Transposons are DNA segments that can move or transpose themselves to new positions within the genome of an organism. Biologists need to predict preferred insertion sites of transposons to devise strategies in functional genomics and gene therapy studies. It has been found that the deformability property of the local DNA structure of the integration sites, called Vstep, is of significant importance in the target-site selection process. We considered the Vstep profiles of insertion sites and developed predictors based on Artificial Neural Networks (ANN) and Support Vector Machines (SVM). We trained our ANN and SVM predictors with the Sleeping Beauty transposonal data, and used them for identifying preferred individual insertion sites (each 12bp in length) and regions (each 100bp in length). Running a five-fold cross-validation showed that (1) Both ANN and SVM predictors are more successful in recognizing preferred regions than preferred individual sites; (2) Both ANN and SVM predictors have excellent performance in finding the most preferred regions (more than 90% sensitivity and specificity); and (3) The SVM predictor outperforms the ANN predictor in recognizing preferred individual sites and regions. The SVM has 83% sensitivity and 72% specificity in identifying preferred individual insertion sites, and 85% sensitivity and 90% specificity in recognizing preferred insertion regions.

Transposons are DNA segments that can move or transpose themselves to new positions within the genome of an organism. Biologists need to predict preferred insertion sites of transposons to devise strategies in functional genomics and gene therapy studies. It has been found that the deformability property of the local DNA structure of the integration sites, called Vstep, is of significant importance in the target-site selection process. We considered the Vstep profiles of insertion sites and developed predictors based on Artificial Neural Networks (ANN) and Support Vector Machines (SVM). We trained our ANN and SVM predictors with the Sleeping Beauty transposonal data, and used them for identifying preferred individual insertion sites (each 12bp in length) and regions (each 100bp in length). Running a five-fold cross-validation showed that (1) Both ANN and SVM predictors are more successful in recognizing preferred regions than preferred individual sites; (2) Both ANN and SVM predictors have excellent performance in finding the most preferred regions (more than 90% sensitivity and specificity); and (3) The SVM predictor outperforms the ANN predictor in recognizing preferred individual sites and regions. The SVM has 83% sensitivity and 72% specificity in identifying preferred individual insertion sites, and 85% sensitivity and 90% specificity in recognizing preferred insertion regions.

Indiana University-Purdue University Indianapolis (IUPUI) / Lentiviral vector mediated gene therapy has made great strides in recent years with several successful clinical trials. However, adverse events encountered with some early trials have highlighted the necessity to improve upon its safety. Improvements can range from early steps in vector production to evaluation of insertion sites post-transduction. We have evaluated an FDA approved DNase for removal of residual plasmid DNA during vector production, developed novel non-integrating lentiviral vectors and employed modified insertion site analysis post-transduction to improve the safety of lentiviral vector mediated gene therapy. To prevent the exposure of gene therapy patients to HIV-1 DNA it is essential to remove residual plasmid DNA during vector production. We evaluated a recombinant human DNase which has been FDA approved for use in patients as an alternative to a bacterially derived DNase. Our results indicate this DNase is an effective alternative with a potentially safer profile for use in patients. The ability of lentiviral vectors to stably integrate their genome into a host cell’s DNA can have negative side-effects due to the risk of insertional mutagenesis. Non-integrating lentiviral vectors have been developed to alleviate this risk in applications where integration is not necessary. However, a low frequency of illegitimate integration persists when using these vectors. We have developed a novel non-integrating vector mutation and evaluated the efficacy of combining it with other mutations for reducing the frequency of illegitimate integration. We demonstrate that combining mutations that inhibit integration can further reduce the frequency of illegitimate integration. Several methodologies have been developed for evaluating the insertion sites of normal integrating lentiviral vectors. Illegitimate integration by non-integrating vectors demonstrates mechanisms which result in insertions and/or deletions at the vector-genome junction. Current methods lack the sensitivity to account for these variables in a high-throughput manner. We have adapted modifications to current methods to improve the capture of these variable insertion sites for analysis. The results of these studies improve the safety of lentiviral vector mediated gene therapy by improving the purity of the vector product, providing a safer vector for non-integrase mediated applications, and allowing more sensitive analysis of insertion sites post-transduction.

No description available.

O uso do Cateter Venoso Central (CVC) viabiliza a assistência de alta complexidade aos pacientes críticos. Contudo, as infecções desses cateteres demonstram uma associação com a migração dos microrganismos da pele do local de inserção até a ponta do cateter. A utilização de novas tecnologias para reduzir a colonização cutânea no sítio de inserção do cateter reforça a questão da antissepsia cutânea e a sugestão do curativo adequado a esta região. As recomendações do Centers for Disease Control and Prevention (CDC) apontam como uma opção de curativo o uso da cobertura de filme transparente de poliuretano (FTP) que protege e facilita a visualização do sítio de inserção. Contudo, o uso do curativo gel de clorexidina (CGCHX), vem apresentado uma resposta superior na prevenção dessas colonizações e infecções. Diante do exposto, propôs-se identificar por meio de um estudo de corte transversal prospectivo, os microrganismos presentes no swab de pele após o uso do CGCHX e do FTP, no momento da retirada do CVC. Fizeram parte do estudo um total de 92 pacientes, destes 45 pacientes em uso do CGCHX e 47 utilizaram FTP, em ambos os grupos, a faixa etária foi em média 60 anos, declararam-se brancos e não houve diferença significativa entre o número de homens e mulheres. A escolha do sítio de inserção preferencial foi a veia jugular interna direita. Houve o crescimento em 13 amostras de Swabs no grupo do CGCHX os microrganismos encontrados foram Acinetobacter baumannii, Pseudomonas aeruginosa, Morganella morganii, Enterobater cloacae, Staphylococcus aereus e Staphylococcus epidermidis. O Staphylococcus aereus apresentou resistência a oxacilina, o que podemos sugerir a possibilidade de uma cepa MRSA. Quanto ao FTP foram seis amostras positivas com o crescimento de Serratia marcescens, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus epidermidis e Staphylococcus haemolyticus. A presença da Klebsiella pneumoniae sensível somente a amicacina sugere a possibilidade da cepa Klebsiella pneumoniae carbapenemas (KPC). Apesar dos achados evidenciarem um número maior de crescimento bacteriano no curativo gel de CHX, este estudo pode contribuir para o olhar e desenvolvimento de novos trabalhos para a nossa população, levando em consideração nosso clima e tipo de pele, como também a importância de medidas educativas permanentes no treinamento da equipe de saúde nos centros onde as novas tecnologias são implementadas / The use of Central Venous Catheter (CVC) enables the high complexity assistance to critically ill patients. However, these catheters infections show an association with the migration of skin microorganisms from the insertion site to the tip of the catheter. The use of new technologies to reduce skin colonization on the catheter insertion site reinforces the issue of skin disinfection and use of suitable dressing to this region. The recommendations from the Centers for Disease Control and Prevention (CDC) show the use of coverages with transparent polyurethane film (TPF) as an option of dressing, which protects and facilitates the visualization of the insertion site. However, the use of chlorhexidine gel dressing (CGD) has presented a higher response in preventing these colonizations and infections. Given the above, this study aimed to identify the microorganisms present on the skin swab after using the CGD and TPF at the time of removal of the CVC from a prospective cross-sectional study. The total of participants were 92 patients, 45 of them were using CGD and 47 were using FTP. In both groups, the average age was 60 years, white, and there was no significant difference between the number of men and women. The choice of preferred insertion site was the right internal jugular vein. There was growth in 13 samples Swabs in the CGD group. Microorganisms found were Acinetobacter baumannii, Pseudomonas aeruginosa, Morganella morganii, Enterobater cloacae, Staphylococcus aereus and Staphylococcus epidermidis. Staphylococcus aereus showed resistance to oxacillin, which can suggest the possibility of a MRSA strain. Related to the FTP group, there were six positive samples with the growth of Serratia marcescens, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus epidermidis and Staphylococcus haemolyticus. The presence of Klebsiella pneumoniae sensitive only to amikacin suggests the possibility of Klebsiellapneumoniaecarbapenemase strain (KPC). Despite findings showing a greater number of bacterial growth in the chlorhexidine gel dressing, this study may contribute to the development of new studies for population, taking into account the climate and skin type, as well as the importance of educational measures for the training of health team in centers where new technologies are implemented

<p>Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 14 months. </p><p>The present thesis describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation. Newborn mice were injected intracerebrally with a Moloney murine leukemia retrovirus carrying the <i>sis</i>/PDGF-B oncogene and a replication competent helper virus. Brain tumors with many characteristics of human glioblastomas developed after 13-42 weeks. </p><p>Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes.</p><p>An array with over 15000 unique cDNAs was used to screen for differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors.</p><p>The gene <i>Prkg2</i>, encoding the cGMP-dependent protein kinase II, was targeted by insertions in two brain tumors. Overexpression of <i>Prkg2</i> in human glioma cell lines led to a reduction in colony formation, cell proliferation and migration. A glioma cell line expressing markers of immature stem cells showed loss of cell adhesion, G1 cell cycle arrest and decreased activation of the survival signaling protein Akt upon stimulation with a cGMP analog that activates the <i>Prkg2</i> protein. The present thesis shows that proviral tagging may be a useful tool in the search for candidate glioma genes.</p>

Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 14 months. The present thesis describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation. Newborn mice were injected intracerebrally with a Moloney murine leukemia retrovirus carrying the sis/PDGF-B oncogene and a replication competent helper virus. Brain tumors with many characteristics of human glioblastomas developed after 13-42 weeks. Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes. An array with over 15000 unique cDNAs was used to screen for differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors. The gene Prkg2, encoding the cGMP-dependent protein kinase II, was targeted by insertions in two brain tumors. Overexpression of Prkg2 in human glioma cell lines led to a reduction in colony formation, cell proliferation and migration. A glioma cell line expressing markers of immature stem cells showed loss of cell adhesion, G1 cell cycle arrest and decreased activation of the survival signaling protein Akt upon stimulation with a cGMP analog that activates the Prkg2 protein. The present thesis shows that proviral tagging may be a useful tool in the search for candidate glioma genes.

O uso do Cateter Venoso Central (CVC) viabiliza a assistência de alta complexidade aos pacientes críticos. Contudo, as infecções desses cateteres demonstram uma associação com a migração dos microrganismos da pele do local de inserção até a ponta do cateter. A utilização de novas tecnologias para reduzir a colonização cutânea no sítio de inserção do cateter reforça a questão da antissepsia cutânea e a sugestão do curativo adequado a esta região. As recomendações do Centers for Disease Control and Prevention (CDC) apontam como uma opção de curativo o uso da cobertura de filme transparente de poliuretano (FTP) que protege e facilita a visualização do sítio de inserção. Contudo, o uso do curativo gel de clorexidina (CGCHX), vem apresentado uma resposta superior na prevenção dessas colonizações e infecções. Diante do exposto, propôs-se identificar por meio de um estudo de corte transversal prospectivo, os microrganismos presentes no swab de pele após o uso do CGCHX e do FTP, no momento da retirada do CVC. Fizeram parte do estudo um total de 92 pacientes, destes 45 pacientes em uso do CGCHX e 47 utilizaram FTP, em ambos os grupos, a faixa etária foi em média 60 anos, declararam-se brancos e não houve diferença significativa entre o número de homens e mulheres. A escolha do sítio de inserção preferencial foi a veia jugular interna direita. Houve o crescimento em 13 amostras de Swabs no grupo do CGCHX os microrganismos encontrados foram Acinetobacter baumannii, Pseudomonas aeruginosa, Morganella morganii, Enterobater cloacae, Staphylococcus aereus e Staphylococcus epidermidis. O Staphylococcus aereus apresentou resistência a oxacilina, o que podemos sugerir a possibilidade de uma cepa MRSA. Quanto ao FTP foram seis amostras positivas com o crescimento de Serratia marcescens, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus epidermidis e Staphylococcus haemolyticus. A presença da Klebsiella pneumoniae sensível somente a amicacina sugere a possibilidade da cepa Klebsiella pneumoniae carbapenemas (KPC). Apesar dos achados evidenciarem um número maior de crescimento bacteriano no curativo gel de CHX, este estudo pode contribuir para o olhar e desenvolvimento de novos trabalhos para a nossa população, levando em consideração nosso clima e tipo de pele, como também a importância de medidas educativas permanentes no treinamento da equipe de saúde nos centros onde as novas tecnologias são implementadas / The use of Central Venous Catheter (CVC) enables the high complexity assistance to critically ill patients. However, these catheters infections show an association with the migration of skin microorganisms from the insertion site to the tip of the catheter. The use of new technologies to reduce skin colonization on the catheter insertion site reinforces the issue of skin disinfection and use of suitable dressing to this region. The recommendations from the Centers for Disease Control and Prevention (CDC) show the use of coverages with transparent polyurethane film (TPF) as an option of dressing, which protects and facilitates the visualization of the insertion site. However, the use of chlorhexidine gel dressing (CGD) has presented a higher response in preventing these colonizations and infections. Given the above, this study aimed to identify the microorganisms present on the skin swab after using the CGD and TPF at the time of removal of the CVC from a prospective cross-sectional study. The total of participants were 92 patients, 45 of them were using CGD and 47 were using FTP. In both groups, the average age was 60 years, white, and there was no significant difference between the number of men and women. The choice of preferred insertion site was the right internal jugular vein. There was growth in 13 samples Swabs in the CGD group. Microorganisms found were Acinetobacter baumannii, Pseudomonas aeruginosa, Morganella morganii, Enterobater cloacae, Staphylococcus aereus and Staphylococcus epidermidis. Staphylococcus aereus showed resistance to oxacillin, which can suggest the possibility of a MRSA strain. Related to the FTP group, there were six positive samples with the growth of Serratia marcescens, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus epidermidis and Staphylococcus haemolyticus. The presence of Klebsiella pneumoniae sensitive only to amikacin suggests the possibility of Klebsiellapneumoniaecarbapenemase strain (KPC). Despite findings showing a greater number of bacterial growth in the chlorhexidine gel dressing, this study may contribute to the development of new studies for population, taking into account the climate and skin type, as well as the importance of educational measures for the training of health team in centers where new technologies are implemented