A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2013. / The forkhead box (FOX) proteins are a family of transcription factors that interact with DNA via a winged helix motif that forms part of the forkhead domain. The FOXP (FOXP1-4) subfamily is unique in the family in that the forkhead domains of these proteins exhibit domain swapping where structural elements are exchanged via extension of the hinge-loop region. The FOXP subfamily members have high sequence homology, yet wild-type FOXP3 is a stable domain-swapped dimer in solution whereas FOXP1 and FOXP2 exist in a monomer/domain-swapped dimer equilibrium. A single amino acid difference is observed in the hinge region of the FOXP subfamily. This corresponds to Tyr540 in FOXP2 and Phe373 in FOXP3. We propose that it is the phenylalanine residue in FOXP3 that shifts the equilibrium towards dimer. Here we use FOXP2 to investigate the effect of a mutation, Y540F, on the structure and dimerisation propensity of the FOXP subfamily. Crystals for the Y540F variant in the presence of DNA have been obtained to demonstrate conclusively that domain swapping occurs. Size-exclusion chromatography indicates that the wild type FOXP2 forkhead domain is almost entirely monomeric at concentrations less than 100 μM. The Y540F variant is shown to stabilise the dimer and the ratio between monomer and dimer is concentration-dependent. DNA binding assays suggest that the Y540F variant binds less favourably to the cognate binding sequence than does the WT FOXP2 forkhead domain. Taken together, these findings suggest that domain swapping may modulate DNA binding.

Degree awarded with distinction on 6 December l995. A dissertation submitted to the Faculty of Science, University the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science. March. 1995 / Hepatitis B virus (HBV) infection resulting in integration of the viral DNA into host liver cell DNA is associated with the development of hepatocellular carcinoma (HCC). This is indicated by epidemiological trends, molecular studies and studies of animal models infected with viruses closely related to HBv. However, little is known about the mechanism by which the integrated HBV DNA includes HCC despite continuing analysis of the integrated HBV DNA and its surrounding cellular sequences. [Abbreviated Abstract. Open document to view full version]

Neste trabalho foram imobilizados nanotubos de carbono de parede simples sobre um eletrodo de ouro policristalino gerando uma camada de nanotubos alinhados verticalmente na superfície do eletrodo. Para isto, foi utilizado um fragmento de DNA (ssDNA tiol-terminado (5-HS-TGG-GGT-TTA-TGG-AAA-TTGGAA-3)) que foi posicionado ao redor do nanotubo de carbono com o procedimento seguinte: 1,0 mg SWCNT funcionalizado foi misturado com 1,0 mL de uma solução de ssDNA de 1,0 &micro;mol L-1, e o ssDNA foi preparado em 0,1 molL-1 de PBS contendo cloreto de sódio a 10% (v / v). Em seguida, a mistura foi sonicada usando uma sonda de ultra-som por 45 min e depois centrifugada a 10000 rpm por 30 min. Finalmente, um eletrodo de Au previamente limpo foi imerso na solução de sobrenadante e monocamadas auto-organizadas (SAM), que consistem de ssDNA/SWCNT foram formadas durante 24 h numa sala refrigerada a 4 &deg;C. As características morfológicas dos eletrodos foram determinadas por microscopia de força atômica, observando-se o alinhamento vertical, que alterou a rugosidade do eletrodo de 1,95 nm para 47,5 nm, com a altura média dos SWCNTs de 260,3 nm, com um desvio padrão relativo de 19,9%. O comportamento eletroquímico do eletrodo de ouro modificado com o hibrido ssDNA/SWCNT foi caracterizado utilizando voltametria cíclica em meio de Na2SO4 0,1 mol L-1 contendo K3Fe(CN)6 5,0 mmol L-1, com velocidade de varredura de potencial de 50 mVs-1. Observou-se que a reversibilidade do par redox Fe(CN)63-/Fe(CN)64- é maior para o eletrodo modificado com ssDNA/SWCNT (&Delta;Epico= 80 mV) quando comparado ao eletrodo de Au (&Delta;Epico = 115 mV). A modificação proporcionou uma resposta mais eletrocatalítica com um deslocamento de 43 mV para valores menos positivos do potencial de oxidação do Fe(CN)63-. A oxidação no eletrodo de Au/ssDNA/SWCNTs ocorre em +417 mV e no eletrodo de Au em +460 mV. Este aumento de reversibilidade foi quantificado por espectroscopia eletroquímica de impedância faradaica, onde se encontrou os valores de constantes de velocidade de 7.56 &times; 10-5 cm s-1 para o eletrodo modificado e apenas 3,36 &times; 10-5 cm s-1 para o de ouro puro. O efeito da modificação da superfície Au com o nanohíbrido ssDNA / SWCNT na oxidação do bisfenol-A (BPA) foi avaliado em Na2SO4 0,1 mol L-1, pH 6,0, contendo 100 &micro;mol L-1 de BPA por voltametria cíclica a 50 mV s-1. Observou-se um processo de oxidação com um pico voltamétrico anódico num valor de potencial de 510 mV. Este processo de oxidação está relacionado com a eletro-oxidação de BPA para íons fenoxeno. O processo ocorreu em um potencial menos positivo do que o valor observado para o eletrodo de Au não modificado, ou seja 720 mV. Além disso, o processo oxidativo referente à superfície modificada mostrou-se mais catalítico, proporcionando um aumento do pico de oxidação de 163%. <br /> Para a metodologia analítica, procurou-se se maximizar o sinal analítico da técnica de voltametria de pulso diferencial, DPV, assim a resposta para o eletrodo de Au/ssDNA/SWCNT foi estudada em relação ao pH, salto de potenciais e a amplitude de pulso. Os valores ótimos encontrados foram 6,0, 2 mV e 50 mV, respectivamente. Nestas condições o eletrodo de Au/ssDNA/SWCNT foi aplicado para a determinação de BPA em uma solução de Na2SO4 0,1 mol L-1, pH 6,0. A resposta analítica tem um comportamento linear na faixa entre 1,0 - 4,5 &micro;mol L-1, de acordo com a seguinte equação: I (&micro;A) = 0.019 (&micro;A) + 5.82 (&micro;A/ &micro;molL-1) [BPA], com um coeficiente de correlação de 0,996 (n = 10) e um limite de detecção (LOD) de 11,0 nmol L-1 (2,51 &micro;g L-1) determinado de acordo com as recomendações da IUPAC. O valor obtido é menor que aqueles disponíveis na literatura. / In the present work, single-walled carbon nanotubes (SWCNT) were immobilized over top a polycrystalline gold electrode. This immobilization assembled a layer of vertically aligned nanotubes on the electrode surface. For this purpose, it was used a DNA probe (ssDNA thiolated (HS-5-TGG-TTA-TGG-GGT-AAA-TTGGAA-3)) that has been used to wrap the carbon nanotube as the following procedure: 1.0 mg of functionalized SWCNT was mixed with 1.0 mL of 1.0 &micro;mol L-1 of a ssDNA solution prepared in 0.1 mol L-1 of PBS containing 10% (v/v) of sodium chloride. Next, the mixture was sonicated using an ultrasonic horn probe and then centrifuged at 10000 rpm; each process took 45 min. Finally, a previously cleaned Au electrode was immersed in the supernatant solution. Self-assembled monolayers (SAMs) consisting of ssDNA/SWCNT were formed after 24 h in a refrigerated room at 4 &deg;C. The morphological characteristics of the electrodes were determined using atomic force microscopy. It was observed that the vertical alignment increased the electrode surface roughness of 1.95 nm to 47.5 nm. The average height of the SWCNT was calculated at 260.3 nm, with a relative standard deviation of 19.9%. The electrochemical behavior of gold electrode modified with the ssDNA/SWCNT hybrid was characterized using cyclic voltammetry (CV) in 0.1 mol L-1 of Na2SO4 containing 5.0 mmol L-1 of [K3Fe(CN)6], with a scan rate of 50 mVs-1. It was observed that the reversibility of the redox couple Fe(CN)63-/Fe(CN)64- decreased using the electrode modified with ssDNA/SWCNT (&Delta;Epeak = 80 mV), when compared with the Au electrode (&Delta;Epeak = 115 mV). The modification provided an electrocatalytic response with a shift of 43 mV to less positive values on the Fe(CN)63- oxidation potential value. The oxidation on the Au/ssDNA/SWCNT electrode occurs at +417 mV and the Au electrode at +460 mV. This improvement on the reversibility was quantified using the electrochemical impedance spectroscopy, in which it was observed an apparent constant rate at 7.56 x 10-5 cm s-1 for the modified electrode and 3.36 x 10-5 cm s-1 for pure gold. The effect of the modification of the Au surface with the nanohybrid ssDNA/SWCNT on the bisphenol A (BPA) oxidation was evaluated 0.1 mol L-1 of Na2SO4 (pH 6.0) containing 100 &micro;mol L-1 of BPA. The system was evaluated using CV at 50 mV s-1. The CV experiments showed an oxidation process with an anodic peak potential at 510 mV. This oxidation process is attributed to the electro-oxidation of the BPA forming the fenoxene ions. The process occurred at a less positive potential value when compared with the unmodified Au electrode, i.e. 720 mV. Moreover, surface modified with the nanohybrid presented more catalytic providing an increase of 163% on the oxidation current peak. For the analytical methodology, the analytical signal was maximized. For this, the differential pulse voltammetry (DPV) parameters such as: pulse amplitude and step potential and pH were optimized. The optimum values found were pH at 6.0, pulse amplitude at 50 mV and step potential at 2 mV. In these conditions, the Au/ssDNA/SWCNT electrode was applied for the BPA determination in 0.1 mol L-1 of Na2SO4. The analytical response showed a linear relationship in a range from 1.0 to 4.5 &micro;mol L-1, in accordance with the following equation: I (&micro;A) = 0.019 (&micro;A) + 5.82 (&micro;A / &micro;mol L-1) [BPA ], with a correlation coefficient of 0.996 (n = 10). The limit of detection (LOD) of 11.0 nmol L-1 (2.51 &micro;g L-1) was determined in accordance with the IUPAC recommendations. The obtained value is smaller than those available in the literature.

No description available.

This study was conducted at the University of Massachusetts Medical Center in the Volkert laboratory. Human alkyl adenine DNA glycosylase (hAAG) is a DNA repair enzyme that repairs alkylated DNA bases. hAAG was cloned in 1991 and a second isoform was classified in 1994. The difference between the two isoforms of hAAG is an alternate spliced first exon. Both isoforms of the hAAG gene were present in the Volkert laboratory collection, however the second isoform (hAAG-2) was phenotypically different than the first and became the first focus of this study. Using the improperly functioning isoform as a template, and constructing a 5' primer with the identical upstream sequence as the functioning isoform (hAAG-1), a phenotypically similar gene was constructed by PCR. The new isoform (hAAG-2) was cloned into an expression vector and its activity as a DNA repair agent was studied. A second version of hAAG-2 was also constructed, incorporating a histidine tag for protein purification and identification purposes. Efforts included using the ability of hAAG to complement glycosylase deficient alkA tagA E. coli double mutant strains to assess and to compare the ability of the two isoforms of hAAG and to determine if the histidine tag affected function. The ability of hAAG to rescue cells from exposure to a variety of DNA damaging agents was studied by inducing each isoform and analyzing the sensitivity of the cells to increased doses of DNA damaging agents. Both hAAG-1 and hAAG-2 were able to restore the wild type resistance of the alkA and tag genes when exposed to the alkylating agents MNNG and MMS. In order to study the ability of hAAG to repair alkyl lesions larger than methyl groups, it was necessary to inactivate the uvrA dependent nucleotide excision repair gene. In E. coli, methyl lesions are repaired primarily by glycosylases, while nucleotide excision repairs bulky lesions. Thus, in order to detect hAAG activity on these types of damage, it was necessary to inactivate the bacterial uvrA gene. Each isoform of hAAG was transformed into a triple mutant strain deficient in alkA tagA and uvrA, then exposed to CNU, BCNU, and Mitomycin C. Each of these DNA damaging agent caused increased toxicity in the presence of hAAG. hAAG-1 expressed in the alkA tag double mutant strain was exposed to Mitomycin C and showed greater resistance than hAAG-1 expressed in the alkA tag uvrA triple mutant. In fact, in the nucleotide excision proficient strain, expression increased Mitomycin C resistance above that seen in the control, suggesting that glycosylase activity may function in a partnership with nucleotide excision repair and that the two isoforms of hAAG have subtle differences. An ompT protease knockout host strain was constructed using P1-transduction and used to examine protein products. hAAG-2 was inserted into the pBlueScript plasmid so that the gene could be regulated by the T7 promoter for use beyond the scope of this thesis. A protein synthesis time course assay was conducted to determine the expression levels of hAAG-1 and hAAG-2 when induced by IPTG. Immunoblot detection of the histidine tag was used to measure expression levels of each isoform.

Fingernail samples are analyzed in forensic casework to determine the source of the nail and/or to recover a foreign profile from beneath the nail. When extracting from a fingernail sample, it is possible to recover deoxyribonucleic acid (DNA) of the nail donor from within the nail and from the surface of the nail; similarly, foreign DNA may also be present on and recovered from the nail surface. When attempting to recover the latter, fingernail samples present particular problems. Often, the foreign component is masked by the greater mass of donor DNA present within and on the nail sample. This masking effect is exacerbated by the use of proteinase K (PK) in DNA extractions, as PK, with an average of 200 cut sites per keratin molecule, is capable of breaking open the keratin matrix of the nail and exposing the nail DNA intercalated in the matrix. Directly extracting nail clippings, in contrast to swabbing or scraping, would further introduce nail DNA when using proteinase K. The present study explores whether utilizing other proteases (ZyGEM, Acrosolv, and Factor Xa) with fewer cut sites than PK or direct lysis methods (IGEPAL® CA-630 and MAWI iSWABTM-ID) would minimize recovery of nail DNA from within the nail and thus mitigate the masking effect often seen with fingernail samples. The endogenous DNA extraction efficiency of each suggested method was compared with the manufacturer’s standard QIAGEN QIAamp® DNA Investigator extraction protocol for hand-washed and/or laboratory-cleaned nails. The extraction results from the hand-washed nails demonstrate variability both within samples from the same donor and between donors. In contrast to previously published literature, a comparison of the results between the hand-washed and cleaned nails suggests that much of the endogenous DNA recovered from fingernail samples is derived from DNA on the surface rather than from within the nail. QIAamp® extraction with the inclusion of dithiothreitol (DTT) recovered significantly more DNA (0.845 ± 0.651 nanograms of DNA per milligram of nail [ng DNA/mg nail]; p = 0.0045) than the same protocol without DTT (0.278 ± 0.253 ng DNA/mg nail). IGEPAL® recovered the least endogenous DNA (0.005 ± 0.012 ng DNA/mg nail) from the nail. The ZyGEM extraction recovered the second lowest amount (0.163 ± 0.161 ng DNA/mg nail) and both the Acrosolv (0.546 ± 0.607 ng DNA/mg nail) and MAWI’s iSWABTM-ID (0.681 ± 0.780 ng DNA/mg nail) methods recovered more DNA than the QIAamp® protocol without DTT. An assessment of the electropherograms resulting from cleaned fingernails across all extraction methods for one donor showed that both IGEPAL® and MAWI failed to recover a complete profile, whereas the remaining methods were able to recover complete profiles of the nail donor. An assessment of donor variability found variations in terms of endogenous nail DNA recovery. Fingernails were also spiked with blood, saliva, or semen to assess the recovery of foreign DNA. The extractions of the spiked nail samples demonstrate variability across all samples, owing, to some degree, to inconsistencies of sample preparation. IGEPAL®’s inability to recover complete foreign profiles suggests that the method may not be viable for extraction of fingernail samples. Conversely, the ZyGEM, Acrosolv, and MAWI extraction methods demonstrate potential as alternative extraction methods for fingernail samples and would benefit from additional experimentation.

Nek1 é uma serina/treonina quinase humana envolvida na ciliogênese, na resposta a danos ao DNA e na regulação do ciclo celular. Devido ao possível envolvimento dessa proteína em patologias como a Doença Policística do Rim (PKD), a Síndrome de Costelas Curtas e Polidáctilia tipo Majewski e no desenvolvimento de tumores, muitos estudos tem sido feitos buscando entender a via de atuação dessa quinase nas células. Em vista disso, o objetivo desse trabalho foi avaliar o papel da Nek1 em resposta a danos ao DNA, principalmente gerados por agentes indutores de Crosslinks (ICLs), como cisplatina, buscando localizar a Nek1 nas vias de sinalização conhecidas e identificar proteínas que interagem com essa quinase. Para isto, foi utilizado um modelo de silenciamento estável de Nek1 por shRNAi na linhagem celular humana Hek293t. Essas células, quando comparadas à linhagem selvagem expressando a proteína, apresentaram um reparo deficiente de lesões induzidas por cisplatina e ausência de quebras de dupla fita através do ensaio cometa, confirmado pelos baixos níveis de fosforilação da histona H2AX. Entretanto, análises com outro agente indutor de ICLs, Nimustina (ACNU), demonstrou que a Nek1 parece atuar nos mecanismos envolvidos no reparo de lesões induzidas por cisplatina. Isso sugere que essa proteína possa estar agindo no reconhecimento da lesão do DNA e até mesmo regulando vias checagem de ciclo celular, uma vez que a linhagem silenciada apresentou modificações no ciclo celular e na sinalização de Chk1 e Chk2, após a exposição com cisplatina. Além disso, essas células também apresentaram alterações na sinalização das proteínas envolvidas no reparo de ICLs, BRCA1 e FANCD2. O estudo de interação proteica por espectrometria de massas destacou algumas proteínas de reparo, como proteínas da via de Fanconi, e principalmente moléculas relacionadas com o processo de ubiquitinação, sugerindo que este pode ser o mecanismo pelo qual essa molécula está atuando e integrando diferentes sistemas. Esse conjunto de dados indica o papel da Nek1 como uma proteína sensora de danos que atua no início das vias de reparo, regulando diferentes moléculas e interagindo em distintos processos celulares como controle do ciclo celular, reparo e morte. / Nek1 is a human serine/threonine protein kinase involved in ciliogenesis, DNA damage response and cell cycle regulation. Because of its possible involvement in human diseases such as Polycystic Kidney Disease (PKD), Short-Rib Polydactyly Syndrome Type Majewski and development of tumors, many studies have been done to understand how this kinase acts in cells. Therefore, the objective of this study was to evaluate a role for Nek1 in response to DNA damage, mainly generated by agents that induce crosslinks (ICLs), such as cisplatin, identifying pathways in which participate and how proteins interact with this kinase. To accomplish that, a stable silencing of Nek1 by shRNAi in human cell line Hek293t was employed. These cells, when compared to the wild type, showed a deficient repair of lesions induced by cisplatin and absence of double strand breaks, when analysed by comet assay and, confirmed by low levels of histone H2AX phosphorylation. However, analysis with another inducing ICLs agent, Nimustine (ACNU) demonstrated that Nek1 seems to act on mechanisms involved in repair of lesions induced by cisplatin. It suggesting that this protein might be acting on the recognition of DNA damage and even regulating checkpoint pathways, since the silenced strain showed changes in cell cycle and in signaling of Chk1 and Chk2 after exposure to cisplatin. Moreover, these cells also showed changes in signaling of proteins involved in the repair of ICLs FANCD2 and BRCA1. The study of protein interaction by mass spectrometry highlighted some repair proteins, like Fanconi pathway proteins, and protein involved in the ubiquitin-related process, suggesting a possible mechanism of action and integration with different systems. This dataset indicates the role of Nek1 as a DNA damage sensor protein that acts at the beginning of the repair pathways regulating and interacting with distinct molecules in different cellular processes such as cell cycle control, repair and death.

Neste trabalho foram imobilizados nanotubos de carbono de parede simples sobre um eletrodo de ouro policristalino gerando uma camada de nanotubos alinhados verticalmente na superfície do eletrodo. Para isto, foi utilizado um fragmento de DNA (ssDNA tiol-terminado (5-HS-TGG-GGT-TTA-TGG-AAA-TTGGAA-3)) que foi posicionado ao redor do nanotubo de carbono com o procedimento seguinte: 1,0 mg SWCNT funcionalizado foi misturado com 1,0 mL de uma solução de ssDNA de 1,0 &micro;mol L-1, e o ssDNA foi preparado em 0,1 molL-1 de PBS contendo cloreto de sódio a 10% (v / v). Em seguida, a mistura foi sonicada usando uma sonda de ultra-som por 45 min e depois centrifugada a 10000 rpm por 30 min. Finalmente, um eletrodo de Au previamente limpo foi imerso na solução de sobrenadante e monocamadas auto-organizadas (SAM), que consistem de ssDNA/SWCNT foram formadas durante 24 h numa sala refrigerada a 4 &deg;C. As características morfológicas dos eletrodos foram determinadas por microscopia de força atômica, observando-se o alinhamento vertical, que alterou a rugosidade do eletrodo de 1,95 nm para 47,5 nm, com a altura média dos SWCNTs de 260,3 nm, com um desvio padrão relativo de 19,9%. O comportamento eletroquímico do eletrodo de ouro modificado com o hibrido ssDNA/SWCNT foi caracterizado utilizando voltametria cíclica em meio de Na2SO4 0,1 mol L-1 contendo K3Fe(CN)6 5,0 mmol L-1, com velocidade de varredura de potencial de 50 mVs-1. Observou-se que a reversibilidade do par redox Fe(CN)63-/Fe(CN)64- é maior para o eletrodo modificado com ssDNA/SWCNT (&Delta;Epico= 80 mV) quando comparado ao eletrodo de Au (&Delta;Epico = 115 mV). A modificação proporcionou uma resposta mais eletrocatalítica com um deslocamento de 43 mV para valores menos positivos do potencial de oxidação do Fe(CN)63-. A oxidação no eletrodo de Au/ssDNA/SWCNTs ocorre em +417 mV e no eletrodo de Au em +460 mV. Este aumento de reversibilidade foi quantificado por espectroscopia eletroquímica de impedância faradaica, onde se encontrou os valores de constantes de velocidade de 7.56 &times; 10-5 cm s-1 para o eletrodo modificado e apenas 3,36 &times; 10-5 cm s-1 para o de ouro puro. O efeito da modificação da superfície Au com o nanohíbrido ssDNA / SWCNT na oxidação do bisfenol-A (BPA) foi avaliado em Na2SO4 0,1 mol L-1, pH 6,0, contendo 100 &micro;mol L-1 de BPA por voltametria cíclica a 50 mV s-1. Observou-se um processo de oxidação com um pico voltamétrico anódico num valor de potencial de 510 mV. Este processo de oxidação está relacionado com a eletro-oxidação de BPA para íons fenoxeno. O processo ocorreu em um potencial menos positivo do que o valor observado para o eletrodo de Au não modificado, ou seja 720 mV. Além disso, o processo oxidativo referente à superfície modificada mostrou-se mais catalítico, proporcionando um aumento do pico de oxidação de 163%. <br /> Para a metodologia analítica, procurou-se se maximizar o sinal analítico da técnica de voltametria de pulso diferencial, DPV, assim a resposta para o eletrodo de Au/ssDNA/SWCNT foi estudada em relação ao pH, salto de potenciais e a amplitude de pulso. Os valores ótimos encontrados foram 6,0, 2 mV e 50 mV, respectivamente. Nestas condições o eletrodo de Au/ssDNA/SWCNT foi aplicado para a determinação de BPA em uma solução de Na2SO4 0,1 mol L-1, pH 6,0. A resposta analítica tem um comportamento linear na faixa entre 1,0 - 4,5 &micro;mol L-1, de acordo com a seguinte equação: I (&micro;A) = 0.019 (&micro;A) + 5.82 (&micro;A/ &micro;molL-1) [BPA], com um coeficiente de correlação de 0,996 (n = 10) e um limite de detecção (LOD) de 11,0 nmol L-1 (2,51 &micro;g L-1) determinado de acordo com as recomendações da IUPAC. O valor obtido é menor que aqueles disponíveis na literatura. / In the present work, single-walled carbon nanotubes (SWCNT) were immobilized over top a polycrystalline gold electrode. This immobilization assembled a layer of vertically aligned nanotubes on the electrode surface. For this purpose, it was used a DNA probe (ssDNA thiolated (HS-5-TGG-TTA-TGG-GGT-AAA-TTGGAA-3)) that has been used to wrap the carbon nanotube as the following procedure: 1.0 mg of functionalized SWCNT was mixed with 1.0 mL of 1.0 &micro;mol L-1 of a ssDNA solution prepared in 0.1 mol L-1 of PBS containing 10% (v/v) of sodium chloride. Next, the mixture was sonicated using an ultrasonic horn probe and then centrifuged at 10000 rpm; each process took 45 min. Finally, a previously cleaned Au electrode was immersed in the supernatant solution. Self-assembled monolayers (SAMs) consisting of ssDNA/SWCNT were formed after 24 h in a refrigerated room at 4 &deg;C. The morphological characteristics of the electrodes were determined using atomic force microscopy. It was observed that the vertical alignment increased the electrode surface roughness of 1.95 nm to 47.5 nm. The average height of the SWCNT was calculated at 260.3 nm, with a relative standard deviation of 19.9%. The electrochemical behavior of gold electrode modified with the ssDNA/SWCNT hybrid was characterized using cyclic voltammetry (CV) in 0.1 mol L-1 of Na2SO4 containing 5.0 mmol L-1 of [K3Fe(CN)6], with a scan rate of 50 mVs-1. It was observed that the reversibility of the redox couple Fe(CN)63-/Fe(CN)64- decreased using the electrode modified with ssDNA/SWCNT (&Delta;Epeak = 80 mV), when compared with the Au electrode (&Delta;Epeak = 115 mV). The modification provided an electrocatalytic response with a shift of 43 mV to less positive values on the Fe(CN)63- oxidation potential value. The oxidation on the Au/ssDNA/SWCNT electrode occurs at +417 mV and the Au electrode at +460 mV. This improvement on the reversibility was quantified using the electrochemical impedance spectroscopy, in which it was observed an apparent constant rate at 7.56 x 10-5 cm s-1 for the modified electrode and 3.36 x 10-5 cm s-1 for pure gold. The effect of the modification of the Au surface with the nanohybrid ssDNA/SWCNT on the bisphenol A (BPA) oxidation was evaluated 0.1 mol L-1 of Na2SO4 (pH 6.0) containing 100 &micro;mol L-1 of BPA. The system was evaluated using CV at 50 mV s-1. The CV experiments showed an oxidation process with an anodic peak potential at 510 mV. This oxidation process is attributed to the electro-oxidation of the BPA forming the fenoxene ions. The process occurred at a less positive potential value when compared with the unmodified Au electrode, i.e. 720 mV. Moreover, surface modified with the nanohybrid presented more catalytic providing an increase of 163% on the oxidation current peak. For the analytical methodology, the analytical signal was maximized. For this, the differential pulse voltammetry (DPV) parameters such as: pulse amplitude and step potential and pH were optimized. The optimum values found were pH at 6.0, pulse amplitude at 50 mV and step potential at 2 mV. In these conditions, the Au/ssDNA/SWCNT electrode was applied for the BPA determination in 0.1 mol L-1 of Na2SO4. The analytical response showed a linear relationship in a range from 1.0 to 4.5 &micro;mol L-1, in accordance with the following equation: I (&micro;A) = 0.019 (&micro;A) + 5.82 (&micro;A / &micro;mol L-1) [BPA ], with a correlation coefficient of 0.996 (n = 10). The limit of detection (LOD) of 11.0 nmol L-1 (2.51 &micro;g L-1) was determined in accordance with the IUPAC recommendations. The obtained value is smaller than those available in the literature.

O DNA repetitivo constitui uma fração considerável do genoma de muitos organismos eucarióticos. Composto tanto por sequências funcionais, como os genes ribossômicos, quanto não codificantes, como é o caso dos elementos transponíveis, mini/microssatélites e o DNA satélite, essa porção do genoma tem sido amplamente utilizada como objeto de estudo, uma vez que sequências repetitivas podem estar associadas, por exemplo, a processos de diferenciação sexual. Esses estudos têm auxiliado tanto na melhor compreensão da dinâmica dessas regiões cromossômicas, como salientado a importância, a conservação e a evolução da porção repetitiva no genoma. O gênero Eigenmannia (Gymnotiformes, Sternopygidae) compreende espécies crípticas do ponto de vista morfológico que exibem variação no número cromossômico e podem apresentar sistemas sexuais XY ou ZW nos quais os elementos do par sexual diferem pela presença de blocos heterocromáticos maiores do que os encontrados em cromossomos autossomos, ou sistemas múltiplos envolvendo translocação Y-autossomo. O presente trabalho tem por objetivos o estudo sobre do gene Citocromo Oxidase I (COI), de forma a verificar a capacidade discriminatória desse gene mitocondrial e sugerir possíveis espécies dos então cariomorfos do gênero Eigenmannia no estado de São Paulo, continuidade do estudo do DNA repetitivo no gênero Eigenmannia, tanto de regiões funcionais do genoma, no caso o gene ribossômico 5S, bem como de elementos transponíveis, permitindo assim uma melhor compreensão sobre a distribuição, conservação nos cariomorfos e verificar sua eventual participação no processo de diferenciação não só de cromossomos sexuais, mas também na evolução cariotípica do grupo. Os resultados obtidos com o gene COI, assim como aqueles obtidos pelo gene ribossômico 5S evidenciam distâncias genéticas consistentes com a hipótese de que os cinco cariomorfos possam ser considerados como espécies distintas. Além disso, a hibridação in situ do gene ribossômico 5S forneceu uma nova evidência para a fusão cromossômica que deu origem ao cromossomo sexual Y, já descrita na literatura, enquanto que a hibridação de sequências teloméricas não forneceu evidências de processos de fusão recentes envolvendo os cariomorfos. Com relação aos elementos transponíveis foi possível verificar padrões distintos nos elementos TC1 e Rex1 no que diz respeito às sequências, uma vez que o elemento TC1 delimitou dois grandes grupos o que pode indicar uma invasão simultânea nos grupos e no retrotransposon Rex1 a invasão tenha ocorrido em um ancestral comum a todos os cariomorfos / The repetitive DNA constitutes a considerable fraction of the genome of many eukaryotic organisms. Compound by both functional sequences, such as ribosomal genes, and non-coding, such as transposable elements, mini / microsatellite DNA and the satellite, this portion of the genome has been widely used as a study object, since the repetitive sequences may be associated with, for example, the processes of sexual differentiation. These studies helped to understand the dynamics of these chromosomal regions, pointing the importance, conservation and evolution of the repetitive portion of the genome. The genus Eigenmannia (Gymnotiformes, Sternopygidae) comprises a morphological cryptic species that exhibit variation in chromosome number and may have sexual XY or ZW systems in which the elements of sexual pair differ by the presence of heterochromatic blocks larger than those found in chromosomes autosomes, or systems involving multiple Y-autosome translocation. The present work aims to study the gene Cytochrome Oxidase I (COI) to verify the discriminatory capacity of this mitochondrial gene and suggest possible species of the so called karyomorphs of the genus Eigenmannia in the state of São Paulo. The study of repetitive DNA in Eigenmannia genus, includes 5S ribosomal gene and transposable elements, thus allowing a better understanding of the distribution, conservation in karyomorphs and verify their possible participation in the process of differentiation not only of sex chromosomes, karyotypic evolution but also in the group. The results obtained with the COI gene, as well as those obtained by the 5S ribosomal gene demonstrate genetic distances consistent with the hypothesis that the five karyomorphs can be regarded as separate species. In addition, in situ hybridization of ribosomal 5S gene provided new evidence for chromosomal fusion which led to the Y sex chromosome, as described in the literature, whereas hybridization of telomeric sequences did not provide evidence of recent fusion events involving the karyomorphs. Regarding transposable elements, it could be verified distinct sequence patterns between TC1 and Rex1 elements, since the TC1 element delimited two groups which may indicate a simultaneously invasion in those groups and retrotransposon Rex1 invasion has occurred in a common ancestor to all karyomorphs

As an Adenovirus infects a host cell a multitude of molecular interactions occur, some driven by the virus and some driven by the cell it is infecting. Many of these areas of Adenovirus biology have been intensely studied over the last half century, however, many questions remain unanswered. The aim of this study was to investigate, more closely, a long studied molecular interaction, namely the role of the tumour suppressor p53 in the Adenovirus life cycle, and also to investigate the related, but much less studied, interaction between Adenoviruses and the host cell DNA repair machinery. Controversy surrounds the role of p53 in the Adenovirus life cycle, with current dogma favouring the view that p53 is inactivated, as it presumably presents an obstacle to a productive infection. In Chapter 3, a standardised infection protocol was developed to examine this area of Adenovirus biology more closely. This was followed with an array of cell viability and western blotting analyses that not only showed p53 was not an antagonist of the Adenovirus life cycle, but in some cases p53 acted as a protagonist. Isogenic cell lines were used to reinforce this point. Following this, data were provided that virus DNA replication was linked to the ability of an Adenovirus to kill cells. Furthermore, p53 was shown by immunofluorescence to be present in infected cells at a time that corresponded with virus DNA replication, albeit at low levels. By adding p53 back into cells, it was shown that the number of Adenovirus progeny could be stimulated to levels produced in genetically wild type TP53 cells. A selection of promoter/reporter assays and infection/transfection assays then showed how p53 might be aiding the virus life cycle. These data showed that low levels of p53 cooperated with the Adenovirus transactivator, E1A, to promote late gene expression, and this translated into a modest increase in virus late antigens in infected cells. Taken together these data show that, contrary to current dogma, p53 generally aids an Adenovirus infection and it may do this through promoting virus late gene expression. Recent data have emerged suggesting Adenoviruses must disable the host DNA double-strand break machinery to achieve a productive infection. As this area of Adenovirus biology is in its infancy, and as p53 has recently been identified as an integral component of these DNA repair processes, the contributions of the host cell repair machinery to Adenovirus biology were examined in Chapters 4 and 5. In Chapter 4, western blotting showed that upon Adenovirus infection, a key component of the homologous recombination repair machinery, Rad51, was markedly up-regulated. This up-regulation occurred independently of other key repair proteins, and was found to be a generalised feature of an Adenovirus infection. Surprisingly, p53 did not appear to be involved in this up-regulation, and neither were several other nodal host regulatory proteins. The up-regulation was then linked to Adenovirus DNA replication using a temperature-sensitive mutant Adenovirus, ts125. In Chapter 5, functional analysis of this up-regulated protein showed that Rad51 colocalised with Adenovirus replication centres. This colocalisation coincided with a time when virus DNA replication was occurring. Furthermore, transient over-expression of Rad51 drastically increased the amount of virus progeny produced. This effect was reproduced in two very different cell types and with a selection of attenuated mutant viruses. Finally, several models were proposed that might account for this newfound effect of Rad51 on the Adenovirus life cycle. The data presented in this thesis shows that Adenovirus not only interacts with key molecular machinery within the host cell, but also manipulates this machinery to its own end. These data add additional layers of complexity to current knowledge of the virus/host cell relationship, and thus reveal new avenues of research for future work.