar o conhecimento sobre o mecanismo molecular de defesa da planta frente a ferrugem asiática da soja, avaliou-se, através da técnica de PCR em tempo real, a expressão de genes que supostamente estão envolvidos no mecanismo de defesa. Dois genótipos foram selecionados para a condução do experimento, PI 230970, que possui o alelo Rpp2 e desenvolve lesões do tipo RB, quando em presença de P. pachyrhizi, e Embrapa-48, suscetível, desenvolvendo lesões tipo TAN. O experimento foi conduzido em sala climatizada (Fitotron) e para a análise de expressão gênica foram extraídos RNAs de folhas da soja inoculadas com o patógeno e falso-inoculadas (água). Dentre os genes avaliados, quitinase apresentou uma expressão 117 vezes maior no genótipo resistente (PI 230970), quando comparado com o calibrador (PI 230970 falso-inoculado), atuando possivelmente como uma barreira de defesa da planta. No genótipo suscetível a expressão deste gene foi 1,75 vezes maior que o seu calibrador (Embrapa-48 falsoinoculado). Foi observada uma expressão diferencial de genes relacionados a espécies reativas de oxigênio, produção de fitoalexinas bem como genes envolvidos na liberação de elicitores. Esses genes podem estar atuando no mecanismo de defesa contra P. pachyrhizi no genótipo resistente, porem sua expressão também no genótipo suscetível pode estar relacionado a uma maior colonização do patógeno no tecido hospedeiro ou uma resposta tardia à infecção. A identificação de uma rota de defesa pode possibilitar o estudo mais aprofundado da mesma, com o objetivo de identificar genes mais específicos no processo de defesa celular e apontar novas alternativas para obtenção de cultivares resistente.

Introduction. The aim of the study was to introduce a new BKV PCR protocol in our centre and to verify its accuracy as well as to assess the prevalence, risk factors of BK virus replication, course of BKV infection and therapeutic approaches in simultaneous pancreas and kidney (SPK) recipients in order to design a screening protocol. Methods. The results analysed by both Affigene® and Transplantation Virology, Basel PCR protocols were compared. Thereafter 183 SPK patients were examined to assess the prevalence of BK viremia, viruria and BKVN and to identify the risk factors of BKV replication. The cases of retransplantation after a graft loss due to BKVN were retrospectively described. Results. 100 of results were analysed according to the Affigene ® and Transplantation Virology, Basel PCR protocols with the accordance of 95%, Rho = 0,946, 95% CI: 0.920 - 0.963, P<0,0001, Bland-Altman plot analyses: bias Basel PCR protocol/Affigene® BKV trender: -0,1 (mean) *±1.96 SD: -1,6 - 1,3] for both methods. Point-prevalence was assessed in 183 patients; Viruria found in 17,3 %, viremia in 3.8% of patients. High-level viruria >107 copies/mL detected in 3,7% of patiets, high-level virémia >104 in 1,6% of patients simultaneously with high-level viruria. BKVN was found in 0,5% of patients. Diabetes duration...

Introduction. The aim of the study was to introduce a new BKV PCR protocol in our centre and to verify its accuracy as well as to assess the prevalence, risk factors of BK virus replication, course of BKV infection and therapeutic approaches in simultaneous pancreas and kidney (SPK) recipients in order to design a screening protocol. Methods. The results analysed by both Affigene® and Transplantation Virology, Basel PCR protocols were compared. Thereafter 183 SPK patients were examined to assess the prevalence of BK viremia, viruria and BKVN and to identify the risk factors of BKV replication. The cases of retransplantation after a graft loss due to BKVN were retrospectively described. Results. 100 of results were analysed according to the Affigene ® and Transplantation Virology, Basel PCR protocols with the accordance of 95%, Rho = 0,946, 95% CI: 0.920 - 0.963, P<0,0001, Bland-Altman plot analyses: bias Basel PCR protocol/Affigene® BKV trender: -0,1 (mean) *±1.96 SD: -1,6 - 1,3] for both methods. Point-prevalence was assessed in 183 patients; Viruria found in 17,3 %, viremia in 3.8% of patients. High-level viruria >107 copies/mL detected in 3,7% of patiets, high-level virémia >104 in 1,6% of patients simultaneously with high-level viruria. BKVN was found in 0,5% of patients. Diabetes duration...

Master of Science / Department of Diagnostic Medicine/Pathobiology / T. G. Nagaraja / Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.

Alstroemeria is an important cut flower and its breeding has been developed focused on aesthetic characteristics and vase life longevity, but little is known about its scent. Five different genotypes were assessed including the non scented cultivars ‘Rebecca’ and ‘Samora’ and the scented cultivars, ‘Sweet Laura’, ‘Ajax’ and the species A. caryophyllaea. The scented Alstroemerias emitted the terpenoids: isocaryophyllene and ocimene as the major floral volatile compounds. Characterization of an Alstroemeria TPS (ALSTER) was based on four ESTs previously found in A. cv ‘Rebecca’. Rapid amplification of cDNA ends (RACE) was performed and the full length ORF was used for characterizations of the genomic organization and amino acid sequences (phylogenetic analysis). ALSTER genomic region contains five introns and six exons. This unique genomic organization classified ALSTER as a member of the class III with a merged 5-6 exon. The deduced amino acid sequence was classified into the subfamily TPS-b. A functional analysis showed enzymatic activity of ALSTER with geranyl diphosphate (GPP) and the monoterpene myrcene was the only product obtained. Gene expression evaluated through real time and semi q RT-PCR on eight different stages of development (SO to S7) showed high expression of ALSTER at around S2 - S4 in the scented Alstroemerias, coinciding with high scent emission perceived and also with the maturation of reproductive organs. Evaluations through surveys focused on level of liking of floral scent, were performed finding positive correlations between floral scent liking and floral appearance liking and between floral scent liking and floral scent intensity. Finally, 17 new lines of A. caryophyllaea were evaluated in terms of their morphology, phenology and productivity. Although none of them were suitable for the market because of their low productivity, short stems and small flowers, they were all scented and identified as promising starting points for breeding purposes.

Thesis (PhD (Microbiology))--University of Stellenbosch 2010. / ENGLISH ABSTRACT: Two isogenic plasmids, pRAS3.1 (11,851-bp) and pRAS3.2 (11,823-bp), were identified as tetracycline resistance plasmids occurring within Aeromonas salmonicida subsp. salmonicida and atypical A. salmonicida subsp. salmonicida strains that were isolated from salmon aquaculture farms in Norway (L'Abee-Lund and Sorum, 2002). Although sequence analysis showed that, except for the repC gene, the replication and mobilization genes of the two pRAS3 plasmids are similar to that of the two IncQ-2 plasmids pTF-FC2 and pTC-F14, incompatibility testing during the course of this study revealed that the replicons of the two pRAS3 plasmids were compatible with the replicons of the IncQ-1α, ß] and y plasmids RSF1010, pIE1107 and pIE1130, as well as with the IncQ-2α and ß plasmids, pTF-FC2 and pTC-F14, respectively. Through sequence analysis it was suggested that the repC gene of the ancestral pRAS3 plasmid was probably acquired during a gene exchange event with a yet to be identified plasmid. The difference in the RepC of the pRAS3 plasmids compared to that of the other IncQ-like plasmids against which the pRAS3 plasmids were tested for incompatibility was thus suggested to be a likely reason for the compatibility of the two pRAS3 plasmid replicons with these IncQ-1 and IncQ-2 plasmids. Two previously unidentified genes, encoding two small 108 and 74-aa proteins distantly related to the PemIK (Bravo et al., 1987; Tsuchimoto et al., 1988) and MazEF (Masuda et al., 1993) TA systems, were found to be present between repB and repA genes of the two pRAS3 plasmids. Cloning of these two genes onto an unstable pOU82-test vector increased the stability of the vector from 35 to 98% after ~72 generations, thus suggesting that like the PasABC and PasAB systems of pTF-FC2 and pTC-F14, these two genes encode proteins which function as a toxin-antitoxin (TA) system. Although located in a similar position on the plasmids, the TA system of the two pRAS3 plasmids and the Pas systems of pTF-FC2 and pTC-F14 are unrelated, suggesting that these two types of TA systems were acquired independently from each other. Based on the sequence similarity and genetic organization of pRAS3 compared to the IncQ-2α and ß] plasmids pTF-FC2 and pTC-F14, respectively, but given that the pRAS3 plasmids were compatible with both pTF-FC2 and pTC-F14, as well as other IncQ-like plasmids, it was suggested that the two pRAS3 plasmids be classified into a new IncQ-2y subgroup. A comparison of the sequences of the two pRAS3 plasmids to each other by L'Abee-Lund and Sorum (2002) revealed that, apart from a number of point mutations within the tetAR tetracycline resistance genes of the two plasmids, the only other differences between them are that pRAS3.1 has 4 tandem copies of 22-bp iteron repeats within its origin of vegetative replication (oriV), and 5 tandem copies of CCCCCG 6-bp repeats near the origin of transfer (oriT), while pRAS3.2 has only three and four copies of each of the two repeated sequences, respectively. As the two pRAS3 plasmids are likely to have arisen from the same ancestor, this raised the question of how the copy numbers of these two different types of repeat sequences affected the ability of pRAS3.1 and pRAS3.2 plasmids to compete within a host cell as well as within a population of host cells, and therefore, why both of these isogenic plasmids have managed to persist in the environment. The plasmid copy numbers (PCN) of pRAS3.1 and pRAS3.2 were estimated to be 45 ± 13 and 30 ± 5 plasmids per chromosome, respectively. By creating a series of pRAS3.1 derivative plasmids with 3 to 7 copies of the 22-bp iterons and 4 or 5 copies of the 6-bp repeats, it was shown that an increase in the number of iterons brought about a decrease in PCN, probably due to an increased ability to bind RepC, while an increase in the number of 6-bp repeats from 4 to 5 brought about an increase in repB transcription, and the higher levels of RepB resulted in an increase in PCN. Thus the reason for pRAS3.1 having a ~1.5-fold higher PCN than pRAS3.2, even though it has 4 x 22-bp iterons compared to the 3 x 22-bp iterons of pRAS3.2, was that it had a higher level of repB transcription due to having 5 x 6-bp repeats in its mobB-mobA/repB promoter region compared to the 4 x 6-bp repeats of pRAS3.2. The differences in the number of iterons and 6-bp repeats, and hence PCN, did not have an effect on the stability of the two wild type (WT) plasmids or their derivatives even when the TA system was neutralized by having a copy of the TA genes present on a vector in trans and it was argued that the relatively high PCN of the two pRAS3 plasmids was sufficient to ensure plasmid stability through random distribution. As the two pRAS3 plasmids were mobilized at similar frequencies difference in PCN and mobB-mobA/repB transcription did not seem to affect their mobilization frequency. When pRAS3.1 and pRAS3.2 were competed intracellularly as coresident plasmids, pRAS3.1 was able to displace pRAS3.2 from 98% of the host cells within ~20 generations. The displacement of pRAS3.2 by pRAS3.1 was found to be as a result of pRAS3.1 having 4 x 22-bp iterons, which enabled pRAS3.1 to titrate of the communal pool of available RepC initiator proteins. Plasmids with 5 or 7 x 22-bp iterons, were however less effective at displacing a plasmid with 3 iterons, and it was speculated that plasmids with more than 4 x 22-bp iterons within their oriV were less successful at initiating replication than was a plasmid with 3 iterons within its oriV. A direct correlation was found between the PCN of a pRAS3 plasmid and the metabolic burden it imposed on its host. Thus pRAS3.1, as a result of its ~1.5-fold higher PCN than pRAS3.2 placed a small but significantly higher (~2.8%) metabolic load on its host compared to pRAS3.2. It was concluded that pRAS3.1 had a competitive advantage over pRAS3.2 when these plasmids were coresident within a single host (as would have been when the two plasmids first diverged from each other) as it was able to displace pRAS3.2. However, as a result of pRAS3.2 having a lower PCN, it placed a smaller metabolic burden on an isogenic host and this resulted in pRAS3.2 having an advantage over pRAS3.1 at the population level. Sequence remnants of pRAS3.2 from horizontal gene transfer suggested that pRAS3.2 was the original pRAS3 plasmid and thus that pRAS3.1 evolved from pRAS3.2. As the pRAS3.1 derivative plasmids that were constructed during the course of this study are likely to have been intermediates in the evolution of pRAS3.1 from pRAS3.2, I was able to speculate on the stepwise evolution of pRAS3.1 from pRAS3.2 based on the characteristics of these plasmids, and thus, how both macro- and microevolutionary events have contributed to the evolution of these two plasmids. / AFRIKAANSE OPSOMMING: Die twee isogeniese plasmiede, pRAS3.1 en pRAS3.2, was geidentifiseer as tetrasiklien weerstandbiedende plasmiede wat in Aeromonas salmonicida subsp. salmonicida en nie-tipiese A. salmonicida voorkom (L'Abee-Lund and Sorum, 2002). DNS volgorde analise deur L'Abee-Lund en Sorum (2002) het gewys dat die gene verantwoordelik vir replisering (uitsluitend die repC) en mobililisering naverwant is aan die van twee IncQ-2 plasmiede, pTF-FC2 en pTC-F14. Eksperimente tydens hierdie studie het egter gewys dat die repliserende sisteme van die twee pRAS3 plasmiede versoenbaar is met die repliserende sisteme van die IncQ-1α, ß and y plasmiede RSF1010, pIE1107 en pIE1130, sowel as die IncQ-2α en ß plasmiede, pTF-FC2 and pTC-F14, onderskeidelik. Analise van die aminosuur volgorde van die pRAS3 RepC proteien het gedui daarop dat die proteien taamlik verskil van die RepC proteiene van die naverwante plasmiede pTF-FC2 en pTC-F14, sowel as die van die IncQ-1 tipe plasmiede, en daar was voorgestel dat die voorsaat pRAS3 plasmied moontlik die repC geen bekom het vanaf 'n ander, nog onbekende, plasmied deur middel van horisontale geen uitruiling. Die verskil in die RepC van die pRAS3 plasmiede teenoor die van die ander IncQ plasmiede waarteen hulle getoets was vir onversoenbaarheid, was waarskynlik die rede waarom die pRAS3 plasmiede versoenbaar was met die IncQ-1 en IncQ-2 plasmiede. DNS volgorde analise tydens hierdie studie het die teenwoordigheid van twee, vantevore ongeidentifiseerde, klein 108 en 74 aminosuur proteiene onthul wat ver langs verwant is aan die PemIK (Bravo et al., 1987; Tsuchimoto et al., 1988) en MazEF (Masuda et al., 1993) toksien-antitoksien sisteme. Die gene wat kodeer vir hierdie toksien-antitoksien proteine kom tussen die repB en die repA gene van die twee pRAS3 plasmiede voor. Klonering van die toksien-antitoksien gene van die pRAS3 plasmiede op 'n ander onstabiele plasmied het die stabiliteit van hierdie plasmied verhoog van 35 tot en met 98% na ~72 generasies. Hierdie experiment het dus bevestig dat, soos die PasABC en PasAB sisteme van pTF-FC2 en pTC-F14 onderskeidelik, die twee gene 'n toksien-antitoksien sisteem kodeer wat die stabiliteit van 'n plasmied binne 'n bakteriese populasie kan verbeter. Alhoewel die toksien-antitoksien gene van pRAS3 op 'n soortgelyke posisie op die pRAS3 plasmiede voorkom as wat die pasABC en pasAB gene op hulle onderskeidelike pTF-FC2 en pTC-F14 plasmiede voorkom, is hulle nie verwant nie en dus was dit voorgestel dat die twee tipe toksien-antitoksien sisteme onafhanklik van mekaar verkry is. Aangesien die DNS volgorde en genetiese rangskikking van pRAS3 teenoor die IncQ-2α en ß plasmiede pTF-FC2 en pTC-F14, onderskeidelik, soortgelyk is, asook die feit dat die pRAS3 plasmiede versoenbaar was met pTF-FC2 en pTC-F14, sowel as ander IncQ tipe plasmiede, word dit voorgestel dat die twee pRAS3 plasmiede in 'n nuwe IncQ-2y subgroep ingedeel word. 'n Vergelyking van die DNS volgorde van die twee pRAS3 plasmiede deur L'Abee-Lund and Sorum (2002) het gewys dat, behalwe vir 'n paar puntmutasies binne die tetAR tetrasiklien weerstandsgene, verskil die twee net in die opsig dat pRAS3.1 het 4 agtereenvolgende kopiee van 22-bp 'iteron' herhalings wat gelee is binne sy replikasie oorsprong en 5 kopiee van 'n CCCCCG 6-bp herhaling wat naby sy oorsprong van oordrag gelee is, terwyl pRAS3.2 net 3 en 4 kopiee het van elk van die onderskeie volgorde herhalings. Dus die bestaan van twee plasmiede met verskillende kopiegetalle van die twee verskillende tipe DNA volgorde herhalings, maar wat vermoedelik afkomstig is vanaf dieselfde stam plasmied, bring die volgende oorhoofse vrae aangaande die plasmiede na vore: hoe beinvloed die DNS volgorde herhalings die vermoe van die twee plasmiede om binne 'n enkele gasheersel te kompeteer vir die beskikbare plasmied repliserings masjinerie, en hoe beinvloed dit die plasmied-gasheersel verhouding en dus hulle vermoe om te kompeteer op die populasie vlak, en laastens, hoekom het beide weergawes van die plasmied bly voortbestaan in die omgewing? Die plasmied kopiegetalle van pRAS3.1 en pRAS3.2 was eksperimenteel beraam by ongeveer 45 ± 13 en 30 ± 5 plasmiede per chromosoom in E. coli, onderskeidelik. Deur 'n reeks van pRAS3.1 derivate te skep met 3 tot 7 'iteron' herhalings en 4 of 5 kopiee van die 6-bp herhalings was dit bewys dat 'n toename in die hoeveelheid 'iterons' 'n afname in die plasmied kopiegetal veroorsaak, vermoedelik deur 'n verbeterde vermoe om RepC te bind, terwyl 'n verhoging van 4 tot 5 kopiee van die 6-bp herhaling 'n afname in die kopiegetal te weeg gebring het. Die repB geen van 'n plasmied met 5 x 6-bp herhalings was ~2-voud hoer uitgedruk as die van 'n plasmied met 4 x 6-bp herhalings, en dit was verder bewys dat 'n verhoogde vlak van repB transkripsie vanaf 'n L-arabinose induseerbare promoter in trans van 'n pRAS3 plasmied met 4 x 6-bp herhalings het 'n ~2-voud verhoging in plasmied kopiegetal teweeg gebring. Die rede dat pRAS3.1 'n ~1.5-voud hoer plasmied kopiegetal gehad het as pRAS3.2, was as gevolg van 'n hoer vlak van repB uitdrukking weens die feit dat pRAS3.1 5 x 6-bp herhalings in die mobB-mobA/repB promoter area het terwyl pRAS3.2 net 4 van die 6-bp herhalings in dieslefde posisie het. Sou pRAS3.1 4 x 22-bp 'iterons' gehad het, maar saam met 4 x 6-bp herhalings soos pRAS3.2, dan sou die plasmied kopiegetal 23 ± 2 plasmiede per chromosoom gewees het. Die verskil in die hoeveelheid 'iterons' en 6-bp herhalings, en dus die plasmied kopiegetal, het nie 'n effek op die stabiliteit van die wilde tipe plasmiede of hulle derivate gehad nie, selfs al was die toksien-antitoksien sisteem geneutraliseer deurdat daar 'n kopie van die toksien-antitoksien sisteem op 'n ander plasmied in trans van die pRAS3 plasmiede en hul derivate geplaas was. Die relatiewe hoe plasmied kopiegetal van die pRAS3 plasmiede, wat moontlik hoog genoeg was om plasmied stabiliteit deur middel van toevallige uitdeling te verseker, was voorgestel as die rede vir die hoe mate van plasmied stabiliteit. Soortgelyke frekwensies van mobilisasie vir pRAS3.1 en pRAS3.2 (0.032 ± 0.014 en 0.021 ± 0.013 transkonjugate per donateur, onderskeidelik) was waargeneem. Dus het dit geblyk dat die verskil in uitdrukking van die mobB-mobA/repB operon, sowel as die plasmied kopiegetal van die twee pRAS3 plasmiede, nie die mobiliserings frekwensie beinvloed het nie. Intrasellulere kompetisie tussen pRAS3.1 en pRAS3.2 het gewys dat pRAS3.1 die vermoe gehad om binne ~20 generasies pRAS3.2 vanuit 98% van die gasheerselle te skop. Daar was gewys dat die teenwoordigheid van 4 x 22-bp 'iterons' in die oorsprong van replikasie van pRAS3.1 die rede was vir die vermoe van hierdie plasmied om pRAS3.2 uit te kompeteer binne die gasheersel, moontlik deurdat die 4 x 22-bp 'iterons' beter in staat was om die RepC protein te bind. Die vermoe van plasmiede met 5 of 7 x 22-bp 'iterons' om te kompeteer met 'n plasmied met net 3 x 22-bp 'iterons' was toenemend swakker in vergelyking met die van 'n plasmied met 4 x 22-bp 'iterons', en hierdie waarneming het gelei tot die voorstel dat plasmiede met meer as 4 x 22-bp 'iterons' nie so suksesvol was om replikasie te inisieer soos ¡¥n plasmied met 3 x 22-bp 'iterons' nie. 'n Direkte korrelasie was gevind tussen die plasmied kopiegetal van 'n pRAS3 plasmied en die metaboliese lading wat die plasmied op die gasheersel geplaas het. Dus het pRAS3.1, met 'n plasmied kopiegetal van ~1.5-voud hoer as die van pRAS3.2, 'n effens hoer (~2.8%) metababoliese lading op die gasheersel as pRAS3.2 geplaas. In gevolge van die inter- en intrasellulere kompetiesie eksperimente, was dit ge-argumenteer dat pRAS3.1 'n mededingende voordeel bo-oor pRAS3.2 binne 'n gasheersel (soos wat dit sou gewees het kort nadat die twee plasmiede van mekaar uiteengevloei het) gehad het omdat dit in staat was om pRAS3.2 vanuit die gasheersel te skop. Aan die ander kant het pRAS3.2 'n laer plasmied kopiegetal en dus 'n laer metaboliese lading op die isogeniese gasheersel geplaas het, en daardeur het pRAS3.2 weer op die populasievlak die kompeterende voordeel bo-oor pRAS3.1 gehad. Die eienskappe van pRAS3.2 was meer soortgelyk aan die van ander IncQ-tipe plasmiede as wat die eienskappe van pRAS3.1 was, en dus word dit voorgestel dat pRAS3.1 vanaf pRAS3.2 afkomstig was. Omdat die derivaat plasmiede wat geskep was vanaf pRAS3.1 tydens hierdie studie moontlike tussengangers in die ontwikkeling van pRAS3.1 vanaf pRAS3.2 was, kan gespekuleer word, gebaseer op die eienskappe van hierdie plasmiede, oor die “stapsgewyse manier” waarmee pRAS3.1 vanaf pRAS3.2 ge-evolueer het, en dus hoe beide makro- en mikro-evolusionere gebeurlikhede bygedra het tot die evolusie van genoemde plasmiede.

<p>Picropodophyllin (PPP) is an analogue of the anti tumour lignan podophyllotoxin with the unique ability to selectively inhibit the receptor of Insulin like growth factor 1(IGF-1). IGF-1 is believed to play an important part in development of the endometrium facing implantation. With PPP treated mice, studies can be made to measure gene expression from tissue of both treated and untreated mice to compare the role of IGF-1 regarding ovulation, implantation and receptivity. The aim of this study was to analyze gene expression of some steroid hormone receptors and cytokines in ovaries from mice treated with PPP. In this study, seven mice were treated with PPP at different times and tissue was collected. PCR-primers for cDNA sequences of estrogene receptor α, estrogene receptor β, progesterone receptor A, progesterone receptor B, growth hormone receptor, interleukin 1 α, interleukin 1 β, tumour necrosis factor α and androgen receptor were used. Real Time PCR was run with the samples and gene expression was measured. The results of this study showed that the inhibition of IGF-1 receptor interacted with IGF-1 which lead to altered levels of estrogene receptor alpha, progesterone receptor, growth hormone receptor and androgen receptor that can decrease ovulation. The results also showed the differences in gene products between treated and untreated samples, suggesting that IGF-1 plays an important role regarding ovulation.</p> / <p>Studier med hjälp av den selektiva insulinlika tillväxtfaktor 1 receptorn (IGF-1R) antagonisten; picropodof?phyllin (PPP), hur samspelet mellan livmoderslemhinnan och implantationsprocessen, samt hur ovulationen påverkas av insulinlika tillväxtfaktorn 1 (IGF-1) kan nu utföras. IGF-1 tros ha en viktig roll för den reproduktiva processen, där den påverkar ovulation, implantation och embryoutveckling. IGF-familjen består av tre ligander; insulin, IGF-1 och IGF-2. IGF transporteras bundet till bindarprotein (IGFBP). Medlemmarna i IGF receptorfamiljen kan binda IGF-1, IGF-2 och insulin fast med olika affinitet. PPP som är en cykloligan, är en analog från podofyllotoxin och fungerar som en syntetisk IGF-1 receptorantagonist, som selektivt inhiberar receptorns aktivitet. PPP tros även kunna nedreglera genexpression av receptorn. Tre tidigare projektarbeten har utförts på vävnader från möss injicerade med PPP. Tyngdpunkterna i dessa arbeten har legat på immunhistokemiska studier av IGF-1 i reproduktionsorgan från möss, uttryck av IGF-1, dess receptor och bindarprotein 1 i ovarier och uterus efter behandling med PPP. I denna studie användes vävnad samt cDNA från sju möss behandlade med PPP, i olika stadier av reproduktionen samt även icke behandlade möss. Studiens syfte var att med sanntids-PCR jämföra genuttryck från östrogenreceptor α och β, progesteronreceptor A och B, tillväxthormonreceptor, Interleukin 1 α och β, ’tumor necrosis’ faktor α samt androgenreceptor i vävnad från PPP-behandlade och obehandlade möss och genom de erhållna resultaten från ovarievävnaden utläsa effekten på ovulationen och från uterusvävnaden effekten på implantation och receptivitet. Studieresultaten visade att IGF-1s frånvaro gav förändrade nivåer av genprodukter, som medförde minskad ovulationen. Studien visade att IGF-1s roll vid ovulationen var väsentlig.</p>

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The South African National Antiretroviral Treatment Guidelines recommend the use of HIV-1 viral load assays for routine monitoring of HIV-1 positive patients receiving highly active antiretroviral therapy (HAART). This thesis describes the innovative approaches to developing more affordable HIV-1 diagnostics and monitoring assays for South Africa, which take into account the tiered laboratory infrastructure of this country. An in-house HIV-1 viral load assay – the LUX assay, was developed and evaluated with a view of implementing this more affordable option in high tier laboratories. The LUX assay represents quantitative real-time RT-PCR that utilizes the LightCycler® technology (Roche) in a novel combination with a LUXÔ primer. The assay showed good analytical sensitivity, specificity and reproducibility of its linear dynamic range of 4x102 to 4x106 RNA copies/ml. Preliminary clinical evaluation (n = 458) of the LUX assay showed good agreement with the COBAS Amplicor assay, and demonstrated its usefulness for long term monitoring of HAART patients. ELISA based viral load testing approaches were investigated as low cost and less technically complex alternatives for medium tier laboratories. The HiSens HIV-1 p24 Ag Ultra (Perkin Elmer) and the ExaVir™ Load Quantitative HIV-RT kits (CAVIDI) were compared with the Roche Amplicor assay. Both assays showed strong association with the Roche Amplicor assay, with R2 = 0.686 and R2 = 0.810, respectively (n = 117). These alternative assays seemed most useful in the serial monitoring of patients on HAART. Major drawbacks included the wide variability of both assays, insufficient sensitivity of the p24 antigen assay and low throughput of the RT assay. Development of a point-of-care HIV-1 RNA assay could address issues related to early and cost effective diagnosis of acute HIV infection. A novel isothermal amplification technique termed the Reverse Transcription Loop Dependant Amplification (RT-LDA) was developed as one component for a potential point-of-care HIV-1 RNA assay. The RT-LDA converted RNA into partially looped ssDNA amplicons, over a wide RNA concentration range (4x103 to 4x108 copies/ml) using a 1 hour incubation at 53ºC. The RT-LDA technology is fully compatible with a lateral flow detection system using dipsticks and highly suitable for point-of-care testing. Overall, this study demonstrates the feasibility of developing novel, more affordable HIV-1 testing options that would be appropriate for the tiered laboratory infrastructure present in South Africa. Evaluation of commercially available, less expensive alternative HIV viral load assays in local settings facilitates their implementation.

Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the availability of anti-viral agents, there is currently no cure. This double stranded DNA virus is hepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA contains a partially synthesised positive DNA stand and a gap region within the negative strand. After infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An important objective of HBV therapy is the elimination of cccDNA, as its persistence within hepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this replication intermediate is crucial. The objective of this study was to develop a method based on real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap were designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV DNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV transgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse livers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA profile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells, only 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results were validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA pools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA detection as it allows for the rapid simultaneous amplification and quantification of a specific DNA target through either non-specific or specific DNA detection chemistries. In conclusion, this HBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy