京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21960号 / 医博第4502号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22729号 / 医博第4647号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 伊達 洋至, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23071号 / 医博第4698号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 YOUSSEFIAN Shohab, 教授 平井 豊博 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are highly effective therapies for sub-populations of non-small cell lung cancers. Specific mutations have been identified in the EGFR gene such as L858R which overstimulate cell pathways that lead to tumor growth. All tumors eventually develop resistance to this treatment, rendering them useless, and tumor growth progresses. Escape mutations in the EGFR gene were first seen in patients undergoing treatment with first-generation TKI erlotinib and gefitinib. T790M is a widely seen gate-keeping mutation which overcomes inhibition from erlotinib and gefitinib. Third-generation irreversible TKI, osimertinib, can inhibit tumor cells with this gate-keeping mutation thus overcoming a major hurdle in containment of tumor growth. Unfortunately, patients eventually develop resistance to osimertinib, exhausting options for managing non-small cell lung cancer. Here we analyzed H1975 cells which harbor L858R + T790M mutations. We aimed to track genomic, transcriptomic, and proteomic changes to uncover mechanisms cells use to develop resistance to osimertinib. We established cell colonies which were able to survive high dose treatment up to 2 µM osimertinib. We also saved cells with IC50 of 30 nM to represent drug-tolerant cells. We conducted single-cell sequencing of mRNA transcription and performed hierarchal gene analysis which identified CD74 as a novel factor which was upregulated in drug-tolerant cells. Further we showed CD74 gene was accessible as open chromatin for easy upregulation. Western blot analysis showed increased expression of CD74 after 24 hours of osimertinib treatment. Using siRNA in H1975 cells, we conducted knockdown experiments of CD74 during osimertinib treatment and showed reduced viability. Next, H1975 cells lines were engineered with deletions in CD74 to knockout its expression. These cells also showed reduced viability in the presence of osimertinib. Quantification of apoptosis using caspase-glo assays showed greater activation of apoptosis in cell populations without CD74 compared to normal H1975 cells. H1975-CD74 knockout cells also took longer to become resistant to osimertinib when compared with control. These results show the role of CD74 in helping tumor cells survive EGFR TKI treatment. / 2023-12-05T00:00:00Z

No description available.

The lung was recently identified to consist of a complex microenvironment made up of microorganisms that interact with one another and the host cells via direct and indirect interactions. As a result, understanding the dynamic of the microbiome in chronic respiratory diseases has become the focus of pulmonary researches. In cystic fibrosis (CF), chronic infections are a comorbidity associated with the genetic disorder. Recently, it was noted that the interactions of the fungus, Aspergillus fumigatus, and the bacterium, Pseudomonas aeruginosa together contribute to more severe disease outcomes in CF patients. In vitro co-cultures show that P. aeruginosa and A. fumigatus can affect one another’s growth and pathogenicity, but very few studies have attempted to model interactions of these microorganisms in vivo. Based on clinical and basic research, we developed a co-exposure model in which we could compare non-allergic and allergic animals co-exposed to Pseudomonas aeruginosa and Aspergillus fumigatus. While both groups had significant neutrophilia and production of acute phase response cytokines and chemokines, the allergic co-exposed group had a greater mortality with 34.8% of the animals expiring by 24h in comparison to 12.5% for the non-allergic co-exposed animals and 100% survival in the controls. A contributing factor to the more severe disease outcomes in the allergic co-exposed group is the increase in eosinophilic inflammation and IL-17A production, which only occurs when both microorganisms are viable. In addition, it was found that viable P. aeruginosa but not A. fumigatus causes interstitial inflammation, significant neutrophilia, and even death during co-exposures. The decline in health of animals co-exposed to the fungus and bacteria could be attributed not only to the host’s inflammatory response, but also to the spatial and temporal co-localization in the lung. To address this, we performed in vitro studies finding an aggregation of the microorganisms that could also be identified in vivo. This current research emphasizes the need for in vivo studies on polymicrobial interactions. / ND Agricultural Experiment Station; National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R155AI137886

Les cancers du poumon non à petites cellules (CPNPC) restent une cause majeure de mortalité par cancer, malgré l’apport de thérapies moléculaires ciblées et des immunothérapies. La survie des patients aux stades avancés reste limitée et la mise au point de biomarqueurs pronostiques permettant de stratifier les patients ou prédictifs de réponse à différents types de traitement constitue un enjeu important pour la prise en charge des patients.La mise au point de biomarqueurs obéit à des enjeux spécifiques tenant à la connaissance de la biologie tumorale dans des domaines complexes tels que celui de la réparation de l’ADN, aux caractéristiques des outils disponibles pour créer ces biomarqueurs et à leur applicabilité dans le contexte clinique.Dans le cadre de cette thèse, il a été étudié la manière dont l’expression de la protéine PARP1 peut s’intégrer aux biomarqueurs pronostiques de réparation de l’ADN dans les CPNPC. Il a par ailleurs été étudié le rôle de la protéine MMS19, identifiée à partir d’études d’expression génique, comme biomarqueur prédictif potentiel de réponse au cisplatine dans les CPNPC. Enfin, l’utilisation des cellules tumorales circulantes pour le développement de biomarqueurs a été étudiée dans le cadre de la détection des remaniements du gène ALK, une altération oncogénique constituant une cible thérapeutique dans les CPNPC. / Non-small cell lung cancers (NSCLC) remain a leading cause of cancer-related death despite the advent of targeted therapies and immunotherapies. At advanced stages, patient survival remains limited and establishment of new biomarkers, either prognostic for patient stratification or predictive of response to various therapies, is an important goal for patient’s treatment.Development of biomarkers is dependent on many components among which: knowledge of cancer cell biology in complex cellular processes such as DNA repair, characteristics of tools available to create biomarkers and applicability in daily medical practice.In this thesis, expression of PARP1 has been evaluated as a prognostic biomarker in NSCLC, in the broader context of DNA repair biomarkers. The biological and clinical relevance of MMS19 protein, identified in gene expression analysis , as a biomarker for cisplatin sensitivity in NSCLC has also been studied. Finally, the use of circulating tumor cells for biomarker development has been studied through the detection of ALK gene rearrangment, an oncogenic targetable alteration in NSCLC.

Thoracic injuries caused by blunt trauma are commonly encountered emergencies in veterinary medicine. However, no literature exists comparing radiography to computed tomography (CT) in blunt thoracic trauma caused by motor vehicle accidents in canine patients. The aim of this prospective case series was to estimate the sensitivity (Se) and specificity (Sp) of thoracic radiography relative to CT for detecting lung contusions, pneumothorax, pleural effusion and rib fractures. The study further aimed to establish a severity scoring system for radiography and CT and to compare the findings between the two modalities. The hypothesis was that radiography would be less sensitive than CT at detecting these injuries and that radiography would underestimate the severity of lung contusions compared to CT. Fifty-nine patients met the inclusion criteria. Radiography underestimated the presence of lung contusions (Se = 69%, 95% Confidence interval (CI)) and overestimated the severity of the contusions relative to CT. There was also high interobserver variability in evaluating lung contusion severity (coefficient of variation = 91%). Both the three-view thoracic and horizontal beam radiography had poor sensitivities for the detection of pneumothorax (Se = 19% and 63% respectively) and pleural effusions (Se = 43% and 71% respectively). Similarly, the sensitivity (56%) of three-view thoracic radiographs for the detection of rib fractures was poor relative to CT. To conclude, three-view thoracic radiography had low sensitivity for pathology related to blunt thoracic trauma caused by motor vehicle accidents and CT could be considered as an additional diagnostic imaging modality in these patients. / Dissertation (MMedVet (Diagnostic Imaging))--University of Pretoria, 2019. / Companion Animal Clinical Studies / MMedVet (Diagnostic Imaging) / Unrestricted

CCAAT/Enhancer Binding Protein Alpha (C/EBPa) is transcription factor protein involved in the differentiation of many cell types, including granulocytes and pulmonary cells. Studies have found that downregulation of C/EBPa leads to tumor formation in the hematopoietic system, bones, lungs, liver, and other organs. Mutations and post translational modifications can also reduce the function of C/EBPa in humans, leading to cancers. Recent studies have made progress in treating acute promyelocytic leukemia (APL), an M3 subtype of acute myeloid leukemia (AML), by incorporating all trans retinoic acid (ATRA) in targeted treatments. ATRA increases C/EBPa expression levels, thus promoting cell differentiation and subsequent apoptosis of leukemia cells. Still, survival rates of AML patients are low. In patients diagnosed with AML subtypes M4 or higher, ATRA does not work. In addition, patients can become resistant to ATRA, making it essential to find an alternative therapy. Therefore, novel drug treatments are necessary. Through a high throughput screening method, we have determined a potent chemical compound, ICCB280, that can enhance C/EBPa expression levels. However, ICCB280’s effective concentration for cell differentiation is relatively high, so we performed a structural-activity relationship (SAR) analysis and discovered a more potent chemical, styryl quinazolinone CCAAT/Enhancer Binding Protein Compound 73 (CEBP- 73). We tested CEBP-73 with two cell lines, HL-60 and A549, which represent leukemia and lung cancer models, respectively. We found that CEBP-73 increased C/EBPa expression levels in a time-dependent, dose-responsive manner in both leukemia cells and lung cancer cells. In western blot analyses, while both ICCB280 and CEBP-73 upregulated C/EBPa protein expression, more protein was expressed in leukemia and lung cancer cells treated with CEBP-73 in a dose-dependent manner than in cells treated with ICCB280. Next, we investigated CEBP-73’s effectiveness in upregulating C/EBPa’s downstream genes. We observed enhanced expression of CEBPe (HL-60 specific downstream gene) in HL-60 cells, and enhanced SPC, NKX2-1 (codes for TTF-1), and HIF-1a (A549 specific downstream genes) expression levels in A549 cells. To investigate the mechanisms of increased C/EBPa expression, we asked whether expression of an extra coding CEBPA (ecCEBPA), a noncoding RNA for C/EBPa that prevents methylation at the CEBPA gene promoter site, will increase. We found that CEBP-73 increased not only C/EBPa expression, but also ecCEBPA in HL-60 and A549 cells. This is the first study to our knowledge that confirms styryl quinazolinone CEBP- 73 can increase ecCEBPA expression. To examine the effectiveness of CEBP-73 in vivo, EGFR-L858R-T790M (EGFRTL/CCSP-rtTA) mice were administered a vehicle solution (control), 1 mg/kg of CEBP-73, or 10 mg/kg CEBP-73. The results showed a trend in CEBP-73 concentrations; higher doses of CEBP-73 induce higher levels of C/EBPa expression in lung tissue. Fewer and smaller tumors were present in lungs treated with CEBP-73 than lungs treated with a control. These findings support the role of CEBP-73 in enhancing C/EBPa expression, including upregulation at the promoter region of the CEBPA gene and at downstream gene loci. In addition, the study’s results affirm the role of C/EBPa as an inducer of cell differentiation in leukemia and lung cancer by showing neutrophils with segmented lobes and granules, indications of cell maturity, in cells treated with compounds that enhanced C/EBPa expression. These data suggest that CEBP-73 could provide novel therapeutic approaches in treating leukemia and lung cancer and could potentially be modified to treat other cancers in targeted drug therapies.

There is a need for a real-time, non-invasive method to monitor blood pH in a patient line during cardiopulmonary bypass, as today’s methods are both invasive and time consuming. Blood pH is an indicator of physiological and biochemical activity in the body and needs to be kept within a relatively narrow range, typically between 7.35-7.45. A pH value outside this range can be critical for the patient and therefore needs to be carefully monitored throughout the course of cardiopulmonary bypass. In this study the feasibility of using spectroscopic methods for indirect measurement of pH was investigated, and both transmission and reflectance spectroscopy were tested. The results showed that NIR reflectance spectroscopy is a feasible technique for blood pH monitoring during cardiopulmonary bypass. A strong correlation was found between measured pH values and spectral output in the wavelength range 800-930 nm. It was suggested that by means of the statistical partial least square regression method, a model could be created with three regression factors with a cross-validated R2 of 0.906 and a prediction error RMSEP of 0.089 pH units. The results presented here form a foundation for further analysis and experiments with larger sample set and more controlled experimental environment.