Diabetes mellitus dramatically increases the risk for atherosclerotic cardiovascular disease. It has been established that chronic hyperglycemia promotes an increase in glucose flux through the hexosamine biosynthesis pathway (HBP). Central to this pathway is glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme controlling the conversion of glucose to glucosamine. We have shown that glucosamine is a potent inducer of endoplasmic reticulum (ER) stress, which is characterized by the accumulation of misfolded proteins in the ER. Chronic ER stress can initiate a multifaceted response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesized that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways. In support of the hypothesis we found that glucosamine-supplemented apoE-/- mice had elevated levels of ER stress and atherosclerosis. Mechanistically, our data showed that glucosamine induced ER stress by interfering with the lipid-linked oligosaccharide biosynthesis pathway and protein N-glycosylation. These findings support a model by which conditions of hyperglycemia promote vascular complications through a glucosamine-intermediate. / Thesis / Doctor of Philosophy (PhD) / Diabetes mellitus dramatically increases the risk for heart attacks and strokes. High blood glucose is utilized in cells through its conversion into metabolites, such as glucosamine. We hypothesized that conditions of high blood glucose can led to an increase in intracellular glucosamine which can initiate pathways involved in accelerating atherosclerosis. Our results show that this is possible in both human cells and mice.

<p> The Notch signalling pathway is a lateral inhibition pathway that serves to limit the number of cells in a proneural cluster (a group of equipotent cells) that will adopt a neural cell fate during neurogenesis in Drosophila. The proper segregation of neural and epidermal progenitor cells during neurogenesis requires the expression of both the proneural genes and the neurogenic genes. Expression of proneural genes, such as achaete, gives cells the potential to commit to a neural cell fate. The neurogenic genes encode proteins that act in the Notch signalling cascade and are required for cell fate determination during Drosophila neurogenests. Notch and Delta are neurogenic genes that encode large transmembrane proteins. Interaction between the extracellular domains of Notch and Delta is thought to transmit a signal to the nucleus by way of the DNAbinding Suppressor of Hairless protein. In response to Notch activation Suppressor of Hairless is translocated to the nucleus where it activates the transcription ofthe neurogenic genes ofthe Enhancer of split complex (E(spl)-C). The products of the E(spl)-C are bHLH transcription factors. They possess a Cterminal tryptophan-arginine-proline-tryptophan (WRPW) motif that interacts with the product of another neurogenic gene, groucho. The groucho gene product encodes a protein containing a WD40 repeat element. When bound to Groucho, E(spl) bHLH proteins are able to repress transcription of proneural genes, such as achaete, thereby directing the cell to adopt a non-neural cell fate.</p> <p> A number of murine groucho homologues have been identified and named Grg's (Groucho related genes). Three full length Grg proteins have been identified which contain all five domains found in the Drosophila Groucho protein. Two short Grg proteins have also been identified which only contain one of the domains found in the full-length Grg proteins. A number of murine homologues of the Drosophila E(spl)-C have also been identified and named Hes (Hairy/Enhancer of split) proteins. Like the gene products of the Drosophila E(spl)-C, the Hes proteins are bHLH proteins containing a C-terminal WRPW motif. One of the Hes proteins, Hes3, is lacking a basic domain and therefore lacks the DNA-binding activity possessed by the other Hes proteins. </p> <p> Attempts were made to detect interactions between Grg and Hes proteins using co-immunoprecipitation techniques. The anti-WD40 antibody, which recognizes the long WD40-containing Grg proteins, was able to specifically immunoprecipitate 35S-labelled Grgl . This antibody was also able to recognize WD40-containing Grg proteins present in Pl9 cell extracts. However, attempts to co-immunoprecipitate radiolabelled Hesl and AMLlb proteins with Grg proteins present in P19 cell extract were unsuccessful due to the low affinity of the antiWD40 antibody and the background caused by the binding of the test proteins to Sepharose. A second method of co-immunoprecipitation was attempted using an HA-tagged Grgl fusion protein and a commercially available anti-HA antibody. The attempt to co-immunoprecipitate 35S-labelled Hesl with radiolabelled HAtagged Grg 1 was unsuccessful due to a high degree of background caused by Hesl binding to protein G Agarose. Using the Yeast Two-Hybrid interaction assay, the WD40-containing Grg proteins, Grgl and Grg4, were found to interact with Hesl. However, using the same assay WD40-containing Grg proteins were found not to interact with Hes3, which lacks DNA-binding activity. A Western blot was performed to determine if the Hes3 fusion proteins were being expressed in transformed yeast but none were detected. This may have been due to the poor affinity of the anti-GAL4 activation domain antibody. A similar Western blot demonstrated that the Grg proteins, fused to the GAL4 DNA binding domain, were being expressed in transformed yeast extract. The WD40-containing Grg proteins, Grgl and Grg4, were also found not to interact with AMLlb, a protein which contains a C-terminal VWRPY domain which is reminiscent of the Cterminal WRPW interaction domain found in Hes proteins and Drosophila E(spl) proteins. However, WD40-containing Grg proteins were able to interact with an AML 1 b mutant in which the VWRPY motif was mutated to VWRPW in the Yeast Two Hybrid assay. </p> / Thesis / Master of Science (MSc)

The myogenic cell line, BC₃H1, upon cell-to-cell contact or serum starvation differentiates as monitored by the appearance of muscle-specific markers, actin, myosin light chain 1 (MLC 1) and tropomyosin (Tm) and morphological changes. The detection of MLC 1 and five Tm isoforms in this cell line is novel. To assess the role of protein kinase C (pk C)-and protein kinase A (pk A) signal transduction pathways in controlling BC₃H1 cell differentiation, activators of pk C (TPA) and pk A (cAMP analogues, dibutyryl-cAMP and 8-Br-cAMP) were used. TPA (500nM) addition caused no deviation from the normal expression patterns of actin, Tm and MLC 1. Addition of cAMP analogues (500μM) delayed the appearance of MLC 1 and muscle-specific isoforms of Tm, as well as α-actin while β- and γ-actin levels remained unchanged. However, α-actin mRNA levels were not affected by cAMP analogues yet the typical β- and γ-actin mRNA downregulation was blocked. cAMP appears to be operating at multiple levels to regulate BC₃H1 cell myogenesis such as post-transcriptional and translational. In addition, given the similarity in mechanisms through which cAMP and adenovirus early region 1A (AdE1A) mediate gene activation, the effect of AdE1A on BC₃H1 cell differentiation was investigated. A stable transfected AdE1A clonal cell line, BC₃E7, was characterized. Together with altered morphology, BC₃E7 cells failed to show the characteristic expression of muscle-specific markers actin, Tm and MLC 1. AdE1A transfection disrupted the synchronous expression of muscle-specific proteins during BC₃H1 cell differentiation. / Thesis / Master of Science (MS)

Monocytes are dynamic innate immune cells that respond differently based upon the dose and duration of an infection. While super low dose endotoxin is found in chronic inflammatory diseases such as atherosclerosis, exposure to high dose endotoxin leads to sepsis. However, clear characterization of monocytes and the underlying mechanisms in these disease conditions is lacking. To elucidate the missing information, we conducted two different projects. In the first project, we investigated the role of super low dose endotoxin in polarizing monocytes to a prolonged low-grade inflammatory state with no resolution, disrupting homeostasis. This low grade inflammatory phenotype was confirmed by sustained induction of inflammatory mediators CD40 and CD11a. In addition, low grade inflammatory monocytes influence neighboring T cells by suppressing T cell regulatory functions. Mechanistically, we showed that the non-resolving inflammatory phenotypes in monocytes is dependent on non-traditional TLR4 adaptor called TRAM. In the second project, we focused on the effects of high dose endotoxin on monocyte phenotypes. We reported that high dose endotoxin give rise to a mix of both immunosuppressive and pathogenic inflammatory monocytes, leading to monocyte exhaustion. While thorough research is conducted to study the immunosuppressive monocytes and underlying long term effects, role of pathogenic inflammatory monocytes is not well addressed. Monocyte exhaustion leads to elevated levels of CD38, an inflammatory mediator, elevated ROS levels, depleted NAD+ and mitochondrial respiration. STAT1 and KLF4 are critical transcription factors in sustaining exhausted phenotypes. Indeed, TRAM adaptor molecule also mediates this exhaustion as TRAM deletion restores monocyte health. Taken together, our work defines novel monocyte phenotypes and mechanism in super-low dose or high dose endotoxin environments. / Doctor of Philosophy / Healthy inflammatory response is represented by initial induction of inflammatory cells in the site of infection and pathogen clearance, followed by resolution of inflammation and damage repair. This balance between inflammation and resolution maintains immune homeostasis. Imbalances in this homeostasis can be a cause or effect of various disease conditions such as atherosclerosis and sepsis, for example. Despite rigorous research, these diseases are still prevalent and treatments are still lacking. It is essential to investigate inflammatory responses at a cellular level and understand how an immune cell responds to a given pathogen. Depending upon the intensity, dose and duration of a pathogen can dictate immune cell functions. Recent discoveries, including the research in our lab have reported that super low dose bacterial endotoxin exacerbates atherosclerosis. Mouse monocytes (innate immune cells) treated with super low dose endotoxin continuously induce mild but sustained inflammatory molecules but are unable to exhibit resolving mediators to dampen the inflammation and hence, monocyte homeostasis is disrupted. Homeostatic imbalance is also in seen in sepsis, when monocytes exposed to high dose bacterial endotoxin. Due to a repetitive exposure to high dose endotoxin, monocytes are unable to respond accurately, where they simultaneously exhibit inflammatory and anti-inflammatory mediators but in a dysregulated manner.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a membrane-associated guanylate kinase (MAGUK) family protein, which is encoded by a gene of identical name present on the X chromosome. CASK may participate in presynaptic scaffolding, gene expression regulation, and cell junction formation. CASK is essential for survival in mammals. Heterozygous mutations in the CASK gene (in females) produce X-linked intellectual disability (XLID) and mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH, OMIM# 300749). CASK mutations are also frequently associated with optic nerve hypoplasia (ONH) which is the most common cause of childhood blindness in developed countries. Some patients with mutations in CASK have been also diagnosed with optic nerve atrophy (ONA) and glaucoma. We have used floxed CASK (CASKfloxed), CASK heterozygous knockout (CASK(+/-)), CASK neuronal knockout (CASKNKO) and tamoxifen inducible CASK knockout (CASKiKO) mouse models to investigate the mechanism and pathology of CASK-linked ONH. Our observations indicate that ONH occurs with 100% penetrance in CASK(+/-) mice, which also displayed microcephaly and disproportionate cerebellar hypoplasia. Further, we found that CASK-linked ONH is a complex developmental neuropathology with some degenerative components. Cellular pathologies include loss of retinal ganglion cells (RGC), astrogliosis, axonopathy, and synaptopathy. The onset of ONH is late in development, observed only around the early postnatal stage in mice reaching the plateau phase by three weeks of birth. The developmental nature of the disorder is confirmed by deleting CASK after maturity since CASKiKO mice did not produce any obvious optic nerve pathology. Strikingly the CASKfloxed mice expressing ~49% level of CASK did not manifest ONH despite displaying a slightly smaller brain and cerebellar hypoplasia indicating that ONH may not simply be an extension of microcephaly. We discovered that deleting CASK in neurons produced lethality before the onset of adulthood. The CASKNKO mice exhibited delayed myelination of the optic nerve. Overall this work suggests that CASK is critical for neuronal maturation and CASK-linked ONH is a pervasive developmental disorder of the subcortical visual pathway. Finally, in a side project, I also described a new methodology of targeting neurons using receptor-mediated endocytosis which would help target retinal neurons for therapeutic purposes in the future. / Ph. D. / 7 in 10,000 children suffer from childhood blindness, for whom all the visual information from the outside world is completely blocked. Although classified as a rare disease, optic nerve hypoplasia (ONH), or the underdevelopment of optic nerve, is the leading cause of childhood blindness in developed countries, accounting for 15% of childhood blindness. Only a handful of genes have been shown to associate with ONH. The CASK gene, whose protein product calcium/calmodulin-dependent serine protein kinase (CASK) plays a role in presynaptic scaffolding, is one of them. Mutations in the CASK gene not only produce ONH, but also microcephaly and intellectual disability. Investigating the mechanism of CASK-linked ONH will provide critical data to understand the molecular basis of optic nerve formation and maturation. Here we have used the CASK heterozygous knockout mouse model to replicate the ONH and microcephaly seen in female human patients. We discovered that the onset of CASK-linked ONH corresponded to the late third trimester developmental stage in humans, thus ONH is developmental in nature. ONH pathologies include thinning of optic nerves, axonal atrophy, and synaptopathy. In contrast to the postnatal death of constitutive CASK loss of function in mice, CASK ablation in adult mice did not lead to lethality. CASK deletion also delays neuronal myelination. Overall, our results indicate that CASK is critical for postnatal maturation of the central nervous system and mutations of the CASK gene is sufficient to lead to ONH. Early intervention and proper gene therapy may treat CASK-linked ONH.

During the past decade, many successful deterministic models of macromolecular regulatory networks have been built. Deterministic simulations of these models can show only average dynamics of the systems. However, stochastic simulations of macromolecular regulatory models can account for behaviors that are introduced by the noisy nature of the systems but not revealed by deterministic simulations. Thus, converting an existing model of value from the most common deterministic formulation to one suitable for stochastic simulation enables further investigation of the regulatory network. Although many different stochastic models can be developed and evolved from deterministic models, a direct conversion is the first step in practice. This conversion process is tedious and error-prone, especially for complex models. Thus, we seek to automate as much of the conversion process as possible. However, deterministic models often omit key information necessary for a stochastic formulation. Specifically, values in the model have to be scaled before a complete conversion, and the scaling factors are typically not given in the deterministic model. Several functionalities helping model scaling and converting are introduced and implemented in the JigCell modeling environment. Our tool makes it easier for the modeler to include complete details as well as to convert the model. Stochastic simulations are known for being computationally intensive, and thus require high performance computing facilities to be practical. With parallel computation on Virginia Tech's System X supercomputer, we are able to obtain the first stochastic simulation results for realistic cell cycle models. Stochastic simulation results for several mutants, which are thought to be biologically significant, are presented. Successful deployment of the enhanced modeling environment demonstrates the power of our techniques. / Master of Science

INTRODUCTION: Porphyromonas gingivalis (P.g), a gram-negative anaerobe, is the major bacterium in the red complex (Socransky et al. 1998) and responsible for the onset and progression of severe periodontal disease. P. gingivalis is currently considered the ‘keystone’ pathogen of periodontal disease. It can produce several virulence factors, such as cysteine proteinases (gingipains), lipopolysaccharide (LPS), capsule and fimbriae. The LPS plays an important role in periodontal disease by inducing inflammation via stimulation of some cytokines such as TNF-ɑ. TNF-ɑ can activate expression of early response genes in macrophages, including Tumor Necrosis Factor-?-Induced Protein 1 (TNFAIP1). However, the role of TNFAIP1 in LPS-induced inflammation is largely unknown. OBJECTIVE: 1. Identification of TNFAIP1 biological functions in response to LPS/TNF-ɑ; 2. Identification of the TNFAIP1 mediated signaling pathway; 3. Determination of factors involved in the TNFAIP-dependent signaling pathway; 4. Analysis of TNFAIP1 promoter activity. MATERIALS AND METHODS: Mouse RAW cells, human THP-1 cells or MC3T3 cells were cultured in RPMI or ɑ-MEM media with 10% FBS at 37°C in 5% CO2. For DNA construction of TNFAIP1 cDNA or its promoter, DNAs were generated by polymerase chain reaction (PCR) with specific primers and templates. The cloned DNA sequences were confirmed by sequencing. Experiments to identify the biological function of TNFAIP1 and its promoter activity, utilized ELISA, DNA recovery, western blot, protein array, and promoter assay. RESULTS: 1. LPS-induced the activation of p-MARK or p-PI3K (but not p-JAK), the production of TNF-ɑ, NFĸB or TNFAIP1 was confirmed by ELISA and western blot analysis; 2. Transfection of TNFAIP1 cDNA for 1-10 hours stimulated TNF-ɑ production in macrophage cells but not after longer exposure; 3. Caspase 1 and 3 were induced by TNFAIP1 after transfection of TNFAIP1 for 20 hours; 4. Overexpression of TNFAIP1 induced apoptotic proteins, such as Bcl-x, Caspase 3, Catalase, Claspin, Cytochromic, HO-1/HMOX1/HSP32, MCL-1, P27/Kip1, or SMAC/Diablo; 5. TNFAIP1 promoter DNA was cloned into pGL3 basic plasmid DNA to determine promoter activity. TNFAIP1 promoter activity was tested via its potential protein-protein interaction using luciferase gene expression. With a MAPK inhibitor, TNFAIP1 promoter activity was increased. In contrast, with an ATK inhibitor, TNFAIP1 promoter activity was reduced. CONCLUSIONS: 1. TNFAIP1 is an important factor of the LPS/TNF-ɑ-dependent pathway; 2. MAPK or PI3K functions as an upstream factor of TNFAIP1, and LITAF is downstream factor of TNFAIP1-mediated signaling pathway in response to LPS; 3. Transfection of TNFAIP1 cDNA stimulated TNF-ɑ production for 1-10 hours exposure but reduced it for 10 - 20 hours exposure; 4. Overexpression of TNFAIP1 can increase expression of apoptotic proteins, Bcl-x, Caspase 3, Catalase, Claspin, Cytochromic, HO-1/HMOX1/HSP32, MCL-1, P27/Kip1, or SMAC/Diablo; 5. AKT and MAPK may act as transcriptional regulators of TNFAIP1 gene by binding to the promoter region. AKT upregulates TNFAIP1 gene expression and MAPK downregulates TNFAIP1 gene expression.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25162号 / 医博第5048号 / 新制||医||1070(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 林 康紀, 教授 髙橋 良輔, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Oral squamous cell carcinoma (OSCC) accounts for majority of head and neck cancers and ranks as the sixth most common cancer in the world. OSCC belongs to the most understudied cancers and little is known about molecular mechanisms underlying its etiology and progression to metastasis. A hallmark of cancer is the enhanced posttranslational modification of cell surface proteins with complex N-glycans. Our studies have shown that induced protein N-glycosylation via activation of the core N-glycosylation-regulating gene, DPAGT1, is associated with reduced E-cadherin adhesion, as well as deregulation of several oncogenic signaling pathways, including Wnt/β-catenin and Hippo. Modest increases in DPAGT1 expression are associated with dramatic amplification of Wnt/β-catenin activity and increased expression and nuclear localization of the Hippo pathway effectors TAZ /YAP. The goal of this study was to align the expression and localization of DPAGT1, complex N-glycans, β-catenin, and TAZ/YAP with the progression of oral cancer in vivo from dysplasia to OSCC. Human oral tissues from different stages of OSCC pathogenesis were characterized for DPAGT1/β-catenin/α-catenin/YAP/TAZ expression and localization and correlated with cell surface expression of complex N-glycans by PHA lectin staining and with expression of primitive cell surface markers, CD44, CD24 and CD29. Results showed that high DPAGT1 expression and nuclear TAZ became increasingly associated with disorganized E-cadherin junctions as oral epithelium progressed from mild to severe dysplasia to OSCC. This correlated with increasing expression of cell surface complex N-glycans and CD44. These studies suggest that DPAGT1/β-catenin/TAZ and high PHA staining represent novel signatures for OSCC pathogenesis.

Schweinfurthins are a family of natural products with significant anti-cancer activities. They were originally identified in the National Cancer Institute (NCI) human 60 cancer cell line screening. The growth inhibition profile of schweinfurthins is distinct from other clinically used anti-cancer agents, indicating that they have a novel mechanism of action or have a previously unrecognized protein target. Previous studies showed that schweinfurthins affect multiple cellular processes in cancer cells. For example, schweinfurthins can alter cytoskeleton organization, induce ER stress and apoptosis, and inhibit the mevalonate pathway. The mevalonate pathway is responsible for the production of isoprenoids and cholesterol, which have been shown to play regulatory roles in the Hedgehog (Hh) signaling pathway. In this study, we found that the Hh signaling pathway in NIH-3T3 and SF-295 cells was inhibited by schweinfurthins. The supplementation of mevalonate and cholesterol partially restored Hh signaling, indicating that schweinfurthins inhibit Hh signaling partially by down-regulating the products from the mevalonate pathway. Interestingly, schweinfurthins in combination with cyclopamine, an inhibitor of the Hh singaling pathway, synergistically decreased cell viability. In order to better understand the underlying mechanism of the anti-cancer action of schweinfurthins, we attempted to identify the protein target of schweifnurthins. Affinity chromatography was performed to pull down the protein target. We found that schweinfurhtins bound to the M2 isoform of pyruvate kinase (PKM2) and inhibit its pyruvate kinase activity. Knockdown of PKM2 by siRNA increased the sensitivity of SF-295 cells to schweinfurthins. The inhibition of PKM2 by schweinfurthins led to a reduction in the rate of glycolysis in cancer cells. Fructose 1,6-bisphosphate (FBP), an activator of PKM2, could alleviate schweinfurthin-mediated inhibition on PKM2 and glycolysis. Notably, FBP could also partially reverse the reduction of cell viability in the presence of schweinfurthins. Taken together, these studies revealed the mechanism by which schweinfurthins inhibit Hh signaling. In addition, we uncovered PKM2 as a schwienfurthin target and highlighted the importance of glycolysis suppression as a mechanism of the anti-cancer action of schweinfurthins.