Using cultured Y-1 mouse adrenal tumor cells which produce the steroid 20(-hydroxypregn-4-en-3-one (20-DHP), it was found that 10-5 M corticosterone and deoxycorticosterone increased basal and inhibited ACTH-induced 20-DHP production. The steroid effects were concentration-dependent, reversible, and specific since six other steroids did not stimulate steroidogenesis and varied in their ability to inhibit ACTH-induced steroidogenesis. Cytochalasin D inhibited steroid-stimulated 20-DHP production, suggesting a mechanism of steroid stimulation similar to that of ACTH. Steroidogenesis stimulated by cholera toxin, (Bu) 2 cAMP, or pregnenolone was not inhibited by exogenous steroid; corticosterone increased basal and inhibited ACTH-induced intracellular cAMP production. Steroids altered cell surface morphology. These findings suggest that steroids alter adrenal steroidogenesis by acting within the plasma membrane.

Cancers of the human skin are divided into melanoma and non-melanoma. Being among the most commonly diagnosed cancer cases globally, non-melanoma skin cancers are comprised of basal and squamous cell carcinomas. In dermato-pathology, basal cell carcinomas (BCCs) are a frequently encountered diagnosis of skin cancer, and most cases are treated with surgical excisions. While sporadic BCC tumors appear primarily due to aging and ultra-violet exposure, the development of numerous BCCs from a young age is one of the main clinical signs in Gorlin syndrome patients. The critical driver of BCC tumor formation is the sonic hedgehog (SHH) pathway, a pivotal developmental signaling pathway that regulates organ development, cell proliferation, and tissue repair. The majority of all sporadic and syndromic BCCs exhibit mutations in two key components in this pathway, the tumor suppressor gene patched 1 (PTCH1) or the proto-oncogene smoothened (SMO), which result in aberrant pathway activation and continued transcription of SHH-dependent genes. In the last decade, SHH inhibitors have emerged as a novel treatment for advanced and metastatic BCCs. Systemic treatment with vismodegib, a potent SMO inhibitor, can effectively reduce BCC tumor burden in adult Gorlin syndrome patients. However, it is associated with chemotherapy-related adverse events, and treatment cessation results in cancer recurrence and formation of a subset of drug resistant BCCs. While aberrant SHH signaling is key, mechanisms that underlie epithelial–stromal crosstalk and reprograming of tumor metabolism can potentially converge with this pathway and promote BCC tumor development. In this study, we investigated the effects vismodegib on the morphology, behavior, and energy metabolism of human BCC cells and human dermal fibroblasts, in individual cultures as well as in co-cultures, that enabled the crosstalk between these two cell types. Computer-assisted bright-field microscopy was used to characterize cell morphology and behavior. Nuclear magnetic resonance (NMR) and metabolomics were used to determine the metabolic activity of these cells. We found that continuous crosstalk between the cells and different concentrations of vismodegib led to distinct changes in cell morphology and growth, as well as consumption of glucose, pyruvate, and glutamine and secretion of acetate, lactate, and glutamate by these cells. Deciphering tumor driver mechanisms that converge with SHH pathway and contribute to changes within the tumor microenvironment are important not only for better understanding of BCC pathobiology, but also for the development of new mechanism-based BCC therapies with improved clinical outcomes. / 2023-02-22T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20701号 / 工博第4398号 / 新制||工||1683(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 小寺 秀俊, 教授 中部 主敬, 教授 安達 泰治, 准教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Polydopamine (PDA) surfaces have attracted much attention, both for their innate capability as a versatile biomaterial and their standalone antibacterial and adhesive properties. However, the mechanics of PDA deposition as well as the attributes of PDA-coated surfaces remain relatively underexplored despite their adaptability and ease of deposition. Two polydopamine surfaces from literature, smooth and rough PDA (sPDA and rPDA), were compared to a novel surface, inverted PDA (iPDA), to further explore their mechanochemical and bioactive properties. The iPDA surface displayed, by design, a smoother topography when compared to sPDA, with smaller aggregate structures covering 2.7% of the overall surface. However, the chemical signature obtained via Raman spectroscopy of these aggregates shared remarkable similarities at the 1370 cm-1 peak with the rougher rPDA surface, leading to the conclusion that gas exchange at the solution surface may play a critical role in determining PDA subunit composition despite dissimilar deposition methods. Atomic force microscopy (AFM) analysis concluded that the iPDA surface was ~17% more adhesive than other PDA types, while also displaying relatively large hysteresis and a small Young’s modulus. Human osteoblastic MG-63 cells cultured on all three surfaces revealed that a smoother surface topography correlated to more pronounced anisotropic spread independent of cell size, while a serum-independent component was also noted. This work provides a clearer insight into the nature of polydopamine surfaces by the creation of a viable new deposition method, providing an analysis of its mechanochemical and bioactive properties as well as a deeper understanding of the PDA coating process.

At present there is an extraordinary need to overcome barriers in regards to discovering novel and enhanced biomaterials for various tissue engineering applications. The need for durable orthopaedic implants is on the rise to limit issues such as revision surgery. A promising pathway to enhance fixation is to accelerate the onset and rate of early cellular adhesion and bone growth through nanoscale surface topography at the implant surface. The main aim of this research project was to investigate cellular response to altered physical and mechanical characteristics of materials suitable for orthopaedic applications. Four injection moulded polymeric substrates were produced, each with varied compositional and topographical characteristics. The four materials fabricated are Polyether-ether-ketone (PEEK), PEEK with 30% glass fibre (GL/PEEK) composite, PEEK and GL/PEEK with grooved topography. SEM and AFM analysis was used to investigate the groove dimensions and surface roughness of all samples followed by mechanical testing using a nano indenter to detect the Young’s modulus, stiffness and hardness of all four substrates. These tests were performed to determine which material has similar characteristics to cortical bone. These tests were followed by wettability and surface energy testing. Cell-substrate adhesion was examined using a cell viability assay to identify if there is a significant difference (p<0.05) between the percentage of viable cells on all four PEEK based materials. Imaging of MG-63 osteosarcoma cells using immunohistochemistry staining kits was conducted to observe the relationship between cell length and surface topography followed by a comparison between HaCaT (skin) cells and MG-63 (bone) cells. Following experimental testing mechanical variations between PEEK and GL/PEEK were identified alongside physical characterization differences. The grooved topography increased the surface roughness of PEEK and GL/PEEK in comparison to the planar surface. After 72 hours a correlation between the increased surface roughness and the percentage of viable MG-63 cells could be identified. When assessing the effect surface topography has on the water contact angles and surface energy, all four substrates showed no correlation. However, the grooved topography did increase the water contact angle and reduced the surface energy of PEEK in comparison to planar PEEK. Images of the four substrates after cell culture observed the grooved topography to affect the cellular orientation of both MG-63 and HaCaT cells. Polycaprolactone (PCL) scaffolds with a concentration of 1, 3, and 5% triclosan (an antimicrobial and antifungal agent) were fabricated using electrospinning. In addition to PCL + Triclosan scaffolds PCL with a concentration of 1% silver (an antimicrobial agent that can reduce the risk of infection) and 1, 3, and 5% triclosan were also electrospun. The pore size and fibre diameters of the scaffolds were investigated using SEM and Image J software followed by wettability and surface energy testing. MG-63 cells were cultured on all PCL scaffolds to study cellular viability percentage after 24 and 72 hours. The findings obtained showed the physical characteristics of PCL scaffolds to affect cellular viability of MG-63 cells. The output from these findings aim to provide data at a proof of concept level in understanding the relationship between the mechanical and physical characteristics of biomaterials and cellular behaviour.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23726号 / 理博第4816号 / 新制||理||1689(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Das Mammakarzinom ist eine der am häufigsten diagnostizierten Krebserkrankungen bei Frauen in Deutschland. Dank neuer Diagnostikverfahren, wie z.B. Mammographie-Screenings, ist die frühzeitige Erkennung von Mammakarzinomen mit einer Reduktion der Mortalität verbunden. Dennoch ist die Inzidenz von Knochenmetastasen bei Mammakarzinom-Patientinnen mit ca. 73 % im Vergleich zu anderen Krebsarten auffällig hoch. Diese können zu skelettbezogenen Komplikationen führen, wie z.B. Rückenmarkskompressionen und pathologischen Knochenbrüchen. Dadurch wird die Lebensqualität der Patientinnen stark eingeschränkt. Es besteht daher ein großes klinisches Interesse, die Pathogenese von Knochenmetastasen bei Mammakarzinomen besser zu verstehen. Somit können neue Strategien für die Prädiktion und die gezielte Therapie von Knochenmetastasen bei Mammakarzinom-Patientinnen entwickelt werden. Frühere Studien haben gezeigt, dass die Zellmorphologie als Resultat der biochemischen und biophysischen Merkmale von Tumorzellen Informationen über das Metastasierungspotential von Mammakarzinomzellen liefern könnte. Der Fokus dieser Arbeit liegt daher auf dem Zusammenhang zwischen Morphologie und Osteotropismus von Mammakarzinom-Zelllinien, um einen ersten deskriptiven Ansatz zu geben. Dabei wurden verschiedene in vitro-Eigenschaften von einem Zelllinien-Modell aus unterschiedlichen osteotropen Mammakarzinomzellen näher beleuchtet und miteinander verglichen. Dieses in vitro-Modell umfasst die hoch osteotropen Zelllinien, MDA-MB-231MET (MET) und MDA MB 231BONE (BONE) sowie die parentale Zelllinie, MDA-MB-231. Die morphologische Untersuchung ergab 7 unterschiedliche Morphotypen, welche in allen untersuchten Zelllinien in einem stabilen Verhältnis vorlagen. Die Verteilung der Morphotypen war für die jeweilige Zelllinie spezifisch. Bei hoher Konfluenz zeigten MDA-MB-231 und MET ein Wachstum in Einzelzellschicht, während BONE-Zellen dazu tendierten, in vertikaler Ausrichtung zu wachsen. Die eng geknüpften Zellkontakte (engl. tight junctions) in MET und BONE waren bei leicht erhöhter Zelldichte stärker ausgeprägt als bei MDA-MB-231. Sobald die Zelldichte hingegen weiteranstieg, verschwanden diese in den hoch osteotropen Zelllinien und blieben bei MDA-MB-231 erhalten. Auf Protein-Ebene war das Adhäsionsprotein E Cadherin in den hoch osteotropen Zelllinien verglichen zu MDA-MB-231 sehr gering exprimiert. Dies konnte auch bei der Proteinexpression von CXCR4 beobachtet werden. Die hoch osteotropen Zelllinien zeigten auch eine geringere Expression von Tetraspanin 29 als die parentale Zelllinie. Dagegen war die Expression des Rezeptors EGFR in BONE stärker ausgeprägt als bei den anderen zwei Zelllinien. Weiterhin wurden Ähnlichkeiten und Unterschiede zu einer nicht osteotropen Zelllinie, MDA MB 231BrM (BRAIN), untersucht, welche ebenfalls von MDA MB 231 stammt. BRAIN zeigte morphologische, molekulare und funktionelle Ähnlichkeiten sowohl zur parentalen als auch zu den hoch osteotropen Zelllinien. Durch diesen Vergleich wurden spezifische Merkmale der hoch osteotropen Zelllinien verdeutlicht. Diese spezifischen Merkmale umfassen den signifikant höheren Anteil an einem fibroblasten-ähnlichen Morphotypen und die geringe Expression von CXCR4 in MET und BONE. Außerdem ergab die Hauptkomponentenanalyse der Transkriptomdaten, dass sich die Zelllinien BONE und BRAIN ähneln. Insgesamt konnte aus der Transkriptomanalyse eine potentiell osteotrope Gensignatur bestehend aus 58 Genen abgeleitet werden. Eines dieser Gene, welches in der Migration involviert ist und möglicherweise eine Rolle bei der Morphologie spielen könnte, wurde ausgeknockt. Dies resultierte in keiner signifikanten Veränderung der Morphologie. Diese Arbeit bietet einen grundlegenden Einblick zu den morphologischen, molekularen und funktionellen Eigenschaften von osteotropen Mammakarzinom-Zelllinien. Somit könnte diese Arbeit eine Basis für die Identifikation von neuen Strategien zur Prädiktion und gezielten Behandlung von Knochenmetastasen darstellen. / Mamma carcinoma is one of the most frequently diagnosed cancers in women in Germany. Due to new diagnostic procedures, such as mammography screening, the early detection of mamma carcinoma is associated with a reduction in mortality. Nevertheless, the incidence of bone metastases in mamma carcinoma patients is strikingly high compared to other cancer types, at around 73%. These can lead to major skeletal complications such as spinal cord compression and pathological bone fractures. Thus, the patients' quality of life is severely impacted. Therefore it is of great clinical interest to better understand the pathogenesis of bone metastasis in mamma carcinoma. This will lead to the development of new strategies for the prediction and targeted treatment of bone metastases in mamma carcinoma patients. Previous studies have shown that cell morphology as a result of the biochemical and biophysical properties of tumor cells could provide information about the metastatic potential of mamma carcinoma cells. Therefore, this work focuses on the relationship between morphology and osteotropism of mamma carcinoma cell lines to provide a first descriptive approach. Various in vitro properties of a cell line model comprising different osteotropic mamma carcinoma cell lines were examined and compared with each other. This in vitro model consists of the highly osteotropic cell lines, MDA MB 231MET (MET) and MDA MB 231BONE (BONE), as well as the parental cell line, MDA MB 231. The morphological examination revealed 7 different morphotypes, which were present in a stable ratio in all examined cell lines. The distribution of morphotypes was unique for each cell line. At high confluence, MDA-MB-231 and MET showed growth in a single cell layer, whereas BONE cells tended to grow in a vertical orientation. At low cell density, the tight junctions in MET and BONE were more pronounced than in MDA MB 231. As soon as the cell density further increased, these junctions disappeared in the highly osteotropic cell lines and remained in MDA-MB-231. At the protein level, the adhesion protein E-cadherin was expressed at very low levels in the highly osteotropic cell lines compared to MDA-MB-231. This was also observed in the expression of CXCR4. The highly osteotropic cell lines also showed a lower expression of Tetraspanin 29 than the parental cell line. In contrast, the expression of the receptor EGFR was more pronounced in BONE than in the other two cell lines. Furthermore, similarities and differences to a non osteotropic cell line, MDA-MB-231BrM (BRAIN), which is also derived from MDA MB 231, were investigated. BRAIN showed distinguished morphological, molecular and functional similarities to both the parental and the highly osteotropic cell lines. This comparison highlighted specific characteristics of the highly osteotropic cell lines. Specific features in MET and BONE include the significantly higher ratio of a fibroblast-like morphotype and low expression of CXCR4 compared to the parental cell line and BRAIN. Principal component analysis of the transcriptome data revealed that the BONE and BRAIN cell lines are similar to each other. Overall, a potentially osteotropic gene signature consisting of 58 genes was derived from the transcriptome analysis. One of these genes, which is involved in migration and could possibly play a role in morphology, was knocked out. This did not result in any significant changes in the cell morphology. This work provides a basic insight into the morphological, molecular and functional properties of osteotropic mamma carcinoma cell lines. Thus, this work could provide a basis for the identification of new strategies for the prediction and targeted treatment of bone metastases.

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer with an average survival time of 15 months. GBM is considered incurable with even the most aggressive multimodal therapies and is characterized by near universal recurrence. Irreversible electroporation (IRE) is a cellular ablation method currently being investigated as a therapy for a variety of cancers. Application of IRE involves insertion of electrodes into tissue to deliver pulsed electric fields (PEFs), which destabilize the cell membrane past the point of recovery, thereby inducing cell death. While this treatment modality has numerous advantages, the lack of selectivity for malignant cells limits its application in the brain where damage to healthy tissue is especially deleterious. In this dissertation we hypothesize that a form of IRE therapy, high-frequency IRE (H-FIRE), may be able to act as a selective targeted therapy for GBM due to its ability to create an electric field inside a cell to interact with altered inner organelles. Through a comprehensive investigation involving experimental testing combined with numerical modeling, we have attained results in strong support of this hypothesis. Using tissue engineered hydrogels as our platform for therapy testing, we demonstrate selective ablation of GBM cells. We develop mathematical models that predict the majority of the electric field produced by H-FIRE pulses reach the inside of the cell. We demonstrate that the increased nuclear to cytoplasm ratio (NCR) of malignant GBM cells compared to healthy brain—evidenced in vivo and in in vitro tissue mimics—is correlated with greater ablation volumes and thus lower electric field thresholds for cell death when treated with H-FIRE. We enhance the selectivity achieved with H-FIRE using a molecularly targeted drug that induces an increase in NCR. We tune the treatment pulse parameters to increase selective malignant cell killing. Finally, we demonstrate the ability of H-FIRE to ablate therapy-resistant GBM cells which are a focus of many next-generation GBM therapies. We believe the evidence presented in this dissertation represents the beginning stages in the development of H-FIRE as a selective therapy to be used for treatment of human brain cancer. / Ph. D. / Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer with an average survival time of 15 months. GBM is considered incurable with even the most aggressive multimodal therapies and is characterized by near universal recurrence. Irreversible electroporation (IRE) is a therapy currently being developed for the treatment of a variety of cancers. Application of IRE involves the delivery of energy directly into the tumor tissue in the form of pulsed electric fields (PEFs). These PEFs destabilize the cell membrane past the point of recovery, thereby inducing cell death. Though this treatment modality has numerous advantages, the lack of selectivity for malignant cells limits its application in the brain where damage to healthy tissue is especially deleterious. In this dissertation we hypothesize that a form of IRE therapy, high-frequency IRE (H-FIRE), may be able to act as a selective targeted therapy for GBM due to its ability to create electric fields inside cells. Because cancer is characterized by alterations in inner organelles compared to healthy cells, electric fields inside the cell may be able to target these alterations resulting in selective malignant cell killing. Through a comprehensive investigation involving experimental testing combined with numerical modeling, we have attained results in strong support of this hypothesis. We have successfully demonstrated selective ablation of malignant GBM cells. We have shown that the increased nuclear to cytoplasm ratio (NCR) of malignant GBM cells compared to healthy brain—evidenced in vivo and in in vitro tissue mimics—is correlated with greater ablation volumes and thus lower electric field thresholds for cell death when treated with H-FIRE. We have enhanced the selectivity v achieved with H-FIRE using a molecularly targeted drug that induces an increase in NCR. We have tuned the treatment parameters to increase selective malignant cell killing. Finally, we have demonstrated the ability of H-FIRE to ablate therapy-resistant GBM cells which are a focus of many next-generation GBM therapies. We believe the evidence presented in this dissertation represents the beginning stages in the development of H-FIRE as a selective therapy to be used for treatment of human brain cancer.

Krüppel-like factors (KLFs) are a family of transcription factors with 3 Cys2/His2 zinc fingers that regulate cell differentiation and developmental processes. EKLF is involved in primitive and definitive erythropoiesis; KLF2 is implicated in the development of primitive erythroid and endothelial cells of the vasculature. Using light and electron microscopy, the yolk sacs and dorsal aortae from EKLF/KLF2 double knockout (KO) E9.5 (embryonic day 9.5) were examined to determine whether these KLFs have compensatory functions in morphology of blood cells and vessels. EKLF/KLF2 double KO E9.5 erythroid, endothelial, and mesothelial cells had more severely abnormal morphology than WT and KLF2-/-. Flow cytometry and cytospins were used to determine maturational effects of single and EKLF/KLF2 double KO primitive erythroid cells double-labeled with anti-TER119 and anti-CD71. EKLF KO and EKLF/KLF2 double KO erythroid cells display defective erythroid maturation. EKLF and KLF2 have overlapping roles in the development of embryonic erythroid and endothelial cells.

Yeasts are unicellular microorganisms which are able to form colonies. The morphology of colonies is characteristic for the strain and differs according to particular growth conditions. Saccharomyces cerevisiae colonies are able to produce volatile ammonia which functions as a signalling molecule alarming nutrient depletion (PALKOVÁ et al. 1997). Changes in gene expression, metabolism and ammonia production occur during the development of giant S. cerevisiae colonies. Genes with changes in expression have been identified to be involved in ammonia transport, amino acid metabolism and also in nucleotide metabolism. Genes, whose deletion may affect the induction of ammonia production have been described (ČÁP et al., 2010), (VÁCHOVÁ a PALKOVÁ 2005), (PALKOVÁ et al. 2002). This work is focused on studying of an impact of deletions of selected genes involved in nucleotide metabolism, as well as of changes in extracellular concentrations of different bases on growth, morphology, ammonia production, cell morphology and differentiation of giant S. cerevisiae colonies. S. cerevisiae strains producing Gfp-tagged proteins (Ade4p-Gfp, Adk1p-Gfp, Urk1p-Gfp, Fcy2p-Gfp, Fur4p-Gfp, Fcy22p-Gfp) have been constructed and analyzed. Development of S. cerevisiae giant colonies with deletions of selected genes (ADE5,7,...