Chk2 is a cell cycle checkpoint kinase that is essential for initiating the DNA damage response in the presence of genetic damage. Its role is highly conserved from budding yeast (where it is named Rad53) to humans. Very few cell cycle checkpoint proteins have ever been studied in fish and the role of Chk2 has never been characterized. Oncorhynchus mykiss (Rainbow trout) was chosen for this project due to its importance in the commercial aquaculture industry and the availability of rainbow trout cell cultures at the University of Waterloo. This study was the first to clone the CHK2 gene in a teleost species, verified through both genomic and cDNA cloning. A section of the CHK2 gene, specifically the forkhead associated domain (FHA), was used to express recombinant Chk2 protein and generate polyclonal anti-Chk2 antibodies. A southern blot was performed and CHK2 was found to exist as a single copy number in the rainbow trout genome. The tissue specificity of Chk2 was also examined both at the mRNA transcript and protein level. Interesting tissue specific differences were discovered with transcript levels moderately low in gill and higher in brain, while protein levels were extremely high in gill and lower in brain tissues. Protein levels were verified in both whole fish tissue samples and in cell culture suggesting that cell cultures accurately reflect the state of checkpoint proteins in vivo. These tissue specific differences suggest that in gill, Chk2 is maintained at a high protein level to combat any toxins in the water attempting to transverse this barrier tissue and gain access to the fish’s circulatory system. Meanwhile, the blood brain barrier offers protection to the highly sensitive brain tissue, suggesting that high levels of Chk2 protein are not constitutively required, but instead remain in a transcript reservoir able to be quickly translated in the event of DNA damage. To determine whether Chk2’s checkpoint role is conserved in O. mykiss, both gill and brain cell cultures were treated with low and high doses of bleocin (a commercially available form of bleomycin) known to cause high levels of double-strand breaks, the most deleterious type of DNA damage and a specific activator of the Chk2 DNA damage response (DSB). Results showed that bleocin had no effect on levels of Chk2 in gill cells, confirming that the protein is constitutively active in this tissue always on alert against potential genetic insult. In contrast, brain cells were able to upregulate Chk2 in a dose-dependent manner to bleocin induced DNA damage demonstrating that Chk2 can act as a biomarker for genetic damage in brain cells. In conclusion, the tissue specific expression of Chk2 and its ability to respond to DNA damage suggests that checkpoint proteins may serve as suitable biomarkers for DNA damage in O. mykiss and other fish species.

Chk2 is a cell cycle checkpoint kinase that is essential for initiating the DNA damage response in the presence of genetic damage. Its role is highly conserved from budding yeast (where it is named Rad53) to humans. Very few cell cycle checkpoint proteins have ever been studied in fish and the role of Chk2 has never been characterized. Oncorhynchus mykiss (Rainbow trout) was chosen for this project due to its importance in the commercial aquaculture industry and the availability of rainbow trout cell cultures at the University of Waterloo. This study was the first to clone the CHK2 gene in a teleost species, verified through both genomic and cDNA cloning. A section of the CHK2 gene, specifically the forkhead associated domain (FHA), was used to express recombinant Chk2 protein and generate polyclonal anti-Chk2 antibodies. A southern blot was performed and CHK2 was found to exist as a single copy number in the rainbow trout genome. The tissue specificity of Chk2 was also examined both at the mRNA transcript and protein level. Interesting tissue specific differences were discovered with transcript levels moderately low in gill and higher in brain, while protein levels were extremely high in gill and lower in brain tissues. Protein levels were verified in both whole fish tissue samples and in cell culture suggesting that cell cultures accurately reflect the state of checkpoint proteins in vivo. These tissue specific differences suggest that in gill, Chk2 is maintained at a high protein level to combat any toxins in the water attempting to transverse this barrier tissue and gain access to the fish’s circulatory system. Meanwhile, the blood brain barrier offers protection to the highly sensitive brain tissue, suggesting that high levels of Chk2 protein are not constitutively required, but instead remain in a transcript reservoir able to be quickly translated in the event of DNA damage. To determine whether Chk2’s checkpoint role is conserved in O. mykiss, both gill and brain cell cultures were treated with low and high doses of bleocin (a commercially available form of bleomycin) known to cause high levels of double-strand breaks, the most deleterious type of DNA damage and a specific activator of the Chk2 DNA damage response (DSB). Results showed that bleocin had no effect on levels of Chk2 in gill cells, confirming that the protein is constitutively active in this tissue always on alert against potential genetic insult. In contrast, brain cells were able to upregulate Chk2 in a dose-dependent manner to bleocin induced DNA damage demonstrating that Chk2 can act as a biomarker for genetic damage in brain cells. In conclusion, the tissue specific expression of Chk2 and its ability to respond to DNA damage suggests that checkpoint proteins may serve as suitable biomarkers for DNA damage in O. mykiss and other fish species.

The cyclin-dependent kinase (CDK) inhibitors have been demonstrated to be an important component in the regulation of plant cell cycle. Although they share a conserved CDK inhibitory region with a family of CDK inhibitors in mammals, the plant CDK inhibitors are very different from the animal and yeast CDK inhibitors. Thus studies of the plant CDK inhibitors could provide insight on the molecular mechanisms regulating the cell cycle in plants as well as the differences between plants and animals. The research described in this thesis investigated the seven Arabidopsis CDK inhibitors ICKs in terms of transgenic expression, phosphorylation, stability and interactions with other proteins. <p>ICKs were expressed in transgenic Arabidopsis plants as fusion proteins with the green fluorescent protein (GFP). Consistent with the previous studies on ICK1, ICK2 and ICK4, overexpression of all seven ICKs inhibited plant growth and resulted in plants with serrated leaves and flowers with altered morphology. A Survey based on large a number of independent transformants showed that GFP-ICK3 and GFP-ICK4 had weaker phenotypic effects compared to other GFP-ICKs. The Western blotting results showed that all GFP-ICKs were expressed at a low level in general. The levels of GFP-ICK3 and GFP-ICK4 were the lowest, suggesting that the weaker effects for ICK3 and ICK4 may partly be due to low protein levels. Treatments with MG132, an inhibitor of the proteasome, resulted in moderate but clear accumulation of fusion proteins for ICK1, ICK5, ICK6 and ICK7 in plants, suggesting that the proteasome is involved in the degradation of these proteins. <p>To study the state of protein phosphorylation, the proteins extracted from the plants were treated with calf intestinal phosphatase (CIP). The CIP treatment caused a faster migration of the GFP fusion protein for ICK1, ICK2, ICK5, ICK6 and ICK7, while the effect was not observed for control GFP and other non-specific proteins, indicating that these proteins can be phosphorylated in plants. The shift also differed among ICKs. Interestingly, dephosphorylation of ICK7 might have rendered it less stable. The protein pulldown experiments using p13Suc1-conjugated agarose beads showed that GFP-ICK4, GFP-ICK5 and GFP-ICK6 could associate with the CDK complex, similar to what has been shown for ICK1 and ICK2. CIP treatments of the p13Suc1 affinity-purified proteins also showed that ICK1, ICK2, ICK5 and ICK6 associated with the CDK complex were phosphorylated. <p>Attempts were also made to isolate peptide aptamers that are able to interact with ICKs for the purpose for expressing such an aptamer in plants. However, an aptamer that has a strong ability to interact with ICKs in two of yeast two-hybrid systems was not identified. In addition, the analysis of Arabidopsis CYCD3;1 for its interaction with ICK1 using a series of deletion mutants showed that the removal of both the N-terminal and C-terminal regions of CYCD3;1 greatly reduced or abolished the interaction with ICK1. <p> In summary, transgenic Arabidopsis plants have been obtained for expressing each of the seven Arabidopsis CDK inhibitors fused to GFP. The results confirmed and extended previous finding that overexpression of a CDK inhibitor inhibits plant growth as well as changes plant morphology. The observation that the ICK fusion proteins were generally at low and often undetectable levels, in comparison to much higher levels of the GFP protein, suggests that ICKs are unstable in the cell. Results from the MG132 experiments indicate that the 26S proteasome may play a role in the degradation of ICK1, ICK5, ICK6 and ICK7. Results from CIP treatments further show that most ICKs, particularly ICK1, ICK2, ICK5, ICK6 and ICK7, can be phosphorylated in vivo. Interestingly, ICK7 stability may depend on the status of protein phosphorylation. This study provides new understanding on how the family of proteins is regulated at the post-transcriptional level and the differences among Arabidopsis CDK inhibitors.

The second law analysis is a powerful tool for assessing the performance of engines and has been employed for few decades now. Turbocharged diesel engines have been explored in much detail with the help of second law analyses. There is also a need to examine the turbocharged spark-ignition engines in greater detail using second law analyses as they are gaining popularity in high performance and conventional automobiles as well. A thermodynamic simulation was developed in order to investigate the effects of turbocharging on spark-ignition engines from second law perspective. The exergy values associated with the components of the turbocharger along with the engine components were quantified as a percentage of fuel exergy. The exergy balance values indicated that turbocharger does not add considerably to the overall irreversibilities and combustion irreversibility is still the major source of exergy destruction. A comprehensive parametric investigation was also performed to investigate the effects of compression ratio, intercooler effectiveness, etc. for the turbocharged spark-ignition engine over the entire load and speed range. The simulation studies helped in understanding the behavior of turbocharged sparkignition engine with these parameters. A simulation study was also performed to compare the turbocharged engine with the naturally aspirated spark-ignition engine. This study examined the engines for operating parameters like bmep and bsfc over the entire speed range and revealed that turbocharging offers higher bmep and lower bsfc values for most of the operating range. In an additional study, these engines were analyzed for the brake thermal efficiency values at part load. The results indicated that turbocharging offers marginally higher brake thermal efficiency at part loads.

Suppose G is a graph and p >= 2q are positive integers. A color-list is a mapping L: V --> P(0, 1,...,p-1) which assigns to each vertex a set L(v) of permissible colors. An L-(p, q)-coloring of G is a (p, q)-coloring h of G such that for each vertex v, h(v) in L(v). We say G is L-(p, q)-colorable if such a coloring exists. A color-size-list is a mapping f: V -->{0, 1, 2,..., p}, which assigns to each vertex v a non-negative integer f(v). We say G is f-(p, q)-colorable if for every color-list L with |{L}(v)| = f(v), G is L-(p, q)-colorable. For odd cycles C, Raspaud and Zhu gave a sharp sufficient condition for a color-size-list f under which C is f-(2k+1, k)-colorable. The corresponding question for even cycles remained open. In this paper, we consider list circular coloring of even cycles. For each even cycle C of length n and for each positive integer k, we give a condition on f which is sufficient and sharp for C to be f-(2k+1, k)-colorable.

Hepatocellular carcinoma (HCC) or hepatoma is the top one cause of death in Taiwan based on the Cause of Death Statistics from the Department of Health, Executive Yuan, Taiwan, for many years. To identify any reliable HCC markers and further applied with the AFP measurement to improve the early diagnosis of HCCs is the most important thing. A high expression level of S-phase protein kinase associated protein 2 (SKP2) protein and its cofactor CDC28 protein kinase regulatory subunit 1B (CKS1B) involved in ubiquitination of some cyclin-dependent kinase (Cdk) inhibitors has been reported in various carcinoma. In this study, we examined the expression of CKS1B in HCC tissues and cell lines, and tested the cell cycle effect caused by specific small interference RNA (siRNA) of CKS1B in SK-hep1 cell line. Up-regulated CKS1B mRNAs in HCC cell lines and tissues were identified in our study, when comparing to the normal liver tissues. But we also found lack of up-regulated CKS1B proteins in our HCC tissues at the same time, indicated that CKS1B proteins might be unstable in HCCs. Down regulation of the Cdk inhibitors p27 was only partially associated with HCCs, and the expressions of CKS1B and p27 were not correlated to each other in HCCs, suggesting other pathway(s) might involve in the regulation(s) of CKS1B and p27 proteins in the HCCs. Down-regulation of the p21 proteins was also found to be not significantly associated with HCCs tissues, this result strongly suggested a post-translational stabilization way might regulate(s) the p21 protein levels in HCCs tissues. On the other hands, in time course experiment, disruption of CKS1B mRNA by si-CKS1B up-regulated the expressions of p27 and SKP2 protein levels and down-regulated the p21 protein level in the SK-hep1 hepatoma cell lines for 24 hrs later. But the mRNA expression level of p21 and p27 were actually both up-regulated for 48 hrs after transfected with si-CKS1B. We also tested the mRNA expression level of many cell cycle regulatory factors for 48 hrs after transfected with si-CKS1B. The results exhibited almost all of the factors (excepted p21, p27 and Cyclin D2) were down-regulated. Furthermore, we saw the apoptosis appearance of SK-hep1 cell after transfected with si-CKS1B for 48 hrs, suggesting the abnormal cell proliferation and tumorigenesis were controlled by siRNA transfection. Taken together, these results suggest that SCFSKP2-CKS1B pathway might not direct involved in ubiquitination of Cdk inhibitors. Another pathway(s), either known or novel, in addition to APC/CCHD1 (G0-G1 phase) and SCFSKP2-CKS1B (G1-S phase) regulation pathways, might regulate the tumorigenesis of HCCs.

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第23回名古屋大学年代測定総合研究センターシンポジウム平成22(2010)年度報告

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The tropical Rainfall Measuring Mission (TRMM) Microwave Imager (TMI) and Precipitation Radar (PR) data are used in this study to reveal diurnal variations of precipitation over the Tropics (30&#9702;S &#8722; 30&#9702;N) from January, 1998, to December 2002. The TMI data were used for the regions over oceans and islands and the PR data was used over continents. The observations are sorted regionally to examine the difference in diurnal cycle of rainfall over ocean, island, and continental regions. The rain rate is averaged over individual two hour intervals of local time in each region to include more observations in order to reduce the sampling error. F-test is used to determine those regions whose diurnal cycle is detected at the 95% confidence level. In most oceanic regions there is a maximum at 0400 LST - 0700 LST. The amplitude of diurnal variation over ocean regions with small total rain is a little higher than that of the ocean regions with heavy total rain. The diurnal cycle peaks at 0700 LST - 0800 LST over islands with rainfall variation similar to surrounding oceanic regions. A maximum at 1400 LST - 1500 LST was found in areas over continents with heavy total rain, while the maximum occured at 1900 LST - 2100 LST over continents with lesser total rain. The amplitudes of variation over continents with heavy total rain and with small total rain do not show significant differences. The diurnal cycle in in JJA (June, July, August) and DJF (December, January, February) varies with latitude over continents. A seasonal cycle of diurnal cycle can also be found in some oceanic regions. The diurnal cycle annual change is not evident over continents, while the diurnal cycle annual change over oceans exists in some regions. Island regions in this paper exhibit no evident seasonal and annual diurnal change.