Fluorescence-based imaging techniques provide a simple, highly sensitive method of studying live cells and whole organisms in real time. Without question, fluorophores such as GFP, fluorescein, and rhodamines have contributed vastly to our understanding of both cell biology and biochemistry. However, most of the fluorescent molecules currently utilized suffer from one major drawback, the use of visible light. Due to cellular autofluorescence and the absorbance of incident light by cellular components, fluorescence imaging with visible wavelength fluorophores often results in high background noise and thus a low signal-to-noise ratio. Fortunately, this situation can be ameliorated by altering the wavelength of light used during imaging. Near-infrared (NIR) light (650-900 nm) is poorly absorbed by cells; therefore, fluorophores excited by this light provide a high signal-to-noise ratio and low background in cellular systems. While these properties make NIR fluorophores ideal for cellular imaging, most currently available NIR molecules cannot be used in live cells. The first half of this thesis addresses the synthetic difficulties associated with preparing NIR fluorophores that can be used within living systems. Small molecule NIR fluorophores are inherently hydrophobic which makes them unsuitable for use in the aqueous environment of the cell. Water-solubility is imparted to these dyes through highly polar sulfonates, which subsequently prevents the dyes from entering the cell. The novel work presented here details vii synthetic routes to aid in the development of sulfonated NIR fluorophores, which can be delivered into live cells through the inclusion of an esterase-labile sulfonate protecting group. Application of these synthetic techniques should allow for the development of novel NIR fluorophores with intracellular applications. The second half of this thesis addresses the need for novel NIR imaging reagents. Although several classes of NIR scaffolds do exist, most NIR probes are derivatives of a single class, heptamethine indocyanines. The work described here increases this palette by displaying the ability of NIR oxazines to function as an imaging reagent in live cells and in vivo and as a molecular sensor of biologically-relevant environmental conditions. Combined, the work contained herein has the capacity to not only advance the current NIR toolkit, but to expand it so that fluorescence imaging can move out of the dark and into the NIR light.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第20294号 / 人健博第42号 / 新制||人健||4(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 二木 淑子, 教授 精山 明敏, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Sustainability has gained a lot of attention in the field of research. Researchers and consumers both prioritize sustainability and environmental issues over previously dominant materials, such as plastic. Packaging and disposable items that used to be made of plastic have largely been replaced with paper. Unfortunately, paper does not perform as well as plastic regarding barrier properties against grease, oxygen, or water vapor. Barrier properties are an important factor when choosing packaging material for food, among other things, as they help maintain the shelf life of the product. In order to improve the properties of the paper packaging and expand its use, the paper is coated with a polymer. However, the polymer contributes to challenges in the recycling of the products as some of the polymer attaches to the fibers, causing difficulties in the separation of each material. Small fragments of plastic may end up in the material streams and the recycled pulp due to the existing challenges in completely removing plastic from cellulosic substrates during recycling. This thesis analyzes the possibilities of identifying and classifying plastic fragments of polyethylene (PE) and polyvinyl alcohol (PVOH) in recycled paper sheets using near-infrared spectroscopy together with multivariate data analysis. The purpose of the work is to develop models that can identify possible residues that may appear in recycled products from various industries. Paper sheets of two different grammages and six different compositions of recycled fiber and virgin fiber were created and scanned by NIR, with and without plastic film under the sheets. The scans were used to develop classification models to identify and categorize scans not included in the calibration data set. The performance of the models was tested by applying them to images of sheets of paper with plastic fragments of different sizes and different type underneath. The results indicated potential in the method. The prediction of the paper sheets with a lower grammage was mostly correct, whereas the classification of polyethylene showed the best performance. There was some noise in the prediction of the plastic fragments, regardless of the grammage of the paper. The noise may be due to a wide variation in the calibration data set since it consisted of paper sheets of six different compositions. A large part of the noise was incorrectly classified as polyvinyl alcohol, which can be due to differences in the manufacturing process of the plastic films. The conclusion of the thesis is that it is feasible to identify and categorize plastic fragments of polyethylene and polyvinyl alcohol in recycled paper sheets with a certain margin of error. It can be stated that the method shows promise, but further research and development in the field is required to build models that can be applied to a wider range of samples.

Aim: To examine local muscle oxygen kinetics when performing wrist extensions till failure in an occluded versus non-occluded state, to compare the perceived ratings of pain and local muscle exertion, and analysing presence of gender differences. Methods: Twenty-one healthy individuals (mean age 25,6±3.2, 10 females) performed wrist extensions at 30% of peak isometric strength till failure. The order in which the states was performed was randomized. The oxygen kinetics in the m. extensor digitorum was analysed using Near-infrared spectroscopy continuously. Ratings of perceived pain and local muscle exertion were collected before exercise, when reaching failure and post exercise recovery. Result: No difference was identified in tissue oxygen saturation (TOI) at the exercise plateau (-24.0±18.3% vs -23.5±13.9%, p=0.609). TOI at rest was lower for the occluded state (64.2±4.5% vs 57.2±5.9%, p<0.001). During the second minute of recovery, there was a greater increase in TOI for the occluded state (0.01±0.01%/sec vs 0.04±0.03/sec, p<0.001). A higher rating of perceived pain was found for the occluded state during baseline (0.0±0.0 vs 0.7±0.9, p=0.005) and first minute of recovery post exercise (2.3±2.0 vs 3.3±2.1, p=0.011). No clinically relevant gender differences were found. Conclusion: Venous occlusion during low-intensity dynamic wrist extensions affects the local oxygen kinetics in the muscle before and after exercise. The oxygenation kinetics does not differ when exercise plateau is reached when both regimes are performed till failure. This suggests that performing low-load dynamic wrist extensions in a no-relaxation manner, may have the same effects no matter whether occlusion is used or not. / Syfte: Att undersöka den lokala syreomsättningen i muskeln när handledsextensioner utförs till failure i ockluderat tillstånd jämfört med icke-ockluderat tillstånd, samt att jämföra den skattade upplevda smärtan och lokala muskelansträngningen, och eventuella skillnader mellan könen. Metod: Tjugoen friska individer (medelålder 25.6±3.2, 10 kvinnor) utförde handledsextentioner på 30 % av maximal isometrisk styrka till failure. Ordningen av ocklusion och icke-ocklusion var randomiserad. Nära-infraröd spektroskopi användes för att undersöka syreomsättningen lokalt i m. extensor digitorum. Skattning av upplevd smärta och lokal muskelansträngning samlades in före träning, vid failure och vid återhämtning efter träning. Resultat: Ingen skillnad identifierades i vävnadssyremättnad (TOI) vid uppnådd träningsplatå (-24.0±18.3% vs -23.5±13.9%, p=0.609). TOI i vila var lägre för det ockluderade tillståndet (64.2±4.5% vs 57.2±5.9%, p <0.001). Under den andra återhämtningsminuten var det en större ökning av TOI för det ockluderade tillståndet (0.01±0.01%/sek vs 0.04±0.03/sek, p <0.001). En högre skattning av upplevd smärta sågs för det ockluderade tillståndet under den första vilominuten (0.0±0.0 vs 0.7±0.9, p=0.005) och första minuten av återhämtning (2.3±2.0 vs 3.3±2.1, p=0.011). Inga kliniskt relevanta skillnader mellan könen hittades. Slutsats: Ocklusion under lågintensiva dynamiska handledsextensioner påverkar den lokala syreomsättningen i muskeln före och efter träning. Den muskulära syreomsättningen skiljer sig däremot inte när träningsplatå uppnås när båda regimerna utförs till failure. Detta tyder på att utförande av dynamiska handledsextensioner på låg belastning utan att muskeln tillåts att slappna av, kan ge samma effekter oberoende av om ocklusion appliceras eller inte.

Title: Effect of cold water immersion on intermittent isometric forearm flexor contractions to failure in rock climbers Objectives: The aim of this study was to determine the effect of cold water immersion on intermittent isometric forearm flexor contraction to failure in rock climbers. Methods: Thirty-two climbers (15 male and 17 female) completed three intermittent trials to failure, on a climbing-specific handgrip dynamometer, on three separate visits to the laboratory. For each visit a different recovery strategy was employed: passive recovery (PAS), cold water immersion at 8 řC (CWI 8) or 15 řC (CWI 15). The force time integral (FTI - time of contraction multiplied by the force of contraction) was determined to assess intermittent performance. The tissue saturation index (TSI) and total haemoglobin (tHb) during isometric contractions from near-infrared spectroscopy (NIRS) were determined to assess hemodynamic changes. Differences between repeated trials and three recovery protocols, derived parameters NIRS, were assessed using repeated measures ANOVA with Bonferroni corrections. Results: There was no significant difference between conditions after trial one. In response to the PAS recovery condition there was a 10% and 22% decrease in FTI in the second and third trials, respectively. The PAS...

The objective of this study was to assess low to high levels of Cognitive Workload by measuring heart rate and cortical blood flow in real-time. Four conditions were implemented into a within-subjects experimental design. Two conditions of difficulty and two conditions of trial order were used to illicit different levels of workload which will be analyzed with psychophysiological equipment. Functional Near-Infrared Spectroscopy (fNIRS) has become more prominent for measuring the blood oxygenation levels in the prefrontal cortex of individuals operating in hazardous work environments, students with learning disabilities, and in research for military training. This is due to the fNIR device being highly mobile, inexpensive, and able to produce a high-spatial resolution of the dorsolateral prefrontal cortex during executive functioning. Heart Rate will be measured by an Electrocardiogram, which will be used in concordance with fNIR oxygenation levels to predict if an individual is in a condition that produces low or high mental workload. Successfully utilizing heart rate and blood oxygenation data as predictors of cognitive workload may validate implementing multiple physiological devices together in real-time and may be a more accurate solution for preventing excessive workload.

We investigate the spectra of asteroids at near- and mid-infrared wavelengths. In 2010 and 2011 we reported the detection of 3 ?m and 3.2-3.6 ?m signatures on (24) Themis and (65) Cybele indicative of water-ice and complex organics [1] [2] [3]. We further probed other primitive asteroids in the Cybele dynamical group and Themis family, finding diversity in the shape of their 3 ?m [4] [5] [6] and 10 ?m spectral features [4]. These differences indicated mineralogical and compositional variations within these asteroid populations. Also in the mid-infrared region we studied a larger population of asteroids belonging to the Bus C, D, and S taxanomic classes to understand the relationship between any mineralogy and hydration inferred in the visible and near- infrared with the shape, strength, and slope of the 10 ?m emission. We have discovered that at least 3 of the main Bus taxanomic groups (Cs, Ds, and Ss as defined by their visible spectra) clearly cluster into 3 statistically distinct groups based on their 8-13 ?m spectra. Additionally we have attempted to simulate in a laboratory the possible organic compounds we have detected on two asteroids, using various mixtures containing aromatic and aliphatic hydrocarbons. We find that asteroid (24) Themis and (65) Cybele have ?CH2/?CH3 and NCH2/NCH3 ratios similar to our 3- methylpentane, propane, and hexane residues, suggesting that the organics on these asteroids may be short chained and/or highly branched. The ?CH2/?CH3 and NCH2/NCH3 for asteroid(24)Themis are most consistent with the DISM, and some carbonaceous chondrites. The band centers of the C-H stretch absorptions indicate that both asteroids may have aliphatic carriers chemically bonded to electronegative groups (i.e. aromatics), and some that are not. We also detect a 3.45 ?m feature in the spectra of both asteroids that is present in several dense molecular clouds. Our results suggest an interstellar origin for the organics on (24) Themis, and likely (65) Cybele. The differences in the organics of Themis and Cybele are likely related to variations in thermal processing, irradiation and/or formation region in the solar nebula.

Speech production not only relies on spoken (overt speech) but also on silent output (inner speech). Little is known about whether inner and overt speech are processed differently and which neural mechanisms are involved. By simultaneously applying electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS), we tried to disentangle executive control from motor and linguistic processes. A preparation phase was introduced additionally to the examination of overt and inner speech directly during naming (i.e., speech execution). Participants completed a picture-naming paradigm in which the pure preparation phase of a subsequent speech production and the actual speech execution phase could be differentiated. fNIRS results revealed a larger activation for overt rather than inner speech at bilateral prefrontal to parietal regions during the preparation and at bilateral temporal regions during the execution phase. EEG results showed a larger negativity for inner compared to overt speech between 200 and 500 ms during the preparation phase and between 300 and 500 ms during the execution phase. Findings of the preparation phase indicated that differences between inner and overt speech are not exclusively driven by specific linguistic and motor processes but also impacted by inhibitory mechanisms. Results of the execution phase suggest that inhibitory processes operate during phonological code retrieval and encoding.

Studies in adults showed differential neural processing between overt and inner speech. So far, it is unclear whether inner and overt speech are processed differentially in children. The present study examines the pre-activation of the speech network in order to disentangle domain-general executive control from linguistic control of inner and overt speech production in 6- to 7-year-olds by simultaneously applying electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS). Children underwent a picture-naming task in which the pure preparation of a subsequent speech production and the actual execution of speech can be differentiated. The preparation phase does not represent speech per se but it resembles the setting up of the language production network. Only the fNIRS revealed a larger activation for overt, compared to inner, speech over bilateral prefrontal to parietal regions during the preparation phase. Findings suggest that the children’s brain can prepare the subsequent speech production. The preparation for overt and inner speech requires different domain-general executive control. In contrast to adults, the children’s brain did not show differences between inner and overt speech when a concrete linguistic content occurs and a concrete execution is required. This might indicate that domain-specific executive control processes are still under development.

In category learning, explicit processes function through the prefrontal cortex (PFC) and implicit processes function through the basal ganglia. Research suggested that these two systems compete with each other. The goal of this study was to shed light on this theory. 15 undergraduate subjects took part in an event-related experiment that required them to categorize computer-generated line-stimuli, which varied in length and/or angle depending on condition. Subjects participated in an explicit "rule-based" (RB) condition and an implicit "information-integration" (II) condition while connected to a functional near-infrared spectroscopy (fNIRS) apparatus, which measured the hemodynamic response (HR) in their PFC. Each condition contained 2 blocks. We hypothesized that the competition between explicit and implicit systems (COVIS) would be demonstrated if, by block 2, task-accuracy was approximately equal across conditions with PFC activity being comparatively higher in the II condition. This would indicate that subjects could learn the categorization task in both conditions but were only able to decipher an explicit rule in the RB condition; their PFC would struggle to do so in the II condition, resulting in perpetually high activation. In accordance with predictions, results revealed no difference in accuracy across conditions with significant difference in channel activation. There were channel trends (p < .1) which showed PFC activation decrease in the RB condition and increase in the II condition by block 2. While these results support our predictions, they are largely nonsignificant, which could be attributed to the event-related design. Future research should utilize a larger samples size for improved statistical power.