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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Development of Fluorescent Turn-on Self-assembled Nanoprobes for Imaging Specific Proteins under Live Cell Conditions. / 生細胞での蛍光オフオン型蛋白質イメージングを可能とする自己組織化ナノ集合体の開発

Mizusawa, Keigo 25 March 2013 (has links)
Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17600号 / 工博第3759号 / 新制||工||1573(附属図書館) / 30366 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 松田 建児, 教授 秋吉 一成 / 学位規則第4条第1項該当
2

Activatable fluorescence imaging of macrophages in atherosclerotic plaques using iron oxide nanoparticles conjugated with indocyanine green / インドシアニングリーン標識酸化鉄ナノ粒子による動脈硬化性プラークにおけるマクロファージのアクチベイタブル蛍光イメージング

Ikeda, Hiroyuki 26 November 2018 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21418号 / 医博第4408号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 髙橋 良輔, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
3

Use of fluorescent imaging to monitor drug responses in mouse models of tumourigenesis

Balderstone, Lucy Anne January 2014 (has links)
As our understanding of the complexities of cancer biology has increased, the ability to exploit unique features of tumour cells with molecularly targeted therapies has become a reality. However, despite unprecedented volumes of new molecules in clinical trials, the number of highly effective drugs approved by the regulatory authorities remains disappointingly low. Moreover, oncology drug development is plagued by high levels of attrition in late phase clinical development. Failure due to poor efficacy and toxicity issues are not believed to be a result of the development of molecules with inadequate pharmaceutical properties, but rather due to a lack of understanding of their full mechanism of action. All of this points to imprecise analysis of the drugs during the preclinical phase, highlighting the need for better preclinical drug development tools. Animal models provide a key preclinical tool, and as a therapeutic area, oncology is characterised by models which are not predictive of the true human pathology. Overexpression of the human epidermal growth factor receptor two (HER2) oncogene, and inactivation of the phosphatase and tensin (PTEN) tumour suppressor, are two important events in human breast cancer. A novel conditional mouse model driven by overexpression of HER2 coupled with / without the loss of PTEN has been characterised to interrogate the importance of these two cellular perturbations. Multifocal tumours arose in mice from both lines, while luminal tumour characteristics were shown to be reduced and basal characteristics increased with a reduction in PTEN expression. Disruption of PTEN rapidly accelerated tumour onset (from 138 to 82 days) and tumour growth (with the time from tumour onset to maximum tumour size reduced from 38 to 21 days), significantly reducing overall survival (from 165 to 102 days). The ability of tumour cells to colonize the lungs was not significantly affected by the loss of PTEN. Tumours arising in both mice genotypes were utilized to generate cell lines. These failed to provide an in vitro representation of the tumours, and found little utility in drug efficacy studies with HER family targeted agents, a situation which could be improved by the use of different culture methods. Since suppression of apoptosis is a hallmark of human cancer, and a desired endpoint of many anticancer therapies is the induction of cell death, the generation of cell lines inherently capable of sensing caspase-mediated apoptotic cell death would be a valuable drug development tool. Given that fluorescence imaging is also emerging as a potentially powerful modality for preclinical drug development, a novel fluorescent in house apoptosis reporter construct was generated (pCasFSwitch). Initial validation of pCasFSwitch by transient transfection into murine mammary carcinoma cells proved difficult due to transfection associated toxicity, yet proof-of-principle was indicated. Transfer of pCasFSwitch into a retroviral backbone vector enabled the generation of stably transfected squamous carcinoma cells more suitable for further analysis. Incubation of lysates from these cells with recombinant enzymes revealed the construct could be cleaved by caspase-3, but not by other members of the cysteine protease family. Furthermore, assessment of apoptosis levels in the cells upon staurosporine treatment proved the utility of the construct to quantify cell death, and was validated against data generated with a commercial competitor, NucView. Further comparison of the specificity of the imaging agents using caspase inhibitors was limited by the functionality of currently available inhibitors, but did reveal that in common with NucView, construct quantified levels of apoptosis were affected by inhibition. This thesis details the development of two preclinical drug development tools. A novel mouse model enables biological interrogation of two key events in human breast carcinogenesis. Since PTEN loss is associated with resistance to HER2 targeted therapies, it is ideally suited for efficacy testing to overcome such resistance. The in house fluorescent apoptosis imaging agent allows a temporal read-out of drug effects in live single cells. While the use of intravital imaging of stable cell lines implanted under imaging windows would allow in vivo validation of in vitro data. Taken together, such facilitation of thorough evaluation of therapies at the preclinical stage, will reduce the adverse effects felt by the pharmaceutical industry of failure late in the drug development pipeline.
4

Optical clearing and deep-tissue fluorescence imaging using fructose / 果糖水溶液による組織透明化及び生体試料の深部観察

Ke, Meng-Tsen 24 March 2014 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18426号 / 生博第306号 / 新制||生||40(附属図書館) / 31284 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松崎 文雄, 教授 渡邉 大, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM
5

Visualization of the Intracellular HIV-1 Replication Cycle

Stultz, Ryan David 07 September 2017 (has links)
No description available.
6

Synthesis and in vitro applications of fluorescent imaging agents

Brunet, Aurelie Claude Laure January 2014 (has links)
Fluorescent imaging technologies that offer new ways to visualise and quantify fluorescently labelled molecules are increasing, necessitating the development of fluorescent molecules that can efficiently and specifically label targets in vitro and in vivo. The first aim of this thesis was the study of human neutrophil elastase. Human neutrophil elastase is an important enzyme in the regulation of inflammation but if over expressed can become part of the cause of inflammation itself. To elucidate this dual function and have a greater understanding of this enzyme, an imaging probe for neutrophil elastase was designed. Firstly, the syntheses of fluorescently labelled three branched dendron core structures were optimised, and studied in neutrophils. The selected core structure was functionalised with an elastase specific peptide sequence and fluorescently labelled. The probe was specifically cleaved by neutrophil elastase in an enzymatic assay and in the presence of activated neutrophils (Chapter 1). Fluorescein and rhodamine are dyes that are readily available, are affordable and have convenient wavelengths for microscopy and flow cytometry. Carboxyfluorescein diacetate N-succinimidyl ester (CFDA-SE) is a commonly used fluorescein derivative, widely used in cell proliferation assay. It is mainly used as a mixture of isomers and its synthesis is not reported. Herein a short and simple synthesis of the two individual isomers of carboxyfluorescein diacetate N-succinimidyl ester as well as the equivalent rhodamine variation (carboxytetraethylrhodamine N-succinimidyl ester) is reported (Chapter 2). The labelling properties of these probes were studied in proliferation assays on mouse and human T lymphocytes. Finally, the nuclear penetration of the dendron structure combined with nuclear localisation sequences (NLS) was investigated. Attachment of nuclear localisation sequences to the probe in the presence of fluorescein demonstrated successful entry into the nucleus in human alveolar adenocarcinoma cell line (A549) (Chapter 3).
7

Protein sorption to contact lenses and intraocular lenses

Luensmann, Doerte January 2009 (has links)
Purpose: To locate protein sorption on the surface and inside the matrix of soft contact lens materials and intraocular lenses (IOL). Methods: The proteins albumin and lysozyme were investigated as they are highly abundant in blood serum and tears, respectively. Proteins were conjugated with organic fluorescent probes and using confocal laser scanning microscopy (CLSM) the sorption profile to contact lenses and IOL could be determined. Radiolabeled protein was used for quantification purposes. • Albumin sorption to etafilcon A and lotrafilcon B was determined (Chapter 3) • Different fluorescent probes were used for conjugation and the impact on albumin sorption behaviour was investigated (Chapter 4) • Lysozyme sorption to nine different pHEMA-based and silicone hydrogel contact lenses was determined using two fluorescent probes (Chapter 5) • The efficiency of protein removal from contact lenses using contact lens care regimens was investigated (Chapter 6) • Albumin sorption to IOL materials was quantified and imaged using a modified CLSM technique (Chapter 7) Results: Albumin and lysozyme sorption profiles differed between materials, and were influenced by the fluorescent probes used for conjugation. After one day of incubation, both proteins could be located within all contact lens materials, except for lotrafilcon A and lotrafilcon B, which primarily allowed deposition on the lens surface. An increase in protein accumulation was found for most materials over the maximum investigated period of 14 days, using CLSM and radiolabel techniques. The efficiency of contact lens care regimens to remove lysozyme and albumin depended on the lens material, care regimen and protein type investigated. PMMA and silicone IOLs showed protein exclusively on the surface, while a hydrophilic acrylic IOL allowed penetration into the lens matrix over time. Despite the albumin penetration depth into hydrophilic acrylic, the highest albumin levels were determined for the silicone IOL. Conclusions: CLSM provides detailed information that can describe the protein distribution in transparent biomaterials, with scanning depths up to a few hundred microns. However, the CLSM data are primarily of qualitative value, which necessitates a quantitative technique (e.g. radiolabeling) to determine the total protein content.
8

Protein sorption to contact lenses and intraocular lenses

Luensmann, Doerte January 2009 (has links)
Purpose: To locate protein sorption on the surface and inside the matrix of soft contact lens materials and intraocular lenses (IOL). Methods: The proteins albumin and lysozyme were investigated as they are highly abundant in blood serum and tears, respectively. Proteins were conjugated with organic fluorescent probes and using confocal laser scanning microscopy (CLSM) the sorption profile to contact lenses and IOL could be determined. Radiolabeled protein was used for quantification purposes. • Albumin sorption to etafilcon A and lotrafilcon B was determined (Chapter 3) • Different fluorescent probes were used for conjugation and the impact on albumin sorption behaviour was investigated (Chapter 4) • Lysozyme sorption to nine different pHEMA-based and silicone hydrogel contact lenses was determined using two fluorescent probes (Chapter 5) • The efficiency of protein removal from contact lenses using contact lens care regimens was investigated (Chapter 6) • Albumin sorption to IOL materials was quantified and imaged using a modified CLSM technique (Chapter 7) Results: Albumin and lysozyme sorption profiles differed between materials, and were influenced by the fluorescent probes used for conjugation. After one day of incubation, both proteins could be located within all contact lens materials, except for lotrafilcon A and lotrafilcon B, which primarily allowed deposition on the lens surface. An increase in protein accumulation was found for most materials over the maximum investigated period of 14 days, using CLSM and radiolabel techniques. The efficiency of contact lens care regimens to remove lysozyme and albumin depended on the lens material, care regimen and protein type investigated. PMMA and silicone IOLs showed protein exclusively on the surface, while a hydrophilic acrylic IOL allowed penetration into the lens matrix over time. Despite the albumin penetration depth into hydrophilic acrylic, the highest albumin levels were determined for the silicone IOL. Conclusions: CLSM provides detailed information that can describe the protein distribution in transparent biomaterials, with scanning depths up to a few hundred microns. However, the CLSM data are primarily of qualitative value, which necessitates a quantitative technique (e.g. radiolabeling) to determine the total protein content.
9

Development of Molecular Tools for Analysis and Imaging of ATP and Other Biomolecules Based on Coordination Chemistry / ATP等の生体分子の解析・イメージングのための配位化学に基づいた分子ツールの開発

Kurishita, Yasutaka 24 March 2014 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18299号 / 工博第3891号 / 新制||工||1597(附属図書館) / 31157 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 梅田 眞郷, 教授 森 泰生 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM
10

<b>DIFFUSION QUANTIFICATION IN SPATIALLY HETEROGENEOUS MATERIALS</b>

Dustin M Harmon (11267964) 08 April 2024 (has links)
<p dir="ltr">Spatial heterogeneity is ubiquitous across life and the universe; the same is true for phase-separating pharmaceutical formulations, cells, and tissues. To interrogate these spatially-varying complicated samples, simple analysis techniques such as fluorescence recovery after photobleaching (FRAP) can provide information on molecular transport. Conventional FRAP approaches localize analysis to small spots, which may not be representative of trends across the full field of view.</p><p dir="ltr">Taking advantage of strategies used for structures illumination, an approach has been developed to use patterned illumination in combination with FRAP for probing large fields of view while representatively sampling. Patterned illumination is used to establish a concentration gradient across a sample by irreversibly photobleaching fluorophores, such as with the simple comb pattern photobleach presented in Chapters 1 and 4. Patterned photobleaching allows spatial Fourier-domain analysis of multiple spatial harmonics simultaneously. In the spatial FT-domain the real-space photobleach signal is integrated into puncta, greatly increasing the signal to noise ratio compared to conventional point-bleach FRAP. The order of the spatial harmonic is directly related to the length-scale of translational diffusion measured, with a series of harmonics accessing diffusion over many length scales in a single experiment. Measurements of diffusion at multiple length scales informs on the diffusion mechanism by sensitively reporting on deviations away from normal diffusion.</p><p dir="ltr">Complementing the physical hardware for inducing patterned illumination, this dissertation introduces novel algorithms for reconstructing spatially-resolved diffusion maps in heterogeneous materials by combining Fourier domain analysis with patterned photobleaching. FT-FRAP is introduced in Chapter 1 for interrogating phase-separating samples using beam-scanning instrumentation for comb-bleach illumination. This analysis allowed disentangling separate contributions to diffusion from normal bulk diffusion and an interfacial exchange mechanism only available due to multi-harmonic analysis. The introduction of a dot-array bleach pattern using widefield microscopy is presented in Chapter 2 for high-throughput detection of mobility in simple binary systems as well as for segmentation in phase-separating pharmaceutical formulations. The analysis becomes more complicated as more components are added to the system such as a surfactant. Introduced in chapter 3, FT-FRAP with dot-array photobleaching was shown to be useful for characterizing diffusion of phase-separating micro-domain smaller than a single pixel of the camera. Supported by simulations, a biexponential fitting model was developed for quantification of diffusion by multiple species simultaneously. Chapter 4 introduces imaging inside of 3D particles comprised of an active pharmaceutical ingredient (API) in microencapsulated agglomerates which exhibited strong interfacial exchange. Multi-photon excited fluorescence enabled imaging a small focal volume within the particles.</p>

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