京都大学 / 新制・課程博士 / 博士(工学) / 甲第23867号 / 工博第4954号 / 新制||工||1774(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 清水 芳久, 教授 米田 稔, 准教授 松田 知成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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<p>The typical High Dose Rate (HDR) lung brachytherapy procedure involves inserting treatment catheters into the bronchi next to the tumour location using a bronchoscope. The anterior-posterior and lateral fluoroscopy images are acquired in order to localize the catheters prior to treatment. Although, these images enable accurate reconstruction of the catheter location, they do not allow for the visualization of the tumour or organs-at-risk due to poor soft tissue contrast. Although CT images offer an improved soft tissue contrast, moving the patient with catheters in place prior to each treatment is impractical.</p> <p>An alternative option is to use prior diagnostic or external beam radiation treatment planning CT images. These images cannot be used for treatment planning directly because of the variation in patient positioning between the CT and orthogonal images acquisition. In order to account for positioning differences, a 2D/3D registration algorithm that registers the orthogonal images with a previously acquired CT data was developed. The algorithm utilizes a rigid registration model based on a pixel/voxel intensity matching approach. A similarity measure combining normalized mutual information (NMI), image gradient, and intensity difference was developed. Evaluation of the algorithm was performed using tissue equivalent phantoms, and, in the clinical setting using data from six patients. The mean registration error was 2.1 mm and 3.2 mm for phantoms and patients respectively.</p> <p>External objects such as the treatment table and ECG leads are often in CT images, affecting the above mentioned 2D/3D registration. To address this, an algorithm for automatic removal of external objects from CT images was developed. This was applied to automatic contouring and removal of the fiducial markers in CT images used for external beam radiation therapy treatment planning for breast cancer. The algorithm was further modified to compute the girth of patients as part of a diagnostic radiology clinical trial.</p> / Doctor of Philosophy (PhD)

Supraphysiologic concentrations of oxygen are used in the management of critically ill patients across the lifespan. However, hyperoxia (HO) results in alveolar- capillary membrane destruction, pulmonary edema, pleural effusions, infiltration and activation of inflammatory cells, altered pulmonary mechanics and gas exchange prompting increased loading of the respiratory muscle. These abnormalities of pulmonary structure and function increase the work of breathing necessitating increased respiratory muscle force production to maintain alveolar ventilation. When the load placed on the respiratory muscle pump exceeds its capacity, respiratory failure develops and is ultimately fatal unless therapeutic interventions are able to reduce the ventilatory load. The use of perfluorochemical (PFC) liquids as a respiratory medium has been effective in the treatment of respiratory distress syndrome and acute lung injury (ALI) requiring mechanical ventilation. Mechanistically, by eliminating the air-liquid interface, PFC liquids reduce surface tension enabling lung volume recruitment at low inspiratory pressures and have high respiratory gas solubility which supports gas exchange. Additionally, through mechanical as well as cytoprotective mechanisms, intrapulmonary PFC liquids reduce inflammatory cell activation and recruitment. Cell culture, animal and human studies have suggested that acute and chronic lung injury secondary to prolonged HO may be ameliorated by administration of antioxidant enzymes (AOE), with superoxide dismutases (SOD) having significant protective effects. Because the lung is exposed to the highest O2 concentrations, a logical strategy to reduce HO-induced damage is to specifically target antioxidant enzymes to the lungs. However, intratracheal delivery of AOE by vehicles like normal saline may transiently impair lung function and be poorly distributed. PFC fluids have previously been shown to be effective respiratory media for pulmonary administration of various drugs. The premise of the proposed studies are to to characterize hyperoxic lung injury in a spontaneously breathing animal model and to develop therapeutic strategies to reduce oxidatative stress and supplement endogenous AOE. With respect to the diaphragm, we reason that HO-induced lung damage and oxidative stress will increase contractile demand of the diaphragm. If AOE activity could be increased in the lungs and respiratory muscles with AOE proteins or the genes encoding these enzymes, then cell damage, inflammatory changes, damage to the lung and respiratory "pump" might be ameliorated or prevented. The results show that PFC and SOD can attenuate the HO- induced decline in lung mechanics and gas exchange, ameliorate the inflammatory and oxidative stress profiles, and promote lung and muscle structural integrity resulting in a survival benefit. These findings support the novel application of PFC liquids in a spontaneously breathing model and support the concept that PFC preconditioning and AOE supplementation play a protective role by reducing mortality and morbidity in hyperoxic lung injury. / Physiology

Infection with "Pneumocystis" causes a ≥ 99% depletion of plasma S-adenosylmethionine (AdoMet) levels in both "Pneumocystis" pneumonia (PcP) animal models and patients. AdoMet is a critical cellular metabolic intermediate, with a pivotal role as methyl donor in a myriad of biochemical processes and necessary for the synthesis of the essential polyamines spermidine and spermine. In the target tissue of "Pneumocystis", the lung, levels of AdoMet were previously shown to be depleted experimentally using nicotine. Here we show that chronic administration of nicotine in an animal model of PcP resulted in decreased lung AdoMet content. Since "Pneumocystis" is dependent on this metabolite, PcP burden was also relived. We hypothesized that the underlying mechanism behind nicotine-induced AdoMet depletion was an increased consumption of AdoMet through the polyamine pathway where the increased activity of N-1-spermidine/spermine acetyl transferase raises the catabolic / anabolic cycling of polyamines, a process that utilizes AdoMet. In a critical test of our hypothesis, we found that blockage of polyamine metabolism via inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) hinders the effect of nicotine on lung AdoMet levels. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of AdoMet toward polyamine synthesis and away from methylation reactions; these shifts are also reversed by inhibition of ODC. Because the nicotine effect on "Pneumocystis" is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection (LCM), we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ODC activity in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on "Pneumocystis" pneumonia. Our studies demonstrate the feasibility of pharmacological manipulation of the polyamine pathway in order to reduce AdoMet levels in the lung and prompted the assessment of compounds alternative to nicotine with the potential to achieve a comparable effect. In vitro evaluation of the polyamine analog DENSPM along with putrescine in type II alveolar cell lines, indicates that although such a combination has the potential to induce polyamine flux, an apparent competition for the same polyamine transport system impairs simultaneous uptake of both compounds at effective concentrations. In conclusion, we showed that chronic nicotine administration causes reduction of AdoMet levels in rat lung following 21 days of treatment, by a mechanism involving the induction of polyamine flux, which is responsible of increased AdoMet utilization for polyamine biosynthesis. According to LCM-based analysis, this effect seems to be confined to the alveolar regions of the lung. / Biochemistry

Background: At the time of endobronchial ultrasound (EBUS) staging, ultrasonographic features can be used to predict mediastinal lymph node (LN) malignancy. Predictive tools have been developed, however they have not gained widespread use due to lack of research demonstrating validity and reliability. We sought to develop a novel predictive tool, the Canada Score, capable of predicting malignancy and potentially guide LN biopsy decision making. Methods: We prospectively analyzed the ultrasonographic features of LNs from patients with NSCLC. Ultrasonographic features were identified by a single experienced endoscopist, this data was used to develop the Canada Score. Pathological specimens were used as the gold standard for determination of malignancy. Videos were then circulated to endoscopists across Canada, who were also asked to identify ultrasonographic features for each LN. Hosmer- Lemeshow test, logistic regression, receiver operator characteristic (ROC) curve, and Gwet’s AC1 analyses were used to test the performance, discriminatory capacity, and inter-rater reliability of the Canada Score. Results: A total of 300 LNs from 140 patients were analyzed by 12 endoscopists across 7 Canadian centres. Backwards elimination was used to create a multivariate model. Hosmer-Lemeshow test and ROC curves indicated the model was well-calibrated (chi2=11.86, p=0.1567) with good discriminatory power (c- statistic= 0.72 ±0.042, 95%CI: 0.64-0.80). Beta-coefficients were used to create a simplified score out of four. Evaluation of the tool showed that LNs scoring 3 or 4 had odds ratios of 15.17 (p<0.0001) and 50.56 (p=0.001), respectively for predicting malignancy. A score of 4/4 was associated with 99.59% specificity and a positive likelihood ratio of 22.78. Inter-rater reliability for a score ≥ 3 was 0.81 ± 0.02 (95%CI: 0.77-0.85). Conclusions: The Canada Score shows excellent performance in identifying malignant LN at the time of EBUS. A cut-off of ≥ 3 has the potential to inform decision-making regarding biopsy or repeat/mediastinoscopy if the initial results are inconclusive. / Thesis / Master of Science (MSc) / During lymph node staging for lung and esophageal cancer, specific features of lymph nodes can be seen. Using diagnostic tools these features can be used to predict whether a lymph node is cancerous or benign. However, many of these diagnostic tools are inaccurate or unreliable. To address this, this thesis aimed to develop a novel diagnostic tool based on lymph node features seen during staging procedures and determine its clinical usefulness and application to the wider lung and esophageal cancer population. This thesis also aimed to use improved methods to develop this diagnostic tool such that patient and clinician experiences would be significantly improved. The results of this thesis may contribute to a reduction in the number of repeat procedures required for patients undergoing staging prior to their treatment for lung and esophageal cancers.

Understanding the cellular and molecular dynamics of lung progenitors is necessary to achieve the ultimate therapeutic goal of tissue regeneration in the context of debilitating lung diseases. Alveolar type 2 cells (AT2s) of the distal lung epithelium are considered the predominant facultative progenitors of adult murine lung alveoli, the tissues responsible for gas exchange in mammals. While normally quiescent at homeostasis, AT2s can enter cell cycle and transdifferentiate into alveolar type 1 cells (AT1s) during lung repair. The kinetics of AT2 transdifferentiation into AT1s following mild insults, where alveolar architectural integrity is preserved and lung function is restored, is not completely understood. Additionally, the transcriptomic programs across all cell lineages that might regulate this process largely remain unknown. To identify putative molecular pathways activated in the epithelium and its overall niche, cell-type specific responses were characterized in a mouse model during adult lung compensatory growth following the mild injury of unilateral pneumonectomy (PNX). Histologic analyses showed proliferation of AT2s as a primary response, followed by differentiation into AT1s that peaked around day 10-12 post-PNX. Time-series single-cell RNA sequencing profiles revealed that AT2s express transitional markers such as Krt8 and Areg, consistent with previous studies. Trajectory analysis using a Continuous State Hidden Markov Model (CSHMM) predicted Hippo pathway effector Tead1 and Notch signaling target Hes1 as active transcription factors associated with the transdifferentiation of AT2s to AT1s. To verify involvement of Notch post PNX, AT2s were assessed for Notch activation using a genetic reporter, which showed an increase in the percentage of Notch-responsive AT2s post-PNX. To test potential function of Notch in AT2 transdifferentiation into AT1s, Notch signaling was inhibited in vitro in a mouse cell co-culture model which showed loss of AT2 transdifferentiation into AT1s. Single-cell RNA sequencing profiles post-PNX revealed an activated lung mesenchymal subset unique to injury that was characterized by high expression of extracellular matrix-related (ECM) genes along with low expression of Acta2 and Pdgfra. Interestingly, this mesenchymal subset included Cthrc1+ fibroblasts that expressed the highest levels of ECM genes and transcripts associated with BMP and TGFβ signaling pathways, including Fstl1, Grem1, and Tgfβ3, factors previously linked to epithelial differentiation. Immunostaining combined with in situ hybridization assays identified Cthrc1+ fibroblasts adjacent to Krt8+ transitional epithelial cells. Genetic lineage tracing of Cthrc1+ cells post-PNX revealed the persistence of their descendants after completion of compensatory lung regrowth. In summary, analysis of AT2 global transcriptomes and differentiation kinetics post-PNX revealed activated/transitional states, nascent AT1 cells, and the emergence of Cthrc1+ mesenchymal subpopulations. These results suggest that AT2s are one of the early responders to PNX stimuli and identified mesenchymal cells states that may assist in overall compensatory lung regrowth post-PNX. / 2025-09-17T00:00:00Z

How CD4 T cells protect against influenza A virus is poorly understood. Here, we address two central questions to better understand how CD4 T cells contribute to immunity during primary and secondary infection. First, we investigate the CD4 T cell-intrinsic requirements for three major transcription factors associated with an antiviral T cell phenotype (termed ‘Th1'): STAT1, STAT4, and T-bet, in directing CD4 T cell responses. We show that STAT4-deficiency does not affect the phenotype or function of wildtype or T-bet-/- CD4 T cells while STAT1-/- cells are virtually undetectable in infected host mice. Depleting NK cells rescues the STAT1-/- cells that phenocopy the compromised Th1 identity of T-bet-/- cells. Finally, we show that cytokine-mediated STAT4 activation enhances infection-induced Th1-polarization and that engaging STAT1 and STAT4 during priming dramatically improves CD4 T cell antiviral capacity. These results are relevant to T cell-based vaccine strategies aiming to promote the most efficient anti-viral T cell responses. Second, we asked the extent to which the recall of influenza-specific lung-resident memory CD4 T cells (TRM) impact the generation of new primary anti-viral T cells. TRM rapidly induce local inflammatory responses that control infection before protective T cells activated in secondary lymphoid organs reach sites of infection. Whether antigen-sensing by TRM can impact T cell priming in secondary lymphoid organs is unclear. We show that activation of influenza-primed lung TRM by antigen delivered into the airways enhances the number and activation status of antigen-bearing dendritic cells in draining lymph nodes. This accelerates the priming of naïve T cells and enhances their recruitment to the lung. Importantly, this TRM-dependent circuit enables productive T cell responses even against levels of airways antigen too low to otherwise activate naïve T cells. This adjuvant-like impact of lung TRM highlights a novel integration of local and regional T cell immunity.

Yes / It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival. Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reductionmediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a prerequisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator. / The authors would like to thank Cancer Research UK for the post-doctoral fellowship to H.F.Y.

No / Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzyme activity, a significant mechanism of action of NSAIDs. Inflammation is associated with increasing cancer incidence. Recent pre-clinical and clinical studies have shown that NSAID treatment could cause an anti-tumour effect in cancers. Such studies are lengthy and expensive. The present study, however, examined DNA damage in the Comet and micronucleus assays in peripheral blood lymphocytes of patients with respiratory diseases and healthy individuals using the nanoparticle (NP) and bulk versions of the NSAIDs, aspirin and ibuprofen. Lymphocytes are suitable surrogate cells for cancers and other disease states. DNA damage decreased in lymphocytes from healthy individuals, asthma, COPD and lung cancer patient groups after treatment with aspirin nano-suspension (ASP N) and ibuprofen nano-suspension (IBU N) compared to their bulk version (micro-suspension) in both assays. However, when ASP N was compared to untreated lymphocytes in all groups in the Comet assay, DNA damage significantly decreased in all groups, except the asthma group. When IBU N was compared to untreated lymphocytes, in healthy individuals and the lung cancer group, DNA damage decreased, but increased in asthma and COPD groups. Similarly, micronuclei (MNi) increased after ASP N and IBU N in the healthy individual and lung cancer groups, and decreased in asthma and COPD groups. Also shows that whilst there are basic similarities with different genetic endpoints in terms of nano and bulk forms, but highlights some differences between the disease states examined. Furthermore, lymphocyte responses after IBU N and ibuprofen bulk were investigated by patch-clamp experiments demonstrating that IBU N inhibited ion channel activity by 20%. This molecular epidemiology approach mirrors pre-clinical and clinical findings, and provides new information using nanoparticles.