A cana-de-açúcar (S. officinarum) é uma gramínea perene de grande importância na economia brasileira. Devido a isto, estudos relativos ao aprimoramento das condições de cultivo são de grande interesse, podendo ser uma destas bases um melhor conhecimento sobre as comunidades microbianas associadas à cana-de-açúcar. Sabe-se que a rizosfera é um ambiente de íntima interação entre as plantas e seus respectivos microbiomas, sendo esta relação intermediada pela exsudação radicular. Este estudo teve como objetivo realizar o cultivo de bactérias da rizosfera e do solo de cana-de-açúcar, sendo os isolados obtidos posteriormente caracterizados genética (BOX-PCR e sequenciamento parcial do gene 16S rRNA) e metabolicamente (BIOLOG®). Os resultados demonstraram que o número de bactérias cultiváveis na rizosfera é maior comparado ao solo, sendo que dentre os isolados destaca-se a maior afiliação taxonômica ao filo Proteobacteria (principalmente as classes γ-proteobacteria e β- proteobacteria). A análise de BOX-PCR mostrou uma grande diversidade genética, mesmo quando comparados isolados obtidos a partir do mesmo meio de cultivo, ou pertencentes ao mesmo grupo taxonômico. Em contrapartida, a análise de sequenciamento parcial do gene 16S rRNA mostrou que muitos isolados preservam grande similaridade do gene ribossomal analisado. A análise de perfil metabólico corroborou com os dados de BOX-PCR, onde isolados com afiliação taxonômica bastante correlata apresentaram capacidades distintas de utilizar as diversas fontes de carbono avaliadas. Por fim, esta diversidade metabólica se traduziu na obtenção de respostas distintas de isolados pertencentes aos mesmos gêneros bacterianos, isolados do solo ou da rizosfera, quando estes foram cultivados na presença de exsudatos radiculares. De maneira geral, este estudo demonstrou que as plantas de cana-de-açúcar podem influenciar o comportamento das comunidades bacterianas presentes no solo, e indicaram que existe uma grande diversificação dos organismos presentes na rizosfera, sendo a resposta a este ambiente diferenciada de forma independente da afiliação taxonômica dos isolados. / The sugarcane plant (S. officinarum) is a perennial gamineous with a great importance for the Brazilian economy. Due to this, studies related to the improvement of cultivation conditions are of great importance, indicating that one of these bases must contribute with the better knowledge about the microbial communities associated with sugarcane. It is known that the rhizosphere is an environment that hosts an intimate interaction between plants and their respective microbiomes, being it mediated by the roots exudates. This study aimed to cultivate bacterial isolates from soil and rhizosphere of sugarcane, followed by the genetic (BOX-PCR and partial sequencing of the 16S rRNA gene) and metabolic (BIOLOG®) characterization of them. Results demonstrated higher numbers of cultivable bacteria in rhizosphere when compared to soil samples, with the prevalent affiliation of the isolates to the phylum Proteobacteria (specially with the classes γ-proteobacteria and β- proteobacteria). The BOX-PCR results showed a great genetic diversity, even when isolates obtained in the same culture medium or affiliated to the same taxa are compared. In counterpart, the analysis of the 16S rRNA gene sequence indicated that several isolates preserve high similarities in the ribosomal gene. The metabolic profiling results corroborated with the BOX-PCR data, which isolates highly correlated in the taxonomical analyses presented distinct capacities to use the carbon sources that were tested. At the end, this metabolic diversity was evidenced by the distinct behavior of isolates belonging to the same genera, isolated from soils or rhizosphere samples as well, when cultivated in the presence of roots exudates. In general, this study demonstrated that sugarcane plants can influence the behavior of bacterial communities present in soils, and indicated a great diversification of organisms present in the rhizosphere being the response to this environment very particular, regardless of the taxonomical affiliation of the isolates.

Neste trabalho foram caracterizadas molecularmente e inferidas as relações filogenéticas de 14 isolados brasileiros de Acanthamoeba, provenientes de casos de ceratite, e 8 isolados da ATCC (4 de ceratite e 4 ambientais). Foram utilizados inicialmente os métodos de RAPD, RFLP de DNA genômico total e RFLP do SSU rDNA. Apesar de revelar a alta variabilidade genética em Acanthamoeba, estes métodos permitiram estabelecer grupos bem definidos de isolados mais similares geneticamente. O seqüenciamento do SSU rDNA permitiu a inferência da filogenia entre os isolados utilizados nesse estudo em relação àqueles presentes na literatura, que estão distribuídos em doze tipos de seqüência deste gene. Dentre os 17 isolados de ceratite presentes em nosso estudo, 16 apresentaram SSU rDNA tipo T4 (anteriormente já fortemente correlacionado à ceratite) e um deles constitui um novo tipo de seqüência. Dois dos 4 isolados ATCC (ambientais) cujas seqüências ainda não haviam sido determinadas também apresentaram novos tipos de SSU rDNA, enquanto outros 2 apresentaram o tipo T4. / In this work we performed the molecular phylogeny and characterization of 22 Acanthamoeba isolates, 14 Brazilian keratitis isolates and 8 from ATCC, 4 keratitis and 4 environmental isolates. In spite of the extensive genetic variability disclosed by RAPD, total genomic DNA RFLP and SSU rDNA RFLP techniques, these methods enabled us to group some isolates in well defined clusters of genetically more related organisms. Sequencing of SSU rDNA allowed inference of the phylogeny of our isolates with those present in the literature, which are distributed through 12 sequence types of this gene. Among the 17 keratitis isolates of our study, 16 presented SSU rDNA of type T4 (previously found to be strongly correlated to keratitis), and one was assigned to a new sequence type. Of the 4 isolates from ATCC whose sequences were previously undetermined, the two environmental isolates also constituted new sequence types, while the two keratitis isolates were assigned to type T4.

Este trabalho foi realizado com a finalidade de avaliar a degradação anaeróbia de benzoato por bactérias redutoras de sulfato (BRS). A primeira fase experimental avaliou essa degradação utilizando como inóculo biomassa de reator anaeróbio horizontal de leito fixo (RAHLF) utilizado no tratamento de água residuária de indústria de peróxidos orgânicos. Os ensaios foram realizados em reatores em batelada mantidos em temperatura de 33ºC ±1 e agitação de 150 rpm. Os reatores foram alimentados com meio para BRS e diferentes concentrações de benzoato e sulfato, estabelecendo as seguintes relações: 0,9 (890 mg/L e 970 mg/L), 0,8 (660 mg/L e 870 mg/L) e 0,6 (242 mg/L e 400 mg/L). Após 13 dias verificou-se valores de remoção do benzoato e utilização do sulfato iguais a: (0,9) 71,2% e 98%, (0,7) 97% e 99,9% e (0,6) 91,3% e 15%, respectivamente. Os exames microscópicos revelaram o predomínio de BRS. Na segunda fase experimental foi realizada a purificação celular, utilizando-se técnica de diluição seriada em meio líquido, obtendo-se cultura com predomínio de cocos, gram-negativos, diâmetro médio de 1,8 mm, não formadores de esporos e desulfoviridina positiva. Sendo identificadas como Desulfococcus multivorans através de seqüenciamento do DNAr 16S. As células foram submetidas a testes fisiológicos visando caracterizar o crescimento com diferentes aceptores de elétrons e substratos orgânicos. Nas condições em que predominaram cocos o consumo das fontes de carbono foram melhores quando as fontes aceptoras de elétron eram possuidoras de enxofre. Na terceira fase experimental foram realizados ensaios com a cultura purificada em reatores anaeróbios em batelada submetidos as seguintes concentrações de benzoato e sulfato estabelecendo relações de: (0,3) 600 mg/L e 2000 mg/L; (0,6) 606 mg/L e 1000 mg/L; (1,2) 600 mg/L e 500 mg/L; (1,8) 910 mg/L e 500 mg/L, respectivamente. Após, 13 dias de operação, verificou-se valores de remoção do benzoato e utilização do sulfato, iguais a: 99% e 61,2% (0,3), 99% e 100% (0,6), 99% e 100% (1,2) e 50% e 100% (1,8), respectivamente. A velocidade de crescimento (&#956) e o tempo de geração (Tg) foram respectivamente: 0,010 h-1 e 66,4 horas, para relação 0,6 e 0,011 h-1 e 66,1 horas, para a relação 0,3. / This work was carried out aiming to assess benzoate anaerobic degradation by sulfate-reducing bacteria (SRB). The first experimental phase consisted of assessing this degradation using biomass provided from a horizontal anaerobic immobilized bed reactor (HAIB) applied in the treatment of wastewater from an organic peroxide industry. The essays were carried out in batch reactors kept under 33ºC ±1 of temperature and 150 rpm of agitation. The reactors were fed with a specific culture medium for SRB and also with different concentrations of benzoate and sulfate, establishing the following relations: 0.9 (890 mg/L and 970 mg/L), 0.8 (680 mg/L and 870 mg/L) and 0.6 (242 mg/L and 400 mg/L), respectively. After 13 days of operation, benzoate removal and sulfate utilization efficiency values equal to: (0.9) 71.2% and 98%, (0.8) 97% and 99.9% and (0.6) 91.3% and 15%, respectively, were verified. The microscopic exams revealed the domain of SRB. In the second experimental phase cellular purification was carried out using the serial dilution technique in liquid medium, obtaining a culture with the domain of gram-negatives cocci, average diameter of 1,8 mm, nonspore-forming and desulfoviridin positive. These cells were identified as Desulfococcus multivorans through the use of ribosomal 16S DNA sequencing technique. This biomass was submitted to physiological tests aiming to characterize the growth with different electron acceptors and organic substrates. It was possible to verify that in the conditions where cocci took place, the consumption of carbon sources were better when the electron accepting sources possessed sulfur. In the third experimental phase essays with culture purified, the reactors were submitted to the following concentrations of benzoate and sulfate: relation (0.3), with 600.0 mg/L and 2000.0 mg/L; relation (0.6) with 606 mg/L and 1000 mg/L; relation (1.2), with 600 mg/L and 500 mg/L; relation (1.8), with 910 mg/L and 500 mg/L, respectively. After 13 days of operation, benzoate removal and sulfate utilization values were equal to: 99% and 61.2% (0.3), 99% and 100% (0.6), 99% and 100% (1.2) and 50% and 100% (1.8), respectively. The specific growth rate (&#956) and the generation time (Tg) of culture, were equal to: 0.010 h-1 and 66.4 hours for relation 0.6 and 0.011 h-1 and 66.1 hours for relation 0.3, respectively.

The incidence of Squamous Cell Carcinoma is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 45% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis lesion and with a small but significant chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. This study had an explicit emphasis on the mitochondrial genome and nuclear genes that encode for subunits in the mitochondrial regulated energy transducing oxidative phosphorylation pathways. More specifically the first aim of this project was to analyse the NDUFA8, PTCH, NDUFAS, SMOH, SDHD, MMPI2, NDUFV1, EMSI, COXVIIc, and RASAI genes via non-specific fluorophoric Real-Time PCR for genetic aberrations in an affected Solar Keratosis and control cohort. The second aim was to analyse two specific genes, SDHD and MMPI2, for copy number aberrations via Dual-Labelled Probe Real-Time PCR in the same affected Solar Keratosis and control cohort. The third aim was to analyse Mitochondrial DNA Depletion syndrome (MDS) in a chemically exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR. The significance of these studies is in their contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. Furthermore, it would determine whether the genes analysed in this study exist in greater prevalence in the affected Solar Keratosis population compared to the control cohort. With regard to the MDS component, identifying the presence of this disease in these individuals was initially undertaken as part of a study to provide evidence in compensation claims. The diagnosis may assist in their medical therapy, insofar as some of them were now suffering from liver malfunctions and atypical male breast cancer. Another application of this effective and low cost method of diagnosing MDS is in populations with high HTV incidences. This is due to the fact that the most common drug used to treat this disease can give rise to the expression of MDS, thus further complicating the health status of HIV infected individuals. The analysis of this research was accomplished via the Real-Time PCR technique, with a non-specific fluorophore component in addition to specific Dual-Labelled Probe components, to ascertain the general nature of any aberration identified in the sample cohort. This project also employed additional methods of analysis such as DHPLC and DNA sequencing to assist in determining the veracity of its aims, particularly in terms of the precise detection of genetic aberrations via Real-Time PCR. Patients exhibiting male breast cancer and liver malftinctions were also analysed via Dual-Labelled Probe RealTime PCR to ascertain the presence of Mitochondrial DNA Depletion syndrome, a disorder characterised by lactic acidosis, liver failure, seizures, and congestive heart failure. Determining the presence of this syndrome in these patients would assist in their medical treatment, and contribute to the analytical methods available to diagnose this syndrome, which is known to occur in HIV sufferers due to the nucleoside drugs used to combat the disease. Real-Time PCR can adequately gauge the integrity of a genetic area in terms of amplicon malformities (non-specific-fluorophoric) and DNA copy number aberrations (Dual-Labelled Probe) via fluorophore signal differentials compared to wild-type samples and housekeeper profiles. The results of the first component of this project, namely the analysis of five gene pairs by non-specific fluorophoric Real-Time PCR, highlighted that a significantly higher incidence of putative aberrants is evident in the affected population when compared to the control cohort. The genes analysed were NDUFA8, PTCH, NDUFA5, SMOH, SDHD, MMP 12, NDUFVI, EMS 1, COXVIIc, and RASA 1. These ten genes were subdivided into five pairs; one of the pair being a gene associated with the development of a non-melanotic skin cancer (NMSC), the other a gene encoding for a subunit of the Electron Transport Chain (ETC). Each of these pairs exists in close proximity to one another on a particular chromosomal locale. Differences were highlighted in the single gene triplicate run population. The ETC genes (NDUFA8, NDUFA5, SDHD, NIDUFVI, COXVIIc) exhibited 10 / 720 (1.37%) as being putative mutants in the control population, compared to 117 / 675 (17.3%) for the affected population (p value less than 0.0001). The NMSC gene analysis (PTCH, SMOH, MMPI2, EMSI, RASA1) produced a 16 / 720 (2.22%) ratio for the control population, with the affected population having an incidence of 97 / 675 (14.4 %) for putative mutants (p value less than 0.0001). The observance of putative aberrants in the NDUFVI (p less than 0.018), EMS1 (p less than 0.003), COXVTIc (p less than 0.001), and RASA I (p less than 0.009) genes in the affected Solar Keratosis (SK) population was significantly higher than that observed in the control population. The majority of aberrations detected via the non-specific fluorophoric Real-Time PCR technique were small nucleotide base insertions and deletions. The analysis of the SK affected and control cohort via Real-Time PCR proved a cost-effective and reliable method in identifying the presence of DNA aberrations such as non-instructional sites. The results of the second component extended the findings of the non-specific fluorophoric analysis. The SDHD and MMPI 2 genes were analysed for copy number aberrations via Dual-Labelled Probe Real-Time PCR for genetic aberrations the same affected and control Solar Keratosis cohort. It was found that 12 of 279 samples had identifiable copy-number aberrations in either the SDHD or MMPI2 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The MMPI2 gene also had nine samples identified as possessing an intronic heterozygous base-pair substitution anomaly via DNA sequencing. The NDUFA8 gene had 12 samples identified as anomalous via the DHPLC technique, 11 of which were identified via non-specific fluorophoric Real-Time PCR, with the analysis performed to verify the accuracy of the Real-Time technique in identifying DNA aberrations. This study identified DNA aberrations in an affected Solar Keratosis and control cohort and ascertained several particular genomic abnomialities in the SDHD, MMPI2 and NDUFA8 genes, with an emphasis on copy-number aberrations and amplicon abnormalities. In the third component of this study, namely the analysis of Mitochondrial DNA Depletion syndrome (MDS) in a jet-fuel exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR, the results indicated that four of the seven patients were expressing MDS. Of the four patients who exhibited a reduction in mitochondrial copy-number the average decrease was of a four-fold level, or approximately a depletion of mitochondrial copies from 200 plus to ~ 54 (74 % reduction in MtDNA). The patients who contributed DNA for investigation into the presence of MDS were suffering from liver malfunction and atypical male breast cancer. The Dual-Labelled Probe technique proved a reliable and cost effective method in identifying the presence of MDS in these patients, with the DNA extracted from fresh white blood cells that had been isolated using the Ficoll-Hypaque method. The importance of this is that accurate levels of Mitochondrial DNA copy numbers can be ascertained in white blood cells as it removes the presence of platelets, which also contain mitochondria but no nucleus. The analysis of ETC and NMSC associated genes in addition to mitochondrial copy number integrity means that this study investigated two aspects of the carcinogenetic pathway i.e. abnormal energy regulation and the regulation of micromolecular and macromolecular cellular homeostatic mechanisms. The mechanism of programmed cell death or apoptosis is regulated by the mitochondria and the ability of a genetically damaged cell to evade the apoptotic process is directly linked to a cell becoming cancerous. It is only after the evasion of apoptosis and the replication of the damaged cells' DNA into daughter cells that neoplastic events can occur. Thus, this study contributed to the understanding of how neo-plastic lesions may develop and progress into invasive tumours. It additionally assisted in proving the effectiveness of the RealTime PCR technique in detecting DNA aberrations and mitochondrial copy number anomalies.

Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations. The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed. Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method. The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms. A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained. Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase. / QC 20100415

Rickettsiae are obligate intracellular gram-negative bacteria transmitted by arthropod vectors. Rickettsiae sometimes cause disease in humans, typically with high fever, headache and occasionally an eschar. In Sweden, Rickettsia helvetica, belonging to the spotted fever group, is the only tick-transmitted rickettsia found free in nature. The pathogenic roll of R. helvetica has not been fully investigated, but it has been implicated in aneruptive fever and cardiac disease. This thesis describes parts of the transmission pathways of rickettsiae in Sweden. Rickettsia infection rates in ticks collected from birds were analysed, and the birds’ role as disseminators and reservoirs was studied. We found that more than one in ten ticks was infected with rickettsia bacteria, predominantly R. helvetica, and that migrating birds contribute not only to long-distance dispersion of bacteria, but also to an inflow of novel and potentially pathogenic rickettsia species, in this case R. monacensis and R. sp. strain Davousti-like species, into Sweden. Further, wild and domestic animals were found to have seroreactivity against R. helvetica, which shows that they are exposed and susceptible to rickettsia. Their role as reservoirs has not been determined, yet they may indirectly be involved in transmission of rickettsia to humans by infected ticks feeding on them. The seroreactivity in humans was also studied. Patients investigated for suspected Borrelioses and blood donors had detectable antibodies against Rickettsia spp., with the highest prevalence detected in the suspected Borreliosis group. This shows that humans in Sweden are exposed to and develop an immune response against rickettsia. The suspicion that R. helvetica may cause severe symptoms was verified by a patient with subacute meningitis where the bacterium was shown for the first time to cause an invasive infection with CNS involvement and where the bacterium was isolated from the patient’s cerebrospinal fluid. Growth characteristics and morphology of R. helvetica were studied to better understand invasiveness and virulence. The findings indicate that the invasiveness is comparable with other rickettsia, though R. helvetica seems to have a stable but slightly slower growth.  Rickettsia helvetica is endemic in Sweden and therefore needs to be considered when investigating disease after a tick bite. / Rickettsia är en liten, strikt intracellulär, gramnegativ bakterie som sprids med vektorer som fästingar, löss och loppor. Bakterien kan orsaka Rickettsios hos människa, en sjukdom där de vanligaste symtomen är hög feber, huvudvärk, muskelvärk och i vissa fall ett bettmärke (eschar). I Sverige är Rickettsia helvetica, som tillhör spotted fever gruppen (SFG), den enda fästingöverförda rickettsia bakterien som hittats allmänt i naturen. Patogeniciteten för R. helvetica är ofullständigt utredd, men ”aneruptive fever” och hjärtmuskelinflammation har rapporterats. Avhandlingen beskriver delar av smittkedjan för SFG rickettsia i Sverige. Bakteriernas förekomst i fästingar plockade från fåglar har studerats, likaså det ekologiska tryck som flyttfåglars bärarskap av infekterade fästingar bidrar med när de korsar olika världsdelar. Mer än var tionde fästing var infekterad med rickettsia bakterier, i huvudsak R. helvetica. Det visade sig att flyttfåglar bidrar inte bara till långväga spridning av bakterier utan även till införsel av nya potentiellt patogena rickettsiaarter, i detta fall identifierades R. monacensis och en R. sp strain Davousti liknande art. Vidare analyserades seroreaktivitet mot Rickettsia helvetica hos både tamdjur och vilda djur, vilket visade på antikroppsutveckling, som uttryck för smittexposition, i mer än vart femte djur. Djurens roll som reservoar för bakterien är inte klarlagd, men oavsett är djuren indirekt involverade i spridningen av bakterien till människa via infekterade fästingar som suger blod. Seroreaktivitet hos människa har också studerats. Patienter, provtagna på grund av misstanke om borreliainfektion, samt blodgivare hade detekterbara antikroppar mot Rickettsiae, med högst prevalens i gruppen med misstänkt borreliainfektion. Fynden visar att människor i Sverige är exponerade för och utvecklar en immunreaktion mot rickettsia. Att R. helvetica skulle kunna ge allvarlig sjukdom verifieras av ett patientfall med subakut meningit där bakterien för första gången visats ge invasiv infektion med påverkan på nervsystemet (CNS engagemang) och där bakterien isolerats från patientens ryggmärgsvätska.  Morfologi och tillväxtegenskaper för R. helvetica undersöktes för att bättre förstå bakteriens invasivitet och virulens. Fynden indikerar att invasiviteten är jämförbar med andra rickettsiaarter men R. helvetica verkar ha en stabil men något långsammare tillväxt. Rickettsia helvetica är endemisk i Sverige och måste tas i beaktande vid sjukdomsutredning efter ett fästingbett.

<p>In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping.</p><p>P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools.</p><p>The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples.</p><p>To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension.</p><p>The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants.</p>

Mycobacteriosis (fish tuberculosis) is a progressive disease of a wide range of wild and captive marine and freshwater fish species. While Mycobacterium marinum, M. fortuitum and M. chelonae are the most frequently reported species to be involved in the disease, several new mycobacteria species have also recently been implicated. Conventional detection / identification of fish mycobacteria is based on histopathology, culture and biochemical characteristics. In this study complementary molecular approaches were developed to assist in Mycobacterium identification. First, a highly specific and sensitive multiplex PCR-based assay, targeting two genes (hsp65 and 16S RNA), was established to simultaneously detect the genus Mycobacterium and identify M. marinum, M. fortuitum or M. chelonae from culture or infected fish tissue, based on presence / absence of specific amplicons. In addition, PCR-restriction enzyme analysis (PRA) and DNA sequence analysis of the 16S-23S internal transcribed spacer (ITS) region and a 441 bp fragment of the hsp65 gene demonstrated the limitations of multiplex PCR (and commercial line probe assays) to differentiate among the species of the M. fortuitum complex. However DNA sequence analysis of the hsp65 gene fragment was found to reliably identify M. fortuitum from closely related species, M. conceptionense and M. senegalense. Reliable identification of novel species (or very similar species) of aquatic mycobacteria requires more extensive DNA sequence comparisons. Thus, multigene (polygenetic) analyses, as used here, provide rapid, accurate and reliable species identification of aquatic mycobacteria. Furthermore, a number of novel species of aquatic mycobacteria, M. stomatepiae, ‘M. angelicum’, ‘M. aemonae’ and M. salmoniphilum were discovered using the polygenetic analysis approach. Correct identification of Mycobacterium species by DNA sequence comparisons relies on accurate database information. Difficulties in this study in assigning M. marine and M. gordonae to their correct taxa suggest errors in the current public sequence repositories. The above methods were successfully applied to detect and identify mycobacteria in field samples including formalin-fixed, paraffin-embedded (FFPE) fish tissue, water and frozen fish tissue.

In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping. P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools. The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples. To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension. The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants. / QC 20101028

The bacterial identification in laboratories of microbiology is achieved by using several morphophysiological and genetic factors. Some members of the actinomycetes group, like the genera Rhodococcus, Gordonia, Nocardia and Dietzia have similar phenotypical characteristics. As result of this, the laboratorial detection of these microorganisms is often faced with bacterial identification problems. The present work analyzed 24 bacterial isolates from milk and skin of buffalo females (Bubalus bubalis), which previously had been identified as Rhodococcus equi, by using a restricted number of phenotypical tests for bacterial characterization. Using additional biochemical tests and molecular tools, the goal of this study was to perform the characterization of these isolates, as well as the differentiation of other microorganisms closely related. The results of the phenotypical tests had not allowed distinguishing definitely the isolates, once that they demonstrated relationship with two correlated genera, Rhodococcus and Dietzia. Despite the fact, these results allowed the separation of the isolates in three distinct biotypes. Only one of the isolates was confirmed as R. equi through the PCR multiplex specifically for this specie, as well DNA sequencing and DNA fragment analysis. All the other isolates only could be precisely identified after the DNA sequencing, where they were characterized as Dietzia maris. The sensitivity profile to antimicrobials demonstrated the biggest resistance of the D. maris isolates to oxacillin and rifampin, 96% and 87% respectively. The R. equi isolate, presented resistance to amikacin, oxacillin, penicillin, rifampin and tetracycline. Alert for the risk of the incorrect identification of the bacterial isolates by using diagnostic analysis based on phenotypical tests in order to differentiate R. equi and D. maris, besides the necessity to use of complementary tests for differentiation of these microorganisms. / A identificação bacteriana em laboratórios de microbiologia é realizada pela observação de um conjunto de fatores morfofisiológicos e genéticos. Alguns membros do grupo dos actinomicetos, como os gêneros Rhodococcus, Gordonia, Nocardia e Dietzia possuem características fenotípicas semelhantes. Por conseqüência, a identificação microbiológica destes agentes pode enfrentar algumas dificuldades. Neste trabalho foram analisados 24 isolados bacterianos oriundos de leite e pele de búfalas (Bubalus bubalis), os quais foram previamente identificados como Rhodococcus equi, com o uso de um número restrito de testes fenotípicos considerados discriminatórios para a caracterização bacteriana. Com o auxílio de testes bioquímicos adicionais e de ferramentas moleculares objetivou-se neste estudo, realizar a caracterização desses isolados, bem como a diferenciação de outros microrganismos intimamente relacionados. Os resultados dos testes fenotípicos não permitiram identificar os isolados com clareza, uma vez que foram condizentes com dois gêneros relacionados, Rhodococcus e Dietzia. Apesar disso, esses resultados permitiram a separação dos isolados em três fenótipos distintos. Apenas um dos isolados foi comprovado como R. equi com a realização da PCR multiplex para esta espécie, por seqüenciamento e análise de fragmentos de DNA. Os demais isolados analisados somente puderam ser identificados com exatidão após o seqüenciamento do DNA, onde foram caracterizados como Dietzia maris. O perfil de susceptibilidade aos antimicrobianos revelou maior resistência dos isolados de D. maris para oxacilina e a rifampicina, 96% e 87% respectivamente. O isolado de R. equi, apresentou resistência à amicacina, oxacilina, penicilina, rifampicina e tetraciclina. Alerta-se para o risco da incorreta identificação dos isolados bacterianos pelo uso de análise diagnóstica baseada em testes fenotípicos, a fim de diferenciar R. equi e D. maris e para a necessidade de utilização de testes complementares para diferenciação destes microrganismos.