突變ras基因造成過度表現在骨髓間質幹細胞啟動氧自由基誘導細胞凋亡
特定基因的表現與不同氧自由基的產生，已知會影響細胞的生長和死亡。我\r 個人有興趣利用間質幹細胞體外培養擴充，以為筋骨組織再生的可能應用。因此\r 利用轉植(transfect)突變ras 基因(Glu61Leu)進入骨髓間質細胞株(HS-5 cells)，來\r 控制ras 基因表現的高低，進而研究這些間質細胞隨著Ras 蛋白質表現的高低，\r 對氧自由基引導細胞生長與死亡之影響。結果發現ras 基因高度表現的間質細胞\r 生長減緩；相較於原生株平均減少62.4%。進一步研究其生長減少是否與細胞凋\r 亡有關，發現ras 基因高度表現的間質細胞凋亡確實比原生株高22.6%。探究其\r 凋亡原因，發現與caspase-3 有關但和粒腺體功能無關：因為caspase-3 有活化，\r 但是以粒腺體膜電位螢光追蹤劑JC-1 測得的膜電位卻沒有改變。追蹤ras 基因高\r 度表現的間質細胞其細胞內氧自由基的產量，發現Ras 高度表現株其細胞內氧自\r 由基明顯增加。當細胞外加入超氧根轉化酵素(SOD, 500 U/ml)去清除超氧根時，\r 對Ras 高度表現細胞的凋亡沒有影響；但是外加觸化酵素(catalase, 500u/ml)於培\r 養液中，卻可以抑制ras 基因高度表現的間質細胞內caspase-3 活化和細胞凋亡；\r 並且增加細胞生長循環促進分子Cyclin D1 的表現。從這些研究我們歸結出兩點\r 重要新發現: 1) ras 基因突變造成Ras 高度表現時，會促成細胞內特定氧自由基產\r 生，使得細胞生長減緩並進行細胞凋亡，只有特定抗氧化酵素(catalase)才能恢復\r 其異常； 2) Ras 蛋白高度表現而引導氧自由基產生的細胞凋亡與caspase-3 活化\r 有關，但與粒腺體功能無關。根據這些發現，未來我們或許可以朝調節特定氧化\r 還原反應或使用caspase-3 抑制劑去調控間質幹細胞的生長，以供筋骨組織再生\r 的應用。Certain gene’s expression as well as different species of oxygen radicals can\r affect cell growth and apoptosis. We are interested in amplifying mesenchymal\r progenitor cells for the application of musculoskeletal tissue regeneration. Thus, we\r transfected a mutant ras gene (61Glu/Leu) to change Ras protein expression in the\r mesenchymal progenitor cell line (HS-5 cells) and studied how ras expressing levels\r influenced intracellular oxygen radicals, and its relationship to cell growth and\r apoptosis. Results showed that Ras over-expressing HS-5 cells grew slower than those\r with wild type ras HS-5 cells and revealed a higher apoptosis rate. The higher\r apoptosis in Ras over-expressing cells was not related to mitochondrial dysfunction\r since mitochondrial membrane potential was normal as determined by flow\r cytometric analysis of JC-1 fluorescent staining assay. The higher apoptosis was\r related to higher caspase-3 activation. Further studies showed that Ras\r over-expressing HS-5 cells revealed a higher production of intracellular oxygen\r radicals in comparison to those with wild type ras HS-5 cells. Addition of catalase\r (500 u/ml) but not superoxide dismutase (SOD; 500 u/ml) specifically revived the cell\r growth associated with increase of cyclin D1 expression, but decrease of apoptosis\r associated with lower caspase-3 activation. Results from these studies demonstrated\r two important findings: 1) the ras gene over-expressing in a ras-mutant HS-5 cell line\r triggers a higher production of intracellular oxygen radicals resulting in higher cell\r apoptosis; and 2) the higher oxygen radicals related cell apoptosis is mediated by\r caspase-3 but not mitochondrial dysfunction. Based on these findings, we may\r propose to regulate mesenchymal progenitor cell growth for musculoskeletal tissue\r regeneration via modulation of redox reactions or caspase-3 inhibitors in the future.
阿拉伯芥AtYAK1 基因5'UTR 中的開放讀序框(uORFs)對基因表現調控之探討
在模式植物──阿拉伯芥（Arabidopsis thaliana）中，AtYAK1（Arabidopsis thaliana Yak1-related protein kinase）是目前發現唯一屬於DYRK（Dual specificity Yak1-Related protein Kinase）的蛋白激?。雖然之前研究已證明，不同物種之DYRKs 和細胞的生長與發育過程有關。然而，其在植物中的生理功能卻尚未被明確地研究報導過。在先前的研究中，為瞭解AtYAK1 在阿拉伯芥內作用之位置，前人選取AtYAK1 基因ATG 上游約2.5 kb 的序列（Upstream Element, 2.5KUSE）建構至一含有GUS（β-glucuronidase）報告基因的質體中，並轉形至阿拉伯芥，進行GUS 組織染色分析。但在初步結果中，並沒有在轉殖株觀察到明顯的GUS 表現。進一步分析，我們發現在2.5KUSE 序列末端約0.5 kb 的5’非轉譯區（5’untranslated region, 5’UTR）中，有四組開放讀序框（Upstream Open Reading Frame, uORF）。有趣的是，許多研究也顯示，uORFs 會影響轉譯過程中的再起始（re-initiation）作用而調控該基因的表現。另一方面，前人亦透過構築好的2KUSE 轉殖株（即不含有5’UTR）進行上述GUS 實驗。結果發現，此2KUSE 轉殖株的GUS 表現非常顯著。本實驗即要瞭解AtYAK1 的uORFs 是否也會影響其蛋白質的合成。首先，我們以點突變的方式將四組uORFs 中之ATG 換成TTG，目的為構築不含有uORFs 之5’UTR（mutated uORFs, ΔuORFs）。在進行原生質體短暫表現分析法（protoplast transient assay）及GUS 組織染色分析後，將結果與含有uORFs 的結果作比較：當缺乏uORFs 後，其3’端報告基因的表現量確實比原來顯著。綜合以上，我們認為此uORFs 對於AtYAK1 蛋白質之表現佔有相當重要的影響地位。最後，我們對5’非轉譯區是否存在開放讀序框進行阿拉伯芥全基因組分析，相關結果亦於本研究報告中分析討論。AtYAK1（Arabidopsis thaliana Yak1-related protein kinase）is the first DYRK（Dual specificity Yak1-Related protein Kinase ） family member identified in the model plant ─ Arabidopsis thaliana and exists as one copy gene in Arabidopsis. Previous studies showed that many eukaryotic DYRKs are involved in regulating the growth and development of cells. However, the study of AtYAK1 in Arabidopsis is lacking to date. In order to understand where AtYAK1 expresses and functions in plants, a 2.5 kb fragment which is located upstream from the major ATG of AtYAK1（termed Upstream Element, 2.5KUSE）was previously constructed to drive the expression of a reporter gene, GUS（β-glucuronidase）, in transgenic Arabidopsis. Much to our surprise, no GUS expression signal could be detected in such transgenic plants. When further analyses were performed, we found that there are four upstream open reading frames （uORFs） in the 5’untranslated region ( 5’UTR ) within the 2.5KUSE. Many studies indicating that the uORFs can regulate the translation of downstream ORF encoding the major gene product through the procedure of translation re-initiation. This action represents a mode of translational regulation for gene expression. Indeed, GUS activity could be readily detected in transgenic plants expression 2KUSE::GUS, a construct lacking the 5’UTR of AtYAK1. In this study, I have tried to elucidate whether the uORFs of AtYAK1 can regulate the translation of the downstream major ORF. First, in order to construct a 5’UTR fragment of which uORFs have been mutated（ΔuORFs）, we apply site-directed mutagenesis to substitute ATG with TTG for the four uORFs and examine the expression of GUS driven by this mutated 2.5KUSE. After analyzing the results in both Arabidopsis protoplast transient assay and transgenic Arabidopsis, stronger expression of reporter genes in both systems were observed when the four uORFs were mutated. We have also confirmed that, in transient expression system, the increase of reporter gene activity was not due to the excess accumulation of the corresponding mRNAs. Rather, it is the four uORFs which play an important role in negatively regulating the translation of AtYAK1, possibly via inhibiting the translation re-initiation of major ORF. A genome-wide examination of uORFs in all Arabidopsis genes was also performed to assess the possible contribution of uORF in regulating gene expression.
Investigation of phytoremediative ability of macrophytes and a design of a phytofiltration system for Singapore’s waterways
"Nitrates and phosphates cause eutrophication when present in high concentrations.\r This project aims to employ macrophytes to reduce such macronutrients in water bodies via growth and kinetic studies, which is a unique fusion of methodologies. It also involves a novel design and analysis of several enclosure prototypes to introduce macrophytes into waterways and their effects on the waterway’s ability to convey storm water rapidly away from flood-prone areas.\r Tropical macrophytes (emergent macrophytes Typha angustifolia and Cyperus haspan, submerged macrophytes Hydrilla verticillata and Cabomba aquatica, floating macrophytes Lemna minor) were grown in simulated wastewater with high nitrate and phosphate concentrations. Analysis of the growth and uptake kinetics of the macrophytes showed a correlation between high growth rate and high nitrates and phosphates uptake. C. aquatica had the highest uptake rate for nitrates and phosphates as well as the highest growth rate of 6.11 ± 1.2 % day-1 . The remaining macrophytes were proven to exhibit good phytoremediative properties, with emergent macrophytes C. haspan and T. angustifolia having great affinity (as indicated by a low Km value) for phosphate and nitrate respectively. An analysis of the phytoremediative abilities of each macrophyte was done to provide recommendations for growth in different aquatic areas.\r A total of eight nettings, made from different materials-linen and plastic, and pore sizes were used to design the enclosure prototypes. These enclosures possess a metal skeletal structure for greater stability. Results show that a combination of 2 different nettings provided the best trade-off between ensuring that the macrophytes were contained within the enclosures and minimising the enclosure’s impact on the flow velocity of the waterways. Enclosures could then be attached to existing infrastructure like the float booms as a platform for large scale phytoremediation locally."
Synthesis and Characterization of Starch-Nanosized Calcium Phosphate Composites
Nano-sized calcium phosphate was used in the synthesis of starch-based composite plastics to provide a cheap but sturdier biodegradable alternative to petroleum derived plastics used in film packaging. Nano-sized calcium phosphates were produced from the precipitation reaction of calcium nitrate tetrahydrate (Ca(NO3)2 ‧ 4H2O) and phosphoric acid (H3PO4). The nanoparticles were co-extruded and molded with thermoplastic starch (TPS) at ratios of 0%, 1%, and 5% by mass. Tensile strength and elongation percentage of the resultant composite plastics were tested in three replicates. The results show that there is a significant difference between the tensile strengths of the 0% and 5% calcium phosphate composites at a 5% level of significance. The trend between the composite’s tensile strength and percentage calcium phosphate follows a geometric progression, enabling a projection for the 10% nano-calcium phosphate to have a tensile strength of 10 MPa, the average tensile strength for low-density polyethylene (LDPE). This shows that it is feasible to synthesize a 10% nano-calcium phosphate-TPS plastic that can be a viable substitute for LDPE plastics in film packaging, paving the way for the commercialization of starch-based plastics. The use of biodegradable plastics with improved physical characteristics will lessen consumer dependence on petroleum derived plastics and solve the environmental issues brought about by the use of such plastics.
微陣列基因分析法探討心肌細胞在機械性展延下的基因表現
高血壓所誘發的機械性展延是造成心肌肥大的基本因子，本實驗即藉由微陣列基因分析法同時大量的分析機械性展延所造成心肌細胞的基因表現。將新生鼠的心肌細胞施以 20﹪的機械性展延，抽取其 mRNA製作成 cDNA 探針與現成的 cDNA 晶片進行雜漬反應 （此晶片上包含了 480個如訊息傳遞、控制細胞生長週期、細胞骨架等的已知基因），在眾多有因為機械性展延而造成基因表現差異的基因中，我們選擇了 eNOS 基因（內皮細胞 NO合成?）進行西方墨點法及 NOS活性和 NO 產生量測定的實驗，進一步證實 eNOS 的基因表現量的確是增加的，此一結果與微陣列基因分析所得之結果不謀而合。 Mechanical stretch induced by high blood pressure is an initial factor laeding to cardiac\r hypertrophy. The use of cDNA microarrays has made it possible to simultaneously analyze\r stretch-induced gene expression in cardiomyocytes. Neonatal rat cardiomyocytes were cultured on\r malleable silicone dishes and were stretched by 20%. We compared the transcript profiles of\r cardiomyocytes under mechanical stretch for 60 minutes by hybridization of cell-derived cDNA to\r DNA probes immobilized on microarrays. The microarrays contained probes for 480 known genes\r including signal transduction, cell cycle regulators, cytoskeleton and cell motility, and so on. Eighteen\r genes were indentified that showed significantly differential expression in response to mechanical\r stretch in cardiomyocytes. Of the represented genes expressed, endothelial nitric oxide synthase (eNOS)\r genes was the most interesting one. Northern blot and western blot analysis further quantified the\r expression of eNOS gene. Mechanical stretch also increased constitutive NOS activity and NO\r production. Our results indicate that mechanical stretch induces eNOS gene expression thus increases\r constitutive NOS activity and NO production in cardiomyocytes.
Going Dotty: The Distribution and Effects of Rust on Highbush Cranberry
Purpose Every fall, I collect highbush cranberries (Viburnum edule) to make jelly. In 2012, for the first time, I observed highbush cranberry leaves covered in striking patterns of raised purple dots (telia) caused by the pathogenic rust fungus Puccinia linkii. I investigated the distribution and effects of this rust for several reasons: little is known about P. linkii, highbush cranberries are an important food source for wildlife and people, and foliar pathogens may increase with climate change in sub-boreal forests. First, I investigated the patterns of telia within plants. Second, I compared the variation in infection severity among plants, hypothesising that younger plants, those in dense populations, and those in low-elevation riparian areas would be most infected. Finally, I studied the effects of P. linkii on its host, hypothesising that highly infected plants would produce fewer berries and produce berries with less sugar, and that infected leaves would die sooner. Procedures This study investigated P. linkii in mixed coniferous-deciduous forest sites near Smithers, British Columbia. I marked a total of 41 randomly-selected focal V. edule plants in four sites. To examine within-plant patterns of infection, I photographed four leaves of each plant and used a graphic analysis program to examine the size, density and coverage of telia. To assess among-plant patterns, I compared infection severity (5 classes of telia coverage), to three ecological variables: host density within 5m, position on a moisture gradient, and plant maturity. To investigate the effects of P. linkii on its host, I compared infection severity to the number of berries produced, the proportion of malformed and infected berries, and the sugar content of mature berries as measured with a handheld refractometer. I measured leaf mortality in fall. Results P. linkii produced a characteristic pattern within each plant: higher leaves consistently had fewer but bigger telia than lower leaves. Across sites, plants were significantly more infected in areas of high host density. Within sites, young plants and plants and in moister ecosystems were significantly more infected (increases of 1.1 ± 0.2; F1,128=44.8, P
The Interplay of Iron and α-synuclein in mediating Neuroinflammation in Parkinson’s Disease
Neuroinflammation is implicated as a contributive factor to neurodegeneration in Parkinson’s disease (PD). Increased iron accumulation and deposition of -synuclein within Lewy Bodies in PD brains have been observed. It has been hypothesized that unbound iron is able to react with H2O2 to generate free radicals. Using the Divalent Metal Transporter-1 (DMT1) as a vehicle to transport iron into the brain, a DMT1 transgenic mouse model (DTg) was generated to recapitulate iron deposition in PD. The DTg was crossbred with the SNCA (synuclein) transgenic mouse to produce a DMT1_SNCA (BTg) mouse model to study the link between iron, -synuclein and neuroinflammation in PD. Our hypothesis predicts that iron exacerbates -synuclein toxicity by inducing larger inflammatory responses and consequently compromising functions of biomolecules. Our study shows that –synuclein triggers a low-grade inflammatory response by microglia and astrocytes while iron exacerbates -synuclein toxicity by eliciting immunological responses mediated by glia cells in the brain observed both in the DTg and BTg mice. Elevated levels of nitrated proteins were observed in the DTg, suggesting the role of iron in inducing nitrosative stress via upregulation of iNOS in glia cells. With the BTg mice, we hope to understand the effect of iron accumulation as an environmental stressor in aggravating -synuclein toxicity which may lead to the selective demise of dopaminergic neurons.
隨機物體轉移過程的實驗時間之初探
有二系統A和B，A中一開始有2k個物體，，B中有0個物體。在一個單位時間內，兩系統可以互相轉移最多一個物體。當B中物體的個數為 i-1，i∈{1,2,...,k+1}，我們稱其為狀態 i，從狀態1﹝初態﹞開始計時，到達狀態 k+1﹝相同態﹞便即刻停止實驗，經過之時間為一隨機變數T，稱之為實驗時間。問當兩個系統的物體數剛好相等時，經過的實驗時間之分佈為何？本文將以上述問題為核心，分別探討不同條件下系統的實驗時間所反映出來的現象，如機率、期望值、變異數等等。
Define two systems, A includes 2k objects, and B has none. They can transfer at most one object from one system to another in a time unit. When the number of objects in B is i-1, i∈{1,2,...,k+1} , we say the system is at state i. As soon as system transfer form state 1 ( initial state ) to state k+1 ( the same state ), the experiment stop. Random variable T, called the experiment time, is the time before stop. What would be the distribution of the experiment time if all systems have the same amount of objects within? This article will focus on the described question and discuss what property the experiment time of the system under various conditions has, such as probability, mean, and variance.