Dissertations / Theses on the topic 'Colon damage'

There is considerable supportive evidence to suggest that increased levels of DNA damage are associated with an increased risk of developing neoplastic lesions in the human colon and rectum. Within this thesis, several different topics related to DNA damage were explored in detail, principally using the single cell gel electrophoresis assay (the comet assay) to measure DNA damage both in cell line studies and in human colorectal mucosal biopsies from patients undergoing routine endoscopic examinations of the colon and rectum.

Bile acids have been linked to the etiology of colon cancer in various studies for over twenty-five years. However, the mechanism by which bile acids act in colon carcinogenesis is not known. By using an assay that can detect induction of the gadd153 stress response to DNA damage, I found that bile acids activate expression of gadd153 promoter. This observation implies that bile acids cause DNA damage. I then hypothesized that exposure of cells to bile acids produces reactive oxygen species which damage DNA. However, experiments to block gadd153 induction by bile acids using antioxidants, and to measure 8-OH-dG induced by bile acids were inconclusive. I also used p53 mutant cell lines to show that bile acid induction of apoptosis is p53-independent. In addition, my experiments showed that bile acid increased expression of gadd153 protein in HT-29 cells, and that gadd153 protein was constitutively overexpressed in HCT-116 cells with or without bile acid treatment. Thus, bile acid action in colon carcinogenesis may involve induction of DNA damage, induction of gadd153 as a protective stress response, and then if the damage is beyond repair, p53-independent apoptosis.

The acute cellular responses to DNA damaging agents are critical in determining the long term outcome of disease. A cell’s susceptibility to damage, or its capacity to remove or repair this damage, all contributes to the eventual health or disease of tissues. This process is especially crucial in colonic epithelial cells and in the development of colorectal oncogenesis. The colonic lumen is constantly subjected to different environmental compounds that may have genotoxic properties that can initiate mutational events and possibly carcinogenesis. Therefore, the study of a regulatory dietary agent that improves the colonic cells ability to withstand damage, improve repair and retain its general health is a significant and practical tool in the fight against colorectal cancer. The health benefits of fish oil, including its potential chemopreventative properties, have been reported in numerous studies. However, the mechanism by which this protective effect occurs remains unclear. A gap in current literature exists that fails to explore the effect of fish oil on the early cellular responses to carcinogenic agents. Therefore, this thesis aims to firstly, better understand the specific host responses to an insult of carcinogen in vivo; secondly, to determine if regulation of these responses can be achieved by dietary fish oil; and lastly, to explore the potential consequences of this regulation for colorectal oncogenesis. All experimental work was carried out using a rat – azoxymethane (AOM) animal model of colorectal carcinogenesis. The key host responses to the carcinogen that were measured included the formation of acute O6methyldeoxyGuanosine (O6medG) DNA damage, the acute apoptotic response to genotoxic carcinogen (AARGC) and cell proliferation rates. A novel immunochemical assay was designed to detect both the levels and distribution of O6medG in colonic cells. With this established, a pattern of these host responses were mapped out over time. A dietary intervention study trialling a range of fish oil diets containing different doses and forms was then carried out to determine if modulation of responses occurred. This study was then followed on by a longer term study that explored the consequences of regulation by fish oil on pre-neoplastic lesions in the colon. The acute host responses to an insult of AOM showed that colonic O6medG formation began 2h post AOM administration and peaked at 6h. The AARGC response followed the pattern of O6medG by a 2h delay, peaking at 8h post AOM administration, while cell proliferation rates decreased significantly after 6h. The inclusion of tuna oil in the diet did not affect either the AARGC or cell proliferation rates when given in any form or at any dose. Animals fed a diet with 15% free tuna oil and 7% encapsulated tuna oil did however have significantly reduced levels of O6medG DNA damage in the distal colon. This reduction in O6medG levels did not translate into a reduction of ACF lesion, with a protective effect against ACF lesions only being observed in animals fed the high dose fish oil groups. Analysis of the data suggest that the acute host responses to an insult of DNA damaging agent appear to be closely related, all reaching their peak level of response 6-8h after the insult. The short time frame between both O6medG and apoptosis also did not support the current popular theory which explains O6medG mediated apoptosis. An alternate hypothesised BER mediated apoptotic pathway was also not supported. Regulation of the acute apoptotic response or the cell proliferation rate was not achieved by dietary fish oil. However, a high dose fish oil diet did regulate the level of O6medG in colonic epithelial cells by significantly reducing the total O6medG DNA damage load. This reduction of O6medG by a high fish oil diet however, was not translated into a protective effect against the formation of pre-neoplastic lesions. These data suggests that regulation of the acute O6medG response to a damaging agent does not necessarily imply protection for longer term colorectal oncogenesis. Additional studies exploring both the effect of fish oil on AOM metabolising enzymes and also a longer term cancer study may help to answer some pertinent questions evolving from this thesis.

A serotonina (5-HT) é um neuro-hormônio com complexos efeitos em humanos e animais. Embora se acredite que a sinalização serotoninérgica promova o desenvolvimento de tumores de cólon, também tem sido observado que a redução dos níveis de 5-HT aumenta o risco de malignidades neste órgão. Assim, é necessário investigar como a síntese de 5-HT modula a carcinogênese de cólon. Esta hipótese será explorada em experimentos mecanísticos envolvendo a exposição ou não de camundongos a um agente carcinogênico (azoximetano). Estes experimentos revelaram que a exposição carcinogênica aumentou a síntese de 5-HT no cólon, uma vez que os processos de liberação e degradação de 5-HT foram reduzidos, enquanto que a sua síntese e recaptação aumentaram. Após 24 semanas da 1° exposição carcinogênica, a deleção da síntese de 5-HT promoveu a multiplicidade tumoral no cólon, enquanto que ao longo de 12 semanas ocorreu um aumento de lesões preneoplásicas e proliferação celular. É interessante que 72 hrs após a sexta e última exposição carcinogênica o número de criptas aberrantes foram detectadas reduzidas na ausência de 5-HT, o que ocorreu em conjunto com uma redução da atividade proliferativa, aumento de células apoptóticas, e maior dano de DNA. Finalmente, a 5-HT modulou mecanismos de reparo genômico. Concluímos que a síntese serotoninérgica parece ser um fator de proteção do cólon intestinal, que caso inibida facilitaria o desenvolvimento tumoral neste tecido.<br>Serotonin (5-HT) is a neurohormone with complex effects in humans and animals. Although serotonergic signaling has been suggested to promote the development of colon tumors, the reduction in 5-HT levels was reported to increase the risk of colon cancer. Here, we sought to investigate how 5-HT synthesis modulates colon carcinogenesis. This hypothesis was explored in mechanistic experiments that carcinogenically exposed or not mice to azoxymethane. It revealed that carcinogenic exposure increased the synthesis of 5-HT in the colon since 5-HT release and decay were inhibited, while its synthesis and reuptake are promoted. After 24 weeks from the first carcinogenic exposure, the deletion of 5-HT synthesis increased tumor multiplicity, while following 12 weeks it promoted the development of preneoplastic lesions, and cell proliferation in the colon. Interestingly, 72 hrs after the sixth and last carcinogenic exposures the number of aberrant crypts were detected reduced in the absence of 5-HT, which occurred together with a reduced proliferation, but increased apoptosis and DNA damage. Finally, 5-HT modulated genomic repair mechanisms. We conclude that serotonergic synthesis seems to be a protective factor of the intestinal colon, which inhibited case facilitates tumor development in this tissue.

Colon cancer is one of the most commonly diagnosed cancers in the US, yet small intestine cancer is a rare event. While there are many similarities between these two tissues, inherent differences such as redox status, may contribute to the variation in cancer occurrence. We examined the difference in reactive oxygen species (ROS) generation, antioxidant enzyme activity and oxidative DNA damage in the small and large intestine of rats under normal conditions and following exposure to exogenous oxidative stress. Basal ROS and antioxidant enzyme activities were greater in the colon than the small intestine, and the balance of ROS to antioxidant enzymes in the colon was more pro-oxidant than in the small intestine. During oxidative stress, ROS and oxidative DNA damage were greater in the colon than the small intestine. Thus the colon responds to oxidative stress less effectively than the small intestine, possibly contributing to increased cancer incidence at this site. We next wanted to understand how diets containing a combination of fish or corn oil and pectin or cellulose may alter the redox environment of the colon. ROS, oxidative DNA damage, antioxidant enzyme activity and apoptosis were measured in colonocytes of rats fed one of four diets containing either corn oil or fish oil and cellulose or pectin. Measurements were madein rats untreated with carcinogen and rats exposed to a chemical carcinogen and radiation. In rats not treated with a carcinogen, fish oil enhanced ROS, and fish oil/pectin suppressed antioxidant enzymes as compared to corn oil/cellulose. Oxidative DNA damage was inversely related to ROS in the fish oil/pectin diet and apoptosis was enhanced relative to other diets. In carcinogen treated and irradiated rats, a similar protective effect was seen with fish oil/pectin as evidenced by a reduction in oxidative DNA damage and enhancement of apoptosis. This suggests that a diet containing fish oil/pectin may protect against colon carcinogenesis by modulation of the redox environment to promote apoptosis and minimize oxidative DNA damage.

In the present study DNA damage was measured in peripheral blood lymphocytes from polyposis coli and colorectal cancer patients, treated with different dietary and environmental compounds and compared with lymphocytes from healthy individuals. In addition, confounding factors such as age, gender, alcohol intake and smoking habits were taken into consideration. The assays used in this study included the Comet assay, the Micronucleus assay, the Micronucleus-FISH assay and the sister chromatid exchange assay. The food mutagens, PhIP and IQ, as well as titanium dioxide nanoparticles (TiO2 NPs) induced a dose dependent increase in the DNA damage and chromosomal abnormalities in all tested groups regardless of confounding factors. Prior to experiments physicochemical characterisation of nanoparticles was conducted. In the presence of the flavonoids, quercetin and rutin that were acting in an antioxidant manner, the DNA damage resulting from the highest doses of food mutagens was significantly reduced. Thus, dietary supplementation with flavonoid-rich vegetables and fruits may prove very effective in protection against oxidative stress. The polyposis coli and colon cancer patients were more susceptible to food mutagens, PhIP and IQ, as well as TiO2 NPs, and in the majority of cases had a higher level of DNA damage in the Comet assay and higher cytogenetic damage in the Micronucleus assay. In the final project, twelve frequently encountered (NewGeneris) chemical compounds were evaluated to establish their damaging potential in lymphocytes and spermatozoa from healthy donors. The highest damage was produced by DNA reactive aldehydes, food mutagens and benzo[a]pyrene when assessed with the neutral and alkaline Comet assay with and without metabolic activation.

Much of the information on carcinogenesis in the intestine derives from studies based on the small intestine, yet the majority of the mutations leading to cancer occur in the large intestine. To overcome some of these issues, the use of a large bowel model and in particular the primary murine colonocytes system was preferred. The objectives were to better understand the importance of p53 and related proteins in the regulation of growth and responses to DNA damage in these cells. In order to investigate both p53 dependent and independent death pathways we exposed primary colonocytes to cisplatin. The initial study showed that the role of p53 in preventing entry into the S-phase following colonocytes DNA damage appears crucial whereas its role in apoptosis seems redundant. This suggests that p53-mediated apoptosis and growth arrest are neither a major nor a unique protector of colonic epithelia cells against mutation following DNA damage. Furthermore, the nuclear translocation of endogenous p73α in response to DNA damage in primary colonocytes is highly suggestive of a functional pro-apoptotic role for p73α in these cells, within the context of p53-independent apoptosis. Moreover, in the context of p53 deficiency, p73α activation is highly suggestive of its involvement in the p53-independent pathway of apoptosis in these cells. Consequently, it seemed plausible that other “early” genetic events could affect the colonic epithelium to lead to cancer. As cancer appears due to abnormal growth control it was of interest to investigate a possible involvement of Hedgehog genes in colorectal neoplasia. Therefore, the second part of this thesis focused on investigating the pattern of expression of Hedgehog signalling pathway members in normal colon versus colonic lesions including hyperplastic polyp, adenoma and adenocarcinoma of the colon. Results showed that Hedgehog signalling pathway members are expressed in normal colonic epithelium with sonic hedgehog (Shh) at the top of the crypts and a few basally located cells, patched in the neuroendocrine cells and smoothened at the brush border of superficial epithelium. RT-PCR analysis of laser-microdissected crypts from normal human colon confirmed that mRNAs encoding these proteins were expressed in colonic epithelium. Expression of Shh, patched and smoothened was upregulated with an increased expression of the proliferation marker Ki-67 in all lesions examined. To address whether the Hh signalling pathway is functional in the gut, the effect of Shh on epithelial cell <i>in vitro</i> was explored by treating primary murine colonocytes with either Shh peptide or neutralising anti-Shh antibody. Results illustrated that exogenous Shh promotes cell proliferation in colonocytes while anti-Shh inhibits proliferation, suggesting that sonic hedgehog is required during proliferation of epithelial cells <i>in vitro</i>. Therefore, the results suggested that SHH is required during epithelial proliferation in the colon and that there is a possible role for Hh signalling in epithelial colon tumour progression <i>in vivo.</i>

Formation of radioactive isotopes is investigated under irradiation by relativistic electrons with energy up to 100 MeV. Radioactive isotopes 87,88Y, 88,89,95Zr, 95Nb, 175Hf are registered after irradiation by relativistic electrons with energy 47.2 MeV. The present data are necessary for the choice of a material for a dielectric wakefield accelerator. The greatest danger at operation of accelerators represents 88Y. Formation of radiation defects in nanoceramics is investigated. The various types of radiation defects are found out at an irradiation by relativistic electrons with energy 47 MeV and 86 MeV. In UV VIS spectra the absorption lines of radiation are registered at 402.2 nm and 635 nm, which correspond to the F and F' centers of monocline lattices of zirconia. It is revealed, that krypton atoms are the centers of segregation of point defects. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/35622

This thesis focuses on two topics: thermal limitations of volume Bragg gratings (VBGs) employed as laser-cavity mirrors and formation of color centers in KTiOPO4 and its isomorphs. To explore the mechanisms of the thermal limitations of VBGs in high power lasers, I designed and constructed a diode-pumped, solid‑state laser with a VBG as cavity mirror that had a significantly higher absorption than what is typical. Thereby I could study the limiting thermal effects by using only moderate intra-cavity power. Additionally, I designed a computer model to numerically investigate the thermal effects in VBGs. Both the experiments and the simulations showed that the laser became successively more unstable when the power was increased. Absorption of the reflected laser beam causes broadening of the grating spectrum accompanied by decreasing diffraction efficiency. The reduced reflectivity leads to a leakage of the radiation through the grating. Moreover, the simulations showed that this increased instability was due to a reshaping of the intensity distribution profile inside the grating, which, in turn, leads to a sharp reduction of the diffraction efficiency. High-intensity visible radiation induces color centers in KTiOPO4, which can lead to severe decrease in the performance of the crystal and can cause catastrophic breakdown. The formation of color centers was investigated by measuring picosecond, blue-light induced infrared absorption (BLIIRA) in periodically-poled KTiOPO4, Rb:KTiOPO4, RbTiOPO4, KTiOAsO4 and RbTiOAsO4 through thermal lens spectroscopy using a common-path interferometer. This setup is capable of measuring absorption as low as 10-5 cm-1. The dependence of the BLIIRA signal on blue light average power and intensity as well as on the crystal temperature was studied. The results show the presence of at least two different types of color centers. A higher level of remnant absorption was observed in the phosphates compared to that of the arsenates. The largest portion of the induced absorption is attributed to photo-generated electrons and holes being self-trapped in the proximity to the Ti4+ and O2- ions, respectively, forming polaron color centers. Stabilization of these centers is aided by the presence of mobile alkali metal vacancies in the crystal.<br>Denna avhandling fokuserar på både volymbraggitters (VBGs) termiska begränsningar, i tillämpning som speglar i laserkaviteter, och på bildandet av färgcentra i KTiOPO4 och isomorfa kristaller. För att undersöka de termiska effekterna i VBGer som medför begränsningar på högeffektlasrar utfördes både experiment och simuleringar. För experimenten konstruerades en diod-pumpad Yb:KYW laser med ett VBG som har betydligt högre absorption än vad som är typiskt. Därmed kunde de termiska effekterna studeras vid måttliga intrakavitetseffekter. Simuleringarna bestod av två delmodeller; gitterstrukturen modelerades med överföringsmatriser och värmeflödet med en tredimensionell modell baserad på finita elementmetoden. Både experimenten och simuleringarna visade att en laser blir successivt mer instabil när den optiska effekten ökar. Absorptionen av laserstrålen i VBGt förändrade dess spektrala egenskaper, vilket i sin tur påverkade laserns stabilitet och prestanda. De huvudsakliga effekterna var en breddning av gittrets spektrum med en minskad reflektans. Simuleringarna visade även att den ökade instabiliteten berodde på en förändring av strålningens intensitetsfördelning inuti gittret, vilket accelererade reduktionen av gittrets reflekterande förmåga. I termer av den effekt som faller in mot gittret, har lasern en tydlig övre effektgräns. När den gränsen har uppnåtts leder vidare ökning av pumpeffekten i huvudsak till ökat läckage genom volymbraggittret, i stället för till ökad uteffekt hos laserstrålen. Kortvågigt synlig ljus av hög intensitet inducerar färgcentra i KTiOPO4, vilket kan leda till kraftigt reducerad transparens och kan orsaka permanent skada i kristallen. För att undersöka skapandet av dessa färgcentra mättes den termiska lins som uppstår vid blå-ljus-inducerad infraröd absorption (Eng: blue-light induced infrared absorption = BLIIRA) inducerad av blåa laserpulser vid en våglängd av 398 nm och vid pulslängder i storlek av pikosekunder i periodiskt‑polad KTiOPO4, Rb:KTiOPO4, RbTiOPO4, KTiOAsO4 och RbTiOAsO4. Den termiska linsen mättes med en metod kallad gemensam-vägsträcka-interferometer (Eng: common-path interferometer), en metod känslig nog för att mäta absorption så låg som 10-5 cm-1. Dessutom undersöktes hur nivån av BLIIRA beror på medeleffekten och intensiteten hos den blåa laserstrålen samt på kristalltemperaturen. Resultaten visar att det bildas minst två typer av färgcentra med olika livslängder. Vidare observerades en högre grad av långsamt avklingande absorption i fosfaterna jämfört med arsenaterna. Den största delen av den inducerade absorptionen tillskrivs fotogenererade elektroner och hål som ”självfångas” i närheten av Ti4+ respektive O2- joner, och bildar färgcentra av polaron karaktär. Stabilisering av dessa centra underlättas av lättrörliga alkalivakanser i kristallerna.<br><p>QC 20150922</p>

Tissue samples from spotted gar (Lepisosteus oculatus) and largemouth bass (Micropterus salmoides) were collected from Caddo Lake. Gar and bass livers were subjected to histological investigation and color analysis. Liver color (as abs at 400 nm) was significantly correlated with total mercury in the liver (r2 = 0.57, p = 0.02) and muscle (r2 = 0.58, p = 0.01) of gar. Evidence of liver damage as lipofuscin and discoloration was found in both species but only correlated with liver mercury concentration in spotted gar. Inorganic mercury was the predominant form in gar livers. In order to determine the role of mercury speciation in fish liver damage, a laboratory feeding study was employed. Zebrafish (Danio rerio) were fed either a control (0.12 ± 0.002 µg Hg.g-1 dry wt), inorganic mercury (5.03 ± 0.309 µg Hg.g-1 dry wt), or methylmercury (4.11 ± 0.146 µg Hg.g-1 dry wt) diet. After 78 days of feeding, total mercury was highest in the carcass of zebrafish fed methylmercury (12.49 ± 0.369 µg Hg.g-1 dry wt), intermediate in those fed inorganic mercury (1.09 ± 0.117 µg Hg.g-1 dry wt), and lowest in fish fed the control diet (0.48 ± 0.038 µg Hg.g-1 dry wt). Total mercury was highest in the viscera of methylmercury fed zebrafish (11.6 ± 1.86 µg Hg.g-1 dry wt), intermediate in those fed inorganic diets (4.3 ± 1.08 µg Hg.g-1 dry wt), and lowest in the control fish (below limit of detection). Total mercury was negatively associated with fish length and weight in methylmercury fed fish. Condition factor was not associated with total mercury and might not be the best measure of fitness for these fish. No liver pathologies were observed in zebrafish from any treatment.

Colorectal cancer (CRC) is the third most common cancer in Western Countries and one of the leading causes of mortality (WHO). CRC is a multistep process involving hyperproliferation of normal epithelial cells, due to an Apc gene mutation, and proceeding through adenomas and adenocarcinomas development due to progressive accumulation of mutations on oncogenes and oncosuppressor genes. These mutations can be due, among others, to increased genomic instability, oxidative stress, and epigenetic changes. Among this last one, DNA methylation changes in CpG islands are involved in 18% of CRC cases (Gallois et al. 2016). Moreover, progress and invasion of CRC are also stimulated by the microenvironment: Cancer Associated Fibroblasts (CAFs) are its main component, responsible for the epithelialmesenchymal transition and inflammation promotion (Wang et al. 2017). A wide range of data are present in literature about CAFs role in colon carcinogenesis: nevertheless, the link between Apc mutation and colon tissue microenvironment has not been clearly defined yet. Three aims have characterized this PhD project: first, the study of natural chemopreventive strategies capable of interrupting CRC carcinogenesis with associated very low toxicity. Indeed, the use of NSAIDs, showed to be a promising chemopreventive strategy in the past decades, is limited by their possible side effects: instead, the use of natural products could allow to overtake this limitation and be useful for secondary and tertiary prevention in individuals at high risk of CRC development. Three different natural compounds/products (a bergamot juice extract from endocarp (BJe), morin, and a pomegranate mesocarp decoction (PMD)) were tested in vivo in the Pirc rat model (F344/NTac-Apcam1137), bearing an Apc gene mutation and spontaneously developing colorectal polyps (Amos-Landgraf et al. 2007) and microscopic preneoplastic lesions (MDFs, Femia et al. 2015) in the normal mucosa (NM): MDFs are also considered useful endpoint for short-term chemopreventive studies. In addition, these in vivo experiments were supported by in vitro tests, aimed to explain the molecular mechanisms of the 3 tested compounds. The second aim was the study of the link between Apc gene mutation and tissue microenvironment at very early stages of colon carcinogenesis, addressing the role of colon fibroblasts within tissue microenvironment, at a stage in which colon carcinogenesis is already ongoing but no macroscopic lesions can be found. We evaluated the effects of this mutation on colon fibroblasts phenotype in established primary cultures from the colon of Pirc and F344-Wt age-matched (one month) rats. The third aim was to add knowledge on the role of Apc mutation on DNA stability at early stages of colon carcinogenesis, with the use of the COMET assay to evaluate DNA strand breaks and oxidative damage in both cultured colon fibroblasts of Pirc and Wt rats and in apparently morphological normal mucosa samples of Pirc and Wt rats aged one month. In addition, we set up a modification of the method, aiming at evaluating the global DNA methylation status. The effects of the treatments tested in Pirc rats were evaluated assessing the number of MDFs, apoptosis and proliferation both histologically and measuring relevant genes and/or proteins expression. Established Pirc and Wt colon fibroblasts primary cultures were characterized for their proliferative and phenotypic profile: both inflammatory and senescence-associated markers were evaluated with immunochemical and cytochemical assays. Concerning BJe, morin and PMD tested in vivo, each of them demonstrated to be capable of perturbing colon carcinogenesis as suggested by the reduction in MDFs number and size, possibly through a combination of proapoptotic and pro-inflammatory actions as suggested by in vitro experiments and also in an ex-vivo model of adenoma (for PMD study) from Pirc rats (Tortora et al. 2018). The novelty of these studies was represented by either the use of non-canonical sources of natural compounds (fruit by-products) or by the possibility to target an oncoprotein (LMW-PTP, low molecular weight phospho-tyrosine phosphatase) consequently enhancing the therapeutic response (study on morin). These data support the idea that a combination of natural compounds acting synergistically with each other, and possibly with drugs, can represent a promising secondary and tertiary chemopreventive strategy. The data obtained on colon fibroblasts mutated in Apc suggest that this mutation determines the development of a proinflammatory phenotype in this tissue microenvironment component, which might be involved in the creation of a protumorigenic environment favoring micro and macro pre-neoplastic lesion development. Moreover, data obtained with the COMET assay on colon fibroblasts and NMs from one month-old Pirc and Wt rats showed a lower level of oxidative damage in Pirc compared to Wt animals: also based on previous data from this lab (Femia et al. 2015), we speculate that Apc mutation could account for a selective advantage for carcinogenesis development through an increase in antioxidants defenses, as reported in the litterature (Ogasawara and Zhang 2008). Finally, we succeeded in the development of a methyl sensitive COMET assay, as proven by the reliability of the methylation changes observed after hypo- and hypermethylating stimuli in two different kinds of normal colon cell lines (epithelial and fibroblasts): this method will be used in the future to address the link between Apc gene and methylation.

no<br>Chemotherapy drugs usually inflict a lethal dose to tumour cells with the consequence that these cells are being killed by cell death. However, each round of chemotherapy also causes damage to normal somatic cells. The DNA cross-linking agent oxaliplatin which causes DNA double-strand breaks and vinflunine which disrupts the mitotic spindle are two of these chemotherapy drugs which were evaluated in vitro using peripheral lymphocytes from colorectal cancer patients and healthy individuals to determine any differential response. Endpoints examined included micronucleus (MN) induction using the cytokinesis-blocked micronucleus (CBMN) assay and pancentromeric fluorescence in situ hybridisation. Also, survivin expression was monitored since it regulates the mitotic spindle checkpoint and inhibits apoptosis. Oxaliplatin produced cytogenetic damage (MN in binucleated cells) via its clastogenic but also previously unknown aneugenic action, possibly through interfering with topoisomerase II, whilst vinflunine produced MN in mononucleated cells because of incomplete karyokinesis. Survivin expression was found to be significantly reduced in a concentration-dependent manner by not only oxaliplatin but surprisingly also vinflunine. This resulted in large numbers of multinucleated cells found with the CBMN assay. As survivin is upregulated in cancers, eliminating apoptosis inhibition might provide a more targeted chemotherapy approach; particularly, when considering vinflunine, which only affects cycling cells by inhibiting their mitotic spindle, and alongside possibly other pro-apoptotic compounds. Hence, these newly found properties vinflunine – the inhibition of survivin expression - might demonstrate a promising chemotherapeutic approach as vinflunine induces less DNA damage in normal somatic cells compared to other chemotherapeutic compounds.<br>Saudi Arabian Government

Quercetin (Q), a water-soluble flavonoid that is ubiquitous to foods of plant origin is postulated to protect against colon cancer due to its antioxidant activity. In contrast, we have shown that a dietary combination of fish oil (FO; n-3 fatty acids) and pectin may protect against colon cancer by decreasing endogenous antioxidant enzyme activities leading to increased reactive oxygen species (ROS), an inducer of apoptosis. We hypothesized that adding an antioxidant to a FO diet may negate the beneficial effects of FO by counteracting FO effects on colonocyte redox status. To test this, we provided 40 rats with FO or CO (fiber = pectin) diets with Q being 0 or 0.45% of the diet for 10 wk. All rats were injected with azoxymethane (AOM) on d 21 and 28. Measurements included: aberrant crypt (AC) enumeration (colon cancer marker); apoptosis (TUNEL assay); catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities; reduced and oxidized glutathione concentrations (GSH/GSSG); and oxidative DNA damage (8-OHdG adducts). AC numbers were lower in FO vs CO rats (p<0.0001), but tended to increase for FO diets containing Q (P<0.098). The apoptotic index was higher (p<0.0001) when Q was added to the FO and CO diets. Total SOD (lipid main effect, p=0.0136) and GPX activity (p=0.0025) was elevated in CO rats. CAT activity was higher (p=0.0204) in FO rats, however Q diminished this effect. GSH was not affected by diet; yet, GSSG accumulated (p=0.0554) in CO rats with Q as compared to CO rats without Q. The GSH/GSSG ratio was lower (p=0.0314) in CO rats than in FO rats. There was no difference in 8-OHdG adduct levels in FO vs CO rats, however, Q decreased 8-OHdG adducts in CO rats (p=0.0428). Despite increasing apoptosis, Q did not significantly lower AC formation. These data suggest that the distinct effects of the CO/Q and FO/Q combinations are functioning through different mechanisms to induce apoptosis. The long-term consequences of adding antioxidants such as Q to a diet thought to exert its anticancer effect through a pro-oxidant mechanism are unknown and deserve further study.

Folate and methionine are critical for one-carbon metabolism, impacting DNA synthesis, repair, and methylation processes, as well as polyamine synthesis. These micronutrients have been implicated in colorectal cancer risk. The aim of this thesis was to examine in greater detail the role of folate and methionine in colon cancer initiation and progression by assessing DNA stability and tumour incidence. Studies were performed in vitro (using human colorectal adenocarcinoma HT29 cell line) and in vivo (using ApcMin/+ [Min/+ superscript] mouse model). The in vitro studies examining the effects of various folic acid and methionine concentrations within the physiological range on cell proliferation and genomic instability of HT29 cells, showed that restriction of folic acid or methionine inhibited cellular proliferation, while supra-physiological folate induced apoptosis. HT29 cells may be resistant to genome instability induced by folic acid or methionine deficiency under the experimental conditions reported for this study because no significant increases in micronuclei, nuclear buds or nucleoplasmic bridges were observed in the Cytokinesisblock micronucleus cytome (CBMN-Cyt) assay. The investigation on the effect of folic acid and methionine depletion on telomere length and DNA methylation in HT29 cells demonstrated that folate and methionine depletion may increase both telomere length and DNA methylation in HT29 cells. The length of telomere was positively correlated with DNA methylation. In the in vivo studies using the ApcMin/+ [Min/+ superscript] mouse model, the effect of supplementing a western-style diet with dietary folic acid and/or methionine on intestinal tumour development was assessed. A total of 113 mice were randomised to receive one of the four diet treatments; New Western Diet (NWD) as control diet, NWD with additional folic acid, NWD with additional methionine, and NWD with additional folic acid and methionine, administered at age of 3 until 13 weeks, with wild type (WT) mice used as controls. Supplementation of folic acid and methionine separately, resulted in marginally lower tumour numbers, when compared to the control diet. However, supplementation with both folic acid and methionine together appeared to annul the marginal protective effect of supplementing individually. The investigation on the effect of supplementing a western-style diet with dietary folic acid and/or methionine on genomic stability (measured via micronucleated erythrocyte assay on blood sample; telomere length and DNA methylation on the colon tissue) showed insufficient evidence that additional folic acid and/or methionine promotes DNA stability or instability in ApcMin/+ [Min/+ superscript] or WT mice. Dietary supplementation with folic acid and/or methionine at the levels and duration used in this study did not substantially promote or protect against DNA damage in WT or intestinal cancer-prone ApcMin/+ [Min/+ superscript] mouse model fed a western-style diet although a marginal effect on tumour number was evident. In conclusion, the results of this thesis support a role of methionine and folate in affecting intestinal cell proliferation and possibly tumour number. However, the impact of supplementation with folate and methionine on genome stability was marginal.<br>Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, Adelaide Medical School, 2017.

PURPOSE: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents. EXPERIMENTAL DESIGN: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts. RESULTS: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. CONCLUSION: Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1.

No<br>Tea catechin epigallocatechin-3-gallate (EGCG) and other polyphenols, such as theaflavins (TFs), are increasingly proving useful as chemopreventives in a number of human cancers. They can also affect normal cells. The polyphenols in tea are known to have antioxidant properties that can quench free radical species, and pro-oxidant activities that appear to be responsible for the induction of apoptosis in tumor cells. The bioavailability of these natural compounds is an important factor that determines their efficacy. Nanoparticle (NP)-mediated delivery techniques of EGCG and TFs have been found to improve their bioavailability to a level that could benefit their effectiveness as chemopreventives. AIM: The present study was conducted to compare the effects of TFs and EGCG, when used in the bulk form and in the polymer (poly[lactic-co-glycolic acid])-based NP form, in oxaliplatin- and satraplatin-treated lymphocytes as surrogate cells from colorectal cancer patients and healthy volunteers. NPs were examined for their size distribution, surface morphology, entrapment efficiency and release profile. Lymphocytes were treated in the Comet assay with oxaliplatin and satraplatin, washed and treated with bulk or NP forms of tea phenols, washed and then treated with hydrogen peroxide to determine single-strand breaks after crosslinking. The results of DNA damage measurements by the Comet assay revealed opposite trends in bulk and NP forms of TFs, as well as EGCG. Both the compounds in the bulk form produced statistically significant concentration-dependent reductions in DNA damage in oxaliplatin- or satraplatin-treated lymphocytes. In contrast, when used in the NP form both TFs and EGCG, although initially causing a reduction, produced a concentration-dependent statistically significant increase in DNA damage in the lymphocytes. These observations support the notion that TFs and EGCG act as both antioxidants and pro-oxidants, depending on the form in which they are administered under the conditions of investigation.

碩士<br>國立東華大學<br>生物技術研究所<br>95<br>Liver diseases, including chronic liver inflammation, liver cirrhosis and liver cancer, have been life-threatening in Asia. The major therapeutic method for terminal chronic liver diseases has been liver transplantation. However, the donated organs are severely in shortage. The majority of patients die while on the waiting list. The hematopoietic stem cells were previously reported to have the potential in cell therapy for liver damages. Because granulocyte colony stimulating factor (G-CSF) has been known effective in mobilizing hemopoietic stem cells, the objective of this study is to investigate the feasibility of its application in the repair of chronic liver damage by a thioacetamide-induced animal model. Firstly, the chronic liver damaged model was successfully established by the repeated intraperitoneal injections of thioacetamide every three days for 60 days with the dosage of 200 mg/kg of body weight of Wistar-Kyoto rats. At day 60, the surviving rats were randomly divided into the experimental group and the sham group. To the experimental group, single dose of G-CSF (150 μg/kg) was introduced intraperitoneally and to the sham group, normal saline was used instead. After one week of G-CSF treatment, it was shown that the liver function indexes including GOT, GPT, serum ammonium, serum albumin and prothrombin time in the experimental group were back to normal values and were significantly different from what was observed in the sham group. It indicates that the recovery of liver function was promoted by the treatment of G-CSF. In addition, the histopathologic examinations on the liver-sectioned samples showed that the vacuolation, nuclear degeneration, bridging necrosis and fibrosis of the liver tissue resulting from the repeated thioacetamide injections were significantly improved in the experimental group due to the G-CSF treatment. In conclusion, both the analysis in biochemical indexes and the histopathologic examination showed consistent results that the single injection of G-CSF could facilitate the repair in the chronic chemical-induced liver damage and fibrosis in the experimental rats and have the potential in the clinical application for the treatment of chronic liver diseases.

碩士<br>國立臺灣大學<br>園藝學研究所<br>99<br>The study investigate the application of color-leaf plants in green land of parks, the layout model of common-used street trees, and the improvement of root-damage from street trees in Kaohsiung city to understand the miss-layout of park plants and street-trees and the improvement methods. Further, to provide a guideline of greening-plants selection and management / maintenance improvement to Kaohsiung city for layout of parks. We surveyed the layout of color-leaf plants of 20 parks in Kaohsiung city and indicated a top 10 color-leaf plants which were frequently used: Duranta repens cv. Golden Leaves, Ficus microcarpa. Cv.’Golden leaves, Cordyline fruticosa , Codiaeum variegatum, Sansevieria trifasciata cv. Laurentii, Ligustrum sinense cv. ‘Variegatum’, Alpinia speciosa cv ‘Variegata’, Acalypha wilkesiana, Breynia nivosa cv. ‘Roseo-picat’, and Crossostephium chinense. The using frequency of color-leaf plants in each park is equal to 4.75. The parks planted with less than 3 kinds of color-leaf plants were accounted for about 55% (11 of 20 parks), which with 4 to 5 species were about 20 % (4 of 20 parks), and which with more than 6 species were about 25 % (5 of 20 parks). The top 3 parks which used the most color-leaf plants were entirely the currently-established park, like “Tropical Botanical Garden” possessing of 17 species, “Labour Park” with 12 species, and “Aozihdi Urban Forest Park” with 10 species. Otherwise, “Gangshan Park” planted without the color-leaf plants. According to classification by plant species, the Duranta repens cv. Golden Leaves has the most planting-area about 412,245 m2 than the second species Ficus microcarpa. Cv.’Golden leaves’with 64,650 m2 and the third species Cordyline fruticosa with 29,325 m2. As a whole, totally only 23 kinds of color-leaf plants were planted in 20 parks of Kaohsiung city we were sampled. So, to enhance the application of color-leaf plants in park design is indeed necessary. The ornamental quality of color-leaf plants was categorized into 5 levels by growth index. Their quality were scored and presented sequentially in accordance with the overall growth performance: Sansevieria trifasciata cv. Laurentii.as 3.5 scores, Duranta repens cv. Golden Leaves as 2.88 scores, Codiaeum variegatum as 2.78 scores, Ficus microcarpa. Cv.’Golden leaves’ as 2.6 scores, and Cordyline fruticosa as 1.65 scores. Furthermore, all the color-leaf plants planted in park of Kaohsiung city were never to be up to the standard of watch according to their lower growth index 2.69. The soil hardness of 20 parks in Kaohsiung city were calculated and averaged into 18.93㎏/cm2 which was belonging to normal hardness range from 10 to 22㎏/cm2. Of 20 parks surveyed in Kaohsiung city, only 1 park yielded a soil hardness value under 15㎏/cm2, 8 parks yielded value 15 to 18㎏/cm2, 4 parks yielded value 18 to 20㎏/cm2, 5 parks yielded value 20 to 22㎏/cm2, and 2 parks yielded value higher than 22 ㎏/cm2that exceed standards. With regard to the soil pH, the average pH of 20 parks was 7.49. Of 20 parks, “Mincyuan Park” and “Tropical Botanical Garden” has mild-acidic soil with pH 6.39 and 6.4, separately; northern and southern part of“Aozihdi Urban Forest Park” has alkaline soil with pH 8.26 and 8.2, separately. All of these 4 parks which without reasonable pH were unfavorable to the growth of plants. Generally, soil is mild acidic, but soils in park of Kaohsiung city were alkaline. We suggested that the waste cement from civil constructions mixed into soil lead to the alkalization of soils. About EC (electrical conductivity) value of soil, average EC value of 20 parks was 498.3 μS/㎝ which was poor in nutrition. Among 20 parks, soil EC of southern part of“Aozihdi Urban Forest Park” and northern part of “Aozihdi Urban Forest Park” were the highest with value of 1296 μS/㎝ and 1296 μS/㎝, separately, which were beneficial to plant growth. Moreover, soil EC of“忠孝 Park”(274.5 μS/㎝), “Siaogang Park” (281.6 μS/㎝) , and“Labour Park” (292.2 μS/㎝) were relative lower than others, which were disadvantageous to plant growth. Therefore, soil EC value is highly correlated to constructive times that the newer park with the higher EC values. About soil water content (SWC), the average water content of soil in 20 parks of Kaohsiung city was 15.98%. Of 20 parks, “Sing Ren Park”has the highest SWC with 31.1%, and“Birthday Park” and northern part of“Aozihdi Urban Forest Park” were the second and third with 22.5% and 20.6%, separately. Conversely, “Mincyuan Park” and“Siaogang Park”were under lower SWC which with 6.28% and 9.36%, separately. Both these two parks with insufficient water content were resulted in low growth quality to plants, like serious leaf-drop and chlorosis on lower-leaf of shrubs and critical withered of turfs. Hence, it is recommended to improve the management of water and fertilizers to enhance ornamental qualities. With regard to the street trees, the maintenance and management of growth were generally well. The questionnaire results about favorite plant layouts indicated that the No.1 layout is the parterre design under the street trees in Speed separation strip in Sihwei 3nd Rd, the No.2 is the green-belt design with colorful seasonal flowers under street trees in Jiouru 1st Rd, and the No.3 is the planting of perennial green plants under boulevard in Minzu 2nd Rd. The tree species applied in street-tree layouts in Kaohsiung city were mainly use of Alstonia scholaris and Pterocarpus indicus, others included the Cinnamomum camphora, Swietenia macrophylla, and Sterculia foetuda. These above tree species were have identical characters which were obtained easily, grew rapidly, with strong adaptation ability, and maintain/manage simply. Recent years, Kaohsiung city government promoted the “multi-layer planting” to layout the street-tree in ten major roadway in Kaohsiung city. For example, open space under street-trees were planted with almost 90% of Duranta repens cv. Golden Leaves and/or perennial Ixora williamsii, two extremities of traffic island were reinforced in layout of seasonal flowers to increase the color and bio-diversity of roadways to promote the sense of urban beauty. In part of root-damage (RD) improvement in Kaohsiung city, the RD of Cinnamomum camphora in Sihwei Rd, Mincyuan Rd, and Minsheng Rd was improved by replacement of RD trees with plants the belt; RD Fious mircocarpa in Jhongiheng Rd, RD Bombax ceiba in Lin SenRd, ChenggongRd, and Zhongxiao Rd were improved by transplanting the RD trees to the nursery of Kaohsiung city government or enlarging the original plant-hole or designing into plants the belt. After inquire about and investigate to the maintenance office of bureau of public works of Kaohsiung city government, the results indicated that the street development, like improvement of street tree and pedestrian space, will continually conducted to decrease the root damage of street from street trees.

Wheat grain quality is defined by several parameters, of which insect and fungal damage and sprouting are considered important degrading factors. At present, Canadian wheat is inspected and graded manually by Canadian Grain Commission (CGC) inspectors at grain handling facilities or in the CGC laboratories. Visual inspection methods are time consuming, less efficient, subjective, and require experienced personnel. Therefore, an alternative, rapid, objective, accurate, and cost effective technique is needed for grain quality monitoring in real-time which can potentially assist or replace the manual inspection process. Insect-damaged wheat samples by the species of rice weevil (Sitophilus oryzae), lesser grain borer (Rhyzopertha dominica), rusty grain beetle (Cryptolestes ferrugineus), and red flour beetle (Tribolium castaneum); fungal-damaged wheat samples by the species of storage fungi namely Penicillium spp., Aspergillus glaucus, and Aspergillus niger; and artificially sprouted wheat kernels were obtained from the Cereal Research Centre (CRC), Agriculture and Agri-Food Canada, Winnipeg, Canada. Field damaged sprouted (midge-damaged) wheat kernels were procured from five growing locations across western Canada. Healthy and damaged wheat kernels were imaged using a long-wave near-infrared (LWNIR) and a short-wave near-infrared (SWNIR) hypersprctral imaging systems and an area scan color camera. The acquired images were stored for processing, feature extraction, and algorithm development. The LWNIR classified 85-100% healthy and insect-damaged, 95-100% healthy and fungal-infected, and 85-100% healthy and sprouted/midge-damaged kernels. The SWNIR classified 92.7-100%, 96-100% and 93.3-98.7% insect, fungal, and midge-damaged kernels, respectively (up to 28% false positive error). Color imaging correctly classified 93.7-99.3%, 98-100% and 94-99.7% insect, fungal, and midge-damaged kernels, respectively (up to 26% false positive error). Combined the SWNIR features with top color image features correctly classified 91-100%, 99-100% and 95-99.3% insect, fungal, and midge- damaged kernels, respectively with only less than 4% false positive error.

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.<br>Every year oak forests become infected by populations of the splendor beetle (Agrilus bigutattus). The detection and monitoring of infected trees is important, because of economic and ecological reasons. Traditional approach to detect the pest infestation level of each tree is performed by using ground-based observation method. It is long and ineffective method because of limitations, such as: poor visibility of the highest trees and impenetrability of some forest plots. The main goal is to identify infected oaks trees by splendor beetle at the 2 study areas. Pest-infested oak trees by splendor beetle are characterized by high level of defoliation and different reflection signatures. These features can be detected by using very high resolution color infrared (CIR) images. In August 2013 it was performed flight campaign by using unmanned aerial systems (UAS). CIR images were covering 2 test sites in rural area, near city Soest (Germany). Study areas represents small, privately owned oaks forest plots. In this research was used a small quadrocopter (Microdrone MD4-200) with vertical takeoff and landing capability (VTOL). Microdrone is carried a digital camera (Canon PowerShot SD 780 IS). Additionally, camera was modified to capture not just a visible spectrum, but also NIR spectrum (400 to 1100 nm) of infected oaks. The proposed workflow includes the CIR image acquisition, image stitching, radiometric correction, georeferencing, modified vegetation indices calculation, pixel based and object-based image classification and accuracy assessment. Images were classified using 5 classes (healthy, low infected, high infected, died trees and canopy gaps). Finally, the results can be integrated with existing WMS service. Applying of UAV make possible to obtain multitemporal data, which facilitates monitoring and detection of infected trees. The work was performed in close cooperation with the Forestry Department of Soest (Germany).

碩士<br>國立中興大學<br>園藝學系所<br>105<br>Summary In Taiwan,Vitis vinifera &apos;&apos;Kyoho&apos;&apos;,gained the popularity among Taiwanese for their rich fruit color, however, they are vulnerable when bird damage occur. People love grapes and birds as well. To solve the costly problem, we tried to improve the bags used for grape bagging to make them capable of preventing bird damage. We have tried four kinds of bags in the experiment; they are green double-layered bags, green single-layered bags, white double-layered bags and white single-layered bags. White single-layered bags are common bags used for grape bagging in Taiwan.Three trials were conducted in total, the first and the third were conducted in winter; the second was conducted in summer. The experiment included the effect of bagging to prevent bird damage and its concerned effect on fruit quality. When summer grapes were harvested, grapes bagged by green double-layered bags, were still green, and the quality were much lower than white double-bagged grapes. On the other hand, the grapes bagged by white double-layered bags seems almost the same to white single-layered ones. However, if we postponed harvest time of the green double-layered ones, we can observed not only anthocyanin content increased significantly from 0.31 μmol / g to 1.81 μmol / g and the soluble tannin content decreased from 2143 ppm to 641 ppm, but also an increase in soluble solids content and the decrease in the titratable acid content. Unfortunately, the quality of green double-layered grapes is still slightly worse than others. In the experiment of effect of preventing bird damage green double-layered bags shows the best effect, followed by white double bagging, but the double-layered white bagging ones are just slightly better than the normal bags. According to the test results, the green bags are better than white ones; and double-layered are better than the single-layered ones when it comes to the effect of prevention bird damage. But we recommend using white double-layered bags in summer, because the temperature is high, if we use green bags will make the temperature in bags higher and effect fruit quality negatively, green bags are more suitable for winter harvest; on the other hand, white double-layered bags are recommended to prevent bird damage and only have slightly influence on fruit quality.