In this dissertation, advances in the fields of Photonics, and Plasmonics, and specifically, single cell analysis and waveguide sensing will be addressed. The first part of the dissertation is on Finite Difference Time Domain (FDTD) optimization and experimental demonstration of a nano-scale instrument that allows sensing at the cellular and subcellular levels. A new design of plasmonic coupler into a nanoscale waveguide is proposed and optimized using FDTD simulations. Following this, a subcellular nanoendoscope that can locally excite fluorescence in labelled cell organelles and collect the emitted fluorescent light for detailed spectrum analysis is fabricated and tested. The nanoendoscope has a sharp tapered tip of diameter ~ 50 nm that permits safe insertion into the cell that was confirmed by a number of viability experiments. FDTD analysis demonstrated that, with an optimized nanoendoscope taper profile, light emission and collection was very local. Thus, signal detection could be used for nano-photonic sensing of proximity of fluorophores. In further experiments, fluorescent signals were collected from individual organelles of living cells including: the nucleus of Acridine orange labelled human fibroblast cells, the nucleus of Hoechst stained live liver cells and the mitochondria of MitoTracker Red labelled MDA-MB-231 cells. In addition, this endoscope was inserted into a live organism, the nematode Caenorhabditis elegans, and in- vivo fluorescence signal was collected. Second, an innovative single step fabrication method of low loss polysilicon waveguides was developed as a potential platform for a number of photonic sensors. Optimization of a capacitively coupled plasma etching for the fabrication of a polysilicon waveguide with smooth sidewalls and low optical loss was demonstrated. A detailed experimental study on the influences of RF plasma power and chamber pressure on the roughness of the sidewalls of waveguides was conducted and waveguides were characterized using a scanning electron microscope. It was demonstrated that optimal combination of pressure (30 mTorr) and power (150 W) resulted in the smoothest sidewalls. The optical losses of the optimized waveguide were 4.1± 0.6 dB/ cm. Finally, an on-chip nanophotonic sensor for continuous blood coagulation analysis was proposed. The system was simulated using three-dimensional FDTD software. At first, the noise due to the presence of cells was calculated. Next, the design of a waveguide cladding-based filtering structure for elimination of the noise from cells was proposed and significantly decreased noise level was theoretically demonstrated.

Cells are the smallest living units and together they form all living organisms on earth. The cells are not only the building blocks of all living things, they also possess the most important information about life. A deeper understanding of these units may reveal hidden secrets about difficultly cured diseases, memory and learning, among others. Today’s techniques have problems such as low sensitivity, lethal preparation steps for the cells and overlaps in result spectra. Microfluidics has shown to be a useful tool allowing improved dynamic control, high throughput and sensitivity in nanoliters. The aim with this project is to design a microfluidic system for future integration with photonic sensors. Three different designs were developed, one design with the aim to integrate with photonic sensors and two for cell trapping only. Simulations and analytical calculations were performed to verify the requirements of single cell trapping. Simulation and analytical calculation results consorted, except for the ladder design. Moreover, strength calculations were performed for the sensor, to verify that it could handle the high pressures. A fabrication process was developed and an OSTE polymer was chosen as a suitable material. The transparency of the OSTE for fluorescent signals was studied. Results from the fabrication show proper lithography and molding as well as flow through channels. However, bubbles tend to appear in the channels. A rough surface of the chip appeared to primarily come from defects and filth on mask and mold. Three different connector solutions were tested, but they could not stand the high pressures. The work in this project has taken the development one step closer to the final goal to integrate photonic biosensors with a microfluidic system enabling single cell sensing.

Alteration in the tone of pulmonary arteries may lead to disease such as pulmonary hypertension often associated with major cardiac complications. This dysfunction is partly in the pulmonary arterial smooth muscle cells (PASMCs) where the excitation-contraction coupling is modified by ion channel behaviour to increase the contractile force. Mathematical models of systemic smooth muscle cells (SMCs) that incorporate electrophysiological and chemomechanical mechanisms to understand the underlying cellular physiology have been successfully employed. Models of pulmonary arterial smooth muscle cells (PASMCs) are only beginning to emerge. Mathematical model prototyping with available experimental data and model investigation from different parameter values is a time-consuming and complex process. This thesis is concerned with the development and validation of mathematical models of excitation-contraction coupling in three types of PASMCs of the rat species, one homogeneous type originating from the distal pulmonary arteries and two from proximal pulmonary arteries. Some key novel additions from previous vascular SMC models include the distinct modelling of Ca2+ in the subplasmalemmal cytosolic region, incorporation of subunit-specific currents from the K+ channel family and a generic G-protein receptor model able to reproduce complex Ca2+ profiles. The main pulmonary and systemic arteries statistically differ in its response to phenylephrine in a wire myograph. The ionic currents of the models were validated against experimental data largely from rat species. The models replicate the recordings of Ca2+ and the resting potential (Em) profiles arising from agonist-induced cytosolic Ca2+ ([Ca2+]i) stimulation (G-protein activation), nifedipine, ryanodine, caffeine and niflumic acid. The distal PASMC model was sensitive to an increase in [Ca2+]i from G-protein activation although were less likely to reproduce Ca2+ oscillations than proximal PASMCs. The proximal models determined the likely proximal PASMC type in literature experiments recording [Ca2+]i and Em. I have developed software that enables other users to simulate Ca2+ and Em changes in SMC studies and the ability to parse a master file describing the mathematical model into different language formats to increase productivity. These models provide a foundation for further studies to better understand PASMC function in the context of normal physiology as well as pathological conditions.

Fish has for a long time been a very important source of protein for human kind and with the world population at an all-time high, 7.5 billion and rapidly growing, the demand for fish as a food source is also at an all-time high and rapidly increasing. This has in turn led to overexploitation of many of the fish stocks of the world ocean’s and in many cases to depletion of fish stocks. The demand for sustainable food sources and sustainable usage of the world ocean’s fish stocks is therefore a subject with great deal of interest today. Much of the fish caught today are used for production of fish meal for usage as fish food at fish farms, which also increases the depletion of fish stocks around the globe. One way of dealing with this problem is to replace the fish meal as protein source in fish feed with protein from agricultural crops which in many cases are done today by usage of soy bean protein. This however poses another problem as the agricultural crops take up vast amount of land, in many cases obtained by diminishing the rainforests in the area. Another usage for the soybean would be as a direct human food source. Agricultural products are also dependent on environmental conditions to ensure reasonable production. The problems related to production of fish meal and soy has sparked the idea of using microorganisms for production of Single cell protein for usage as protein source in fish feed. Single cell protein can be produced in closed fermentation vessels and can be produced at a controlled rate and under controlled manners, while taking up negligible land space. During this thesis, the production of single cell protein from spent sulfite liquor using the filamentous fungi Paecilomyces variotii was examined. The aim of the project was to examine the effect of cultivation parameters (i.e., pH, temperature and nutrients) on the production of biomass as well as the protein content of the biomass. The correlation of the biomass growth and protein content have also been examined. The project was carried out by performing several experiment cultivations using spent sulfite liquor provided by Domsjö Fabriker in Örnsköldsvik. This process enables the utilization of a residual stream from the pulp industry which gives this process a huge environmental upside compared to similar processes as for example the commercial production of Quorn (a Single cell based food product) which utilizes pure glucose. The results showed that the protein content will steadily decrease as the biomass production increases hence it is desirable to keep the cultivation time at a minimum while maximizing biomass production during that time frame. It also points towards that the highest protein content is present in the young cell mass. The key conclusion from this thesis is however that it is possible to lower the pH of the cultivation from pH 6 down to pH 4.5 while still maintaining the biomass production and increasing the protein content. The highest obtained protein content was 62.7% at pH 4.5. The high protein content might be due to a slightly longer lag phase in the beginning of the cultivation which yields a higher number of younger cells in the final broth thus increasing protein content. Running the process at a lower pH is a huge advantage for industrial implementation as this on large scale means significant lower amounts of chemicals needed for pH adjusting of the spent sulfite liquor which renders the process much more economical. This is because pH adjustment today is one of the most costly process steps in the production of bioethanol from spent sulfite liquor.

Disruptions in the response of endothelial progenitor cells to changes in oxygen environment may present a possible mechanism behind multiple pediatric pulmonary disease models, such as bronchopulmonary dysplasia. Using high-throughput fixed single-cell protein and RNA imaging, we have created "stop-motion" movies of Thymosin. 4 (T beta 4) and Hypoxia Inducible Factor 1 alpha (HIF-1 alpha) protein expression and vascular endothelial growth factor (vegf) and endothelial nitric oxide synthase (eNOS) mRNA in human umbilical cord-derived endothelial colony-forming cells (ECFC). ECFC were grown in vitro under both room air and hyperoxia (50% O-2). We find elevated basal T beta 4 protein expression in ECFC derived from prematurely born infants versus full term infants. T beta 4 is a potent growth hormone that additionally acts as an actin sequestration protein and regulates the stability of HIF-1 alpha. This basal level increase of T beta 4 is associated with lower HIF1 alpha nuclear localization in preterm versus term ECFC upon exposure to hyperoxia. We find altered expression in the pro-angiogenic genes vegf and eNOS, two genes that HIF-1 alpha acts as a transcription factor for. This provides a potential link between a developmentally regulated protein and previously observed impaired function of preterm ECFC in response to hyperoxia.

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Made available in DSpace on 2016-05-02T13:54:42Z (GMT). No. of bitstreams: 1 Dissertacao completa Adriana Pereira Freire Junqueira.pdf: 220979 bytes, checksum: 17532e7ed387e5a189086db336b604ca (MD5) Previous issue date: 2006-06-09 / Coordenacao de Aperfeicoamento de Pessoal de Nïvel Superior / Piper cubeba widely distributed in the tropical and subtropical regions of the world is used medicinally in various manners without genetic toxicity evaluation In this study we investigated the clastogenic and genotoxic potential of the crude extract of the fruits of P cubeba in rodents cells using the micronucleus and single cell gel electrophoresis (SCGE) test systems The animals were treated by gavage with 3 concentrations of the extract 25 50 and 75 per cent of the LD50 (2 g/kg) From Swiss mice peripheral blood was collected at 24 h after the treatment for SCGE assay and at 48 and 72 h for micronucleus test when the animals were sacrificed From Wistar rats peripheral blood and hepatic cells were collected for SCGE assay and bone marrow cells for micronucleus test 24 h after the treatment and then the animals were submitted to euthanasia At the 75% of the LD50 extract concentration a statistically significant increase in the mean number of cells with micronuclei and with DNA damage was observed in all rodents cell types analyzed and on three of the analyzed animal groups genetic toxicity was observed also at the 50% of the LD50 concentration Under our experimental conditions P cubeba extract showed moderate clastogenic and genotoxic effect in the rodents cells / A Piper cubeba é distribuída nas regiões tropicais e subtropicais do mundo e é usada de várias maneiras na medicina como nos ensaios de genética toxicológica Neste estudo foi investigado o potencial clastogênico e genotóxico do extrato cru dos frutos da P cubeba em células de roedores usando o teste do micronúcleo e o ensaio single cell gel electrophoresis SCGE (ensaio cometa) Os animais foram tratados por gavage com 3 concentrações do extrato 25 50 e 75% da LD50 (2 g/Kg). Dos ratos Swiss foi coletado sangue periférico 24h após o tratamento para o ensaio SCGE e 48 e 72h para o teste do micronúcleo, quando os animais foram sacrificados Para os camundongos Wistar coletou-se sangue periférico e células hepáticas para o ensaio SCGE e células de medula óssea para o teste do micronúcleo 24h após o tratamento quando os animais foram submetidos à eutanásia No extrato na concentração de 75% da LD50 foi observado um aumento estatisticamente significativo no número de células com micronúcleos e danos no DNA nas células de roedores analisadas e no terceiro grupo de animais analisados foi observada também toxicidade genética na concentração de 50% da DL50 Conforme nossa condição experimental o extrato de P cubeba mostrou efeito clastogênico e genotóxico moderado em células de roedores

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22065号 / 工博第4646号 / 新制||工||1724(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 井上 康博, 教授 中部 主敬, 教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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Useful properties render titanium dioxide nanomaterials (NMs) to be one of the most commonly used NMs worldwide. TiO2 powder is used as food additives (E171), which may contain up to 36% nanoparticles. Consequently, humans could be exposed to comparatively high amounts of NMs that may induce adverse effects of chronic exposure conditions. Visualization and quantification of cellular NM uptake as well as their interactions with biomolecules within cells are key issues regarding risk assessment. Advanced quantitative imaging tools for NM detection within biological environments are therefore required. A combination of the label-free spatially resolved dosimetric tools, microresolved particle induced X-ray emission and Rutherford backscattering, together with high resolution imaging techniques, such as time-of-flight secondary ion mass spectrometry and transmission electron microscopy, are applied to visualize the cellular translocation pattern of TiO2 NMs and to quantify the NM-load, cellular major, and trace elements in differentiated Caco-2 cells as a function of their surface properties at the single cell level. Internalized NMs are not only able to impair the cellular homeostasis by themselves, but also to induce an intracellular redistribution of metabolically relevant elements such as phosphorus, sulfur, iron, and copper.