BACKGROUND: Postoperative hypoparathyroidism due to damage to the parathyroid glands during a thyroidectomy is a well-known surgical complication. The current gold standard intraoperative method for identifying parathyroid glands is surgical judgment; other methods are limited in both their sensitivity and convenience. There is a clear need for an objective diagnostic tool that is effective in its identification of the parathyroid glands and of their viability. We hypothesized that an intraoperative tool using Elastic Scattering Spectroscopy (ESS) has the potential to differentiate optical signatures of the parathyroid gland from surrounding tissues in real-time. METHODS: ESS optical signatures were collected intraoperatively from perfused parathyroid, thyroid, fat, muscle, and nerve tissue during thyroidectomy. The ESS data was collected using a portable, handheld, noninvasive, "cold" fiber optic probe able to detect spectra in a non-ionizing, broadband spectrum of light (320 to 920 nm). Five measurements were collected at each tissue site over a total of 1.2 seconds. Visual analysis of tissue was analyzed and compared to pathology, and spectral measurements attained with ESS. RESULTS: No complications resulted from the use of the ESS probe. Of the 10 parathyroid glands evaluated to be at risk in the study, the optical probe utilizing ESS was able to detect unique spectral features associated with the parathyroid gland. All 10 parathyroid glands are optically differentiable from surrounding tissues with ESS. CONCLUSIONS: Utilizing spectroscopy in the form of a hand-held probe is feasible for intraoperative differentiation between tissue types. Preliminary results of this spectral technique warrant further investigation. If successful, implementation of a portable ESS probe to identify the parathyroid tissue during endocrine surgical procedures could reduce surgical complications and improve patient safety.

Thesis (M.A.)--Boston University, 2011. / OBJECTIVE: Thyroid cancer is the most common endocrine malignancy and patients presenting with thyroid nodules often undergo surgery solely for diagnostic purposes. The goal of our study was to examine the accuracy of Elastic Scattering Spectroscopy (ESS) in distinguishing between benign and malignant thyroid nodules in fresh ex vivo specimens and to design an in vivo ESS probe and device, manufacture it and conduct a clinical trial. METHODS: Patients already undergoing thyroidectomy surgery were consented for the ex vivo study. ESS data was obtained from ex vivo specimens by recording 5 readings per nodule with five repetitive readings per each site. Final pathology reports were used to confirm the diagnosis. The spectra were analyzed using principal component analysis, linear discriminant analysis and leave one out technique. The in vivo ESS study was conceptually designed and IRB approval from Boston Medical Campus was obtained. RESULTS: The ex vivo study showed that ESS could predict the difference between benign and malignant tumors with a sensitivity of 74%, specificity of 90%, positive predictive value of 82% and negative predictive value of 85%. 193 spectra were analyzed from 64 patients, 120 spectra were from benign nodules and 73 from malignant nodules. Subanalysis examined only indeterminate nodules showed sensitivity of 65%, specificity of 79%, PPV 77% and NPV 67%. The in vivo ESS probe was designed and 12 identical instruments were manufactured. Initial experimental readings were taken and parameters were adjusted for the in vivo tissue environment. The clinical trial is underway. CONCLUSIONS: ESS is a practical tool that can accurately identify malignancy in ex vivo thyroid specimens with high specificity and sensitivity. Initial in vivo experimental trials have been conducted and show promise for similar results.

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Aseptic loosening of artificial joints is caused by an osteolytic reaction to wear debris mediated by macrophages and other cells. Imaging these wear particles within cells can be a key process in understanding particle-cell interactions. However, the compounds used in surgical implants are not easily visualised as no tagging molecule can be added without altering the properties of the material. We were interested in using a label free optical technique known as coherent anti-Stokes Raman scattering spectroscopy (CARS) to image these particles in cells. In this thesis we studied how to use CARS to image physiologically relevant wear particles within cells. We characterised the responses from our CARS system and found them to be in good agreement to the Raman spectra we obtained for the same materials. We showed that the forward scattered CARS signal was consistently larger than the backwards scattered signal for the same size particles, and also generated a larger contrast, especially between sub-micron sized particles and the non-resonant background. Wear particles of polyethylene isolated from a pin-on-plate wear simulator were shown to be in a similar size range to those retrieved from revision tissue. When incubated in our model macrophage cells we were able to image areas of CARS signal that indicated the location of these particles in the cell. Furthermore, using multiple CARS images taken at different Raman resonances we were able to distinguish between three different polymeric compounds added to cells, showing the specificity of the technique. The inherent 3D sectioning capabilities of multiphoton microscopy were used to generate projected images of the cells and contents, as well as estimating the particle loads within cells. These results show that CARS could be an important tool in imaging intra-cellular polyethylene and characterising the interactions of wear particles with cells and the surrounding tissue.

Nous montrons comment des dimères de nanoparticules d'or, assemblés sur un brin d'ADN unique présentant un site de reconnaissance spécifique, fournissent une réponse optique macroscopique dépendant de leur environnement chimiqueLa séparation électrophorétique nous permet de préparer des suspensions purifiées de dimères de nanoparticules d'or de diamètre allant de 8 à 60 nm, et possédant différentes chimies de surface, avec une pureté pouvant atteindre 90%. L'échafaudage d'ADN contient une structure tige-boucle permettant de modifier réversiblement la structure du dimère en présence d'un brin d'ADN cible. Les distances interparticules (D), estimées en TEM cryogénique, peuvent varier de façon réversible jusqu'à un facteur 3 pour des sphères d'or de 8 nm de diamètre.Afin de traduire le changement conformationnel de l'échafaudage d'ADN en un signal optique mesurable, nous mesurons les spectres de diffusion de dimères uniques de nanoparticules d'or de 40 ou 60 nm de diamètre, en chambre microfluidique.Nous avons pu, en augmentant la concentration saline locale, diminuer progressivement D à l'échelle nanométrique, de 20 à 1 nm, ce qui se traduit par un décalage spectral de la résonance vers le rouge. La bonne corrélation entre les réponses spectrales de dimères uniques, estimées avec un spectromètre ou une caméra CCD couleur, nous permet de démontrer une méthode de détection en champ large et à bas coût, de la déformation nanométrique de ces nanostructures. L'utilisation de ligands amphiphiles nous a permis d'optimiser la stabilité colloïdale des dimères de particules d'or pour minimiser leur sensibilité à la force ionique locale et aux changements de température. / We demonstrate how gold nanoparticle dimers assembled around a single DNA template exhibiting a specific recognition site, provide a macroscopic optical signal depending on their chemical environment. Electrophoresis enables us to produce purified suspensions of gold nanoparticle dimers, with particle diameters ranging from 8 to 60 nm, with different surface chemistries and sample purities as high as 90%. The DNA template features a hairpin loop in order to switch its shape reversibly when binding a target DNA strand. Interparticle distances are estimated using cryo-electron microscopy and indicate a reversible change of the surface-to-surface distance by a factor of 3 in the case of 8 nm diameter gold particles. In order to translate the dynamic switching of a single DNA scaffold in a measurable optical signal, we study the scattering cross-sections of single 40 nm or 60 nm diameter gold nanoparticle dimers, in microfluidic conditions. We are able to progressively decrease the interparticle distance, at the nanometer scale, by increasing the local salt concentration. This deformation results in a spectral shift of the resonance (up to 100 nm) corresponding to a decrease of the interparticle distance from 20 to 1 nm. Moreover, the good correlation between the spectral responses of individual dimers, estimated using a spectrometer or a CCD color camera, enables us to demonstrate a wide-field low-cost detection method of the nanometric deformation of these nanostructures. Using amphiphilic ligands enables us to optimize the colloidal stability of gold nanoparticle dimers in order to minimize their sensitivity to the local ionic strength and temperature changes.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第18611号 / エネ博第307号 / 新制||エネ||63(附属図書館) / 31511 / 京都大学大学院エネルギー科学研究科エネルギー応用科学専攻 / (主査)教授 大垣 英明, 教授 白井 康之, 教授 松田 一成 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

No presente trabalho, o peptídeo inseticida recombinante Jaburetox-2Ec, derivado da urease de Canavalia ensiformis (Jack bean) foi estudado, principalmente através de técnicas de espectroscopia de espalhamento de luz e raios-X. Foi analisada a tendência do peptídeo a se agregar em diferentes condições físico-químicas quando em solução aquosa, e a sua interação com membranas de lipossomas miméticos de plaquetas humanas. A partir dos dados de raio hidrodinâmico, raio de giro, e massa molar ponderal média obtidos do peptídeo em solução, foi possível observar uma tendência para maior agregação numa faixa em torno de pH 5,5, bem como ausência de agregação em pH inferior a 4,0 e superior a 7,0. Não se verificou nenhum efeito visível no nível de agregação com a adição de agente redutor ou íons, sendo que a exposição ao oxigênio do ar levou a um aumento da mesma. Os estudos de interação do Jaburetox-2Ec com lipossomas revelaram uma significante mudança estrutural na membrana, que se mostrou mais intensa com uma maior concentração do peptídeo. Foram, através de software, feitos ajustes teóricos das curvas de espalhamento de raios-X à baixos ângulos dos lipossomas, a fim de quantificar as mudanças nos parâmetros físicos das membranas causados pela ação do peptídeo. A formação de poros na membrana pelo peptídeo foi proposta como explicação para os resultados encontrados. / In the present work, the insecticide recombinant peptide Jaburetox-2Ec, derived from Canavalia ensiformis (Jack bean) urease, was studied, primarily by light and X-ray scattering techniques. Its tendency to aggregate in aqueous solution under different physical-chemical conditions, and its interaction with human platelet mimetic liposome membranes were analyzed. From the data relative to hydrodynamic radii, radii of gyration and mean molecular weight obtained from the peptide in aqueous solution, it was possible to observe a tendency for greater aggregation around pH 5.5, as well as an absence of aggregation at values of pH below 4.0 and above 7.0. No visible effect on the level of aggregation was verified with the addition of reducing agent or ions, whereas the exposure to oxygen in the air resulted in an increase in this level. The studies of Jaburetox-2Ec interaction with liposomes revealed a significant structural change in the membrane, which was shown to be more intense in higher peptide concentrations. Software-assisted theoretical fits were made for the experimental small-angle X-ray scattering curves aiming to quantify the changes in the physical parameters of the membranes caused by peptide action. The formation of pores in the membrane was proposed as an explanation to the results found.

No presente trabalho, o peptídeo inseticida recombinante Jaburetox-2Ec, derivado da urease de Canavalia ensiformis (Jack bean) foi estudado, principalmente através de técnicas de espectroscopia de espalhamento de luz e raios-X. Foi analisada a tendência do peptídeo a se agregar em diferentes condições físico-químicas quando em solução aquosa, e a sua interação com membranas de lipossomas miméticos de plaquetas humanas. A partir dos dados de raio hidrodinâmico, raio de giro, e massa molar ponderal média obtidos do peptídeo em solução, foi possível observar uma tendência para maior agregação numa faixa em torno de pH 5,5, bem como ausência de agregação em pH inferior a 4,0 e superior a 7,0. Não se verificou nenhum efeito visível no nível de agregação com a adição de agente redutor ou íons, sendo que a exposição ao oxigênio do ar levou a um aumento da mesma. Os estudos de interação do Jaburetox-2Ec com lipossomas revelaram uma significante mudança estrutural na membrana, que se mostrou mais intensa com uma maior concentração do peptídeo. Foram, através de software, feitos ajustes teóricos das curvas de espalhamento de raios-X à baixos ângulos dos lipossomas, a fim de quantificar as mudanças nos parâmetros físicos das membranas causados pela ação do peptídeo. A formação de poros na membrana pelo peptídeo foi proposta como explicação para os resultados encontrados. / In the present work, the insecticide recombinant peptide Jaburetox-2Ec, derived from Canavalia ensiformis (Jack bean) urease, was studied, primarily by light and X-ray scattering techniques. Its tendency to aggregate in aqueous solution under different physical-chemical conditions, and its interaction with human platelet mimetic liposome membranes were analyzed. From the data relative to hydrodynamic radii, radii of gyration and mean molecular weight obtained from the peptide in aqueous solution, it was possible to observe a tendency for greater aggregation around pH 5.5, as well as an absence of aggregation at values of pH below 4.0 and above 7.0. No visible effect on the level of aggregation was verified with the addition of reducing agent or ions, whereas the exposure to oxygen in the air resulted in an increase in this level. The studies of Jaburetox-2Ec interaction with liposomes revealed a significant structural change in the membrane, which was shown to be more intense in higher peptide concentrations. Software-assisted theoretical fits were made for the experimental small-angle X-ray scattering curves aiming to quantify the changes in the physical parameters of the membranes caused by peptide action. The formation of pores in the membrane was proposed as an explanation to the results found.