Biological evidence may contain any number of cells in any proportion. Extreme low-template DNA samples are often very difficult to interpret due to complex signal or peaks which may be indistinguishable from baseline noise. Current solutions focus on increasing the amount of amplicon detected by adjusting PCR cycle number or capillary electrophoresis injection parameters. Consensus profiling is an additional option. However, the aforementioned solutions are often not helpful for extreme low-template samples due to the high occurrence of allelic drop-out. Additionally, PCR is a destructive technique that causes one amplification to completely exhaust this type of sample, making further typing and analysis impossible. Therefore, a technique that allows for the re-generation of a DNA template in order to amplify it multiple times would be an extremely useful tool. This study outlines the development of a method that allows for the recursive amplification of a DNA sample. Amplification was performed using biotinylated primers for an STR locus and the resulting product was cleaned using streptavidin-coated magnetic beads to sequester the amplicons. Subsequent centrifugal filtration was used to remove the remaining PCR components, thus isolating the original genomic DNA. Re-amplification was then successfully performed at a different STR locus. Though successful, multiple run-throughs of the method indicated retention of signal from the original amplification as well as significant genomic DNA loss during the process. This study outlines experiments seeking to characterize the cause(s) of these imperfections in order to effectively direct method optimization. A computer generated dynamic model was also created and used to simulate the recursive amplification process to assist in development. When optimized, it is expected that recursive amplification can significantly reduce the difficulties associated with low-template DNA analysis and eradicate the concept of an ‘exhaustive’ DNA sample.

Copy number variation (CNV) is an important polymorphism that is associated with a wide range of traits in human, wild and livestock species. In chicken, an important source of animal protein and a developmental model organism, CNV is associated with several phenotypes and evolutionary footprints. However, identification and characterization of CNV inheritance on chicken genome lacks further investigation. We screened CNVs in chicken using two distinct populations with known pedigree. In 826 broilers we identified 25,819 CNVs (4,299 deletions and 21,520 duplications) of which 21,077 were inherited, 201 showed no inheritance and 4,541 were classified as de novo CNVs. In 514 F2 animals (layer and broiler cross) we identified 21,796 CNVs (2,254 deletions and 19,543 duplications) of which 18,230 were inherited, 587 not inherited and 2,979 were classified as de novo CNVs. After a strict filtering step to remove potential false positives and negative CNVs, only 220 (4.84%) and 430 (14.43%) de novo CNVs remained in the broiler and F2 populations, respectively. A total of 33.11% (50 out of 151) of the inherited CNVs identified in ten animals were validated by sequencing data. From the validated CNVs, 64% had more than 80% of their size (bp) validated. A total of 59% and 48.8% were classified as novel CNVs regions (CNVRs) in the broiler and F2, respectively. Considering the Bonferroni-corrected p-values for multiple testing and statistically significant p-values ≤ 0.01, we found two CNV segments significantly associated with breast weight, one with breast weight yield, six with breast meat weight, 18 CNV segments with breast meat yield, four with breast filet weight and two with breast yield. These CNV segments that were significantly associated overlapped with 181 protein-coding genes. The CNVseg 300, that was associated with all traits and encompass six CNVRs, overlapped a total of 26 protein-coding genes. Among these genes, the gene MYL1 (Myosin Light Chain 1) is expressed in the fast skeletal muscle fibers, and the genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) and RAB17 (Member RAS Oncogene Family), that were associated with the lavender phenotype (feather blue-grey color) and regulation of homeothermy and the metabolism. The present study improves our knowledge about CNV in the chicken genome and provides insight in the distribution and of different classes of CNVs, i.e. inherited and de novo CNVs, in two experimental chicken populations. In addition, the genome-wide association analyses were the first performed on broiler population with breast muscle traits, that are important characteristics for poultry production. The GWAS results allow us to understand the probably relationship between some genes and CNVRs that are significantly associated with breast muscle traits. / A variação de número de cópias (CNV) é um polimorfismo importante que está associado a uma ampla gama de características em seres humanos, espécies selvagens e domésticas. Em frango, que é uma importante fonte de proteína e considerado um modelo biológico, CNVs foram associados a vários fenótipos e passos evolutivos. No entanto, nenhum estudo foi realizado para a identificação e caracterização da herança da CNV no genoma da galinha. Identificamos as CNVs no genoma da galinha usando duas populações experimentais e com pedigree conhecido: uma população de frangos de corte e uma F2. Em 826 frangos de corte, identificamos 25.819 CNVs (4.299 deleções e 21.520 duplicações), dos quais 21.077 foram herdados, 201 não foram herdados e 4.541 foram CNVs denominados de novo. Em 514 animais F2, identificamos 21.796 CNVs (2.254 deleções e 19.543 duplicações) das quais 18.230 foram herdadas, 587 não foram herdadas e 2.979 foram de novo CNVs. Após a etapa de filtragem nos de novo CNVs, apenas 220 (4,84%) e 430 (14,43%) permaneceram nas populações de frango de corte e F2, respectivamente. Um total de 33,11% (50 de 151) das CNV identificadas por dados de genotipagem em dez animais foram validados por dados de sequenciamento. Dos validados, 64% tinham mais de 80% do tamanho (pb) validados. Um total de 59% e 48,8% foram classificados como novas regiões de CNVs (CNVRs) nas populações de frango de corte e F2, respectivamente. Considerando os p-values corrigidos por Bonferroni para testes múltiplos e estatisticamente significativos (≤ 0,01), encontramos dois segmentos de CNV significativamente associados ao peso do peito, um ao rendimento de peso de peito, seis ao peso de carne de peito, 18 ao rendimento de carne de peito, quatro ao peso de filé de peito e dois ao rendimento do filé de peito. Esses segmentos de CNV significativamente associados estão sobrepostos com 181 genes codificadores de proteínas. O CNVseg 300, que foi associado a todas as características e abrange seis CNVRs, foram sobrepostos a um total de 26 genes codificadores de proteínas. Entre estes genes, o gene MYL1 (Myosin Light Chain 1) é expresso nas fibras rápidas do músculo esquelético, e os genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) e RAB17 (Member RAS Oncogene Family), que foram anteiromente associados ao fenótipo de cor azul acinzentado de penas e à regulação da homeotermia e do metabolismo. O presente estudo melhora o conhecimento sobre CNVs no genoma de frango, especialmente sobre a distribuição de CNV herdadas, não herdadas e de novo, em duas populações experimentais de frango. Além disso, a associação genômica foi a primeira realizada na população de frangos de corte com características do músculo do peito, que são muito importantes para a avicultura. Os resultados do GWAS nos permitem compreender a provável relação entre alguns genes e CNVRs que foram significativamente associados às características do músculo do peito.

Espécies de Malassezia fazem parte da microbiota de humanos e de animais. Essas leveduras lipofílicas são microrganismos oportunistas que estão associados com muitas doenças superficiais como: pitiriase versicolor, dermatite seborréica, foliculite, dermatite atópica e algumas infecções sistêmicas. As espécies de Malassezia têm sido identificadas através de procedimentos morfológicos e bioquímicos, no entanto, pequenas semelhanças entre algumas espécies estão presentes em algumas regiões do genoma. Foi avaliado o PCR-RFLP, método molecular para genotipar espécies de Malassezia obtidas de 55 amostras clinicas isoladas. Foram analisados 23 pacientes com dermatite seborréica, pitiriase versicolor e com a síndrome de Gourgerot- Carteaud. Encontramos quatro espécies diferentes: M. furfur, M. globosa, M. sympodialis and M. slooffiae. A identificação fisiológica e o PCR-RFPL foram compatíveis em 83% das amostras. M. furfur esteve presente em 52% dos casos, o que sugere que é o agente causador da pitiriase versicolor e da dermatite seborréica. Os resultados sugerem que a utilização do PCR-RFLP em amostras clínicas é importante no diagnóstico de algumas micoses causadas por esta levedura. / Malassezia species are part of the microbiota of humans and other animals. These lipophilic yeasts are opportunistic microorganisms that are associated with some dermatoses including pityriasis versicolor, seborrheic dermatitis, folliculitis ptirospórica, atopic dermatitis, some systemic infections among others. The species of Malassezia have been identified by morphological and biochemical and molecular techniques currently. We identify the species of Malassezia obtained from 55 clinical samples and 15 samples that formed the control group, by conventional techniques using a medium with not described in the literature, the mixture of media and Dixon Kimming, which was higher than each when used separately. Used the molecular method of PCR-RFLP to genotype 23 of the 55 clinical samples of Malassezia spp. from patients with seborrheic dermatitis, pityriasis versicolor and Gourgerot - Carteaud syndrome. Could the isolation of four species: M. furfur, M. globosa, M. Sympodialis and M. slooffiae. The physiological identification obtained with the PCRRFLP was consistent in 83% of the samples. M. furfur was present in 52% of cases, suggesting that is the main causative agent of pityriasis versicolor and perhaps the agent most frequently found in seborrheic dermatitis. The results suggest that the use of PCR-RFLP method in clinical samples is of great use for the identification of Malassezia species associated with diseases in humans.

Copy number variation (CNV) is an important polymorphism that is associated with a wide range of traits in human, wild and livestock species. In chicken, an important source of animal protein and a developmental model organism, CNV is associated with several phenotypes and evolutionary footprints. However, identification and characterization of CNV inheritance on chicken genome lacks further investigation. We screened CNVs in chicken using two distinct populations with known pedigree. In 826 broilers we identified 25,819 CNVs (4,299 deletions and 21,520 duplications) of which 21,077 were inherited, 201 showed no inheritance and 4,541 were classified as de novo CNVs. In 514 F2 animals (layer and broiler cross) we identified 21,796 CNVs (2,254 deletions and 19,543 duplications) of which 18,230 were inherited, 587 not inherited and 2,979 were classified as de novo CNVs. After a strict filtering step to remove potential false positives and negative CNVs, only 220 (4.84%) and 430 (14.43%) de novo CNVs remained in the broiler and F2 populations, respectively. A total of 33.11% (50 out of 151) of the inherited CNVs identified in ten animals were validated by sequencing data. From the validated CNVs, 64% had more than 80% of their size (bp) validated. A total of 59% and 48.8% were classified as novel CNVs regions (CNVRs) in the broiler and F2, respectively. Considering the Bonferroni-corrected p-values for multiple testing and statistically significant p-values ≤ 0.01, we found two CNV segments significantly associated with breast weight, one with breast weight yield, six with breast meat weight, 18 CNV segments with breast meat yield, four with breast filet weight and two with breast yield. These CNV segments that were significantly associated overlapped with 181 protein-coding genes. The CNVseg 300, that was associated with all traits and encompass six CNVRs, overlapped a total of 26 protein-coding genes. Among these genes, the gene MYL1 (Myosin Light Chain 1) is expressed in the fast skeletal muscle fibers, and the genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) and RAB17 (Member RAS Oncogene Family), that were associated with the lavender phenotype (feather blue-grey color) and regulation of homeothermy and the metabolism. The present study improves our knowledge about CNV in the chicken genome and provides insight in the distribution and of different classes of CNVs, i.e. inherited and de novo CNVs, in two experimental chicken populations. In addition, the genome-wide association analyses were the first performed on broiler population with breast muscle traits, that are important characteristics for poultry production. The GWAS results allow us to understand the probably relationship between some genes and CNVRs that are significantly associated with breast muscle traits. / A variação de número de cópias (CNV) é um polimorfismo importante que está associado a uma ampla gama de características em seres humanos, espécies selvagens e domésticas. Em frango, que é uma importante fonte de proteína e considerado um modelo biológico, CNVs foram associados a vários fenótipos e passos evolutivos. No entanto, nenhum estudo foi realizado para a identificação e caracterização da herança da CNV no genoma da galinha. Identificamos as CNVs no genoma da galinha usando duas populações experimentais e com pedigree conhecido: uma população de frangos de corte e uma F2. Em 826 frangos de corte, identificamos 25.819 CNVs (4.299 deleções e 21.520 duplicações), dos quais 21.077 foram herdados, 201 não foram herdados e 4.541 foram CNVs denominados de novo. Em 514 animais F2, identificamos 21.796 CNVs (2.254 deleções e 19.543 duplicações) das quais 18.230 foram herdadas, 587 não foram herdadas e 2.979 foram de novo CNVs. Após a etapa de filtragem nos de novo CNVs, apenas 220 (4,84%) e 430 (14,43%) permaneceram nas populações de frango de corte e F2, respectivamente. Um total de 33,11% (50 de 151) das CNV identificadas por dados de genotipagem em dez animais foram validados por dados de sequenciamento. Dos validados, 64% tinham mais de 80% do tamanho (pb) validados. Um total de 59% e 48,8% foram classificados como novas regiões de CNVs (CNVRs) nas populações de frango de corte e F2, respectivamente. Considerando os p-values corrigidos por Bonferroni para testes múltiplos e estatisticamente significativos (≤ 0,01), encontramos dois segmentos de CNV significativamente associados ao peso do peito, um ao rendimento de peso de peito, seis ao peso de carne de peito, 18 ao rendimento de carne de peito, quatro ao peso de filé de peito e dois ao rendimento do filé de peito. Esses segmentos de CNV significativamente associados estão sobrepostos com 181 genes codificadores de proteínas. O CNVseg 300, que foi associado a todas as características e abrange seis CNVRs, foram sobrepostos a um total de 26 genes codificadores de proteínas. Entre estes genes, o gene MYL1 (Myosin Light Chain 1) é expresso nas fibras rápidas do músculo esquelético, e os genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) e RAB17 (Member RAS Oncogene Family), que foram anteiromente associados ao fenótipo de cor azul acinzentado de penas e à regulação da homeotermia e do metabolismo. O presente estudo melhora o conhecimento sobre CNVs no genoma de frango, especialmente sobre a distribuição de CNV herdadas, não herdadas e de novo, em duas populações experimentais de frango. Além disso, a associação genômica foi a primeira realizada na população de frangos de corte com características do músculo do peito, que são muito importantes para a avicultura. Os resultados do GWAS nos permitem compreender a provável relação entre alguns genes e CNVRs que foram significativamente associados às características do músculo do peito.

Espécies de Malassezia fazem parte da microbiota de humanos e de animais. Essas leveduras lipofílicas são microrganismos oportunistas que estão associados com muitas doenças superficiais como: pitiriase versicolor, dermatite seborréica, foliculite, dermatite atópica e algumas infecções sistêmicas. As espécies de Malassezia têm sido identificadas através de procedimentos morfológicos e bioquímicos, no entanto, pequenas semelhanças entre algumas espécies estão presentes em algumas regiões do genoma. Foi avaliado o PCR-RFLP, método molecular para genotipar espécies de Malassezia obtidas de 55 amostras clinicas isoladas. Foram analisados 23 pacientes com dermatite seborréica, pitiriase versicolor e com a síndrome de Gourgerot- Carteaud. Encontramos quatro espécies diferentes: M. furfur, M. globosa, M. sympodialis and M. slooffiae. A identificação fisiológica e o PCR-RFPL foram compatíveis em 83% das amostras. M. furfur esteve presente em 52% dos casos, o que sugere que é o agente causador da pitiriase versicolor e da dermatite seborréica. Os resultados sugerem que a utilização do PCR-RFLP em amostras clínicas é importante no diagnóstico de algumas micoses causadas por esta levedura. / Malassezia species are part of the microbiota of humans and other animals. These lipophilic yeasts are opportunistic microorganisms that are associated with some dermatoses including pityriasis versicolor, seborrheic dermatitis, folliculitis ptirospórica, atopic dermatitis, some systemic infections among others. The species of Malassezia have been identified by morphological and biochemical and molecular techniques currently. We identify the species of Malassezia obtained from 55 clinical samples and 15 samples that formed the control group, by conventional techniques using a medium with not described in the literature, the mixture of media and Dixon Kimming, which was higher than each when used separately. Used the molecular method of PCR-RFLP to genotype 23 of the 55 clinical samples of Malassezia spp. from patients with seborrheic dermatitis, pityriasis versicolor and Gourgerot - Carteaud syndrome. Could the isolation of four species: M. furfur, M. globosa, M. Sympodialis and M. slooffiae. The physiological identification obtained with the PCRRFLP was consistent in 83% of the samples. M. furfur was present in 52% of cases, suggesting that is the main causative agent of pityriasis versicolor and perhaps the agent most frequently found in seborrheic dermatitis. The results suggest that the use of PCR-RFLP method in clinical samples is of great use for the identification of Malassezia species associated with diseases in humans.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: S. aureus causes serious infections in the hospital and community settings. The rate of MRSA infections are rapidly increasing worldwide. Currently, at Tygerberg hospital, approximately a third of S. aureus isolates are MRSA. This was the first epidemiological study of S. aureus conducted at Tygerberg Hospital that included prospective clinical data on patients with S. aureus bacteraemia together with spa typing of strains and the detection of the mecA and pvl genes in a multiplex PCR. Clonal cluster groups of S. aureus isolates were obtained by BURP analysis and compared to international important clones. The molecular epidemiology of hospital acquired (HA), health-care associated (HCA) and community acquired (CA) S. aureus bacteraemic strains at this hospital was examined. Lastly, repeat isolates of patients were collected to analyse any possible organism-related factors associated with persistent and recurrent bacteraemia. We investigated a total number of 113 S. aureus strains from 104 patients (70% MSSA, 30% MRSA). Repeat strains consisted of nine isolates (from 5 patients). All isolates were obtained from blood cultures collected during the period March 2008 to May 2009. Phenotypic and genotypic detection of methicillin resistance correlated well. According to the literature, most CA-MRSA strains are distinguishable from HA-MRSA strains based upon the presence of the PVL toxin. However, no CA-MRSA was detected in our study, therefore the association between HA-MRSA versus CA-MRSA strains could not be analysed. In this study, CA-MSSA was identified in 22% of all MSSA isolates versus 0% CA-MRSA. PVL positive strains were found in 22.7% of all MSSA isolates with no detection in MRSA isolates. It was noted that MRSA strains clustered in spa CC-701 and CC-012, whereas CC-002 only contained MSSA strains. Likewise HA-strains representing the majority of MRSA strains also clustered in spa CC-701 and CC-012. Forty nine spa types were identified in 89.3% of all isolates, whereas 9.7% of these strains were non-typeable. Five novel spa types were revealed. We detected a diverse number of spa-types that correlated to international clones. The most predominant spa type found in our setting was t037 (only in MRSA), followed by t891. According to the literature, t037 is associated to the Brazilian/Hungarian clone (SCCmec type III; ST 239). Our findings, as well as other South African studies, indicate that t037 has been identified in clinical strains from numerous provinces in South Africa. Interestingly, all isolates from spa type t891 were PVL positive MSSA. Bacteraemia cases were predominantly related to catheter sepsis, followed by skin and soft tissue infections (SSTI). Only one persistent bacteraemia case was identified related to a HA-SSTI. Recurrent bacteraemia cases were found in patients on dialysis for chronic renal failure and in burns patients related to intravascular catheter infections. The local epidemiology of S. aureus and the prevalence rate of different strains are important to investigate. The information provided contributes to the epidemiology of staphylococcal strains causing bacteraemia in our setting. These insights are useful for optimal diagnostic and therapeutic measures. The techniques developed can be used to identify outbreaks and recurrent infections. / AFRIKAANSE OPSOMMING: S. aureus veroorsaak ernstige infeksies in die hospitaalomgewing en in die gemeenskap. Wêreldwyd, neem metisillien-weerstandige S. aureus (MRSA) infeksies vinnig toe. Huidiglik by Tygerberg hospitaal is ongeveer ‘n derde van S. aureus isolate MRSA. Hierdie is die eerste epidemiologiese studie by Tygerberg hospitaal wat prospektiewe kliniese data van pasiënte met S. aureus bakteremie saam met spa tipering en aantoning van die mecA en pvl gene in ‘n multipleks PKR insluit. Klonale groepe (spa-CC) van MRSA en MSSA isolate is deur BURP analise verkry, en vergelyk met internasionaal belangrike klone. Die molekulêre epidemiologie van hospitaalverworwe (HA), gesondheidsorgverworwe (HCA) en gemeenskapsverworwe (CA) S. aureus bakteremie by hierdie hospitaal is ondersoek. Laastens, oorspronklike en daaropvolgende herhaal isolate is gekollekteer om moontlike organisme- faktore geassosieerd met persisterende en herhalende bakteremiese episodes te analiseer. Ons het in totaal 113 S. aureus isolate van 104 pasiënte ondersoek (70% MSSA, 30% MRSA). Nege isolate (van 5 pasiënte) was herhaal isolate. Alle isolate was afkomstig vanaf bloedkulture wat gedurende die periode Maart 2008 tot Mei 2009 gekollekteer is. Fenotipiese en genotipiese aantoning van metisillien weerstandigheid het goed gekorreleer. Volgens die literatuur kan die meeste CA-MRSA isolate van HA-MRSA isolate onderskei word op grond van die teenwoordigheid van die PVL toksien. Geen CA-MRSA is egter in ons studie gevind nie, dus kon die assosiasie tussen HA-MRSA en CA-MRSA isolate nie ondersoek word nie. CA-MSSA was in 22% van alle MSSA geidentifiseer teenoor 0% CA-MRSA. PVL is in MSSA isolate gevind (22.7% van alle MSSA) maar glad nie in MRSA nie. Dit is opgemerk dat MRSA isolate hoofsaaklik in spa CC 701 en CC-012 kloongroepe voorkom, teenoor kloongroep CC-002 wat slegs MSSA isolate bevat het. Soortgelyk het HA-isolate wat die meerderheid van MRSA isolate verteenwoordig het ook in kloongroepe 1 & 2 gegroepeer. Nege-en-veertig spa tipes is geïdentifiseer in 89.3% of alle isolate en 9.7% was nie-tipeerbaar. Vyf nuwe spa tipes is getoon. Ons het ‘n diverse aantal spa-tipes geïdentifiseer wat met internasionale klone gekorreleer het. Die mees dominante spa tipe in ons omgewing was t037 (slegs in MRSA), gevolg deur t891. Volgens die literatuur word t037 met die Brasiliaanse/Hongaarse kloon geassosieer (SCCmec tipe III; ST 239). Ons bevindings, asook ander Suid Afrikaanse studies, dui aan dat t037 in kliniese isolate vanaf talle provinsies in Suid-Afrika aangetoon is. Van belang is dat al die isolate van spa tipe t891 MSSA en PVL positief was. Bakteremiese gevalle was hoofsaaklik geassosieer met kateter-sepsis, gevolg deur vel en sagteweefsel infeksies (SSTI). Slegs een persisterende bakteremiese geval was geïdentifiseer geassosieer met HA-SSTI. Herhalende bakteremiese episodes is in pasiënte op dialise vir kroniese nierversaking en in brandwonde pasiënte met intra-vaskulêre kateter infeksies aangetoon. Die lokale epidemiologie van S. aureus en die prevalensie koers van verskillende stamme is van belang. Hierdie inligting dra by tot kennis van die epidemiologie van stafilokokkale stamme wat in ons omgewing bakteremie veroorsaak. Hierdie insigte is nuttig vir optimale diagnostiese en terapeutiese riglyne. Die tegnieke wat ontwikkel is, kan gebruik word om uitbrake en herhalende infeksies te identifiseer.

<p>Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets.</p><p>This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized.</p><p>The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set.</p><p>In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained.</p> / QC20100907

Brassica napus L. is an economically important oilseed species cultivated across Western Canada. Hybrid B. napus cultivars compose the majority of the market due to their seed yield and agronomic quality. It is important to attempt to predict high-yielding parental combinations in order to conserve resources during experimental hybrid evaluation. Genetic distance between parents has been implicated in producing high-yielding hybrids and is used as one criteria for determining parental combinations.In the current study, the genetic distance between high erucic acid rapeseed (HEAR) genotypes of B. napus was established using both phenotypic and genotypic criteria. Phenotypic criteria took the form of nine agronomic and seed quality traits gathered from 318 distinct B. napus genotypes over the 2013 and 2014 field seasons in Southern Manitoba. Genotypic criteria took the form of either 291,782 SNP markers identified in 231 distinct B. napus genotypes using genotyping-by-sequencing (GBS) or 230 polymorphic sequence-related amplified polymoprhism (SRAP) markers identified in 160 B. napus genotypes. The genetic distance between available pollinators and a single male-sterile female was established using each set of criteria in an attempt to correlate genetic distance with hybrid yield. Regression analysis was conducted with yield data from hybrid genotypes gathered from 37 field sites from 2011-2014. Using the phenotypic-derived genetic distance, a significant correlation between genetic distance and hybrid yield was uncovered explaining either 22 % or 42 % of the variation in hybrid yield depending upon whether hybrids were grown at three or more, or five or more sites in the analysis, respectively. No significant link was found between GBS or SRAP-derived genetic distance and hybrid yield. These results provide evidence that that phenotypic criteria can be used to establish genetic distance with utility in the selection of high-yielding hybrid genotypes. / February 2017

L'objectif de la thèse était d'enrichir les connaissances sur les poux de tête et les poux de corps de l'homme. Les poux de tête vivent et pondent leurs œufs à la base des cheveux et sont très répandus chez les enfants dans les écoles. Les poux de corps vivent dans les vêtements et infestent les personnes de milieux sociaux très défavorisés ne permettant pas une hygiène vestimentaire adéquate. Les poux de corps sont vecteurs de trois grandes maladies : le typhus épidémique, la fièvre des tranchées et la fièvre récurrente à poux. Ces insectes hématophages sont étudiés depuis des décennies afin de déterminer si ils constituent deux écotypes de la même espèce ou deux espèces distinctes. Avant l'avènement des techniques de biologie moléculaire, la taxonomie des poux était basée sur leur morphologie et leur biologie. Bien que les différences biologiques décrites entre les poux de tête et les poux de corps ne sont pas toujours cohérentes, il est légitime de penser que leur séparation physique puisse mener à une différenciation spécifique. Certaines études expérimentales ont cependant montré certains cas de migrations de poux de tête vers les vêtements et inversement. Puis, grâce au séquençage du génome du pou, beaucoup de progrès ont été réalisés ces dernières années sur leur phylogénie. Ainsi, sur base de l'ADN mitochondrial, les poux de l'homme sont séparés en trois Clades phylogénétiques : le Clade A qui comprend à fois des poux de tête et des poux de corps et les Clade B et C qui comprennent uniquement des poux de tête. / The objective of this thesis work was to increase the knowledge of human head lice and body lice. Head lice live and lay their eggs at the base of hair shaft and are found frequently in school going children. Body lice live in clothes and are usually associated with low income persons that do not have adequate clothes hygiene. Body lice are the vector of three major diseases: epidemic typhus, trench fever and relapsing fever. These blood feeding insects have been studied over the past decades to determine whether they are two ecotypes of the same species or two distinct species. Before the advent of molecular biology, taxonomical classification of lice was based on their morphology and biological activities. Although described biological differences between head lice and body lice are not always consistent, their physical separation could lead to species differentiation. However, some experimental studies have shown that in certain cases head lice could migrate to the clothes and vice versa. In the recent years many progress were made in the body louse genome sequencing, and further their phylogenetic classification. Thus, on the basis of mitochondrial DNA, human lice are classified into three phylogenetic clades: Clade A that comprise both head lice and body lice and the Clades B and C that comprise only head lice. This phylogenetic organization clustered into three clades surprisingly shows that head lice of Clade A are closer to body lice than to head lice of Clade B or C. Since body lice serve as vectors of several diseases, in view of this, it is crucial to understand human lice epidemiology.

La leishmaniose à Leishmania infantum est une zoonose transmise de mammifère à mammifère par la piqûre d'un insecte vecteur, le phlébotome femelle. S'il est classiquement décrit la leishmaniose viscérale avec la triade classique fièvre, pâleur et splénomégalie, de nombreuses formes cliniques peuvent être associées à ce parasite. Le portage asymptomatique est la forme la plus fréquente et la plus répandue dans l'Ancien Monde ou le Nouveau Monde. Entre la leishmaniose viscérale et le portage asymptomatique, plusieurs formes cliniques sont présentes. Ainsi certains sujets vont exprimer la leishmaniose sous forme d'adénopathies isolées. Ces formes sont intermédiaires entre une expression pauci symptomatique et une leishmaniose viscérale larvée. Alors que L. infantum n'est pas classiquement retrouvé dans des formes muqueuses, des cas ont été récemment décrits. Ces formes muqueuses isolées ne sont pas rares puisque sur les 3 CHU Marseille, Montpellier et Nice, entre 1997 et 2009, 10 cas de leishmaniose muqueuse à L. infantum ont été diagnostiquées principalement chez des sujets immunodéprimés. Il est important de faire le diagnostic de ces formes cliniques particulières puisqu'elles ont pour diagnostic différentiel des néoplasies (lymphome pour la première et néoplasie de la sphère ORL pour la seconde). Afin d'appréhender le rôle du parasite dans l'expression clinique, il a été réalisé le typage par les microsatellites de neuf souches isolées de sujets porteurs asymptomatiques. Il s'avère que ces neuf souches sont très peu polymorphes et que sept d'entre elles possèdent un génotype unique. / Leishmaniasis due to Leishmania infantum is a zoonotic disease transmitted from mammal to mammal through the bite of an insect vector the sandfly female. Beside the classical triad of visceral leishmaniasis symptoms: fever, pallor and splenomegaly, many clinical forms could be associated with this parasite infection. Asymptomatic carriage of L. infantum is the most common and the most widespread in the Old World and New World. Many other clinical forms are present and some subjects will develop only isolated lymphadenopathy. These forms are intermediate between pauci symptomatic and visceral leishmaniasis forms. Whereas L. infantum is not typically associated with mucosal forms, several cases have been described. Indeed, in the 3 academic hospitals of Marseille, Montpellier and Nice from 1997 to 2009, 10 cases were revealed mainly in immunocompromised patients. To understand the role of parasite in clinical expression, nine strains isolated from asymptomatic carriers were genotyped using microsatellite. The nine strains have few polymorphisms and seven of them are identical with a unique genotype. In addition, those strains are very different from strains of HIV-positive subjects. If the strains genetic appears to have a role in the clinical expression of the disease, the environment in which individuals live in endemic areas is associated with an excess of risk to develop visceral leishmaniasis. While in Marseille, cases of visceral leishmaniasis occur in an urban environment, they take place in Nice in a rural environment, as it is classically described. To investigate differences between parasite strains form Nice and Marseille studies with microsatellites are ongoing.