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Thesis (M.S. in M.E.)--School of Mechanical Engineering, Georgia Institute of Technology, 2004. Directed by Thomas R. Kurfess. / Includes bibliographical references (leaves 110-112).

Nanotechnology is a rapidly growing field focused on the manipulation and control of materials with dimensions under 100 nm. The novel electronic, optical and mechanical properties observed at the nanoscale have resulted in a number of applications in catalysis, light emitting devices, solar power, self-cleaning surfaces and medicine. Medical applications of nanotechnology (“nanomedicine”) are particularly promising for rapid clinical diagnosis and targeted treatments. Understanding the interactions of nanoparticles with living matter is of fundamental importance for all application areas: manufacture, use and disposal of the growing number of nanoproducts will result in increased environmental exposure in addition to direct exposure through nanomedical applications. However, there is a lack of standard methodologies for assessing these interactions. In this work the stability of silver-based nanoparticles was established by UV- Visible (UV-Vis) spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM). The presence of a higher valence metal or polymer on the nanoparticle surface was demonstrated to improve stability. A standard methodology was developed to study nanoparticle-cell interactions: an “atlas” of the effects of known drugs on a cell is created, and compared with the effects of a nanoparticle. Escherichia coli was selected as a model organism and the effects of a range of antibiotics were characterised through a combination of microbiological assays and AFM. Susceptibility, population cell growth and individual heights, widths, lengths and volumes of bacteria were obtained on a 2% agarose substrate in air. The methodology was applied and adjusted for silver nanoparticles due to the interactions of silver with the bacterial growth medium. 10 and 30 nm silver nanoparticles and ions were found to kill E. coli through an internal mechanism of action, with a size-specific effect on the height of bacteria. Finally, a novel AFM characterisation method is described to examine the mechanical properties of live bacterial and human cells in liquid.

La particule de reconnaissance du signal (signal recognition particle-SRP) et son récepteur (FtsY chez Escherichia coli) médiatise le processus simultané de traduction-ciblage de la protéine en dirigeant le complexe ribosome-nascent chain (RNCs) vers la membrane de destination. La reconnaissance par la SRP d'une charge RNC à transporter dépend de la présence de la partie N-terminale. L'assemblage de Ftsy au complexe RNC-PRS entraine plusieurs changements de configuration de SRP et de FtsY durant le cycle de direction. D'abord un stade « précoce » sans GTP est adopté. Celui-ci est stabilisé par le RNC. Ensuite une configuration « fermée » avec GTP est formée. Cette dernière peut s'activer pour hydrolyser GTP, elle entre alors dans sa configuration « active ». La succession de ces trois étapes conduit à la libération du complexe SRP-récepteur d'avec le ribosome et de sa protéine en cours de traduction, et leur mise à disposition au pore de la membrane. Dans ce projet, notre intérêt se limite à la traduction par le ribosome de la séquence signale EspP (RNCEspP). In vivo, EspP est une protéine dont le ciblage vers le récepteur membranaire se réalise après la traduction. Cependant il arrive que RNCEspP se lie au complexe SRP-FtsY, faisant échouer le ciblage. Nous avons étudié les bases structurales du rejet de RNCEspP par SRP et FtsY. Pour cela nous avons effectué la comparaison de la structure RNCEspP-SRP-FtsY obtenue par observation au cryo-microscope électronique avec d'autres complexes ribosome-SRP-récepteurs traduisant la charge FtsQ, qui est elle normalement ciblé par SRP. Nous avons cherché à observer la différence de structure entre les complexes SRP-FtsY dans les deux cas. Deux différences majeurs entre les complexes de ciblages contenants les séquences RNCFtsQ et RNCEspP ont été observés. Premièrement, dans le cas de la structure de RNCEspP le domaine M -Ffh est attaché à l'hélice 59 du ribosome, alors que celui-ci est détaché dans le cas de la structure de RNCFtsQ. Nous pensons que le domaine M empêche la libération de la séquence de signal, étape nécessaire à la réalisation du ciblage. Deuxièmement, dans le cas de la structure du complexe avec RNCEspP l'arrangement Ffh-FtsY avec le domaine NG était flexible. Ceci indiquerait que le complexe “précoce” formé sur RNCEspP est moins stable que celui formé sur RNCFtsQ. Une étude biochimique utilisant le transfert d'énergie via résonance fluorescente a corroboré ce résultat, montrant que FTS Y est lié avec une affinité moindre dans le cas du complexe précoce formé sur RNCEspP et que la reconfiguration au stade de complexe fermé est moins efficace. Une analyse biochimique plus poussée des variantes de la séquence de EspP montre que la partie N-Terminale de la séquence est la principale cause de rejet du cycle de ciblage via SRP.Dans un second projet, nous avons étudié la configuration “fermée” de SRP et ftsY en complexe avec une charge RNC stabilisée par un analogue non-hydrolysable de GTP (GMP-PCP). Pour franchir la barrière cinétique qui permet de passer du complexe précoce au complexe fermé, nous avons utilisé une version tronquée de FtsY, à laquelle la séquence terminale avait été amputée de tout le domaine acide (A-) ainsi que de la première hélice alpha du domaine NG. De plus, pour la formation du complexe, nous avons utilisé une construction contenant les 50 premiers acides aminés du leader peptidase (RNCLep50). En l'absence de nucléotides, notre reconstruction au cryo-EM a montré une configuration similaire à celle du stade précoce, dans laquelle Ftsy et Ffh- domaine NG, sont proche du tetraloop de la 4.5 S ARN. Une incubation avec GMP-PCP induit un détachement du domaine NG d'avec la queue du tetraloop. Il semblerait que les domaines NG soient flexibles dans l'état clos, et non attaché à la terminaison ouverte de l'ARN. / The signal recognition particle (SRP) and its receptor (FtsY in Escherichia coli) mediate co-translational protein targeting by delivering ribosome nascent chain complexes (RNCs) to the target membrane. Recognition of an RNC cargo by SRP is dependent on an N-terminal signal sequence. Binding of FtsY to the RNC-SRP complex leads to several conformational changes of SRP and FtsY during the targeting cycle: first, an “early” GTP-independent state is adopted which is stabilized by the RNC, subsequently a “closed” GTP- dependent conformation is formed which can activate itself to hydrolyze GTP (the “activated” state). Faithful completion of all three steps leads to release of the cargo from SRP-FtsY and hand over of the RNC to the translocation pore.It has been shown for E. coli that cargos can be rejected from the SRP pathway during all targeting steps. In the first project, our interest concentrates on ribosomes translating the EspP signal sequence (RNCEspP). In vivo, EspP is a post-translationally targeted protein, but RNCEspP has been shown to be bound by SRP and FtsY leading to a non-productive “early”-like RNCEspP-SRP-FtsY complex. Using single particle cryo-electron microscopy (EM), we analysed the structural basis for the rejection of RNCEspP by SRP and FtsY. Comparison of our RNCEspP-SRP-FtsY cryo-EM structure to other available cryo-EM structures of co-translational targeting complexes containing the correct cargo RNCFtsQ unravelled differences in the SRP-FtsY structure between a correct cargo and an incorrect cargo. Two major differences between the targeting complexes containing the cargos RNCFtsQ and RNCEspP were observed: first, the Ffh M-domain was attached to ribosomal RNA helix 59 of RNCEspP, while it was detached from this site in the case of RNCFtsQ. It could be that such an ordered M-domain is hampering the release of the signal sequence which is required for successful completion of targeting. Second, the Ffh-FtsY NG-domain arrangement was flexible in the complex with RNCEspP in comparison to RNCFtsQ indicating that the "early"-like complex formed on RNCEspP is less stable. Biochemical data using fluorescence resonance energy transfer corroborated these results, showing that FtsY is bound with lower affinity in the RNCEspP “early” complex and that the rearrangement to the “closed” conformation is less efficient. Further biochemical analysis of EspP signal sequence variants showed that mainly the N-terminal extension of the EspP signal sequence is responsible for its rejection from the SRP pathway.

Introdução: Os lasers fracionados não ablativos são efetivamente utilizados no rejuvenescimento da pele. As novas tecnologias a laser permitem uma remodelação dérmica seletiva, sem ablação da epiderme. Objetivo: Avaliar a eficácia do laser Erbium Glass (Sellas Evo) fracionado não ablativo 1550nm no rejuvenescimento facial através do estudo da quantificação histomorfométrica de fibras colágenas e elásticas, a expressão da molécula de adesão intercelular 1 (ICAM-1) por imuno-histoquímica , a análise das fases do ciclo celular, o potencial elétrico da membrana mitocondrial, a expressão de interleucina-1 (IL-1), o marcador celular endotelial CD34, o receptor do fator transformador de crescimento beta (TGF-beta), atividade da caspase-3 por citometria de fluxo e as alterações ultraestruturais na pele por microscopia eletrônica de varredura, 4 meses após o tratamento com laser. Materiais e Métodos: Quinze indivíduos (média de 56,4 anos, fototipo II-IV), com fotoenvelhecimento cutâneo na face fizeram 3 tratamentos com laser Erbium Glass fracionado não ablativo 1550nm usando uma fluência de 70 mJ e uma densidade de 100 cm2. Foram avaliados biópsias na região pré-auricular em 15 pacientes no início e 4 semanas após o tratamento final. Os níveis de expressão dos receptores e a atividade do potencial elétrico mitocondrial foram analisados na suspensão de células da derme obtida a partir da digestão por colagenase. A avaliação clínica e fotográfica foi analisada quatro semanas após o final do tratamento. Resultados: Após 4 meses do início do tratamento foi observada melhora clínica moderada, com uma média de satisfação de 8,8, as fibras de colágeno aumentaram em 6,68%, as fibras elásticas mostraram uma diminuição de 12,85%, o ICAM-1 aumentou 4,47% significativamente na área dos vasos. Houve aumento significativo dos níveis de expressão do receptor de IL-1 e TGF-beta após o tratamento com laser Erbium Glass. As respostas proliferativas e a ausência de apoptose dependente da caspase-3 foram observadas nas suspensões de células após o tratamento. Conclusão: Rejuvenescimento facial com laser Erbium Glass fracionado não ablativo 1550nm demonstrou melhora visível no fotoenvelhecimento, assim como, aumento significativo da expressão de IL-1 e receptores de TGF-beta, que estão envolvidos na remodelação e indução da proliferação de componentes da matriz extracelular da pele. O grau de satisfação foi alto especialmente em relação à textura, rugas e discromias / Background: Non-ablative fractional lasers have been effectively used in skin rejuvenation. The new laser technologies allow selective dermal remodeling without ablation of the epidermal surface. Objectives: to evaluate the efficacy of the 1550nm Erbium Glass Laser (Sellas Evo) for facial rejuvenation through study of the histomorphometric quantification of colagen and elastic fibers; the expression of intercellular adhesion molecule 1 (ICAM-1) by immnohistochemistry; and analysis of the cell cycle phases, the electrical potential of the mitochondrial membrane and the expression of interleukin 1 (IL-1), the endothelial cell marker CD34 and transforming growth factor beta (TGF-beta) receptors, caspase-3 activity by flow cytometry and the ultrastructural changes in the skin by scanning electron microscopy 4 months after the laser treatment. Materials and methods: fifteen subjects (mean age 56,4 skin typoes II-IV) with photodamage on the face and wrinkles had 3 treatments with the 1550 nm Erbium Glass Laser using a fluence of 70 mJ and a density of 100 cm2. Pre-auricular biopsies from 15 subjects were evaluated at baseline and 4 weeks after the final treatment. Receptor expression levels and the presence of potentially functional mitochondria were analyzed in a cell suspension obtained from collagenase digestion of the skin. Data from the photo assessments and the subjects\' self-assessed improvements were analyzed 4 weeks after the final treatment.Results: Four months after the last treatmenbt application, clinical improvement was observed, with an average satgisfation score of 8.8, corresponding to moderate improvement. After 4 months of treatment, collagen fibers had increased up to 6.68%, while the average proportion of collagen fibers in the dermis, the elastic fibers, showed a 12.85% decrease of ICAM-1 and a mean increase in vessel area of 4.47%.Significantly enhanced IL-1 and TGF-beta receptor expression levels were identified after Erbium Glass Laser treatment. Proliferative responses and non-apoptosis-dependent caspase-3 activity were both observed in the cell suspensions after dermal treatment. Conclusion: Non-ablative rejuvenation with the 1550 mn Erbium Glass Laser was of the IL-1 and TGF-beta receptors, that are involved in remodeling and induced proliferation components in the extracellular matrix of skin fibers. The subjects were highly satisfied, especially regarding texture, rhytids and dyschromia

Introdução: Os lasers fracionados não ablativos são efetivamente utilizados no rejuvenescimento da pele. As novas tecnologias a laser permitem uma remodelação dérmica seletiva, sem ablação da epiderme. Objetivo: Avaliar a eficácia do laser Erbium Glass (Sellas Evo) fracionado não ablativo 1550nm no rejuvenescimento facial através do estudo da quantificação histomorfométrica de fibras colágenas e elásticas, a expressão da molécula de adesão intercelular 1 (ICAM-1) por imuno-histoquímica , a análise das fases do ciclo celular, o potencial elétrico da membrana mitocondrial, a expressão de interleucina-1 (IL-1), o marcador celular endotelial CD34, o receptor do fator transformador de crescimento beta (TGF-beta), atividade da caspase-3 por citometria de fluxo e as alterações ultraestruturais na pele por microscopia eletrônica de varredura, 4 meses após o tratamento com laser. Materiais e Métodos: Quinze indivíduos (média de 56,4 anos, fototipo II-IV), com fotoenvelhecimento cutâneo na face fizeram 3 tratamentos com laser Erbium Glass fracionado não ablativo 1550nm usando uma fluência de 70 mJ e uma densidade de 100 cm2. Foram avaliados biópsias na região pré-auricular em 15 pacientes no início e 4 semanas após o tratamento final. Os níveis de expressão dos receptores e a atividade do potencial elétrico mitocondrial foram analisados na suspensão de células da derme obtida a partir da digestão por colagenase. A avaliação clínica e fotográfica foi analisada quatro semanas após o final do tratamento. Resultados: Após 4 meses do início do tratamento foi observada melhora clínica moderada, com uma média de satisfação de 8,8, as fibras de colágeno aumentaram em 6,68%, as fibras elásticas mostraram uma diminuição de 12,85%, o ICAM-1 aumentou 4,47% significativamente na área dos vasos. Houve aumento significativo dos níveis de expressão do receptor de IL-1 e TGF-beta após o tratamento com laser Erbium Glass. As respostas proliferativas e a ausência de apoptose dependente da caspase-3 foram observadas nas suspensões de células após o tratamento. Conclusão: Rejuvenescimento facial com laser Erbium Glass fracionado não ablativo 1550nm demonstrou melhora visível no fotoenvelhecimento, assim como, aumento significativo da expressão de IL-1 e receptores de TGF-beta, que estão envolvidos na remodelação e indução da proliferação de componentes da matriz extracelular da pele. O grau de satisfação foi alto especialmente em relação à textura, rugas e discromias / Background: Non-ablative fractional lasers have been effectively used in skin rejuvenation. The new laser technologies allow selective dermal remodeling without ablation of the epidermal surface. Objectives: to evaluate the efficacy of the 1550nm Erbium Glass Laser (Sellas Evo) for facial rejuvenation through study of the histomorphometric quantification of colagen and elastic fibers; the expression of intercellular adhesion molecule 1 (ICAM-1) by immnohistochemistry; and analysis of the cell cycle phases, the electrical potential of the mitochondrial membrane and the expression of interleukin 1 (IL-1), the endothelial cell marker CD34 and transforming growth factor beta (TGF-beta) receptors, caspase-3 activity by flow cytometry and the ultrastructural changes in the skin by scanning electron microscopy 4 months after the laser treatment. Materials and methods: fifteen subjects (mean age 56,4 skin typoes II-IV) with photodamage on the face and wrinkles had 3 treatments with the 1550 nm Erbium Glass Laser using a fluence of 70 mJ and a density of 100 cm2. Pre-auricular biopsies from 15 subjects were evaluated at baseline and 4 weeks after the final treatment. Receptor expression levels and the presence of potentially functional mitochondria were analyzed in a cell suspension obtained from collagenase digestion of the skin. Data from the photo assessments and the subjects\' self-assessed improvements were analyzed 4 weeks after the final treatment.Results: Four months after the last treatmenbt application, clinical improvement was observed, with an average satgisfation score of 8.8, corresponding to moderate improvement. After 4 months of treatment, collagen fibers had increased up to 6.68%, while the average proportion of collagen fibers in the dermis, the elastic fibers, showed a 12.85% decrease of ICAM-1 and a mean increase in vessel area of 4.47%.Significantly enhanced IL-1 and TGF-beta receptor expression levels were identified after Erbium Glass Laser treatment. Proliferative responses and non-apoptosis-dependent caspase-3 activity were both observed in the cell suspensions after dermal treatment. Conclusion: Non-ablative rejuvenation with the 1550 mn Erbium Glass Laser was of the IL-1 and TGF-beta receptors, that are involved in remodeling and induced proliferation components in the extracellular matrix of skin fibers. The subjects were highly satisfied, especially regarding texture, rhytids and dyschromia