The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response.

Traditionally, response to treatment in hematological malignancies is evaluated by light microscopy of bone marrow (BM) smears, but due to more effective therapies more sensitive methods are needed. Today, detection of minimal residual disease (MRD) using immunological and molecular techniques can be 100 times more sensitive than morphology. The main aim of this thesis was to compare and evaluate three currently available MRD methods in childhood acute lymphoblastic leukemia (ALL): (i) real-time quantitative PCR (RQ-PCR) of rearranged antigen receptor genes, (ii) multicolor flow cytometry (FCM) of leukemia-associated immunophenotypes and (iii) real-time quantitative PCR of fusion gene transcripts (RT-PCR). In paper I, we assessed the applicability of RQ-PCR in a population-based cohort of childhood ALL diagnosed in Sweden between 2002-2006. Clonal IG/TCR rearrangements were identified in the 96% of the 279 ALL cases. Using RQ-PCR, the quantitative range of 10-3 was reached in 93% of B-cell precursor (BCP) ALL and 86% of T-cell ALL (T-ALL) by at least one target gene. In paper II, we compared MRD detection using both RQ-PCR and FCM in the context of NOPHO ALL-2000 protocol. By applying the stratification threshold of ≥0.1% MRD late during induction therapy (day 29), we could demonstrate that both methods can predict the risk of BM relapse but not extramedullary relapse. However, the threshold of ≥0.2% MRD appears to be more optimal using RQ-PCR in BCP ALL, whilst in T-ALL, the results indicate that RQ-PCR is preferable for MRD assessment. The stability of RNA in vitro is a critical factor when using sensitive molecular techniques such as MRD detection. In paper III, we evaluated the influence on MRD detection when blood is collected in tubes with RNA stabilization reagents (PAX gene Vacutatiner®) compared to collection in EDTA-tubes (non-stabilized). We analyzed 68 matched samples from chronic myeloid leukemia patients and the results indicated that non-stabilized blood processed within 30 hours is preferable for MRD detection. In paper IV, follow-up samples from eight children with Philadelphia positive (Ph+) ALL were evaluated with the three available MRD methods. MRD measured by the fusion gene transcripts (BCR-ABL1) appeared to be the most sensitive method, however, precise quantification can be difficult and the other methods are thus complementary. In conclusion, all three applied MRD methods are useful and correlate to each other, although not necessary exchangeable in individual patients. We also conclude that MRD assessment by RQ-PCR, based on rearranged IG/TCR genes and multicolor FCM are predictive for identification of high risk childhood ALL patients.

Huntington’s disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which translocates, and accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients and is consistently observed in all animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression and an understanding of the effects of mHtt may lead to strategies to slow the progression of the disease. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of these models using two N-mHtt-affected promoters in in vitro transcription assays and in two cell models of HD using techniques including overexpression of known N-mHtt-interacting transcription factors, chromatin immunoprecipitation, promoter deletion and mutation analyses and in vitro promoter binding assays. Based on our results and those in the literature, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at affected promoters. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as the observation that some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.

This thesis is an investigation of detecting and quantifying airborne inoculum of Sclerotinia sclerotiorum (Lib.) de Bary to improve the Sclerotinia rot of carrot (SRC) forecast model. A quantitative polymerase chain reaction (qPCR) assay was developed to specifically detect and quantify DNA from airborne ascospores of S. sclerotiorum. The qPCR assay was evaluated on air samples collected using a Burkard Sampler, and showed that ascospores of S. sclerotiorum were specifically detected among a pool of foreign DNA. The concentration of detected ascospores was related to the observed incidence of SRC to suggest a preliminary threshold of 2 to 4 ascospores m-3 of air for SRC development. Evaluation of an Andersen Sampler, the blue plate test (BPT) and the qPCR assay showed that the latter two methods were equally effective in detecting and quantifying ascospores of S. sclerotiorum and consistently detected greater numbers of ascospores than an Andersen Sampler. Three days are required to confirm the presence of S. sclerotiorum using the BPT, while results from the qPCR assay can potentially provide results within five hours of air sampling. The choice of detection method depends on the available resources and need for a quick result. Analysis of data from nine years of air sampling using the BPT indicated that a single air sampling site is sufficient to detect ascospores when counts are low, increasing to two sites during periods when ascospores are detected near threshold levels and crop and environmental conditions are conducive to disease. Chitosan and canopy trimming were evaluated to manage SRC under field conditions. Chitosan reduced area under the disease progress curve (AUDPC) by 55 and 42% in 2009 and 2011, respectively, which was comparable to a standard fungicide. Trimming enhanced chitosan efficacy, reducing AUDPC by 88 and 82% in 2009 and 2011, respectively. Trimming as a stand-alone treatment reduced AUDPC by 66% in 2011. Under controlled environmental conditions, chitosan inconsistently enhanced defense responses against S. sclerotinia. The results show that chitosan has potential to be integrated into SRC management systems, particularly when combined with foliar trimming in years with moderate to high disease risk. / National Research Council of Canada; University of Guelph; Department of Plant Agriculture; Ontario Ministry of Agriculture, Food and Rural Affairs

The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response.

結核病是目前全球傳染病中死亡率最高的疾病，由於抗藥菌株的產生以及與愛滋病的共同感染而使得結核病的控制面臨更嚴厲的挑戰。目前，結核病的預防主要是施打卡介苗，卡介苗是世界上使用最廣泛的疫苗之一，其優點是沒有施打時間的限制、便宜、副作用小、穩定性高。卡介苗目前仍然是採用培養後統計可培養之菌落數來判定疫苗之效價。但由於卡介苗菌的生長十分緩慢，培養時間有時甚至長達二個月，製程監控不易達到效果。本研究的目的即是利用同步定量聚合酶連鎖反應技術檢測BCG 16S ribosomal RNA及IS6110基因，並分別比較檢測BCG菌之DNA及RNA檢體的敏感度及專一性，以建立一個快速、靈敏度高且專一性高的BCG效價檢測方法。在研究中已成功建立檢測之標準曲線，並能在8天培養後檢測出BCG菌的生長，可以有效率的減少BCG疫苗菌數的檢測時間。因此，期望未來能夠將此技術應用做為BCG疫苗生產製程中管控的工具，以取代曠日費時的傳統檢測方式，並期望將來可以將此檢測系統應用於結核病之臨床檢測。 / Tuberculosis is one of the important infectious diseases with the highest mortality rate worldwide. The control of tuberculosis infection is facing an even more strict challenge because of the evolving of drug-resistant clones and the co-infection of AIDS. Currently, the prevention of tuberculosis is mainly done by BCG vaccination, which is one of the most widely used vaccines. The advantages of BCG vaccine are its low cost, low side effects, and high stability. At present, the BCG potency is analyzed by calculating the growth of colonies after culture. However, the growth of BCG is pretty slow, sometimes it can take as long as two months and has made the in process control very difficult. The aim of this study was to establish a rapid, sensitive, and specific real-time quantitative PCR (Q-PCR) method for the detection of BCG potency and separately compares BCG DNA and RNA samples for BCG 16S ribosomal RNA and IS6110 genes for Q-PCR detection system. We have successfully set up the standard curves for the detection of BCG using Q-PCR, and the method could detect the growth of BCG after 8 day of cultivation. Using Q-PCR system, the detection of BCG vaccine is more efficient and the detection time can be largely reduced. We hope we will be able to apply this method as a tool for the in process control during the BCG vaccine production and to replace the time-consuming traditional culture method. / 目錄 誌謝 ------------------------------------------------------------------------ i 中文摘要 ------------------------------------------------------------------------ ii 英文摘要 ------------------------------------------------------------------------ iii 目錄 ------------------------------------------------------------------------ iv 表目錄 ------------------------------------------------------------------------ vi 圖目錄 ------------------------------------------------------------------------ viii 第一章 緒論------------------------------------------------------------------ 1 第二章 文獻回顧------------------------------------------------------------ 4 第一節 結核病 (Tuberculosis)-------------------------------------------- 4 第二節 分歧桿菌 (Mycobacteria)---------------------------------------- 6 第三節 卡介苗 (Bacille Calmette-Guérin vaccine ; BCG vaccine) 的發展--------------------------------------------------------------- 8 第四節 聚合酶連鎖反應 (Polymerase chain reaction；PCR) 及其應用------------------------------------------------------------------ 9 第五節 同步定量聚合酶連鎖反應 (Real-time quantitative polymerase chain reaction; 定量PCR)------------------------- 10 第三章 材料與方法--------------------------------------------------------- 13 一 BCG菌製備與培養----------------------------------------------- 13 二 定量PCR的primer與probe設計----------------------------- 13 三 定量PCR的primer與probe最適化-------------------------- 14 四 BCG DNA檢體萃取---------------------------------------------- 14 五 BCG RNA萃取與反轉錄---------------------------------------- 15 六 定量PCR反應條件----------------------------------------------- 16 七 傳統PCR反應條件----------------------------------------------- 16 八 洋菜膠體 (agarose gel) 電泳分析----------------------------- 16 九 BCG定量標準曲線製作與製圖-------------------------------- 17 第四章 結果與討論--------------------------------------------------------- 18 一 定量PCR primer 與 probe的設計----------------------------- 18 二 16S rRNA及IS6110 定量PCR中primers與 probes 之最適化--------------------------------------------------------------- 19 三 建立BCG疫苗之定量PCR檢測標準曲線------------------- 20 四 以16S rRNA primer進行BCG疫苗培養分析--------------- 22 五 以定量PCR檢測BCG疫苗菌株之最佳檢測濃度--------- 23 六 依16S rRNA及IS6110定量PCR建立BCG疫苗效價之檢測------------------------------------------------------------------ 25 第五章 結論與建議--------------------------------------------------------- 27 參考文獻 ------------------------------------------------------------------------ 30 附錄一 16S ribosomal RNA 基因序列---------------------------------- 81 附錄二 IS6110基因序列--------------------------------------------------- 83 表目錄 表一 臨床中重要的分歧桿菌------------------------------------------ 37 表二 分歧桿菌之致病力------------------------------------------------ 38 表三 Primer Express軟體設計之16S rRNA及IS6110 primers與probes------------------------------------------------------------ 39 表四 定量PCR primer與probe最適化測試濃度組合------------ 40 表五 反轉錄反應藥品配方--------------------------------------------- 41 表六 定量PCR試劑配方------------------------------------------------ 42 表七 定量PCR反應條件------------------------------------------------ 43 表八 傳統PCR試劑配方------------------------------------------------ 44 表九 16S rRNA定量PCR之primer與probe最適化結果------- 45 表十 IS6110定量PCR之primer與probe最適化結果------------- 46 表十一 以16S rRNA及IS6110定量PCR檢測DNA檢體結果---- 47 表十二 以16S rRNA及IS6110定量PCR檢測RNA檢體結果---- 48 表十三 16S rRNA及IS6110定量PCR標準曲線之有效檢測範團及靈敏度比較------------------------------------------------------ 49 表十四 16S rRNA定量PCR檢測BCG疫苗在不同培養條件下之生長情形------------------------------------------------------------ 50 表十五 以16S rRNA定量PCR檢測BCG疫苗之最佳檢測濃度--- 51 表十六 以IS6110定量PCR檢測BCG疫苗之最佳檢測濃度-------- 52 表十七 16S rRNA定量PCR檢測BCG疫苗之效價------------------ 53 表十八 IS6110定量PCR檢測BCG疫苗之效價---------------------- 55 圖目錄 圖一 TaqMan Probe之定量PCR的作用原理反應流程--------- 57 圖二 ABI 7700 Sequence Detection System之同步偵測光學路徑原理--------------------------------------------------------------- 58 圖三 定量PCR偵測螢光後之Threshold cycle圖---------------- 59 圖四 PCR過程及定量PCR偵測原理------------------------------- 60 圖五 Primer Express軟體中，設計primer與probe的 Preferences操作界面---------------------------------------------- 61 圖六 以傳統PCR測試16S rRNA primer之PCR產物agarose gel電泳分析-------------------------------------------------------- 62 圖七 以傳統PCR測試IS6110 primer之PCR產物agarose gel電泳分析------------------------------------------------------------ 63 圖八 16S rRNA定量PCR primer與probe 最適化結果----------- 64 圖九 IS6110定量PCR primer與probe 最適化結果--------------- 65 圖十 16S rRNA定量PCR primer與probe最適化結果產物agarose gel 電泳分析--------------------------------------------- 66 圖十一 16S rRNA定量PCR以DNA建立檢測標準曲線之結果--- 67 圖十二 16S rRNA定量PCR以DNA檢體所建立之標準曲線----- 68 圖十三 16S rRNA定量PCR以RNA檢體建立標準曲線之結果-- 69 圖十四 16S rRNA 定量PCR以RNA檢體所建立之標準曲線---- 70 圖十五 IS6110定量PCR以DNA檢體建立標準曲線之結果------ 71 圖十六 IS6110 定量PCR以DNA檢體所建立之標準--------------- 72 圖十七 IS6110定量PCR以RNA檢體建立標準曲線之結果------ 73 圖十八 IS6110定量PCR於RNA檢體所建立之標準曲線---------- 74 圖十九 以16S rRNA 定量PCR檢測BCG疫苗DNA檢體之生長------------------------------------------------------------------------ 75 圖二十 以16S rRNA 定量PCR檢測BCG疫苗RNA檢體之生長------------------------------------------------------------------------ 76 圖二十一 建立16S rRNA 定量PCR之BCG菌最佳檢測濃度-------- 77 圖二十二 建立IS6110定量PCR之BCG菌最佳檢測濃度------------ 78 圖二十三 以16S rRNA定量PCR檢測BCG疫苗效價----------------- 79 圖二十四 以IS6110定量PCR檢測BCG疫苗效價---------------------- 80

Highbush blueberry (Vaccinium corymbosum L.) has one of the highest antioxidant capacities and flavonoid concentrations of any fruit or vegetable, and regular consumption of blueberries has been connected to a wide range of health benefits. A diversity of flavonoids (flavonols, anthocyanins, proanthocyanidins) are likely responsible for many of the health benefits, and these compounds also significantly contribute to the organoleptic properties of ripe blueberries. Despite the potential importance of these flavonoids in diet, there has been little investigation into the molecular genetics of blueberry flavonoid biosynthesis. Therefore, I developed a real-time quantitative PCR protocol to monitor expression of flavonoid genes throughout development and ripening. Following evaluation of five reference genes, expression profiling of biosynthetic genes revealed that flavonoid synthesis is tightly controlled at the transcriptional level in a biphasic developmental pattern. These results are discussed in relation to flavonoid metabolite accumulation profiles, which were produced as part of a collaboration. Finally, in conjunction with a second group of collaborating scientists, some promising preliminary evidence is provided suggesting that the hormone abscisic acid might have a role in regulating ripening initiation in blueberry. / Graduate

The aims of this study were to identify Chloroviruses in two different Ontario freshwaters and to determine if the seasonal abundance patterns of Chloroviruses in different environments are similar. Gene fragments nearly identical to cultivated Chloroviruses were obtained from Lake Ontario and a nearby pond at the University of Toronto Mississauga (UTM) and novel Chlorovirus gene fragments were obtained from Lake Ontario. Quantification of these two Chlorovirus genes over several seasons revealed the presence of persistent viruses with different seasonal dynamics suggesting that different Chloroviruses replicate by infecting different hosts. Additionally, patterns of seasonal abundance and timings of peak abundances for individual viruses differed between Lake Ontario and the UTM pond, demonstrating the critical role of the environment in Chlorovirus dynamics. The observation of different Chloroviruses with different seasonal dynamics allows speculation that these viruses and their hosts stably coexist in Ontario freshwater environments.

To initiate algal virus research in the Bay of Quinte, three stations were sampled biweekly throughout 2011. By targeting algal virus DNA polymerase, major capsid protein genes (MCP), and a Microcystis aeruginosa cyanophage (Ma-LMM01) tail sheath protein gene, PCR amplification revealed diverse and unique Phycodnaviruses (viruses of eukaryotic algae) and cyanophage. When analysed statistically, patterns of virus abundance suggested that the seasonality of any one virus cannot be generalised to predict that of other viruses, even among closely related viruses. This study also demonstrated a strong relationship between algal virus abundance and host biomass. It was found that despite the apparent heterogeneity of virus abundance across the Bay, virus abundance patterns clustered by sampling date and geographic location. By providing evidence for diverse algal viruses with complex seasonality, this work highlights significant gaps in the current understanding of Bay of Quinte phytoplankton ecology.

The aims of this study were to identify Chloroviruses in two different Ontario freshwaters and to determine if the seasonal abundance patterns of Chloroviruses in different environments are similar. Gene fragments nearly identical to cultivated Chloroviruses were obtained from Lake Ontario and a nearby pond at the University of Toronto Mississauga (UTM) and novel Chlorovirus gene fragments were obtained from Lake Ontario. Quantification of these two Chlorovirus genes over several seasons revealed the presence of persistent viruses with different seasonal dynamics suggesting that different Chloroviruses replicate by infecting different hosts. Additionally, patterns of seasonal abundance and timings of peak abundances for individual viruses differed between Lake Ontario and the UTM pond, demonstrating the critical role of the environment in Chlorovirus dynamics. The observation of different Chloroviruses with different seasonal dynamics allows speculation that these viruses and their hosts stably coexist in Ontario freshwater environments.