Lors de l'élaboration des aciers plats inoxydables, un recuit haute température de courte durée (60 s environ), sous atmosphère oxydante (O2/H2O/CO2/N2) est mis en place après laminage à froid. Ce recuit entraîne la formation d'une couche d'oxyde de faible épaisseur qui est éliminée par un décapage. Ces étapes de recuit et décapage peuvent entraîner une perte de qualité du produit fini, se traduisant par une perte de brillance. L'acier bistabilisé Ti, Nb (AISI 441) est sensible à la dégradation de surface. L'objectif de la thèse est d'identifier les phénomènes à l'origine de la perte de brillance. Les travaux menés consistent à étudier les couches d'oxyde et le comportement de l'acier lors du décapage. Une morphologie originale de l'interface interne avec la présence de protrusions métalliques a été observée. L'étude des protrusions par la microscopie électronique à balayage couplée à une sonde ionique focalisée : MEB-FEG/FIB, a permis de proposer un mécanisme décrivant leur formation. Ce mécanisme s'appuie sur la précipitation de la silice qui semble également être un élément clé pour comprendre la dégradation de surface. Un lien entre perte de brillance et teneur en chrome dans le substrat a été établi. La déchromisation de l'acier liée au recuit est susceptible d'être facilitée par les protrusions.

Sensory, cognitive, and emotional processing are rooted in the cerebral cortex. The cerebral cortex is comprised of six layers defined by the neurons within them that have distinctive connection, both within cortex itself and with other subcortical structures. Although still far from solving the mysteries of the mind, it is clear that networks form by neurons in the cerebral cortex provide the computational substrate for a remarkable range of behaviours. This neuron to neuron activation is mediated through dendritic spines, the postsynaptic target of most excitatory synapses in the cerebral cortex. Dendritic spines are small protrusions found along dendrites of neurons, and their number, as well as structural changes, accompany the development of synapses and establishment of neuronal networks. In fact, dendritic spines undergo rapid structural and functional changes guided by neuronal activity. This marriage between structural and functional plasticity, makes dendritic spines crucial in fine-tuning of networks in the brain; not surprisingly, dendritic spine aberrations are a hallmark of multiple neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. With this in mind, I considered Pannexin 1 (Panx1) an interesting novel candidate for a regulatory role on cortical neuronal network and dendritic spine development, for the following reasons. First, Panx1 transcripts are enriched in the brain and in the cortex are most abundant during the embryonic and early postnatal period. . This timing roughly corresponds to a period of synaptogenesis in the postnatal brain. Second, Panx1 localizes to postsynaptic compartments in neurons and its disruption leads to enhance excitability and potentiation of neuron-to neuron communication. Third, disruption of Panx1 (either knockdown or pharmacological blockade) leads to neurite outgrowth in neuron-like cells. Lastly, genetic variants in PANX1 have been associated with neurodevelopmental disorders. This dissertation explores the role of Panx1 in the development of dendritic spines and neuronal networks, furthering our understanding on cortical development and placing Panx1 as a novel regulator of structural and functional plasticity in the brain. Chapter 1 discusses core concepts on cortical development, with an emphasis on pyramidal neuron, the most abundant and only known projection neurons in the cerebral cortex. Here, I review the embryonic origin of pyramidal neurons, their postnasal development, and how cortical circuits are assembled. I finish this chapter with a brief review on Panx1 and its known expression and involvement in neuronal function. In Chapter 2 I explore the functional properties of neuronal networks and synaptic composition of cortical neurons in the absence of Panx1. Using live cell imaging and analysis of Ca2+ transients in dense primary cortical cultures, revealed that Panx1 knock-out (KO) cultures exhibit more and larger groups of significantly co-activated neurons, known as network ensembles. These network properties were not explained by differences in cell viability or cell-type composition. Examination of protein expression from cortical synaptosome preparations revealed that Panx1 is enriched in synaptic compartments, while also confirming a developmental downregulation. This analysis also revealed increased levels of the postsynaptic scaffolding protein PSD-95, along with the postsynaptic glutamate receptors GluA1 and GluN2A. Lastly, ex vivo tracing of dendritic spines on apical dendrites of Layer 5 pyramidal neurons in global and glutamatergic-specific Panx1 KO brain slices revealed higher spine densities in early and late postnatal development, with no differences in spine length. Analysis of dendritic spine densities in fixed cultured cortical neurons revealed an increase associated with Panx1 KO. Altogether, this work presents for the first time a connection between Panx1 and structural development of dendritic spines and suggest that Panx1 regulates cortical neuronal networks through changes in spine density. Chapter 3 examines the influence of Panx1 on spiny protrusions growth and movement. Spiny protrusion are long, thin, highly dynamic spine precursors. Taking advantage of a fluorescent signal localized to the plasma membrane, I visualized spiny protrusions and quantified their dynamics in wildtype (WT) and Panx1 KO developing cortical neurons, both under fixed and live conditions. I found that transient Panx1 expression is associated with decreased spiny protrusion density both in WT and Panx1 KO neurons. Using live cell imaging, I found that spiny protrusions are more stable and less motile in Panx1 KO neurons, while its transient expression reversed both of these phenotypes. These results suggest that Panx1 regulation of dendritic spines development is rooted partly in the regulation of spiny protrusion dynamics. Overall, this dissertation demonstrates that Panx1 negatively regulates dendritic spines in part by influencing spiny protrusion dynamics. It is reasonable to speculate that Panx1 regulation of dendritic spines underlies its newly discovered role in the formation network ensembles, providing a putative mechanism for previously described roles of Panx1 in synaptic plasticity. Likewise, this body of work furthers our understanding of Panx1 by filling some of the gaps attached to its developmental expression and association with neurodevelopmental disease. / Graduate / 2021-04-16

Die Planung korrektiver Eingriffe an der Ohrmuschel bei Vorliegen eines als zu groß empfundenen aurikulocephalen Winkels basierte bislang auf anthropometrisch gewonnenen Empfehlungen in der Literatur. Die menschliche Ohrmuschel ist jedoch messtechnisch schwierig zu untersuchen und weist zudem eine außerordentlich große individuelle Variabilität auf. Insoweit ist es nicht einfach, eine ideale Ohrmuschelposition zu definieren. Anthropometrische Idealwerte beruhen meist auf Morphologien, die in der Bevölkerung häufiger vorkommen und eine gewisse Durchschnittlichkeit aufweisen. Sie bewirken jedoch nicht zwangsläufig, dass Individuen mit derartigen Maßen auch als ansprechend bzw. attraktiv von ihrem Umfeld wahrgenommen werden. Chirurgische Korrekturen des abstehenden Ohres gehören heute zu den häufig durchgeführten Eingriffen innerhalb des Spektrums plastisch-ästhetischer Operationen im Kopf-Hals-Bereich. Für die Planung solcher Eingriffe ist es daher von großer Bedeutung, Zielwerte zur Verfügung zu haben, deren chirurgische Umsetzung ein gefälliges Ergebnis erwarten lässt. Das Anliegen der im Rahmen dieser Dissertation durchgeführten Studie war es, die Wirkung unterschiedlicher Ohrmuschelpositionen auf ein Jurorenkollektiv zu untersuchen. Hierfür wurden 44 Studierende der Zahnmedizin der Universität Leipzig als Juroren gewonnen. Sie erhielten Klone eines weiblichen und eines männlichen Modells zur Bewertung vorgelegt. Die Erfassung der Ergebnisse erfolgte mittels Fragebögen. Die Klone wurden nach Anfertigung von Porträtfotografien eines freiwilligen weiblichen bzw. männlichen Probanden mit Hilfe von metrisch definierten, am Ohr zu befestigenden Distanzkeilen erstellt und durch digitale Nachbearbeitung komplettiert. Sie simulierten aurikulocephale Winkel von 0° bis 90° in Schritten von jeweils 6°. Die Juror*innen hatten die Möglichkeit, einen positiven (maximale Attraktivität) und negativen (minimale Attraktivität) Favoriten jeweils für die weiblichen und männlichen Klone auszuwählen und einen Übergangsklon für wiederum beide Geschlechter zu definieren, der die Grenze zwischen diesen Extremen repräsentiert. Die Ergebnisse der Studie zeigen, dass weibliche und männliche Juroren bei der Bewertung von Ohrmuschelpositionen übereinstimmende ästhetische Präferenzen haben. Bevorzugt werden eher unauffällige, enganliegende Ohren. Extremwerte des aurikulocephalen Winkels wie 90° oder auch 0° werden demgegenüber als besonders unattraktiv empfunden. Neben diesen übereinstimmenden Bewertungen konnten auch signifikante Unterschiede im Urteilsverhalten festgestellt werden. Der beim Menschen bekannte Geschlechtsdimorphismus beeinflusst offenbar auch die Erwartungshaltung von Juror*innen in Bezug auf das für Frauen bzw. Männer ästhetisch „zulässige“ Ausmaß des Abstehens der Ohrmuschel. So wird Männern insgesamt ein etwas stärker abstehendes Ohr eher zugestanden als Frauen. Anhand der Gesamtheit aller Bewertungen des Jurorenkollektivs wurde zudem als Idealmaß für den aurikulocephalen Winkel der Bereich zwischen 21–24° ermittelt. Limitationen der Studie ergeben sich aus der begrenzten Anzahl der Juror*innen, die nicht repräsentativ für eine größere Gesellschaft ist. Auch stellt die virtuelle Herstellung symmetrischer Ohrmuschelpositionen eine Abstraktion bzw. Vereinfachung der Realität dar. Außerdem ist die Vermessung und Positionierung der flexiblen und nicht plan geformten Ohrmuschel schwierig, so dass kleinere Toleranzen von 2-3° auftreten können.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Intervertebral disc disease (IVD) is a common pathology in clinical neurology of dogs, representing 45.8% of neurological cases treated at Veterinary Hospital of the Universidade Federal de Santa Maria. The most affected segments are the thoracolumbar (T3-L3) and cranial cervical (C1-C5) of the spinal cord. The clinical sign occurs due a combination of the compressive effect of the disc material and the injury of impact on spinal cord, probably due to an extrusion. A clinical sign varies according to the affected segment of the spinal cord and the severity of the injury. It could be presented only by spinal hyperesthesia, whereas more severe injuries can lead to tetra / paraplegia with no nociception (deep pain) caudal to the lesion. Clinical management for IVD is generally indicated for dogs with hyperesthesia with or without minimal neurological deficits and consists of absolute rest in cage between four to six weeks. Surgery is the treatment of choice for dogs with severe neurological deficits (not ambulatory tetraparesis, tetraplegia, paraplegia with or without nociception in less than 48 hours) in dogs with unsuccessful of clinical management, or dog that have recurrence of disease. In contrast to the numerous studies evaluating the efficacy of surgical treatment in dogs with thoracolumbar and cervical IVD, studies demonstrating the effectiveness of conservative treatment are rare. The aim of this study was to identify dogs with presumptive diagnosis of thoracolumbar and cervical IVD who underwent clinical management and evaluate the response to therapy; and to analyze the effect of age, gender, duration of clinical signs, neurological degree and therapy, as prognostic factors in clinical outcome of the patient. Five hundred six neurological records were used to identify affected dogs (n = 379 thoracolumbar; n = 127 cervical), and was selected those patients with presumptive diagnosis of IVD submitted to clinical management as a first option. The outcome was satisfactory in 73.3% of cases of thoracolumbar IVD, and 92.7% of cases of cervical IVD, demonstrating that clinical management (cage rest, anti-inflammatory and analgesic opioid administration) is effective, especially in milder disease. Conservative treatment has a substantial rate of recurrence and neurological signs may be more severe than the first clinical presentation. The gender, age and duration of clinical signs has no prognostic effect on clinical outcomes of patients IVD of thoracolumbar and cervical, in the sample of the study. / A doença do disco intervertebral (DDIV) é uma afecção frequente na clínica neurológica de cães, representando 45,8% dos casos neurológicos atendidos pelo Serviço de Neurologia do Hospital Veterinário Universitário da Universidade Federal de Santa Maria. Os locais mais acometidos pela doença são os segmentos toracolombar (T3-L3) e cervical cranial (C1-C5) da medula espinhal. A manifestação clínica ocorre devido a uma combinação do efeito compressivo do material de disco e da lesão de impacto na medula espinhal, decorrente principalmente da extrusão do disco e varia de acordo com o segmento da medula espinhal afetado e da severidade da lesão. Pode ser evidenciada apenas por hiperestesia espinhal, enquanto as lesões mais graves podem levar a tetra/paraplegia com ausência da nocicepção (dor profunda) caudal a lesão. O tratamento clínico para DDIV geralmente é indicado para cães com hiperpatia associada ou não a mínimas deficiências neurológicas e consiste em repouso absoluto em gaiola entre quatro a seis semanas. Já a cirurgia é o tratamento de eleição para cães com deficiências neurológicas graves (tetraparesia não ambulatória, tetraplegia, paraplegia com ou sem nocicepção em menos de 48 horas), em cães refratários ao tratamento clínico, ou que apresentem recidiva da doença. Em contraste com os inúmeros estudos avaliando a eficácia do tratamento cirúrgico em cães com DDIV toracolombar e cervical, estudos demonstrando a eficácia do tratamento conservativo são escassos na literatura. Diante desses fatores, o objetivo desse estudo foi identificar cães com diagnóstico presuntivo de DDIV toracolombar e cervical que foram submetidos ao tratamento clínico e avaliar a resposta à terapia instituída; além de avaliar a relação da idade, do gênero, da duração dos sinais clínicos e do tratamento de acordo com o grau neurológico como fatores prognósticos na evolução clínica desses pacientes. Foram analisados 506 registros neurológicos (n=379 toracolombar; n=127 cervical), e selecionados aqueles pacientes com diagnóstico presuntivo de DDIV submetidos ao tratamento clínico como primeira opção. A evolução clínica foi satisfatória em 73,3% dos casos de DDIV toracolombar, e 92,7% dos casos de DDIV cervical, demonstrando que o tratamento clínico com repouso absoluto, administração de anti-inflamatórios e analgésicos opióides é efetivo, principalmente em graus mais leves da doença. O tratamento conservativo apresenta um índice considerável de recidiva, cujos sinais neurológicos poderão ser mais graves do que a primeira apresentação clínica. O gênero, a idade e a duração dos sinais clínicos não apresentam efeito prognóstico na evolução clínica dos pacientes de DDIV toracolombar e cervical, na amostra estudada.

Made available in DSpace on 2016-12-06T17:07:27Z (GMT). No. of bitstreams: 1 Gilmar.pdf: 1680429 bytes, checksum: 6eb062d33cfa1169c08c1dc12e076ae5 (MD5) Previous issue date: 2010-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The purpose of this study is to construct a biofeedback device to monitor the posture of shoulder protrusion. The research subjects were five normal adult individuals. The choice was intentional, with one being male and four female, with an average age of 26.6 years (20/326). A device was built to detect posture of the shoulder protrusion by means of sensors with gyroscopes fixed to the acromia. Based on a pre-established posture, the device detects variations in the coordinates. When the individual transgresses from the pre-established posture the device issues a vibratory alert signal by biofeedback. This device was tested in a laboratory and in the field. In the laboratory the vibratory response was checked in the movements of clavicular abduction and trunk rotation movements in different angular positions. The vibratory response was also found at pre-established times that represented tolerance of activation in relation to daily activities. In the laboratory, all of these measures of the device functions were virtually simulated with the Proteus Labcenter Electronics software. In the field, in working activities, the viability of the use of the device was verified when programmed with a tolerance of 30 seconds and allowing a variation of 7 degrees in clavicular abduction. The laboratory tests for validation demonstrated that the precision of the coordinate measurements is <1°. The coefficients of correlation of the tests and retests were = 1. The results measured in the tests show that this device measures what it was proposed to measure and can be used in the detection and orientation of posture of shoulder protrusion. / Este estudo teve como objetivo a construção de um aparelho de biofeedback para a monitoração da postura de protusão de ombros. Os sujeitos da pesquisa foram cinco indivíduos adultos normais. A escolha foi intencional, sendo um do gênero masculino e quatro femininos, média de idade = 26,6 anos (20/326). Foi desenvolvido um aparelho de detecção da postura de protusão de ombros por meio de sensores com giroscópios fixados nos acrômios. A partir de uma postura pré-estabelecida o aparelho detecta as variações de coordenadas. Quando o indivíduo transgride a postura pré-estabelecida o aparelho emite por biofeedback um sinal de alerta vibratório. Este aparelho foi testado em laboratório e em campo. Em laboratório foi aferida a resposta de vibração nos movimentos de adução clavicular e rotação de tronco em diferentes posições angulares. Também foi verificada a resposta de vibração em tempos pré-estabelecidos que representam a tolerância de ativação frente as atividades de vida diária. Em laboratório, todos estes aferimentos de funções do aparelho foram simulados virtualmente no software Proteus Labcenter Eletronics. Em campo, em atividades laborais, foi verificada a viabilidade de uso do aparelho programado com um tempo de tolerância de 30s e permitindo a variação de 7° graus de movimento de adução clavicular. Os testes de validação de laboratório demonstraram que a precisão das medidas de coordenadas do aparelho é <1°. Os coeficientes de correlação dos testes e retestes foram =1. Os resultados aferidos nos testes mostraram que este aparelho mede o que se propõe a medir e pode ser utilizado na detecção e orientação na postura de protusão de ombros.

This work is dedicated to small distance contact-less measurement. Especially the surface detection of the commutator or collector of the electrical machines was described. The probes utilising the induction principle of the distance measurement have been developed. The part of the work described the history of the induction distances sensing in FEEC BUT and the using of the induction principle in the industry small distances sensing nowadays. The paper concretely determines the possibilities of the small distances measurement using mentioned probes. The sensitivity for different materials was investigated and the error of the measurement for different measuring object was investigated, too. The effects, that could affected the measurement error, were described in the work. The contribution of the each effect for the total measuring error was specified. Next part is including the description of the measuring workstation and of the hardware and software. At the end the verification of the measuring principle and measuring workstation is presented. The verification has been made on three different commutator types. The verification results are in the conclusion.

O formato, posição e alinhamento corretos dos componentes oculares são definidos através de uma série de mudanças morfológicas complexas durante sua embriogênese. A retina se origina de células da vesícula óptica do tubo neural enquanto as células precursoras do cristalino surgem do ectoderma que reveste o ápice da vesícula óptica. Este ectoderma é delimitado molecularmente como pre-placoidal, sofre uma série de eventos morfogênicos durante o seu desenvolvimento inicial para formar o placóide do cristalino e, posteriormente, a vesícula do cristalino. O placóide do cristalino surge a partir do espessamento do ectoderma pre-placoidal. Posteriormente, o placóide invagina para formar a vesícula do cristalino. Durante a invaginação do placóide, as células da ectoderme que circundam o placóide (células periplacodais) também se movem para fechar a abertura do cristalino que invaginou e reconstruir o ectoderma da superfície. Aqui, nos concentramos em dois processos do desenvolvimento do olho. Nós investigamos o papel da matriz extracelular no espessamento do placóide do cristalino e a dinâmica da emissão de protrusões de membrana pelas células periplacodais durante a invaginação do placóide. A matriz extracelular desempenha papel relevante na morfogênese placodal. Por exemplo, a Fibronectina na matriz extracelular entre a vesícula óptica e o ectoderma pré-placoidal é necessária para a formação de placóide do cristalino. No entanto, a dinâmica da arquitetura de Fibronectina durante a formação do placóide é desconhecida. Assim, nosso primeiro objetivo aqui foi investigar a arquitetura da Fibronectina e da Laminina, dois importantes componentes da matriz extracelular, durante o espessamento do placóide do cristalino através de imagens confocais em 3D. Nossos dados sugerem que um padrão de Fibronectina e Laminina difuso e pontuado é restrito à região do placóide. Este padrão é mantido durante o espessamento e invaginação do placóide. Encontramos um padrão similar de Laminina na região do placóide de embrião de camundongo, sugerindo a conservação desta arquitetura neste contexto. Também demonstramos que a inibição mediada por Noggin (inibidor da sinalização de BMP), que interrompe o desenvolvimento do olho, afeta a organização da Fibronectina e da Laminina, sugerindo que a sinalização de BMP regula a organização da matriz extracelular durante o desenvolvimento do placóide do cristalino. Nosso segundo objetivo foi analisar a emissão de 5 protrusões celulares finas por células periplacodais correlacionando com o movimento de invaginação. Aqui, nós investigamos a dinâmica e composição do citoesqueleto dessas protrusões para entender sua função durante o desenvolvimento do olho. Observamos uma grande quantidade de protrusões em células periplacodais de embriões de galinha e de camundongo. Nossos resultados de quantificação com protrusões de embriões de galinha não mostraram correlação entre comprimento e direção de emissão ou com meia-vida. Nós também analisamos a diversidade na composição do citoesqueleto, uma vez que encontramos protrusões positivas para Cofilina e Tubulina. Estes dados sugerem uma população heterogênea de protrusões finas de membrana periplacodais. Finalmente, também identificamos essas protrusões em outras superfícies ectodérmicas de embriões de galinha e de camundongo, sugerindo que elas desempenham um papel no desenvolvimento de ectoderme superficial. / The correct shape, position and alignment of optic components are defined through a series of complex morphological changes during the embryogenesis of the eye. The retina originates from the neural tube´s optic vesicle while the lens precursor cells arise from the ectoderm that overlie the apex of the optic vesicle. This ectoderm is molecularly delimited as preplacodal and undergoes a series of morphogenic events during its initial development to form the lens placode and subsequently the lens vesicle. The lens placode arises from the thickening of the pre-placodal ectoderm. Subsequently, the placode invaginates to form the vesicle of the lens. During the invagination of the placode, the ectodermal cells that surround the placode (peri-placodal cells) also move to close the opening of the lens that invaginated and reconstruct the surface ectoderm. Here we focus on two processes of eye development. We investigated the role of the extracellular matrix in the lens placode thickening and the dynamics of the emission of membrane protrusions by the peri-placodal cells during the lens placode invagination. The extracellular matrix plays a relevant role in placodal morphogenesis. For example, Fibronectin in the extracellular matrix between the optic vesicle and the preplacodal ectoderm is required for the formation of lens placode. However, the dynamics of the Fibronectin architecture during placode formation is unknown. Thus, our first aim here was to investigate the architecture of Fibronectin and Laminin, two important components of the extracellular matrix, during thickening of lens placode through confocal 3D images. Our data suggest that both Fibronectin and Laminin present a diffuse and punctate pattern restricted to the placodal region. This pattern is maintained during thickening and invagination of the placode. We found a similar pattern of Laminin in the placodal region of the mouse embryo, suggesting the conservation of this architecture in this context. We also demonstrate that Noggin-mediated inhibition of BMP signalling, which disrupts the development of the eye, affects the organization of Fibronectin and Laminin, suggesting that BMP signalling regulates the organization of the extracellular matrix during the lens placode development. Our second objective was to analyse the emission of thin cellular protrusions by peri-placodal cells correlating with the lens invagination movement. Here we investigated the dynamics and 3 composition of the cytoskeleton of these protrusions to understand their function during the development of the eye. We observed a large number of protrusions in peri-satellite cells of chicken and mouse embryos. Our quantification results with chicken embryo protrusions showed no correlation between length and direction of emission or with half-life. We also analysed the diversity in the composition of the cytoskeleton, and we found protrusions positive for Cofilin and Tubulin. These data suggest a heterogeneous population of periplacodal protrusions. Finally, we have also identified these protrusions on other ectodermal surfaces of chicken and mouse embryos, suggesting that they play a role in the development of surface ectoderm.

In this study nano-patterning was carried out using two different nano-optic devices namely- the NSOM and Fresnel zone plate. In the first study, NSOM was used to generate nano-patterns on selected semiconducting (Si and Ge) and metallic (Cr, Cu and Ag) targets under different laser pulse durations, laser energies and number of laser pulses. Based on the experimental results, femtosecond laser pulses, provided lower pattern generation thresholds on targets but higher damage thresholds to the NSOM probes at the wavelength (~400-410 nm) studied, compared with nanosecond laser pulses. Three different mechanisms were identified as the dominant processes for pattern generation under different conditions, namely nano-scale laser ablation, nano-scale thermal oxidation and nano-scale melting/recrystallization of the targets. Furthermore, the resulting nano-patterns also showed a significant dependence on the optical properties (i.e., absorption coefficient and surface reflectivity) of the target material. By comparing the obtained experimental results, it was concluded that the optical energy transport from the NSOM probe to the target dominates the pattern generation when femtosecond laser is applied to the NSOM system. When nanosecond laser is applied, both the thermal and optical energy transported from the NSOM probe to the targets attribute to the obtained morphology of nano-patterns on different targets under the experimental conditions studied. In the second study, a traditional Fresnel zone plate with a focus length of 3 micrometres was fabricated with a novel lift-off process in e-beam lithography. The fabrication process involved, using a HSQ/PMMA bi-layer in a negative tone lift-off process with a layer of conducting polyaniline for charge dissipation. HSQ was used as the high resolution negative resist for e-beam patterning and the PMMA under-layer was used to enable a HSQ lift-off process. The fabricated Fresnel zone plate was used to generate nano-patterns on a UV sensitive photoresist using nanosecond laser light with lamda~409nm. The smallest pattern sizes generated was close to the diffraction limit. Nano-pattern sizes generated on the photoresist were comparable with a numerically calculated intensity distribution at the focus spot of the designed Fresnel zone plate obtained from Scalar Diffraction Theory.

No description available.

Cancer metastasis, the spread of cancer from the primary site to distant regions in the body, is the major cause of cancer mortality, accounting for almost 90% of cancer related deaths. During metastasis, cancer cells from the primary tumor initially probe the surrounding fibrous tumor microenvironment (TME) prior to detaching and subsequently migrating towards the blood vessels for further dissemination. It has widely been acknowledged that the biophysical cues provided by the fibrous TME greatly facilitate the metastatic cascade. Consequently, there has been a tremendous wealth of work devoted towards elucidating different modes of cancer cell migration. However, our knowledge of how cancer cells at the primary tumor site initially sense their fibrous surroundings prior to making the decision to detach and migrate remains in infancy. In part, this is due to the lack of a fibrous in vitro platform that allows for precise, repeatable manipulation of fiber characteristics. In this study, we use the non-electrospinning, Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with exquisite control on fiber dimensions and network architecture and use these networks to investigate how single cancer cells biophysically sense fibers mimicking in vivo dimensions. Using high spatiotemporal resolution imaging (63x magnification/1-second imaging interval), we report for the first time, that cancer cells sense individual fibers by coiling (i.e. wrapping around the fiber axis) at the tip of a cell protrusion. We find that coiling dynamics are mediated by both the fiber curvature and the metastatic capacity of the cancer cells with less aggressive cancer cells showing diminished coiling. Based on these results, we explore the possibility of using coiling in conjunction with other key biophysical metrics such as cell migration dynamics and forces exerted in the development of a genetic marker independent, biophysical predictive tool for disease progression. Finally, we identify the membrane curvature sensing Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) as a key regulator of protrusive activity with IRSp53 knockout (KO) cells exhibiting significantly slower protrusion dynamics and diminished coil width compared to their wild-type (WT) counterparts. We demonstrate that the hindered protrusive activity ultimately translates to impaired contractility, alteration in the nucleus shape and slower migration dynamics, thus highlighting the unique role of IRSp53 as a signal transducer – linking the protrusive activity at the cell membrane to changes in cytoskeletal contractility. Overall, these findings offer novel perspectives to our understanding of how cancer cells biophysically sense their fibrous surroundings. The results from this study could ultimately pave the way for elucidating the precise fiber configurations that either facilitate or hinder cancer cell invasion, allowing for the development of new therapeutics in the long term that could inhibit the metastatic cascade at a relatively nascent stage and yield a more promising prognosis in the perennial fight against cancer. / Doctor of Philosophy / Cancer is a leading cause of death worldwide. Almost ninety percent of cancer related deaths arise from the spreading of cancer cells from the primary tumor site to secondary sites in the body – a processed termed as metastasis. The environment surrounding a tumor (tumor microenvironment) is highly fibrous in nature and can assist in the metastatic process by providing biophysical cues to the cells at the tumor boundary. These cells sense the presence of the surrounding fibers by extending "arms" termed as protrusions, and then eventually detach from the primary tumor and start migrating through the fibrous microenvironment. While numerous studies have investigated the various modes of cell migration in fibrous environments, there is very little information regarding how cancer cells use protrusions to initially sense the fibers prior to detaching. In this study, we used the Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with robust control on fiber diameter and network architecture and use these networks to systematically investigate how single cancer cells biophysically sense fibers that mimic in vivo dimensions. We discovered that cancer cells sense individual fibers by "wrapping-around" the axis of the fiber at the tip of the protrusion – a phenomenon we refer to as coiling. We found both the fiber diameter as well as the invasive capacity of cells can influence the coiling mechanics. Based on these results, we explored the use of coiling in conjunction with other key biophysical metrics such as the cell migration speed and how much force a cell can exert to develop a biophysical predictor for cancer cell aggressiveness. Finally, given that cells sense the fiber curvature by coiling, we explored the role of a key curvature sensing protein Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) in mediating coiling activity and found that knocking out (KO) IRSp53 results in reduced coiling and slower protrusions compared to wild-type (WT) cells. Furthermore, IRSp53 KO cells showed impaired contractility which led to an alteration in the nucleus shape and slower migration dynamics thus highlighting the role of IRSp53 in linking changes at the cell membrane to the underlying cell cytoskeleton. The results from this study could ultimately help us understand what type of fiber conditions around a primary tumor would either help or delay the emergence of the tumor boundary cells and thus allow for the development of therapeutics that could significantly slow down the metastatic process at a relatively early stage.