Studies have discovered higher frequencies of single nucleotide polymorphisms (SNPs) in different mitochondrial genes are associated with subnormozoospermia. However, the frequencies of SNPs in ND1 and ND2 are not unknown. The present research was aimed to determine the frequencies of SNPs in ND1 and ND2 genes of the mitochondrial genome in fertile and subfertile men and whether changes in codon usage was associated with fertility phenotypes. Total genomic DNA from 157 semen samples was extracted using the proteinase K/SDS digestion procedure, followed by phenol/chloroform purification and ethanol precipitation. ND1 and ND2 genes were amplified respectively from 80 and 92 DNA samples from different fertility groups. Each PCR product was sequenced to identify mutations. Codon change resulting from a nucleotide substitution was determined by comparison with a reference mtDNA sequence obtained from the NCBI database. The frequency of codon usage in the reference mtDNA was determined by the computer program MEGA version 2.1. Eleven synonymous nucleotide substitutions and two non-synonymous substitutions were found in this study. Four SNPs were previously characterized; all SNPs were homoplasmic. None of the SNPs were likely to affect the function of the proteins on the basis of the hydrophobicity plots or secondary structure predictions. Sixty two percent of synonymous mutations were found to change from a high to a low relative codon usage values; 37% of synonymous mutations changed from a low to a high relative usage value. Chi-square (χ²) test (χ²= 0.067 with 1 d.f.) showed that there was no significant difference at the 5% level between these changes. Thus, change in codon usage was not related to semen quality in men. Further, there were no statistically significant differences in the observed frequencies of SNPs of fertile and subfertile men. However, the sample size was small and this study was only focused on a single NZ Caucasian population. Further study including larger and more diverse population samples may provide further insight into the functional importance of codon usage and its relevance to fertility

Studies have discovered higher frequencies of single nucleotide polymorphisms (SNPs) in different mitochondrial genes are associated with subnormozoospermia. However, the frequencies of SNPs in ND1 and ND2 are not unknown. The present research was aimed to determine the frequencies of SNPs in ND1 and ND2 genes of the mitochondrial genome in fertile and subfertile men and whether changes in codon usage was associated with fertility phenotypes. Total genomic DNA from 157 semen samples was extracted using the proteinase K/SDS digestion procedure, followed by phenol/chloroform purification and ethanol precipitation. ND1 and ND2 genes were amplified respectively from 80 and 92 DNA samples from different fertility groups. Each PCR product was sequenced to identify mutations. Codon change resulting from a nucleotide substitution was determined by comparison with a reference mtDNA sequence obtained from the NCBI database. The frequency of codon usage in the reference mtDNA was determined by the computer program MEGA version 2.1. Eleven synonymous nucleotide substitutions and two non-synonymous substitutions were found in this study. Four SNPs were previously characterized; all SNPs were homoplasmic. None of the SNPs were likely to affect the function of the proteins on the basis of the hydrophobicity plots or secondary structure predictions. Sixty two percent of synonymous mutations were found to change from a high to a low relative codon usage values; 37% of synonymous mutations changed from a low to a high relative usage value. Chi-square (χ²) test (χ²= 0.067 with 1 d.f.) showed that there was no significant difference at the 5% level between these changes. Thus, change in codon usage was not related to semen quality in men. Further, there were no statistically significant differences in the observed frequencies of SNPs of fertile and subfertile men. However, the sample size was small and this study was only focused on a single NZ Caucasian population. Further study including larger and more diverse population samples may provide further insight into the functional importance of codon usage and its relevance to fertility

O câncer de mama é uma das neoplasias mais freqüentes em mulheres, conseqüentemente muitos esforços têm sido feitos na tentativa de se chegar a um diagnóstico mais precoce ou até mesmo na obtenção de maiores índices de cura. Neste sentido, o presente trabalho avaliou três tipos de alterações em nível de DNA mitocondrial com a intenção de verificar relações entre a apresentação clínico-patológica do tumor, na ocasião do ato cirúrgico, a ocorrência de mutações no DNA mitocondrial do tecido neoplásico primário e linfonodos axilares metastásicos. Para tanto, 82 amostras foram analisadas quanto à presença de alterações na alça D (D310 e microsatélite (CA).) e a ocorrência de grandes deleções na região 8295-13738. Os resultados mostraram que variações na região correspondente a alça D do mtDNA, tanto na região D310 quanto no microsatélite (CA)., são freqüentes, confirmado esta região como um "hot-spot" mutacional. Por outro lado, a deleção de 4977pb foi rara (4%) nas amostras analisadas, diferindo de resultados obtidos em outros trabalhos. Uma nova deleção de 5247pb na região 8295¬13738 foram constatadas numa amostra de linfonodos metastásico. Comparação direta entre tumores primários e linfonodos metastásicos mostrou que em 53% dos linfonodos comprometidos apresentam alterações em nível de mtDNA. Estas alterações correspondem a deleções no microsatélite (CA). na região D310 e um caso de deleção de 4977pb. Considerando que as alterações em nível de mtDNA refletem a instabilidade do genoma, tal constatação pode ser tomada como indicativo do maior grau de malignidade das células presentes nos linfonodos metastásicos, e a ocorrência seleção de grupos celulares durante o processo de metastização. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-06-11T12:50:15Z No. of bitstreams: 1 Dissertacao Leandro Brust.pdf: 1590408 bytes, checksum: 18029852219e400f87c980617a20da0d (MD5) / Made available in DSpace on 2014-06-11T12:50:15Z (GMT). No. of bitstreams: 1 Dissertacao Leandro Brust.pdf: 1590408 bytes, checksum: 18029852219e400f87c980617a20da0d (MD5) / Breast cancer is one ofthe most ftequent diseases among women, consequently much effort has been done in order to have earlier diagnosis or even to achieve higher cure rates. In this way, the present work had assessed three kinds of alterations in mitochondrial DNA aiming to verify possible relations between clinical and pathological data at the time of surgery and the occurrence of mitochondrial DNA mutations in the primary neoplasic tissue and metastasic axillary lymphonodes. In this sense, 82 samples were analyzed with respect to alterations at the D¬loop region (D310 and microsatelite (CA)n) and occurrence of large deletions at the 8295-3738 region. The results showed that variations at the D-Ioop region of the mtDNA, both at the 0310 location and the microsatellite (CA)n are frequent, confirming this region as a mutational hot-spot. Conversely, the deletion of 4977bp was rare (4%) in the analyzed samples, differing from results obtained in other works. A new deletion of 5247 pb within the region 8295-\3738 was found in a metastatic lymphonode sample. Direct comparison between primary tumor and metastatic lymphonodes showed that 53% of the compromised lymphonodes exhibit alterations at the mtDNA level. This alterations correspond to deletions at the microsatellite (CA)n, the D31O region, and one case of 4977bp delection. Considering that alterations at mtDNA level reflect the genomic instability, the present results can be considered as indicative of the higher degree of malignance of the cells present at the metastatic lynphonodes, and the occurrence of clonal selection during the metastatic process.

O câncer de mama é uma das neoplasias mais freqüentes em mulheres, conseqüentemente muitos esforços têm sido feitos na tentativa de se chegar a um diagnóstico mais precoce ou até mesmo na obtenção de maiores índices de cura. Neste sentido, o presente trabalho avaliou três tipos de alterações em nível de DNA mitocondrial com a intenção de verificar relações entre a apresentação clínico-patológica do tumor, na ocasião do ato cirúrgico, a ocorrência de mutações no DNA mitocondrial do tecido neoplásico primário e linfonodos axilares metastásicos. Para tanto, 82 amostras foram analisadas quanto à presença de alterações na alça D (D310 e microsatélite (CA).) e a ocorrência de grandes deleções na região 8295-13738. Os resultados mostraram que variações na região correspondente a alça D do mtDNA, tanto na região D310 quanto no microsatélite (CA)., são freqüentes, confirmado esta região como um "hot-spot" mutacional. Por outro lado, a deleção de 4977pb foi rara (4%) nas amostras analisadas, diferindo de resultados obtidos em outros trabalhos. Uma nova deleção de 5247pb na região 8295¬13738 foram constatadas numa amostra de linfonodos metastásico. Comparação direta entre tumores primários e linfonodos metastásicos mostrou que em 53% dos linfonodos comprometidos apresentam alterações em nível de mtDNA. Estas alterações correspondem a deleções no microsatélite (CA). na região D310 e um caso de deleção de 4977pb. Considerando que as alterações em nível de mtDNA refletem a instabilidade do genoma, tal constatação pode ser tomada como indicativo do maior grau de malignidade das células presentes nos linfonodos metastásicos, e a ocorrência seleção de grupos celulares durante o processo de metastização. / Breast cancer is one ofthe most ftequent diseases among women, consequently much effort has been done in order to have earlier diagnosis or even to achieve higher cure rates. In this way, the present work had assessed three kinds of alterations in mitochondrial DNA aiming to verify possible relations between clinical and pathological data at the time of surgery and the occurrence of mitochondrial DNA mutations in the primary neoplasic tissue and metastasic axillary lymphonodes. In this sense, 82 samples were analyzed with respect to alterations at the D¬loop region (D310 and microsatelite (CA)n) and occurrence of large deletions at the 8295-3738 region. The results showed that variations at the D-Ioop region of the mtDNA, both at the 0310 location and the microsatellite (CA)n are frequent, confirming this region as a mutational hot-spot. Conversely, the deletion of 4977bp was rare (4%) in the analyzed samples, differing from results obtained in other works. A new deletion of 5247 pb within the region 8295-\3738 was found in a metastatic lymphonode sample. Direct comparison between primary tumor and metastatic lymphonodes showed that 53% of the compromised lymphonodes exhibit alterations at the mtDNA level. This alterations correspond to deletions at the microsatellite (CA)n, the D31O region, and one case of 4977bp delection. Considering that alterations at mtDNA level reflect the genomic instability, the present results can be considered as indicative of the higher degree of malignance of the cells present at the metastatic lynphonodes, and the occurrence of clonal selection during the metastatic process.

Bacterial diversity has always been associated with micro-evolutionary events such as horizontal gene transfer and DNA mutations. Such events influence the rapid evolution of bacteria as a result of the environmental conditions which they encounter. They further establish beneficial phenotypic effects that allow bacteria to specialize in new habitats. Due to the increase in number of bacterial genomic sequences, studying microbial evolution has been made possible, and the impact of micro-evolution on bacterial diversity is becoming more apparent. To gain biological information from this ever increasing genomic data, a variety of computational tools are required. This thesis therefore, focuses on the development and application of computational approaches to identify genomic regions of divergence which have resulted from horizontal gene transfer or small mutational changes. The first and major part of the thesis describes the application of DNA patterns, termed oligonucleotide signatures to identify horizontally acquired genomic regions in prokaryotes. These DNA patterns are demonstrated to differentiate between signatures of the core genome and those which have been acquired through horizontal transfer events. DNA patterns are further demonstrated to: reveal the distribution patterns of horizontally acquired genomic elements, determine their acquisition periods, and predict their putative donor organisms. The second part of the thesis focuses on the evaluation of modern short read sequence data of geographically unrelated Pseudomonas aeruginosa to study their intraclonal genomic diversity. The work described in the thesis was purely in silico driven and performed at Hannover Medical School and the Bioinformatics and Computation Biology Unit at the University of Pretoria. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Biochemistry / unrestricted

Indiana University-Purdue University Indianapolis (IUPUI) / DNA suffers constant damage, leading to a variety of lesions that require repair. One of the most devastating lesions is a double-strand break (DSB), which results in physical dissociation of two pieces of a chromosome. Necessarily, cells have evolved a number of DSB repair mechanisms. One mechanism of DSB repair is break-induced replication (BIR), which involves invasion of one side of the broken chromosome into a homologous template, followed by copying of the donor molecule through telomeric sequences. BIR is an important cellular process implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities. Furthermore, BIR uniquely combines processive replication involving a replication fork with DSB repair. This work employs a system in Saccharomyces cerevisiae to investigate genetic control, physical outcomes, and frameshift mutagenesis associated with BIR initiated by a controlled HO-endonuclease break in a chromosome. Mutations in POL32, which encodes a third, non-essential subunit of polymerase delta (Pol delta), as well as RAD9 and RAD24, which participate in the DNA damage checkpoint response, resulted in a BIR defect characterized by decreased BIR repair and increased loss of the broken chromosome. Also, increased incidence of chromosomal fusions determined to be half-crossover (HCO) molecules was confirmed in pol32 and rad24, as well as a rad9rad50S double mutant. HCO formation was also stimulated by addition of a replication-inhibiting drug, methyl-methane sulfonate (MMS), to cells undergoing BIR repair. Based on these data, it is proposed that interruption of BIR after it has initiated is one mechanism of HCO formation. Addition of a frameshift mutation reporter to this system allowed mutagenesis associated with BIR DNA synthesis to be measured. It is demonstrated that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2800-fold higher than normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. Pol  proofreading and mismatch repair (MMR) are confirmed to correct BIR errors. Based on these data, it is proposed that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR. Pif1p, a helicase that is non-essential for DNA replication, and elevated dNTP levels during BIR also contributed to BIR mutagenesis. Taken together, this work characterizes BIR as an essential repair process that also poses risks to a cell, including genome destabilization and hypermutagenesis.

Tumors are one of the leading causes of death worldwide. Generally, the prognosis is better if the treatment begins at an early stage. Nowadays, the conventional chemotherapy treatment of cancer, known for its limited efficacy and side effects, is being gradually replaced by targeted biological treatment, which is used when specific genetic mutations are found. A part of the treatment is a detection of a potential progression, which is mainly based on the tumor biomarkers monitoring. Currently, further investigation of a so-called liquid biopsy method are ongoing, on which this thesis is focused. The main aim of this work was the experimental development and validation of the method for detection of the ctDNA in the plasma samples based on the somatic mutations presence. For the development and optimization of the system on the principle of denaturation capillary electrophoresis, the samples of cancer patients with KRAS mutation were used. Subsequently, a clinical part of the research was performed on a pilot set of 21 plasma samples. Finally, the method was optimized for the detection of BRAF and EGFR markers. A partial objective was to improve the detection sensitivity and increase the capture of the ctDNA in patients with advanced stage of the disease. The results of this work suggest the...

Circulating tumor DNA (ctDNA) in peripheral blood of patients with metastatic colorectal cancer appears to be a promising molecular marker that provides various applications. ctDNA levels vary depending on the presence, alternatively on the volume of tumor mass within patient's body, which can be used primarily for early detection of disease progression or recurrence and moreover for evaluating radicality of surgical treatment, all within long-term postoperative follow-up of the patient. Due to minimal invasivity of ctDNA analysis from peripheral blood (so-called liquid biopsy), it is possible to perform it repeatedly at relatively short time intervals. On account of very low fraction of ctDNA in total cell-free DNA (cfDNA) ranging between units and hundreds of percent, the key factor is optimal methodology covering all steps from the isolation process to a sufficiently sensitive detection technology. In this thesis I focus on an optimization of isolation process and analysis of ctDNA obtained from tumor tissue and plasma of selected patients with metastatic colorectal cancer in connection with surgical radicality and correlation with a clinical status of the patients.