No description available.

Methoden zur direkten Detektion oder Anreicherung von doppelsträngiger DNA (dsDNA) bieten ein hohes Potential zum Einsatz in der molekularen Diagnostik. Bereits etablierte Methoden für die Nukleinsäure - Detektion (NAD) basieren in der Regel auf der Hybridisierung des komplementären Stranges gefolgt von der optischen Detektion oder enzymatischer Amplifikation. DNA - bindende oder organisierende Proteine (z.B. endogene Transkriptionsfaktoren) bieten im Kontrast zu den Hybridisierungsreaktionen eine überaus interessante Alternative um dsDNA direkt und zugleich spezifisch zu detektieren oder diese aus einem komplexen Gemisch heraus anzureichern. Im Rahmen der Entwicklung von neuartigen NAD - Assays zur direkten Detektion oder Anreicherung von Nukleinsäuren wurden vier DNA - bindende Proteine kloniert und in HEK293 und E. coli exprimiert. Der Cys2His2 - Zinkfinger (ZFD) vom humanen Transkriptionsfaktor Sp1 wurde mit MBP und 9×Lys - MBP fusioniert. Das MBP - Derivat 9×Lys - MBP ist eine erweiterte Variante mit neun aufeinanderfolgenden Lysinen im N - terminalen Bereich, welche eine regioselektive Immobilisierung ermöglichen soll. Der humane Sp3 - ZFD wurde mit EGFP fusioniert. Die Mitglieder der Sp - Familie binden spezifisch die Konsensussequenz 5’ - GGG GCG GGG - 3’ (GC - Box). Zusätzlich wurde die C - terminale DNA - bindende Domäne der E. coli DNA - Gyrase Untereinheit A (gyrA - CTD) ebenfalls mit MBP fusioniert. Die Domäne bindet spezifisch repetitive extragene Palindrome (REP), welche bislang nur auf bakteriellen Chromosomen vorkommen. Sämtliche MBP - Fusionsproteine liegen nach der Expression löslich vor und konnten über eine native Strategie aufgereinigt werden. Transiente Transfektionsexperimente in HEK293 zeigten einen destabilisierenden Effekt der Sp3 - ZFD und eine massive einhergehende Degradierung des EGFP - Fusionsproteins nach 120 h. Die Analyse der mRNA - Integrität nach Transfektion des Expressionsplasmids, sowie zellbiologische und proteinbiochemische Untersuchungen mit Durchflusszytometrie bzw. Western Blots deuten auf eine posttranslationale Modulierung von EGFP - Sp3 hin. Um die Hypothese der proteasomalen Degradierung von EGFP - Sp3 zu belegen, wurden transfizierte HEK293 mit dem reversiblen Proteasominhibitor MG132 behandelt. In Gegenwart von 1 µM MG132 konnte das zytosolische Fusionsprotein stabilisiert werden. Die hier präsentierten Daten offenbaren die humane Sp3 - ZFD als ein neues Substrat für das 26S - Proteasom. Lediglich die SUMOylierung von Wildtyp - Sp3 im Bereich der inhibitorischen Domäne (ID) ist bislang beschrieben worden. Die Funktionalität, Affinität und kinetische Parameter der mit MBP fusionierten Sp1 - ZFD und gyrA - CTD wurden anhand von Oberflächenplasmonresonanz (BIAcore) bzw. EMSAs analysiert. Sämtliche gewonnenen MBP - Fusionsproteine sind funktionell und interagieren mit dsDNA. Fusionsproteine mit Sp1 - Domäne zeigten in EMSAs ebenso eine Bindung an unspezifische dsDNA. In sensitiveren BIAcore - Assays mit immobilisierter dsDNA wurden (um den Faktor 2) geringere Assoziations (ka) - und Dissoziationsraten (kd) von MBP - Sp1 ermittelt, wenn bestimmte Basen innerhalb der GC - Box ausgetauscht wurden. Die Affinität (Kd) von MBP - Sp1 mit 4×10 - 9 M zur GC - Box und deren Derivate ist vergleichbar mit der Kd von nativem Sp1. Die EMSA - Experimente für MBP - gyrA zeigen eine deutliche Präferenz zum spezifischen dsDNA - Oligo in Gegenwart von humaner gDNA, eine interessante Eigenschaft die durchaus zur Anwendung in einem Assay zur Anreicherung von bakterieller DNA dienen kann. Nach der vorausgehenden Charakterisierung der MBP - Fusionsproteine wurden diese auf verschiedenen gängigen festen und semifesten Substraten über physische Adsorption, kovalent oder Affinität immobilisiert um das Konzept der direkten Detektion von dsDNA mit funktionellen Proteinen als neuartige Komponente in NAD - Assays umzusetzen. Lediglich MBP - Sp1 zeigte auf Glas und Polystyren - Mikrotiterplatten nach kovalenter oder adsorptiver Immobilisierung eine ausgeprägte Funktionalität hinsichtlich der Bindung von dsDNA. Die Immobilisierung von 9×Lys - MBP - Sp1 über identische Strategien führten zum massiven Verlust der ZFD - Funktion. Aus dieser Datenlage heraus wurde erfolgreich ein simples Lumineszenz - basiertes Mikrotiterplatten - Assay mit MBP - Sp1 entwickelt um PCR - Amplikons direkt aus einer analytischen PCR auf gDNA von S. aureus, welche die GC - Box beinhalten, nachzuweisen. Das spezifische Amplikon konnte mittels des simplen Assays in Gegenwart von 100fachem Überschuss an humaner gDNA nachgewiesen werden. Mit einem höheren Anteil an humaner gDNA wurde die PCR massiv inhibiert, ein negativer Effekt der bislang im Bereich der diagnostischen NAD - Assays nicht optimal adressiert wurde. Die magnetische Separation von bakterieller und humaner gDNA wurde dazu mit MBP - gyrA umgesetzt. Zunächst erfolgte die regioselektive Immobilisierung von MBP - gyrA auf Protein A - funktionalisierte magnetische Nanopartikel mittels MBP - Antikörper, wodurch die Funktionalität hinsichtlich der Bindung von dsDNA gewährleistet werden konnte. Dieses System eignet sich insbesondere für die Separation von bakterieller DNA (E. coli oder S. aureus) aus einem komplexen Gemisch mit bis zu 100fachem Überschuss an humaner gDNA. Die Kombination von MBP - gyrA - basierter magnetischer Separation mit NAD - Assays könnte deren Sensitivität signifikant erhöhen. Durch simple Verfahrensweise bietet das System einen wesentlichen Beitrag zur Verringerung des zeitlichen Aufwands für die Generierung therapierelevanter Resultate. / Methods for direct detection or enrichment of double - stranded DNA (dsDNA) possess tremendous potential for use in molecular diagnostics. Already established methods for nucleic acid detection (NAD) are generally based on the hybridization of two complementary strands followed by optical detection or enzymatic amplification. In contrast, DNA - binding or organizing proteins (e.g. endogenous transcriptions factors) are able to read the sequence information directly from dsDNA without prior denaturation of the double strand and subsequent hybridization. In order to develop novel NAD assays or assays for sample preparation, four artificial DNA - binding proteins were cloned, expressed and purified in HEK293 cells or E. coli. The Cys2His2 zinc finger domains (ZFD) from human Sp1 were fused to maltose binding protein (MBP) and its derivate 9×Lys - MBP, an extended variant with nine successive lysine residues in the N - terminal region of the protein to facilitate site - directed immobilization. The human Sp3 - ZFD was fused to green fluorescent protein (EGFP). The family of Sp - transcription factors was known to bind specifically the consensus sequence 5\' - GGG GCG GGG - 3 \'(GC - box). Moreover, the C - terminal DNA - binding domain of E. coli DNA Gyrase subunit A (gyrA - CTD) was fused to MBP. The CTD binds specifically repetitive extragenic palindromes (REP), which were only found on prokaryotic chromosomes. All MBP fusion proteins were soluble after expression and could be purified to homogeneity. Surprisingly, transient transfection experiments in HEK293 revealed a destabilizing effect of the Sp3 - ZFD accompanied by massive degradation of the EGFP fusion protein after 120 h post transfection. Analysis of mRNA integrity in combination with western blots indicates a posttranslational modulation of EGFP - Sp3. To confirm the hypothesis of proteasomal degradation of EGFP - Sp3, transfected cells were treated with the reversible proteasome inhibitor MG132. In the presence of 1µM MG132 the fusion protein could be stabilized. Taken together, the data presented here identified the human Sp3 - CTD as a new substrate for the 26S proteasome. Only SUMOylation of wild type human Sp3 within the inhibitory domain (ID) has been described so far. Initial EMSA experiments showed that purified MBP - ZFD fusion proteins were functional in terms of interacting with dsDNA containing the specific sequence motiv. However, all proteins bound to unspecific dsDNA as well. Therefore MBP - Sp1 was subjected to BIAcore analysis to determine the rate constants for association ka, dissociation kd and the dissociation constant Kd of the GC - Box - Protein complex as well as mutants of the GC - Box. The determined Kd (4 × 10 - 9 M) for MBP - Sp1 associated with GC - box or its derivatives were found to be comparable with the Kd of native Sp1, however the rate constants were reduced 2 fold in presence of the modified GC - boxes. EMSA experiments with MBP - gyrA revealed functionality and a clear preference for specific dsDNA in the presence of unspecific human genomic DNA (gDNA). After preliminary functional characterization, MBP fusion proteins were immobilized by physical adsorption, covalent or by affinity on various solid substrates or nanoscaled magnetic beads to implement the concept of direct detection of dsDNA or specific enrichment of bacterial DNA, respectively. MBP - Sp1 remains functional after adsorptive or covalent immobilization on different chemical modified glas surfaces. 9×Lys - MBP - Sp1 shows significantly reduced functionality after immobilization on the same glas substrates by similar strategies. Moreover, a simple NAD - assay with adsorptive immobilized MBP - Sp1 on polystyrene in microtiter format was established for direct detection of GC - boxes within PCR - products from S. aureus gDNA. By using the assay, specific PCR - products could be detected in presence up to 100 - fold excess of human gDNA in relation to 10 ng bacterial DNA. Separation of bacterial DNA from human DNA from clinical samples may have an important impact on downstream applications, involving NAD assays. To address this often underestimated technical problem, a new functional protein MBP - gyrA was introduced to overcome some limitations of already established methods. MBP - gyrA was site - directed coupled on nanoscaled magnetic beads by affinity. This system enabled the fast and specific separation of gDNA of E. coli or S.aureus from a huge background of human gDNA. The combination of MBP - gyrA - based magnetic separation with NAD assays could significantly increase the sensitivity and shorten the time for initiation of effective treatment.

Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2017-12-15T11:25:42Z No. of bitstreams: 1 PGEF17MA082.pdf: 2704849 bytes, checksum: 402c8ed2d802138ba830b989401226b5 (MD5) / Made available in DSpace on 2017-12-15T11:25:42Z (GMT). No. of bitstreams: 1 PGEF17MA082.pdf: 2704849 bytes, checksum: 402c8ed2d802138ba830b989401226b5 (MD5) Previous issue date: 2017-04-28 / FAPESC / The use of the fiber reinforcement in structural elements of Glued Laminated Timber (Glulam) is advantageous because of increase its strength and stiffness. However, few studies have evaluated the performance of reinforcement on finger joints, which, even under excellent manufacturing conditions, shows less strength than solid wood, making this kind of joint one of the main weaknesses of Glulam. In this context, the present dissertation aims to evaluate the performance of reinforcement with fibers in finger-joints for use in Glulam. The performance analysis was developed under two aspects: (i) the tensile strength parallel to grain performance in two geometries, "A" and "B", on joints, with three reinforcement compositions, "Glass", "Glass2" and "Carbon", in two species of wood, Pinus taeda and Eucalyptus spp. and (ii) the performance of Glulam beams of Eucalyptus spp. with three reinforcement compositions, "Glass", "Glass2" and "Carbon", in tensile strength parallel to grain, normal tensile, strength to shear and three points bending test. All tests were performed according to the guidelines of NBR 7190/1997, using the Scott-Knott and Tukey tests for statistical analysis with 95% of confidence interval. The results show that the finger-joints, even within normative requirements, significantly reduces (up to 43%) the tensile strength parallel to grain, regardless of geometry or species. For the samples of Eucalyptus spp., the treatments "B-Glass2", "B-Carbon" and "A-Glass2" presented average strength equivalent to solid wood. For specimens of P. taeda, the treatments with the same performance as solid wood were "A-Glass2", "A-Carbon", "B-Glass2", "B-Carbon" and "B-Glass". It can be concluded that the application of two layers of reinforcement of glass fibers or one layer of carbon, concentrated in the region of the finger-joints with the geometry B, increases significantly (in up to 71% for P. taeda and 25% for Eucalyptus spp. in the treatments with better performance) the tensile strength parallel to grain in both species evaluated. It was also concluded that the apparent densities (Pinus taeda ρap,m= 0.49 and Eucalyptus spp. ρap,m= 0.60) show poor correlation with the tensile strength parallel to the fibers. The performance of the glulam beams of Eucalyptus spp. did not present significant differences for the evaluation of the bonding lines. However, in the flexural strength, the treatments "Glass 2" and "Carbon" were significantly superior to the glulam beams without reinforcement, reaching at increments of 37.8% and 40.5%, respectively, in the bending strength. The modulus of elasticity did not differ significantly among. It was observed that the mode of rupture was by tensile in the finger-joints region in all the beams evaluated in bending test, however, the bending strengths were higher than tensile strengths parallel to grain, indicating influence of the thickness of the blades and thickness of the reinforcements in the performance of the reinforced finger-joints. In this way, it is possible to conclude that the application of concentrated reinforcement in finger-joints significantly improves the performance of glulam beams of Eucalyptus spp. to bending, as well as the tensile strength parallel to grain for P. taeda / A aplicação de reforço com fibras em elementos estruturais de Madeira Laminada Colada (MLC), na linha de colagem de maior esforço, se apresenta de maneira vantajosa por aumentar sua resistência mecânica e sua rigidez. Todavia, poucos estudos avaliam o desempenho do reforço sobre as emendas longitudinais, que, mesmo em condições excelentes de manufatura, têm resistência menor que a da madeira maciça, tornando as emendas um dos principais pontos de fragilidade da MLC. Nesse contexto, a presente dissertação tem por objetivo avaliar o desempenho do reforço com fibras em emendas por entalhes múltiplos (finger-joints) para utilização em MLC. A análise de desempenho foi desenvolvida sob dois aspectos: (i) desempenho à tração paralela às fibras de duas geometrias, “A” e “B”, para emendas, com três composições de reforço, “Vidro”, “Vidro2” e “Carbono”, em duas espécies de madeira, o Pinus taeda e o Eucalyptus spp. e (ii) o desempenho de vigas em MLC de Eucalyptus spp.com três composições de reforço, “Vidro”, “Vidro2” e “Carbono” quanto à resistência à tração paralela às fibras, tração perpendicular às fibras, cisalhamento e flexão estática. Todos os ensaios foram executados de acordo com as diretrizes da NBR 7190/1997, sendo empregados os testes de Scott-Knott e Tukey para as análises estatísticas com 95% de confiabilidade. Os resultados mostram que as emendas por entalhes múltiplos, mesmo dentro dos referenciais normativos, diminuem significativamente (em até 43%) a resistência à tração paralela às fibras, independentemente da geometria ou da espécie utilizada. Para as amostras de Eucalyptus spp., os tratamentos “B-Vidro2”, “B-Carbono” e “A-Vidro2” apresentaram resistência mecânica média equivalente à madeira maciça. Para as amostras de P. taeda, os tratamentos com desempenho mecânico similar à madeira maciça foram “A-Vidro2”, “A-Carbono”, “B-Vidro2”, “B-Carbono” e “B-Vidro”. Pode-se concluir que a aplicação em duas camadas de reforço de fibras de vidro ou uma camada de carbono, ambos concentrados na região das emendas por entalhes múltiplos com a geometria B, aumenta significativamente (em até 71% para o P. taeda e 25% para o Eucalyptus spp. nos tratamentos com melhor desempenho) a resistência à tração paralela às fibras em ambas espécies avaliadas. Conclui-se ainda que a densidade aparente (P. taeda ρap,m= 0,49 e Eucalyptus spp. ρap,m =0,60 ) apresenta fraca correlação com a resistência à tração paralela às fibras. O desempenho mecânico das vigas MLC de Eucalyptus spp. não apresentou diferenças significativas para a avaliação das linhas de colagem. Entretanto, na resistência à flexão estática, os tratamentos “Vidro 2” e “Carbono” foram significativamente superiores às vigas MLC sem reforço, chegando a incrementos de 37,8% e 40,5%, respectivamente, à flexão estática. Os módulos de elasticidade não diferiram significativamente entre si. Foi constatada ruptura por tração na região das emendas em todas as vigas avaliadas à flexão estática, entretanto as resistências à flexão foram superiores às resistências à tração paralela às fibras, indicando influência da espessura das lâminas e da espessura dos reforços no desempenho mecânico das emendas reforçadas. Desta forma, é possível concluir que a aplicação de reforço concentrado na região das emendas por entalhes múltiplos melhora, significativamente, o desempenho de vigas MLC de Eucalyptus spp. à flexão, bem como a resistência à tração paralela às fibras para o P. taeda

Made available in DSpace on 2016-03-15T19:40:36Z (GMT). No. of bitstreams: 1 Aline Rodrigues Sorcinelli.pdf: 545203 bytes, checksum: 537c38908ca66dbcaceb6f9079a917e5 (MD5) Previous issue date: 2008-08-29 / The shortage of normative data in instruments and specific tests for the Brazilian population is a challenge to the professionals that intend to measure the effectiveness of its work. At the practical clinic, many diagnoses, preventions and treatments are established with the use of tests or measuring instruments. Instruments and tests that cover the motor developing aspects of manual coordination ability are even harder to find on the Brazilian children s norms and are important to notice the motor coordination issues that may affect their ability to take care of themselves, social activities and school performance. This study intended to find validation evidences of three manual motor ability tests, Purdue Pegboard, Finger Tapping and Simple Reaction Time in 134 right-handed children of 5 and 6 years old from two schools based in São Paulo, one of which is a private school and the other one is a public school with different socioeconomic levels. The specific objectives were intended to compare the performance of the manual motor ability tests between sexes and ages of 5 and 6 years olds, and to verify whether there was a performance difference in the tests between the children from the public and private school. The results showed a significant correlation between the three tests helping to validate the theory. Regarding the specified objectives, it was observed that between all the manual motor ability, only on the Purdue Pegboard 3 and 4 sub-tests did the girls perform better than the boys; the older children achieved a better performance on the tests; and the manual motor ability tests performance doesn t depend on which type of school the children attend. / A escassez de dados normativos em instrumentos e testes específicos para a população brasileira é um desafio aos profissionais que propõem mensurar a efetividade de seu trabalho. Na prática clínica, muitos diagnósticos, assim como prevenções e tratamentos podem ser estabelecidos com o uso de testes ou instrumentos de mensuração. Instrumentos e testes que abrangem aspectos do desenvolvimento motor como habilidade motora manual são ainda mais difíceis de estarem disponíveis nas normas para crianças brasileiras. Estes são importantes para a detecção de problemas de coordenação motora, que podem influenciar nas atividades de auto-cuidado, nas atividades sociais e no desempenho escolar. Este estudo propôs buscar evidências de validação de três testes de habilidade motora manual, Purdue Pegboard, Finger Tapping e Tempo de Reação Simples em 134 crianças de cinco e seis anos de idade. Todas destras, de duas escolas paulistanas, sendo uma escola particular e outra pública, com níveis sócioeconômicos distintos. Como objetivos específicos, a pesquisa propôs comparar o desempenho dos testes de habilidade motora manual entre os sexos, e entre as idades de cinco e seis anos. Além de verificar se existe diferenças no desempenho dos testes entre os sujeitos da escola pública e particular. Os resultados apontaram correlações significantes entre os três testes, o que auxiliou no processo de validade da construção. Em relação aos objetivos específicos observamos que entre todos os testes de habilidade motora manual, apenas nos sub-testes 3 e 4 do Purdue Pegboard, as meninas tiveram melhor desempenho que os meninos, os sujeitos com maior idade obtiveram melhor desempenho nos testes e o desempenho dos testes de habilidade motora manual independe do tipo de escola que o sujeito freqüenta.

<p>Duchenne muscular dystrophy (DMD) is a severe hereditary disorder caused by a loss of dystrophin, an essential musculoskeletal protein. Decades of promising research have yielded only modest gains in survival and quality of life for these patients and there have been no approved gene therapies for DMD to date. There are two significant hurdles to creating effective gene therapies for DMD; it is difficult to deliver a replacement dystrophin gene due to its large size and current strategies to restore the native dystrophin gene likely require life-long administration of a gene-modifying drug. This thesis presents a novel method to address these challenges through restoring dystrophin expression by genetically correcting the native dystrophin gene using engineered nucleases that target one or more exons in a mutational hotspot in exons 45-55 of the dystrophin gene. Importantly, this hotspot mutational region collectively represents approximately 62% of all DMD mutations. In this work, we utilize various engineered nuclease platforms to create genetic modifications that can correct a variety of DMD patient mutations.</p><p>Initially, we demonstrate that genome editing can efficiently correct the dystrophin reading frame and restore protein expression by introducing micro-frameshifts in exon 51, which is adjacent to a hotspot mutational region in the dystrophin gene. Transcription activator-like effector nucleases (TALENs) were engineered to mediate highly efficient gene editing after introducing a single TALEN pair targeted to exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from DMD patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. We show that our engineered TALENs have minimal cytotoxicity and exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein coding regions of the genome, as predicted by in silico target site analysis. </p><p>In an alternative approach, we capitalized on the recent advances in genome editing to generate permanent exclusion of exons by using zinc-finger nucleases (ZFNs) to selectively remove sequences important in specific exon recognition. This strategy has the advantage of creating predictable frame restoration and protein expression, although it relies on simultaneous nuclease activity to generate genomic deletions. ZFNs were designed to remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript, a method that can potentially restore the dystrophin reading frame in up to 13% of DMD patients. Nucleases were assembled by extended modular assembly and context-dependent assembly methods and screened for activity in human cells. Selected ZFNs had moderate observable cytotoxicity and one ZFN showed off-target activity at two chromosomal loci. Two active ZFN pairs flanking the exon 51 splice acceptor site were transfected into DMD patient cells and a clonal population was isolated with this region deleted from the genome. Deletion of the genomic sequence containing the splice acceptor resulted in the loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin expression in vitro. Furthermore, transplantation of corrected cells into the hind limb of immunodeficient mice resulted in efficient human dystrophin expression localized to the sarcolemma. </p><p>Finally, we exploited the increased versatility, efficiency, and multiplexing capabilities of the CRISPR/Cas9 system to enable a variety of otherwise challenging gene correction strategies for DMD. Single or multiplexed sgRNAs were designed to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing either intraexonic small insertions and deletions, or large deletions of one or more exons. Significantly, we generated a large deletion of 336 kb across the entire exon 45-55 region that is applicable to correction of approximately 62% of DMD patient mutations. We show that, for selected sgRNAs, CRISPR/Cas9 gene editing displays minimal cytotoxicity and limited aberrant mutagenesis at off-target chromosomal loci. Following treatment with Cas9 nuclease and one or more sgRNAs, dystrophin expression was restored in Duchenne patient muscle cells in vitro. Human dystrophin was detected in vivo following transplantation of genetically corrected patient cells into immunodeficient mice. </p><p>In summary, the objective of this work was to develop methods to genetically correct the native dystrophin as a potential therapy for DMD. These studies integrate the rapid advances in gene editing technologies to create targeted frameshifts that restore the dystrophin gene around patient mutations in non-essential coding regions. Collectively, this thesis presents several gene editing methods that can correct patient mutations by modification of specific exons or by deletion of one or more exons that results in restoration of the dystrophin reading frame. Importantly, the gene correction methods described here are compatible with leading cell-based therapies and in vivo gene delivery strategies for DMD, providing an avenue towards a cure for this devastating disease.</p> / Dissertation

No description available.

<p>The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.</p><p>Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible.</p><p>The recent emergence of the field of optogenetics--the ability to control gene expression using light--has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.</p><p>Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific three base-pair DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or eGFP transgene enabled expression of the transgene in response to blue-light. Gene activation was both reversible and tunable based on duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be spatially patterned by illuminating the cell culture through photomasks containing various patterns.</p><p>Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a light-activated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the N-terminal fragment of CIB1 to the N-, C-, or N- and C- terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask. </p><p>Finally, we engineered a light-activated genetic "on" switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase-mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system,1 could be used to express MyoD or the synthetic transcription factor VP64-MyoD2 in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated upregulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T½ cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased microvessel density compared to mice that were not illuminated. Analysis of human VEGF and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.</p><p>In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.</p> / Dissertation

As an extension of previous studies (Urbanczyk, Angel, & Kennelly, 1988) examining the effects of unimanual finger tapping on lateralized cognitive tasks, lateral body orienting was added to an established dual task paradigm to generate differential hemispheric activation and shifts of attention. One hundred twenty university students retained sequences of digits or spatial locations for 20 seconds either alone or during finger tapping. By turning both head and eyes left or right, the hemisphere congruent with the sequences (LH for digits, RH for locations) or incongruent (vice versa) was activated. Activation had little effect on retention means but greatly affected resource composition supporting task performance. Congruent orientation produced significantly higher positive correlations between digit and location tasks than incongruent orientation. Females showed higher sequence retention correlations than males across both orienting groups. For females, congruent activation enhanced tapping rates and retention-tapping correlations. For males, activation affected neither of these. Discussed in light of neuroanatomical research, these results suggest that congruent attentional orienting may integrate regions of the less activated hemisphere into networks of the more activated hemisphere. This unification may occur more readily across the female corpus callosum, producing a greater dependence upon a general attentional resource than for males, who appear to depend more upon hemispheric resources.

The problem was to design and construct the necessary equipment to cut and fasten short stock end to end using finger-joints. The study was divided into five chapters: I contained the introduction to the problem; II was concerned with the design and construction of the equipment; III detailed the operation of the equipment; IV contained the presentation of the data; and V covered the summary and findings. The study concluded that the equipment could be constructed inexpensively, and would perform a useful service. Also, a student using waste wood salvaged through use of this equipment could expect a smaller bill for materials than if he purchased new wood.

Bioinorganic chemistry strives to understand the roles of metals in biological systems, whether in the form of naturally occurring or addition of non-essential metals to natural systems. Metal ions play vital roles in many cellular functions such as gene expression/regulation and DNA transcription and repair. The study of metal-protein-DNA/RNA interactions has been relatively unexplored. It is important to understand the role of metalloprotein interactions with DNA/RNA as this enhanced knowledge may lead to better understanding of diseases and therefore more effective treatments. A major milestone in the development of this field was the discovery of the cytotoxic properties of cisplatin in 1965 and its FDA approval in 1978. Since then, two other chemotherapeutic drugs containing platinum, carboplatin and oxaliplatin, have been used in the clinic. These three compounds are all bifunctional with the ligands surrounding platinum In the cis conformation and rearrangement of the ligands to the trans orientation results in a loss of cytotoxic properties due to rapid deactivation through binding to S-containing proteins. This enhanced reactivity yields new opportunities to study the reactions between proteins and DNA. One of the first crosslinking experiments used transplatin to crosslink NCp7 to viral RNA in order to understand how/where the protein bound to RNA. We have studied the interaction between cis and trans dinuclear platinum complexes and the C-terminal zinc finger (ZF). The trans complex reacts at a faster rate than the cis isomer and causes N- terminal specific cleavage of the ZF. The dinuclear structure plays a critical role in the peptide cleavage as studies with transplatin (the mononuclear derivative) does not result in cleavage. Monofunctional trans platinum-nucleobase complexes (MPNs) serve as a model for the binding of transplatin to DNA. This provides an interesting opportunity to study their reactions with S-containing proteins, such as HIV1 NCp7. MPNs have been shown to bind to the C-terminal ZF of HIV1 NCp7, resulting in zinc ejection. This occurs through a two-step process where the nucleobase π-stacks with Trp37 on the ZF, followed by covalent binding at the labile Cl site to Cys. MPNs have also shown antiviral activity in vitro. The labile Cl on MPNs reduces specificity of these compounds, as it leaves an available coordination site on the platinum center for binding to other S-proteins or DNA. Therefore, we have moved to an inert PtN4 coordination sphere, [Pt(dien)L]2+ (dien= diethylenetri- amine). Due to the strong bond between platinum and nitrogen, covalent reactions are highly unlikely to occur at rapid rates. The strength of the pi-stacking interaction between nucleobases (free and platinated) and the aromatic amino acid, tryptophan (Trp), showed an enhanced binding constant for platinated nucleobases. This was confirmed by density functional theory (DFT) calculations as the difference in energy between the HOMO of Trp and the LUMO of the nucleobase was smaller for the platinum complex. The studies were extended to the Trp-containing C-terminal ZF of HIV1 NCp7 and an increase in association constant was seen compared to free Trp. Reaction of PtN4 nucleobases compounds with a short amino acid sequence con- taining either Ala (no pi-stacking capabilities) or Trp (pi-stacking interactions) revealed an enhanced rate of reactivity for the Trp-containing peptide. This result supports the theory of a two-step reaction mechanism where the platinum-nucleobase complex recognizes the pep- tide through a pi-stacking interaction with Trp followed by covalent binding to the platinum center. The [Pt(dien)L]2+ motif allows for systematic modification of the structural elements surrounding platinum in a search for the most effective compound. Methylation of the dien ligand should, in theory, increase lipophilicity of the compounds, however, due to 2+ charge of the compounds, this simple association does not hold true. Analysis of the cellular accumulation profiles showed little change in the uptake with the addition of methyl groups to the dien ligand, in agreement with the non-linear change in lipophilicity. Modification of L using different nucleobases allows for the tuning of the strength of the π-stacking interaction between Trp and the platinum complex. The addition of inosine (which lacks a H-bonding donor/acceptor at the C2 position) resulted in a lower association constant with both N-AcTrp and the C-terminal zinc finger of HIV1 NCp7. Interestingly, the addition of xanthosine resulted in an ehanced pi-stacking interaction with the C-terminal zinc finger of HIV1 NCp7; likely as a results of the addition of a H-bonding donor (double-bonded O) at the C2 position. The ability of PtN4 nucleobase complexes to inhibit formation of the NCp7 complexation with viral RNA was studied by mass spectrometry and gel electrophoresis. Dissociation of the NCp7-RNA complex was seen upon addition of PtN4 compounds. These compounds were also able to retard formation of the NCp7-RNA complex when pre-incubated with the protein. These results have important implications as inhibition of complex formation between NCp7 and viral RNA has negative implications for viral replication. Despite the success of platinum-nucleobase compounds, it is important to evaluate all potential pi-stacking ligands. A series of pyridine- and thiazole-based compounds were evaluated for the strength of the pi-stacking interaction with N-AcTrp and the C-terminal ZF of HIV1 NCp7. There was notable increase in association constant for the platinum- DMAP (4-dimethylaminopyridine) complex compared to other ligands studied. This result highlights the importance of exploring multiple avenues for the design of specifically targeted inhibitors and further confirms the viability of the medicinal chemistry dual approach of target recognition (non-covalent) followed by target fixation (covalent).