abstract: In this work, atomic force microscopy (AFM) and time resolved confocal fluorescence microscopy are combined to create a microscopy technique which allows for nanometer resolution topographic and fluorescence imaging. This technique can be applied to any sample which can be immobilized on a surface and which can be observed by fluorescence microscopy. Biological problems include small molecular systems, such as membrane receptor clusters, where very high optical resolutions need to be achieved. In materials science, fluorescent nanoparticles or other optically active nanostructures can be investigated using this technique. In the past decades, multiple techniques have been developed that yield high resolution optical images. Multiple far-field techniques have overcome the diffraction limit and allow fluorescence imaging with resolutions of few tens of nanometers. On the other hand, near-field microscopy, that makes use of optically active structures much smaller than the diffraction limit can give resolutions around ten nanometers with the possibility to collect topographic information from flat samples. The technique presented in this work reaches resolutions in the nanometer range along with topographic information from the sample. DNA origami with fluorophores attached to it was used to show this high resolution. The fluorophores with 21 nm distance could be resolved and their position on the origami determined within 10 nm. Not only did this work reach a new record in optical resolution in near-field microscopy (5 nm resolution in air and in water), it also gave an insight into the physics that happens between a fluorescent molecule and a dielectric nanostructure, which the AFM tip is. The experiments with silicon tips made a detailed comparison with models possible on the single molecule level, highly resolved in space and time. On the other hand, using silicon nitride and quartz as tip materials showed that effects beyond the established models play a role when the molecule is directly under the AFM tip, where quenching of up to 5 times more efficient than predicted by the model was found. / Dissertation/Thesis / Ph.D. Physics 2012

Orientadora: Carmem Silvia Fontanetti Christofoletti / Coorientadora: Roberta Cornélio Ferreira Nocelli / Banca: Elaine Cristina Mathias da Silva Zacarin / Banca: Thaisa Cristina Roat / Resumo: Os diplópodos configuram-se como organismos do solo ecologicamente importantes, mas também como espécies economicamente ameaçadoras em áreas rurais e urbanas quando ocorrem explosões populacionais; assim tais organismos configuram-se ora como alvo ora como vítima da ação de agroquímicos. Nesse sentido, alguns inseticidas, tal como a deltametrina, têm sido aplicados com o intuito de controlar a invasão de diplópodos nas residências. Tal inseticida é genericamente caracterizado por ter ação no sistema nervoso, no entanto, os efeitos deste sobre diplópodos ainda não foram analisados. Adicionalmente, existem poucos estudos sobre a morfologia e estrutura do sistema nervoso de diplópodos. Sendo assim, o presente trabalho objetivou primeiramente elucidar a anatomia e morfologia do sistema nervoso da espécie Gymnostreptus olivaceus por meio da observação durante a dissecção em estereomicroscópio e de análise histológica, histoquímica e citoquímica. Observou-se que o sistema nervoso dessa espécie é composto por um cérebro e um cordão nervoso, envolvidos por perineuro, lamela neural e mais externamente por um envoltório celular externo que foi descrito pela primeira vez em Diplopoda. Foram descritos os detalhes histológicos observados em cada estrutura que compõe o sistema nervoso da espécie. Além disso, uma formulação comercial do inseticida deltametrina foi aplicada com o intuito de avaliar a efetividade da utilização popular de tal inseticida no combate a infestações de diplópodos. Para a observação de efeitos neurotóxicos causados pelo inseticida foi utilizada a marcação de sinapsina avaliada por microscopia confocal. Foram aplicadas topicamente em indivíduos da espécie G. olivaceus, quatro concentrações de deltametrina; a menor concentração baseou-se nas recomendações do fabricante para o combate de outros artrópodes, enquanto as demais concentrações corresponderam a um quinto da DL50... / Abstract: Diplopods are ecologically important soil organisms that may also act as economically damaging species in urban and rural areas when their populations undergo outbreaks. They are, therefore, sometimes targetedfor agrochemicals action. Thus, some insecticides (e.g., deltamethrin) have been applied in order to control diplopods home invasions and crop attacks. Deltamethrin is characterized by its action on the nervous system; however, its effects on diplopods have yet to be analyzed. Additionally, there are few studies on the morphology and structure of the nervous system of diplopods. The present study aimed to elucidate the anatomy and morphology of the nervous system of the species Gymnostreptus olivaceus through direct observation during dissection under a stereomicroscope and by histological, histochemical, and cytochemical analyses. It was observed that the nervous system of the species consists of a brain and ventral nerve cord that are both wrapped by a perineurium, a neural lamella, and, more externally, by an external cell sheath that was first described in Diplopoda. Histological details were described for each of this species' nervous system structures. Furthermore, a commercial formulation of deltamethrin was applied to evaluate the effectiveness of the popular use of this insecticide to combat diplopods infestations. In order to observe neurotoxic effects caused by the insecticide, a synapsin labeling was used and assessed by confocal microscopy. Four concentrations of deltamethrin were applied topically to G. olivaceus individuals; the lowest concentration was based on the manufacturer's recommendations for controlling other arthropods, while the other concentrations correspond to one fifth of the LD50 previously established for the species, the LD50 itself, and twice the LD50. Death numbers were evaluated and synapsin levels were assessed in the brain at 12, 24 and 48 hours after insecticide treatment. The insecticide was... / Mestre

PEIXOTO, M. E. B. Histomorfometria das terminações nervosas das conchas nasais inferiores de humanos por imunofluorescência e microscopia confocal a laser. 2016. 64 f. Dissertação (Mestrado em Cirurgia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-05-02T12:39:45Z No. of bitstreams: 1 2017_dis_mebpeixoto.pdf: 2390705 bytes, checksum: fef4a7f0436fecf6ad1553ff47d370e3 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-05-02T12:39:53Z (GMT) No. of bitstreams: 1 2017_dis_mebpeixoto.pdf: 2390705 bytes, checksum: fef4a7f0436fecf6ad1553ff47d370e3 (MD5) / Made available in DSpace on 2017-05-02T12:39:53Z (GMT). No. of bitstreams: 1 2017_dis_mebpeixoto.pdf: 2390705 bytes, checksum: fef4a7f0436fecf6ad1553ff47d370e3 (MD5) Previous issue date: 2017-04-18 / Nasal obstruction is one of the main complaint of patients with rhinitis, which is frequently associated with increased nasal airway resistance caused by hypertrophic changes of the inferior turbinates, however, studies have demonstrated that the objective measurement of nasal airway resistance does not always correlate with the subjective perception of the degree of nasal obstruction. The inferior turbinates are elongated, paired structures situated at the lateral nasal wall and made of a central core of osseous skeleton and a mucosal layer on each side almost exclusively covered with a pseudostratified ciliated columnar epithelium, with a well-defined basement membrane zone and a thick lamina propria. They play an important function in nasal physiology through reflex responses. The sensory nerves monitor the conditions of the mucosal microenvironment and initiate protective mechanisms immediately via axon responses. These nerve endings also have an important role on the perception of nasal patency. Despite the importance of these structures, little is known about their morphology and distribution in the normal nasal mucosa of human inferior turbinates. In order to obtain morphological and distribution data of nerve endings in inferior turbinates, specimens obtained from six individuals (three men and three women) with ages ranging from 16 to 76 years were submitted to fluorescent antibody technique with the marker pan -axonal anti-protein gene product 9.5 and confocal laser scanning microscopy. Free nerve endings were identified, mainly in the superficial layers of the lamina propria, some very close to the basement membrane. No complex nerve endings of the corpuscular type were identified. There were no differences in the distribution of nerve endings when compared to the septal and meatal faces or according to the gender of the individuals evaluated. Free nerve endings maintain an intimate relationship with seromucous glands and their ducts in the more superficial layers of the lamina propria, as well as with blood vessels, especially in their deeper portions, pointing to the role they play in the neuromodulation of glandular secretion and vasomotor control. Surgical techniques that allow the preservation of these free nerve endings may provide better postoperative outcomes, with a higher resolution of the clinical complaint of nasal obstruction and a lower rate of complications such as empty nose syndrome. / Obstrução nasal é uma das principais queixas em pacientes com rinite, sendo frequentemente associada ao aumento da resistência nasal pela hipertrofia das conchas nasais inferiores, contudo, estudos têm demonstrado que mensurações objetivas da resistência de via aérea nasal nem sempre se correlacionam à percepção subjetiva do grau de obstrução nasal. As conchas nasais inferiores são estruturas alongadas, pareadas e situadas nas paredes nasais laterais, constituídas de um eixo ósseo central envolto, quase exclusivamente, por epitélio respiratório pseudoestratificado cilíndrico ciliado, com uma membrana basal bem definida e uma espessa lâmina própria. Possuem um papel importante na fisiologia nasal através de respostas reflexas. Os nervos sensoriais monitoram o microambiente da mucosa nasal e iniciam os mecanismos protetores imediatamente, via respostas axonais. Essas terminações nervosas também possuem importante ação na percepção da patência nasal. A despeito do importante papel dessas estruturas, pouco é conhecido acerca de sua morfologia e distribuição na mucosa nasal normal de conchas inferiores de humanos. Com o objetivo de obter dados morfológicos e de distribuição das terminações nervosas em conchas nasais inferiores, espécimes retirados de seis indivíduos (três homens e três mulheres) com idades variando de 16 a 76 anos foram submetidos a estudo de imunofluorescência com o marcador pan-axonal antiproduto gênico protéico 9,5 e microscopia confocal de varredura a laser. Foram identificadas terminações nervosas livres, principalmente, nas camadas mais superficiais da lâmina própria, algumas bem próximas à membrana basal. Não foram identificadas terminações nervosas complexas, do tipo corpusculares. Não houve diferenças na distribuição das terminações nervosas quando comparadas as faces septal e meatal ou em função do sexo dos indivíduos avaliados. As terminações nervosas livres mantém relação íntima com glândulas seromucosas e seus ductos nas camadas mais superficiais da lâmina própria, bem como com vasos sanguíneos, sobretudo, em suas porções mais profundas, apontando para o papel que possuem na neuromodulação dos fenômenos de secreção glandular e controle vasomotor. Técnicas cirúrgicas que permitam a preservação dessas terminações nervosas livres talvez possibilitem desfechos pós-operatórios melhores, com maior resolução da queixa clínica de obstrução nasal e menor índice de complicações como a síndrome do nariz vazio (empty nose).

Este estudo teve como objetivo avaliar, em microscopia confocal à laser (CLSM), a ação antimicrobiana do Neo MTA Plus, do Biodentine e do MTA Angelus, utilizando um modelo de biofilme multi-espécies microcosmo, e descrever a morfologia dos microrganismos encontrados no biofilme, através da análise em microscópio eletrônico de varredura (MEV). Cinquenta e seis blocos de dentina, confeccionados a partir de raízes de dentes humanos, foram cortados em dimensões de 5 x 4 x 4 mm, limpos e esterilizados. Dois blocos foram utilizados para o controle do processo de esterilização. Os demais 54 foram fixados em placas de Hawley confeccionadas para 6 voluntários. Cada voluntário utilizou uma placa por 72 horas. Após esse período, os blocos foram mantidos em poços de cultura celular, contendo BHI, em estufa à 37ºC, por um período de 21 dias. Ao final do processo de maturação do biofilme, as amostras foram divididas aleatoriamente em 5 grupos, conforme os cimentos a serem testados e o método de análise: Neo MTA Plus (n=12), Biodentine (n=12), MTA Angelus (n=12), controle negativo (n=12) e grupo para análise em MEV (n=6). A seguir, os cimentos foram colocados em contato com o biofilme dos blocos dos grupos-teste e os conjuntos (bloco + cimento) ou os blocos do grupo controle e para análise em MEV foram recolocados em poços de cultura contendo BHI, onde permaneceram por 7 dias à 37ºC. A análise em CLSM foi realizada nos blocos de dentina e nos cimentos, separadamente. A analise em MEV foi realizada nas seis amostras do grupo correspondente. Todos os grupos apresentaram mais de 50% de bactérias viáveis. O Neo MTA Plus foi o único material a apresentar diferença significativa quando comparado ao grupo controle negativo (P=.0379). A análise em MEV mostrou um biofilme composto por bactérias de forma circular e de bastonetes, circundadas por matriz extra-celular. Em conclusão, todos os grupos apresentaram bactérias viáveis e o Neo MTA Plus teve a maior ação antimicrobiana. / This study aimed to assess, by confocal laser scanning microscopy (CLSM), the antimicrobial effect of Neo MTA Plus, Biodentine and MTA Angelus, using a microcosmo biofilm model, and to describe the microrganisms morphology, by SEM. Fifty-six dentin blocks were made from human teeth roots, cut in a size 5 x 4 x 4 mm, cleaned and sterelized. Two blocks were used to sterelization control. The others 54 blocks were fixed in Hawley retainers made for 6 volunteers that used it for 72h. After this period, they were placed in wells with BHI for 21 days, at 37ºC. At the end of the biofilm growth, the samples were randomly divided in 5 groups: Neo MTA Plus (n=12), Biodentine (n=12), MTA Angelus (n=12), negative control (n=12), and SEM analysis (n=6). The cements were placed in contact with the blocks of the tested groups. All samples were replaced in wells with BHI by 7 days at 37ºC. CLSM analysis evaluated the dentin blocks and cements isolated. SEM observation were performed in the six samples of the respective group. All groups presented a level of viable bacteria higher than 50%. Neo MTA Plus was the only one that showed significant difference from the negative control group (P=.0379). SEM analysis showed a biofilm composed of spherical and rod-shaped bacteria surrounded by extracellular matrix. All groups presented viable bacteria and Neo MTA Plus had the highest antimicrobial effect.

Made available in DSpace on 2015-06-17T19:33:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-25. Added 1 bitstream(s) on 2015-06-18T12:47:57Z : No. of bitstreams: 1 000832708_20170225.pdf: 185166 bytes, checksum: 03d714a9483efdb12391afe44d94377d (MD5) Bitstreams deleted on 2017-03-03T11:01:34Z: 000832708_20170225.pdf,. Added 1 bitstream(s) on 2017-03-03T11:02:42Z : No. of bitstreams: 1 000832708.pdf: 940445 bytes, checksum: 83c176e5777a5bb9d647ba70aa10ea1b (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os diplópodos configuram-se como organismos do solo ecologicamente importantes, mas também como espécies economicamente ameaçadoras em áreas rurais e urbanas quando ocorrem explosões populacionais; assim tais organismos configuram-se ora como alvo ora como vítima da ação de agroquímicos. Nesse sentido, alguns inseticidas, tal como a deltametrina, têm sido aplicados com o intuito de controlar a invasão de diplópodos nas residências. Tal inseticida é genericamente caracterizado por ter ação no sistema nervoso, no entanto, os efeitos deste sobre diplópodos ainda não foram analisados. Adicionalmente, existem poucos estudos sobre a morfologia e estrutura do sistema nervoso de diplópodos. Sendo assim, o presente trabalho objetivou primeiramente elucidar a anatomia e morfologia do sistema nervoso da espécie Gymnostreptus olivaceus por meio da observação durante a dissecção em estereomicroscópio e de análise histológica, histoquímica e citoquímica. Observou-se que o sistema nervoso dessa espécie é composto por um cérebro e um cordão nervoso, envolvidos por perineuro, lamela neural e mais externamente por um envoltório celular externo que foi descrito pela primeira vez em Diplopoda. Foram descritos os detalhes histológicos observados em cada estrutura que compõe o sistema nervoso da espécie. Além disso, uma formulação comercial do inseticida deltametrina foi aplicada com o intuito de avaliar a efetividade da utilização popular de tal inseticida no combate a infestações de diplópodos. Para a observação de efeitos neurotóxicos causados pelo inseticida foi utilizada a marcação de sinapsina avaliada por microscopia confocal. Foram aplicadas topicamente em indivíduos da espécie G. olivaceus, quatro concentrações de deltametrina; a menor concentração baseou-se nas recomendações do fabricante para o combate de outros artrópodes, enquanto as demais concentrações corresponderam a um quinto da DL50... / Diplopods are ecologically important soil organisms that may also act as economically damaging species in urban and rural areas when their populations undergo outbreaks. They are, therefore, sometimes targetedfor agrochemicals action. Thus, some insecticides (e.g., deltamethrin) have been applied in order to control diplopods home invasions and crop attacks. Deltamethrin is characterized by its action on the nervous system; however, its effects on diplopods have yet to be analyzed. Additionally, there are few studies on the morphology and structure of the nervous system of diplopods. The present study aimed to elucidate the anatomy and morphology of the nervous system of the species Gymnostreptus olivaceus through direct observation during dissection under a stereomicroscope and by histological, histochemical, and cytochemical analyses. It was observed that the nervous system of the species consists of a brain and ventral nerve cord that are both wrapped by a perineurium, a neural lamella, and, more externally, by an external cell sheath that was first described in Diplopoda. Histological details were described for each of this species' nervous system structures. Furthermore, a commercial formulation of deltamethrin was applied to evaluate the effectiveness of the popular use of this insecticide to combat diplopods infestations. In order to observe neurotoxic effects caused by the insecticide, a synapsin labeling was used and assessed by confocal microscopy. Four concentrations of deltamethrin were applied topically to G. olivaceus individuals; the lowest concentration was based on the manufacturer's recommendations for controlling other arthropods, while the other concentrations correspond to one fifth of the LD50 previously established for the species, the LD50 itself, and twice the LD50. Death numbers were evaluated and synapsin levels were assessed in the brain at 12, 24 and 48 hours after insecticide treatment. The insecticide was... / FAPESP: 2012/24562-5

Made available in DSpace on 2016-06-02T19:06:26Z (GMT). No. of bitstreams: 1 DissMS.pdf: 2267976 bytes, checksum: dfd0f34aeb56928031a68733947fbf11 (MD5) Previous issue date: 2005-09-23 / Computer Optical Sectioning Microscopy (COSM) is widely used in morphological studies of cellular structures. COSM appears as a great challenge to digital image processing, because the large size of the three-dimensional files. The major problem in COSM is the blurring, which practically turn the image unusable without the use of digital image processing techniques. In this work, based on projection onto convex set were developed image processing techniques to restore COSM images, obtained by a confocal microscope. Although confocal microscope presents less blurring than the wide field microscope, it has a larger Poisson noise level. To reduce the Poisson noise the Anscombe Transform (AT) was used as well a pointwise Wiener Filter. The POCS methods were evaluated by means of simulations, using synthetic images (phantom), and the results was evaluated by ISNR (Improvement in Signal Noise Ratio) and UIQ (Universal Quality Index). The developed algorithms, using RAP (Row Action Projection) present satisfactory results, but the computational costs were expensive. / A técnica de Microscopia de Seccionamento Óptico por Computador (COSM) é muito utilizada no estudo morfológico de estruturas celulares. Essa técnica apresenta um grande desafio para a área de processamento de imagens, pois os arquivos tridimensionais possuem grande volume de dados para serem processados. O principal problema do COSM é o borramento existente, que praticamente inviabiliza a utilização dessa imagem sem o uso de técnicas de processamento de imagem. Nesse trabalho, foram desenvolvidas técnicas de projeção em conjuntos convexos POCS (Projection Onto Convex Sets) de processamento de imagens para a restauração das imagens de COSM, obtidas por microscópio confocal. Embora os microscópios confocais apresentem menor borramento da imagem que os microscópios wide field, ele possuem um nível maior de ruído Poisson. Para atenuar o ruído Poisson foi utilizada a Transformada de Anscombe (AT) e o filtro de Wiener pontual. Os métodos POCS, foram avaliados através de simulações utilizando imagens sintéticas (phantoms), e os resultados foram avaliados através do ISNR(Improvement Signal to Noise Ratio) e do UIQ(Universal Quality Index). Os algoritmos desenvolvidos, utilizando RAP (Row Action Projection) apresentaram bons resultados, mas o custo computacional para imagem tridimensional é bastante elevado.

Este estudo teve como objetivo avaliar, em microscopia confocal à laser (CLSM), a ação antimicrobiana do Neo MTA Plus, do Biodentine e do MTA Angelus, utilizando um modelo de biofilme multi-espécies microcosmo, e descrever a morfologia dos microrganismos encontrados no biofilme, através da análise em microscópio eletrônico de varredura (MEV). Cinquenta e seis blocos de dentina, confeccionados a partir de raízes de dentes humanos, foram cortados em dimensões de 5 x 4 x 4 mm, limpos e esterilizados. Dois blocos foram utilizados para o controle do processo de esterilização. Os demais 54 foram fixados em placas de Hawley confeccionadas para 6 voluntários. Cada voluntário utilizou uma placa por 72 horas. Após esse período, os blocos foram mantidos em poços de cultura celular, contendo BHI, em estufa à 37ºC, por um período de 21 dias. Ao final do processo de maturação do biofilme, as amostras foram divididas aleatoriamente em 5 grupos, conforme os cimentos a serem testados e o método de análise: Neo MTA Plus (n=12), Biodentine (n=12), MTA Angelus (n=12), controle negativo (n=12) e grupo para análise em MEV (n=6). A seguir, os cimentos foram colocados em contato com o biofilme dos blocos dos grupos-teste e os conjuntos (bloco + cimento) ou os blocos do grupo controle e para análise em MEV foram recolocados em poços de cultura contendo BHI, onde permaneceram por 7 dias à 37ºC. A análise em CLSM foi realizada nos blocos de dentina e nos cimentos, separadamente. A analise em MEV foi realizada nas seis amostras do grupo correspondente. Todos os grupos apresentaram mais de 50% de bactérias viáveis. O Neo MTA Plus foi o único material a apresentar diferença significativa quando comparado ao grupo controle negativo (P=.0379). A análise em MEV mostrou um biofilme composto por bactérias de forma circular e de bastonetes, circundadas por matriz extra-celular. Em conclusão, todos os grupos apresentaram bactérias viáveis e o Neo MTA Plus teve a maior ação antimicrobiana. / This study aimed to assess, by confocal laser scanning microscopy (CLSM), the antimicrobial effect of Neo MTA Plus, Biodentine and MTA Angelus, using a microcosmo biofilm model, and to describe the microrganisms morphology, by SEM. Fifty-six dentin blocks were made from human teeth roots, cut in a size 5 x 4 x 4 mm, cleaned and sterelized. Two blocks were used to sterelization control. The others 54 blocks were fixed in Hawley retainers made for 6 volunteers that used it for 72h. After this period, they were placed in wells with BHI for 21 days, at 37ºC. At the end of the biofilm growth, the samples were randomly divided in 5 groups: Neo MTA Plus (n=12), Biodentine (n=12), MTA Angelus (n=12), negative control (n=12), and SEM analysis (n=6). The cements were placed in contact with the blocks of the tested groups. All samples were replaced in wells with BHI by 7 days at 37ºC. CLSM analysis evaluated the dentin blocks and cements isolated. SEM observation were performed in the six samples of the respective group. All groups presented a level of viable bacteria higher than 50%. Neo MTA Plus was the only one that showed significant difference from the negative control group (P=.0379). SEM analysis showed a biofilm composed of spherical and rod-shaped bacteria surrounded by extracellular matrix. All groups presented viable bacteria and Neo MTA Plus had the highest antimicrobial effect.

A novel colloidal model system is developed for the purpose of achieving simultaneous three-dimensional (3D) confocal imaging and optical tweezing of impurity probe particles embedded in a dense material of colloidal host particles. First, the colloidal host particles are developed from 3-trimethoxysilyl-propylmethacrylate (TPM), and the solvent mixture is tuned to match the refractive index and the density of the particles. A sedimentation-diffusion equilibrium profile of the TPM particles was imaged in 3D to establish suitability of the system for 3D confocal laser scanning microscopy, and to study its phase behaviour and particle dynamics. Then, core-shell particles, which consist of a high refractive index core and a TPM shell, are synthesised to be used as the impurities. The versatility of the two-step coating procedure with TPM has been demonstrated on several core materials, and the optical properties of the core-shell particle are established using digital holographic microscopy and their optical trapping strengths. Together with the TPM host particles, the core-shell particles are shown to be suitable impurity probes in dense colloidal materials, as they can be manipulated using optical tweezers in all three-dimensions, whilst the structure and dynamics of the surrounding host particles can be imaged simultaneously using fast confocal laser scanning microscopy. Subsequently, to demonstrate the capability of the TPM-based colloidal model system, the depletion potential of a pair of core-shell probe particles embedded in a sea of TPM host particles has been measured using optical tweezing. This is derived from comparing the direct pair potential between the core-shell particles in a TPM refractive index matching solvent, and the potential of mean force for the core-shell particles embedded in a dense fluid region of the TPM host particles. Direct visualisation of the liquid structures of the TPM host particles in the binary system around the probe particles has been linked to the form of the depletion potential, and its oscillatory nature as a function of the particle separation. Lastly, the formation of colloidal dumbbell particles is discussed. The dumbbell formation has been rationalised in terms of the total surface energy of droplet formation on spherical surfaces and the calculations are compared with experimental results from coating various seed particles with TPM. Using optically anisotropic dumbbell particles and tuning the refractive index of the dispersion medium, a preliminary two-trap experiment has been conducted which shows unusual trapping behaviour when a time-delayed feedback trapping trajectory has been applied.

Two corneal dystrophies, posterior polymorphous corneal dystrophy (PPCD) and keratoconus, have been associated with missense mutations found in the transcription factor-encoding gene Visual System Homeobox 1 (VSX1). Despite this association, the pathogenic link between VSX1 and these diseases remains controversial. To address this issue, I utilized a variety of in vitro approaches to study how seven VSX1 missense mutations found in disease populations that span two highly conserved domains, the homeodomain (HD) and CVC domain affect VSX1 transcriptional activity, protein expression levels and subcellular localization. I also carried out an in vivo investigation by generating a mouse line carrying a mutation in Vsx1: P254R. Corneal morphology was examined through histology and ex vivo whole eye confocal imaging which was used to assess corneal thickness. Quantification of immunocytochemistry was used to characterize terminal marker expression in the inner retina compared to previously described phenotypes in Vsx1-null mice. My in vitro results showed that mutations found in both the HD and CVC domain alter the normal transcriptional repression activity in Vsx1. These changes were not due to changes to protein expression or subcellular localization. Characterization of corneal and retinal phenotypes in vivo revealed no significant differences in Vsx1 P254R mice when compared to wild-type and Vsx1-null controls. In conclusion, my work shows that Vsx1 P254R is not pathogenic for corneal dystrophies in a mouse model. However, my in vitro studies show that Vsx1 mutations have the ability to alter transcriptional activity and therefore still have the potential to be pathogenic in humans. Further investigation is needed to determine whether VSX1 mutations found in disease populations are, in fact, causative for corneal dystrophies. / Graduate / 2019-04-25

No description available.