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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

The effectiveness of HS-72 variants in inhibition of heat shock protein 72

Fraile, Katherine 17 June 2016 (has links)
Heat shock proteins (HSPs) play important roles in the process of maintaining proteostasis in a cell. HSP72, the inducible form of the HSP70 family, is expressed in response to stress on the cell or tissue, including those stresses caused by tumor growth. Increasing evidence suggests that HSP72 is necessary for a cancerous cell to survive under the stresses of a tumor microenvironment. This has naturally raised interest in identifying an inhibitor selective for HSP72. The Haystead Laboratory at Duke University identified such a small-molecule inhibitor, referred to as HS-72, and proposed the scaffold as an ideal starting point to develop a family of therapeutic agents targeting HSP72. This work examines the potency and effectiveness of HS-72 and a number of its analogs developed by the Haystead Laboratory. These results suggest that HS-159 is a more effective inhibitor of HSP72 on a range of human tumor cell lines than HS-72. Further studies are needed to quantify how much more potent HS-159 is than HS-72 and potentially identify even more potent compounds.
2

蛋白激酶 CK2 在大鼠腦部之抗細胞凋亡機制的探討 / The anti-apoptotic mechanisms of protein kinase CK2 in the brain of rat

張家銘 Unknown Date (has links)
蛋白激酶 CK2 是一種具有多種功能的絲胺酸/蘇胺酸蛋白質激酶,CK2 作用的受質眾多且廣泛表現在哺乳類動物細胞中,對於細胞週期的發展、轉錄作用以及抗細胞凋亡等機制扮演非常重要的角色。在神經系統中,CK2 已知可以保護神經細胞以抵抗外來的傷害,但是其分子層面的機制目前尚未釐清。本篇論文的研究重點在於探討 CK2 保護作用可能參與的細胞分子機制。血清反應因子 SRF 是一種哺乳類動物細胞的轉錄因子,調控基因的轉錄作用來促進細胞的存活。Mcl-1 是抗細胞凋亡家族 Bcl-xL 家族蛋白成員之一,可以促進細胞的存活能力。先前研究指出,SRF 會受到 CK2 的磷酸化作用而增加本身的 DNA 結合能力。在其他研究也指出,Mcl-1 會受到 SRF 的調控。在本篇論文的第一部份,著重於 Mcl-1 的表現是否會受到 CK2 調控 SRF 的路徑所影響,實驗結果顯示,轉染野生型 CK2α 質體 DNA 可以增加海馬迴 CA1 腦區的 SRF 磷酸化,而轉染不活化的突變型 CK2αΑ156 質體 DNA 則會減少 SRF 的磷酸化。更進一步,轉染野生型 CK2α 會增加 Mcl-1 的 mRNA 及蛋白質表現,轉染突變型 CK2αΑ156 則減少 Mcl-1 的表現。此外,轉染突變型 SRF99A 也會減少 Mcl-1 的 mRNA 及蛋白質表現;而且在共同轉染實驗中,SRF99A 會拮抗野生型 CK2α 對促進的 Mcl-1 蛋白質表現的作用。 另一方面,DARPP-32 是一個在新紋狀體神經細胞中具有調控多巴胺訊息效力的訊息傳遞分子。先前研究指出,DARPP-32 具有抗細胞凋亡的功能,且發現在 DARPP-32 Ser102 氨基酸會受 CK2 的磷酸化作用。因此,本篇論文的第二部份主要是探討 CK2 的抗細胞凋亡能力是否是透過磷酸化 DARPP-32 來調控。實驗結果顯示,轉染野生型 CK2α 可以增加紋狀體 DARPP-32 的磷酸化,而轉染不活化的突變型 CK2αΑ156 則會減少 DARPP-32 的磷酸化。此外,轉染 CK2α 的小干擾 RNA (siRNA) 可以抑制內生性的 CK2 表現,同時也會減少 DARPP-32 的磷酸化以及抗細胞凋亡蛋白, Bcl-xL 的表現。綜合這些實驗結果,CK2α可以分別透過 SRF 或 DARPP-32 調控的訊息傳遞來促進 Mcl-1 或 Bcl-xL 的表現進而調控神經系統的抗細胞凋亡機制。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrares and is ubiquitously expressed in mammalian cells to play an important role in cell cycle progression, transcription, and anti-apoptosis. In the nervous system, CK2 is shown to protect neurons against injury, but the cellular mechanisms are not well studies. In the present studies, we investigate which cellular mechanism might involve in the CK2 protection effects. The serum response factor (SRF) is a mammalian transcription factor which mediates some gene transcriptions relevent to promote the cell survival. The Myeloid cell leukemin 1 (Mcl-1) is one of the anti-apoptotic Bcl-2 family members and is involved in promoting cell viability. Previous studied have revealed that the SRF phosphorylation by CK2 can enhance its DNA-binding activity. The regulation of Mcl-1 by SRF has also been reported in other studies. In the first part of the present studies, we investigate whether the Mcl-1 expression is regulated by CK2 through SRF mediated pathway. The results from wildtype CK2α plasmid DNA transfection revealed that the phosphorylated SRF were increased in hippocampus CA1 region, whereas transfection of the catalytically inactive CK2αA156 mutant plasmid DNA decreased phosphorylated SRF. Further, wildtype CK2α increased, whereas CK2αA156 mutant decreased the mRNA and protein levels of Mcl-1. Moreover, transfection of the mutant SRF99A also decreased the mRNA and protein levels of Mcl-1. Furthermore, the mutant SRF99A antagonized the upregulatory effects of wildtype CK2α on Mcl-1 protein level in the co-transfection experiments. In the other side, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Previous studies have revealed that DARPP-32 might involve in the anti-apoptosis and its Ser102 residue is phosphorylated by CK2. Therefore, in the second part of this study, we investigate whether one of the anti-apoptotic effects of CK2 is through DARPP-32 phosphorylation by CK2 in the present study. The results revealed that the phosphorylated DARPP-32 is increased in stratum by wildtype CK2α transfection and decreased by catalytically inactive CK2αA156 mutant transfection. Further, transfection of CK2α siRNA can inhibit endogenous CK2 expression and also decrease phosphorylation of DARPP-32 as well as the anti-apoptotic protein, Bcl-xL. These results together suggest that CK2α-mediated anti-apoptotic effects are partially through SRF mediated or DARPP-32 mediated signaling to regulate Mcl-1 or Bcl-xL expression, respectively.
3

蛋白激酶 CK2 與轉錄因子 SRF 所調控之抗細胞凋亡蛋白 Mcl-1 對 PC12 神經細胞之保護機制的探討 / Anti-apoptotic effects of Mcl-1 through CK2-mediated SRF pathway in PC12 cells

曾惠敏, Tseng, Hui Min Unknown Date (has links)
蛋白質激酶 CK2 是一種多功能的絲胺酸/蘇胺酸蛋白激酶,且普遍存在於哺乳類動物細胞中,CK2 受質眾多,對於細胞週期的發展、轉錄作用以及抗細胞凋亡等過程中扮演很重要的角色。SRF 是一種哺乳類動物的轉錄因子,它會結合到血清反應元素 SRE 上進而調控一些促進細胞存活的基因轉錄作用。Mcl-1歸類於抗細胞凋亡 Bcl-2 家族,具有促進細胞存活的能力。過去研究顯示 SRF 的 DNA 結合活性會受到蛋白激酶 CK2 的磷酸化而增加,且 SRF 對 Mcl-1 的活性調控作用也被描述在其他的研究中,然而,對於細胞的訊息目前還沒有更詳細的研究。在本實驗中,我們探討是否可以藉由 CK2 調控 SRF 的路徑來影響 Mcl-1 的表現以作為抗細胞凋亡的機制。利用 CK2 抑制劑 TBB 處理的結果顯示,在 4 hr 後,phospho-SRF 蛋白質表現的降低具有劑量相關性。而相似的降低也可以從 Mcl-1 的 mRNA 和蛋白質表現量觀察到。處理 24 hr 後,phospho-SRF 的蛋白質表現量有顯著降低,而 Mcl-1 的 mRNA 表現量相較 Mcl-1 的蛋白質影響層面微弱。另一方面,轉染野生型 CK2α 會增加 phospho-SRF,相反的,轉染抑制催化活性的突變型 CK2αA156 則會顯著降低 phospho-SRF 的表現。更進一步,野生型 CK2α 同時增加 Mcl-1 的 mRNA 及蛋白質層級,而 CK2αA156 則會降低 Mcl-1 的表現。突變型的 SRF99A 轉染作用降低 Mcl-1 的 mRNA 及蛋白質,並經由共同轉染的實驗顯示具有抵抗上游野生型CK2α 對 Mcl-1 蛋白質的影響。綜合這些結果我們認為 CK2α對 SRF 的訊息調控影響包括對 Mcl-1 的表現。且這條訊息路徑所促進的 Mcl-1 蛋白質表現可能對魚藤酮處理所引發的細胞凋亡作用具有保護的效果。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrates and is ubiquitously expressed in mammalian cells to play an important role in cell cycle progression, transcription, and anti-apoptosis. The serum response factor (SRF) is a mammalian transcription factor which binds to serum response element (SRE) and mediates some gene transcriptions relevent to promote the cell survival. The Myeloid cell leukemia 1 (Mcl-1) belongs to the anti-apoptotic Bcl-2 family and its effect are involved in promoting cell viability. Previous studies have revealed that the DNA-binding activity of SRF is enhanced when it is phosphorylated by protein kinase CK2. The activation regulation of Mcl-1 by SRF has also been reported in other studies. However, the detailed cellular signaling has not been studied well. In the present study, we investigate whether the regulation of Mcl-1 expression through CK2-mediated SRF pathway is involved in its anti-apoptotic effects. The results from CK2 inhibitor TBB revealed that the phosphorylated SRF were reduced in a dose-dependent manner after 4 hr of TBB treatments in PC12 cells. The similar decreases were also observed in the mRNA and protein levels of Mcl-1. After a 24 hr exposure of PC12 cells to TBB, a decreased in phosphorylated SRF and Mcl-1 mRNA were observed; a decreased in Mcl-1 protein level was also detected, albeit to a lesser extent. On the other hand, transfection of the wildtype CK2α increased, whereas transfection of the catalytically inactive CK2αA156 mutant decreased phosphorylated SRF. Further, wildtype CK2α increased, whereas CK2αA156 mutant decreased the mRNA and protein levels of Mcl-1. Furthermore, the mutant SRF99A transfection decreased, the mRNA and protein levels of Mcl-1 and antagonized the up-regulatory effects of wildtype CK2α on Mcl-1 protein level in the co-transfection experiments. These results together suggest that CK2α-mediated SRF signaling is involved in the regulation of Mcl-1 expression, and this signaling pathway may involves the anti-apoptotic effects of Mcl-1 against rotenone treatment.
4

蛋白激酶 CK2 調控受質蛋白 DARPP-32 磷酸化對 PC12 細胞株之抗凋亡機制的探討 / DARPP-32 phosphorylation by protein kinase CK2 mediates the anti-apoptotic effects in PC12 cells

李曉怡, Lee, Hsiao Yi Unknown Date (has links)
蛋白激酶 CK2 是一種具有多種功能的絲胺酸/蘇胺酸蛋白質激酶,其作用的受質眾多且普遍存在於哺乳類動物細胞中。從許多的研究結果顯示,蛋白激酶 CK2 參與調節許多的神經系統功能其中包括有神經保護作用,但是其分子層面的機制目前尚未釐清。DARPP-32(Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa)主要表現在紋狀體中型多刺狀 GABA 神經元中的蛋白質,參與調控與藥物成癮相關的多巴胺訊息傳遞路徑,不過,近年來的一些研究報告指出DARPP-32亦參與了細胞的抗凋亡作用。雖然先前已有研究發現DARPP-32 Ser102胺基酸是CK2的磷酸化作用受質,但是並沒有進一步的研究證實,該胺基酸的磷酸化作用是否參與CK2所調控的細胞機制。屬於抗細胞凋亡蛋白Bcl-2 家族成員之ㄧ的bcl-x基因會經由pre-mRNA選擇性剪裁機制(alternative splicing)而產生兩種異構蛋白Bcl-xL和Bcl-xS,其中Bcl-xL蛋白被證實會促進細胞存活;而Bcl-xS蛋白則會造成細胞死亡。實驗室先前的研究結果發現,在神經滋養因子BDNF的刺激下,CK2可以促進Bcl-xL基因的表現,因此本論文欲進一步探討CK2對DARPP-32 Ser102的磷酸化作用是否參與CK2的抗細胞凋亡訊息傳遞,進而影響Bcl-xL和Bcl-xS的表現。實驗結果顯示,轉染野生型CK2α DNA質體會增加DARPP-32 Ser102的磷酸化現象、Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例;而處理 CK2 抑制劑 TBB 或轉染 CK2α siRNA則會降低 DARPP-32 Ser102的磷酸化現象、Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例。此外,轉染 DARPP-32 siRNA會降低 Bcl-xL的蛋白質表現。轉染模擬之磷酸化構型的DARPP-32 S102D DNA質體會增加Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例;但是,轉染突變型DARPP-32 S102A DNA質體則會降低Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例。進一步利用野生型CK2α和DARPP-32 S102A DNA質體進行細胞共同轉染的實驗結果則發現,DARPP-32 S102A會拮抗野生型 CK2α對促進Bcl-xL蛋白質表現的作用;另外,利用過氧過氫產生細胞氧化逆境下,CK2α或DARPP-32 siRNA處理可以顯著降低 DARPP-32 Ser102的磷酸化現象、Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例,同時會顯著造成細胞凋亡。綜合本論文的實驗結果,顯示CK2會透過DARPP-32 Ser102的磷酸化作用而調控Bcl-xL以及Bcl-xS的表現,而且在氧化逆境下,此條細胞訊息傳遞路徑應參與了細胞的抗凋亡機制。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrares and is ubiquitously expressed in mammalian cells. Many studies have shown that CK2 is involved in many neuronal functions including neuroprotection, but its cellular mechanisms are not well-studied. DARPP-32 (Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa) is highly enriched in striatal medium-size spiny GABA neurons and is a prominent mediator of dopamine signalling which relates with drug abuse. Beside its well-known function in drug abuse, recent studies also reveal that DARPP-32 may be involved in the anti-apoptotic effects. Although the Ser102 residue of DARPP-32 is a phosphorylation site for CK2, this phosphorylation-mediated CK2 signaling has not been studied yet. The bcl-x gene, one member of the Bcl-2 family, encodes two isoform proteins Bcl-xL and Bcl-xS by the pre-mRNA alternative splicing. The former increases cell survival and the later enhances cell apoptosis. Our previous study found that CK2 can increase Bcl-xL expression by BDNF treatment. In the present study, we investigate whether DARPP-32 ser102 phosphorylation also mediates the CK2 signaling for cell survival. Our results revealed that DARPP-32 Ser102 phosphorylation, Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio were all increased by wild-type CK2α plasmid DNA transfection. Meanwhile, CK2 inhibitor TBB treatment or CK2α siRNA transfection decreased DARPP-32 Ser102 phosphorylation, Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio. On the other hand, DARPP-32 siRNA transfection decreased Bcl-xL protein level. Furthermore, transfection of DARPP-32 S102D, which mimics the constitutive phosphorylation form, increased whereas transfection of mutant S102A decreased the Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio. Further, the mutant DARPP-32 S102A antagonized the up-regulatory effects of wild-type CK2α on Bcl-xL protein level in the co-transfection experiments. From the results of H2O2-induced oxidative stress experiments, we also found that prior knock-down of CK2 or DARPP-32 can aggravate the decrease in DARPP-32 Ser102 phosphorylation, Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio by H2O2 treatment. These results together suggest that DARPP-32 mediates CK2α signaling in regulating Bcl-xL/Bcl-xS expression and this signaling pathway might be involved in cell survival under oxidative stress.

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