Dehydropyrrolizidine alkaloids (DHPAs) are a large group of globally important plant-derived pro-toxins that can contaminate or are naturally present in animal feed and the human food supply as well as herbal supplements. Their bioactive metabolites are potentially hepatotoxic, pneumotoxic, genotoxic and carcinogenic. Due to the difficulty in obtaining sufficient quantities of purified DHPAs, toxicity studies have largely relied on single intraperitoneal injections in rodent models, and carcinogenicity studies have been limited to a small handful of the hundreds of isolated DHPAs. To assess the relative toxicity of structurally diverse DHPAs in a more biologically relevant manner, male California White chicks were dosed orally with 0.01, 0.04, 0.13, or 0.26 mmol of seven different DHPAs and three DHPA N-oxides kg-1 bodyweight for 7 days. DHPAs were grouped in relation to their toxicity based on clinical, serum biochemical, and histopathological evaluations as well as tissue adduct accumulation rates. Using the same model, a reduced extract from comfrey, a commonly used DHPA containing herb, was compared to its two major constituent DHPAs, intermedine and lycopsamine. Based on the same parameters, the comfrey extract was more toxic than pure lycopsamine or intermedine. Addressing the need for a more sensitive carcinogenicity model, male heterozygous p53 knockout mice were treated with riddelliine 5, 15 or 45 mg kg–1 bodyweight day-1 by oral gavage for 14 days, or given a long-term treatment of riddelliine 1 mg kg-1 bodyweight day–1 in pelleted feed for 12 months. Exposure to riddelliine increased the odds of tumor development in a dose-responsive manner (odds ratio 2.05 and Wald 95% confidence limits between 1.2 and 3.4). The most common neoplasm was hepatic hemangiosarcoma, which is consistent with previously published lifetime rodent studies. The results of this research demonstrate that the California White chick model is sensitive for comparison of DHPA toxicity, and data obtained from this research can be used to validate previous DHPA toxicity research. It also demonstrates that comfrey toxicity may have been previously underestimated. The heterozygous p53 knockout mouse model is beneficial for further investigation of comparative carcinogenesis of structurally and toxicologically different DHPAs and their N-oxides.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21692号 / 医科博第96号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 渡邉 大, 教授 伊佐 正, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Protocadherine spielen eine wichtige Rolle bei der Entwicklung des Nervensystems und sind an Prozessen der Zellmigration und -differenzierung, sowie der Hemmung von Zellwachstum beteiligt. Um die Funktion und Regulation von Protocadherin gamma C3 (PcdhγC3) an mikrovaskulären Endothelzellen des Großhirns (cEND) und des Kleinhirns (cerebEND) zu untersuchen, wurden die PcdhγC3-Knock-out (KO) Zelllinien mit der CRISPR/Cas9 Methode etabliert. Der KO führt zu verminderten Barriereeigenschaften der Blut-Hirn-Schranke (BHS), was sich in einer erhöhten Permeabilität für Fluoreszein und einem verringerten transendothelialen elektrischen Widerstand (TEER) widerspiegelt. Es konnte eine Veränderung der Wachstumsrate und dem Adhäsionsverhalten der KO-Zellen nachgewiesen werden. Auch die Expression der Tight-Junction-Proteine, sowie einiger Komponenten des Wnt und mTOR Signalwegs wurden durch den KO von PcdhgC3 beeinflusst. / Protocadherins (Pcdhs) play an important role in neuronal development, cell migration and differentiation, as well as inhibition of cell growth. To investigate the function and regulation of Protocadherin gamma C3 (PcdhgC3) on microvascular endothelial cells of the cerebrum (cEND) and cerebellum (cerebEND), PcdhgC3 knock-out (KO) cell lines were generated, using the CRIPS/Cas9 method. The Knock-out lowers the barrier function oft he blood-brain-barrier (BBB), which can be seen in lower transendothelial electrical resistance (TEER) and higher permeabilitiy for fluorescein. We detectet a change in the growth rate and the cellular adhesion of the KO-cells. Also the expression of tight-junction proteins, as well as parts oft the Wnt and mTOR signalling pathway are altered by the KO of PcdhgC3.

Indiana University-Purdue University Indianapolis (IUPUI) / There are many clinical diseases involving abnormal differentiation of smooth muscle, such as atherosclerosis, hypertension and asthma. In these diseases, one important pathological process is the disruption of the balance between differentiation and proliferation of smooth muscle cells. Serum Response Factor (SRF) has been shown to be a key regulator of smooth muscle differentiation, proliferation and migration through its interaction with various accessory proteins. Myocardin Related Transcrition Factors (MRTFs) are important co-activators of SRF that induce smooth muscle differentiation. Elucidating the mechanism of how MRTFs and SRF discriminate between genes required to regulate smooth muscle differentiation and those regulating proliferation will be a significant step toward finding a cure for these diseases. We hypothesized that SWI/SNF ATPdependent chromatin remodeling complexes, containing Brg1 and Brm, may play a role in this process. Results from western blotting and quantitative reverse transcription - polymerase chain reaction (qRT-PCR) analysis demonstrated that expression of dominant negative Brg1 or knockdown of Brg1 with silence ribonucleic acid (siRNA) attenuated expression of SRF/MRTF dependent smooth muscle-specific genes in primary cultures of smooth muscle cells. Immunoprecipitation assays revealed that Brg1, SRF and MRTFs form a complex in vivo and that Brg1 directly binds MRTFs, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated SRF binding and the ability of MRTFs to increase SRF binding to the promoters of smooth muscle-specific genes, but not proliferation-related early response genes. The above data suggest that Brg1/Brm containing SWI/SNF complexes play a critical role in differentially regulating expression of SRF/MRTF-dependent genes through controlling the accessibility of SRF/MRTF to their target gene promoters. To examine the role of SWI/SNF in smooth muscle cells in vivo, we have generated mice harboring a smooth muscle-specific knockout of Brg1. Preliminary analysis of these mice revealed defects in gastrointestinal (GI) development, including a significantly shorter gut in Brg1 knockout mice. These data suggest that Brg1-containing SWI/SNF complexes play an important role in the development of the GI tract.

Lysine-specific Demethylase 1B (KDM1B) is a chromatin regulator which functions as a histone eraser through the removal of the post-translational modifications mono and dimethylation of histone 3 on lysine 4 (H3K4me1/2). This process is enhanced by the formation of a complex with Nuclear Protein Glyoxylate Reductase (NPAC). NPAC resolves the sequestration of the nucleosome histone tail to allow robust demethylation of H3K4me2 by KDM1B, during transcriptional elongation by RNA polymerase 2 (RNAP II). KDM1B is involved in many crucial processes during development. Its physiological functions include the establishment of maternal genomic imprints, reset of the epigenome during somatic cell reprogramming, and regulation of brown adipogenic differentiation. In light of this, the role of KDM1B in human embryonic stem cells (hESCs) is examined through CRISPR/Cas9-editing to further dissect its biological functions during embryogenesis. CRISPR-induced knockouts of KDM1B exhibited similar cell proliferation rate and expression of OCT4 and NANOG pluripotency markers to wildtype cells. Furthermore, KDM1B-/- clones were able to maintain their pluripotency potential by differentiating to all germ layers in teratoma and embryoid body formation assays. In addition, RNA-seq of KDM1B-/- clones showed enrichment of mesoderm lineage-related gene ontology (GO) terms in the downregulated differentially expressed genes. Thus, KDM1B is believed to be dispensable during the pluripotent stage of the cell but proved fundamental during later stages of development.

The 52 kDa intermediate filament protein desmin plays an important role in force transmission in skeletal muscle by connecting myofibrils at Z-lines and to the sarcolemma. Desmin content in muscle adapts to contractile activity and may be involved in cellular signaling mechanisms responsible for muscle growth. Purpose: To compare signaling responses of the mTOR pathway in wild type (WT) vs desmin knock out (KO) mice. Methods: WT (n=12) and KO (n=12) mice were exposed to high frequency electric stimulation of the left hindlimb to elicit an acute response of the mTOR pathway. Non-stimulated right hindlimbs were used as a within animal control. Right and left TA and EDL muscles were dissected 30 min post-stimulation and examined for changes in mTOR, 4E-BP1 and p70S6K. Results: Relative to WT control samples, total mTOR and total 4E-BP1 content was higher in KO control samples. Electrical stimulation resulted in an increase p70S6K phosphorylation in WT and KO animals however there was no difference between the groups. 4E-BP1 phosphorylation was increased in WT but not KO following electrical stimulation. There was no change in mTOR phosphorylation in response to stimulation in WT or KO. Conclusion: The absence of desmin in skeletal muscle does not impair the phosphorylation of p70S6K demonstrating that a tensile load on the muscle will likely result in an increase in protein synthesis. Elevated levels of total mTOR and 4E-BP1 may imply an adaptation to increase sensitivity to growth stimuli in the muscle.

Tissue Factor Pathway Inhibitor (TFPI) is an anticoagulant protein containing three Kunitz domains, K1, K2 and K3. K1 inhibits Factor VIIa, K2 inhibits Factor Xa, and K3 enhances the Factor Xa inhibition by its interaction with Protein S. Since zebrafish is an excellent genetic model, we hypothesized that TFPI regulation could be studied using this model. As a first step, we confirmed the presence of tfpia in zebrafish. Subsequently, we performed knockdown of tfpia, and knockout of tfpia in K3 domain using CRISPR/Cas9. Both the tfpia knockdown and tfpia homozygous deletion mutants showed increased coagulation activities. Our data suggest that zebrafish tfpia is an orthologue for human TFPIα, and silencing it results in a thrombotic phenotype. We then optimized the piggyback knockdown method, where we could simultaneously piggyback 3 or 6 ASOs corresponding to 3 or 6 genes, respectively, using one VMO. These multiple gene knockdowns will increase the efficiency of genome-wide knockdowns. Since there are no studies on chromatin remodeling that control TFPI expression, we hypothesized that the genome-wide knockdowns of the Chromatin Binding and Regulatory Proteins (CBRPs) in zebrafish could help identify novel tfpia gene regulators. We chose 69 CBRPs and subjected them to simultaneous gene knockdowns. Our results have identified 5 novel regulators for tfpia. We exploited this information to discover UNC6852, a drug that enhances tfpia mRNA levels. This could be used as an antithrombotic drug. The approach developed here could be used to study the regulation of other coagulant and anticoagulant factors.

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