生物化學

TDP-43是漸凍症(ALS)以及額顳葉失智症(FTLD)等神經退化性疾病的致病蛋白，在患者腦內會有不正常的類澱粉蛋白TDP-43錯誤折疊而產生蛋白質堆積，而不同堆積階段依序為單體(monomer)、多倍體(oligomer)、纖維(fiber)。其中纖維為許多神經退行性疾病的病理標誌，且相較於已有的 TDP-43 oligomer 單株抗體(Yu-Sheng Fang et al.(2014))，目前沒有對於 TDP-43 纖維抗體的研究，因此本實驗利用單株抗體技術所製造出對應TDP-43纖維的不同抗體進行純化及濃縮，找出對TDP-43 fiber專一性最高、結合效率及結合率皆最好抗體，以作為能夠檢測漸凍症患者血液中TDP-43 fiber的生物標誌(Biomarker)。 同時為了增加偵測的靈敏度，本實驗成功創新利用PMCA ( protein misfolding cyclic amplification) 技術快速放大纖維的數量以提高檢測準確度，搭配高專一性、高結合效率的編號3-2自製抗體作為Biomarker，只需五個小時便可以將極少量纖維的訊號約提高至原先的17倍，具有極高的靈敏性可以準確辨認出有無TDP-43纖維的樣本差別。

Infectious diseases caused by bacteria from biological pollutants pose a great burden in terms of diagnosis and treatment, and millions of people worldwide die from bacterial infections. Detection of bacteria plays a critical role in clinical diagnosis and control of contamination, but is not accessible due to the high cost, complex devices and equipment required. In the project, an alternative to existing methods, a paper-based biosensor for the detection of model organism E. coli bacteria, which is visible, low cost, easy to use, can be integrated with a smartphone, is based on rapid color change in the exposed environments, drinking and pool water, wastewater, beverage products. platforms were developed. For the specific detection of E.coli bacteria, two different biosensors have been developed that can perform colorimetric detection in a user-friendly origami design, minimizing microchip and processing steps based on antibody-bound PVDF membrane and filter paper-based immunological method. In the presence and absence of target bacteria E.coli, the lowest detection limit of the biosensors obtained by using paper-based platforms that create a distinctive color on them, depending on the concentration, was 0.9x103 bacteria/ml for origami biosensor, 2.7x103 bacteria/ml for microchip biosensor and the widest dynamic linear operating range was calculated as 103-107 bacteria/ml. With the biosensor platforms we have developed, the use of only one smartphone for both qualitative and quantitative, visible results and analysis within minutes constitutes the originality of our project. With these promising results, the biosensors we have developed can also be used for the detection of different biological pollutants, do not contain complex devices and can be easily produced in large scales. We believe that the biosensors we have developed for the detection of biological pollutants in water and beverages, especially in regions where test laboratory infrastructure is not available, will contribute to the literature, public health, health economy and sustainable development goals such as clean water and sanitation, health and quality life, and life in water.

The emergence of antibacterial resistance has necessitated the development of alternative treatments, such as antimicrobial peptides (AMPs). AMPs are part of the innate immune systems of various organisms such as Momordica charantia L., a known medicinal plant in Southeast Asia. In this study, potential novel AMPs from M. charantia were derived in silico to provide prospective antibiotic alternatives using promising plant-based peptides. M. charantia protein sequences that were 500 amino acids long were digested using proteolytic enzymes, resulting in 3,621 peptides. Each resulting sequence was characterized as either AMP or Non-AMP using four statistical analysis tools, and those identified as AMPs were analyzed. This led to 102 AMPs, 53 of which were unregistered on the Data Repository for Antimicrobial Peptides, indicating that they have yet to be derived from other species. Six of the eight studied physicochemical properties show strong correlations with each other, suggesting that subsequent AMP design studies may focus on these six properties. As such, M. charantia may be a rich source of potential AMPs and, thereby, alternative antibiotics. The in vitro examination of these novel AMPs is also recommended to further understand their potential as alternative antibiotics sourced from locally available plants.

本研究針對臺灣恆春半島海域所採集的櫟葉指形軟珊瑚Sinularla querciformis進行天然物化學成分研究，由此珊瑚中分離出兩個新型菸草烷類型天然化合物，分別是Querciformolide G (1)與Querciformolide H (2)，以及兩個已知菸草烷類型天然化合物，分別是Sinulaparvalide B (3)與3,4:8,11-Bisepoxy-7-hydroxycembra-15(17)-en-1,12-olide (4)。上述化合物的物理性質和化學結構皆是由核磁共振儀、紅外線光譜儀和質譜儀等數據，以及比對相關化合物的文獻來分析確認。 針對化合物1-4進行抗發炎測試，對超氧陰離子產生和人中性粒細胞彈性蛋白酶釋放的抑製作用，發現化合物2針對彈性蛋白酶，有顯著的抑制效果。

神經細胞成熟的過程中可分成數個階段，每個階段間的轉換都伴隨著蛋白質的種類，RNA異構體、細胞結構與功能等全面性的轉變。但控制神經細胞在確切的時序下成熟的分子機制尚待研究。本研究發現 Paxillin 的新功能：當腦神經細胞在活體外培養至第七天時， Paxillin 的位點 Serine119 會被磷酸化 (p-PaxillinS119)，並從細胞質轉位進入至細胞核。我們使用 N2a 細胞以神經分化的模式來探討 p-PaxillinS119 進核的分子機制與功能，發現 p-PaxillinS119 進入細胞核需要位點 Serine119 被磷酸化，且分析後確認 Paxillin 的 LIM 結構域中帶有 PY-NLS 序列，分別為 P516/Y517 及 P575/Y576。我們發現 Paxillin 藉由轉運蛋白 Importin β2 辨識其 PY-NLS序列，進行蛋白間的交互作用後進入細胞核中。從螢光影像的分析，我們觀察到神經細胞的 p-PaxillinS119 在細胞核中會呈現顆粒狀，並與 RNA 剪接因子 P-SR 共定位在核斑點上。經由免疫共沉澱與細胞轉染的方式，我們證實位點 Serine119 突變，會影響 Paxillin 與 RNA 剪接因子的交互作用，及降低細胞分化與 RNA 剪接的程度。

在幹細胞治療中，並無法去偵測打入之幹細胞在人體內的位置及存在狀態。本研究創新在於(1)藉由使用新型奈米氧化鐵(IOP, iron oxide nanoparticles)來標定間葉幹細胞(MSCs, mesenchymal stem cells)，並能順利在磁振造影下成像 (2)同時確認IOP其安全性及效能。在實驗中先以X光繞射確認IOP粒徑平均10.4 nm，磁滯曲線證實不會產生磁性，CCK-8細胞測試中證實高濃度IOP(400 μg/mL)對間葉幹細胞存活影響小於17%，接著以普魯士藍染色觀察到IOP濃度愈高被幹細胞吸收愈多，平均每個細胞攝入2.79顆粒增加至3.47顆粒，並同時在磁振造影成像證實。最後高濃度IOP (400 μg/mL)會造成PGE2顯著性的下降55%，證實IOP可影響免疫調節但其角色仍待進一步研究。

硫磺菇（Laetiporus sulphureus）和桑黃菇（Sanghuangporus sanghuang）是東亞，特別是台灣森林中的兩種真菌。這些真菌的次級代謝物，特別是多醣，具有抗炎和抗癌的生物效應；其地面子實體長期被當地人作為傳統藥物使用。然而，這些藥用特性及其機制尚未充分研究。本研究旨在分析和量化這些真菌多醣的抗炎效果。從硫磺菇中提取硫酸化多醣，從桑黃菇中提取非硫酸化多醣，並使用水和乙醇進行多步純化。隨後，將純化後的產品餵給巨噬細胞進行體外測試以檢查其抗炎性。硫酸化多醣的最佳濃度為150 ppm，能夠最大程度地降低自由基濃度21.6%，且不影響細胞活力。相比之下，桑黃菇的所有多醣濃度均顯示出增強的細胞炎症，顯示其作為藥物無效，因為沒有去除真菌毒素。相比之下，硫磺菇的硫酸化多醣顯示出其藥用潛力，對生物醫學和生物探索領域具有新啟示。

GATD3A 是粒線體中的蛋白質，被推測可能具有「去糖化」的能力，能移除AGEs。糖尿病、帕金森氏症、阿茲海默症皆與人體中過高濃度的AGEs有關，因此GATD3A具有相當高的研究價值。 本研究探索GATD3A(麩醯胺酸轉移酶樣1 類結構域 3A)，經基因合成與蛋白表現後，大量製備蛋白質並探討其結構與功能。目前已得到蛋白質最佳製備環境、成功培養出蛋白質晶體，進行了結構分析，了解其結構、保守性、親水性及電性等，對於酵素適合反應的溫度、pH值也有初步了解。未來也會利用酵素動力學計算酵素活性、並製備糖化蛋白質，進行去糖化測試，探討 GATD3A 是否具有臨床運用於糖尿病患者之糖化血紅素去糖化的可能性。

Previous research observed increased TNTs formation between hypoxic and normoxic neuroblastoma cells, aiding hypoxic cell survival. CHGA was identified as a potential factor in this process. This study compared CHGA expression and whether CHGA exists in TNTs in five cell lines, with SH-SY5Y showing the highest levels, followed by SK-N-BE(2)C, while the other three showed lower expression. Future studies will focus on the impact of CHGA on cell survival and its mechanisms.

本研究旨在比較人類間葉幹細胞 (human Mesenchymal Stem Cells, hMSCs) 在三維球形微孔和二維平面聚丙烯醯胺 (polyacrylamide, PA) 水凝膠上的生長和分化特性。我們製備了具有60μm直徑球形微孔陣列的PA水凝膠平面PA水凝膠，將hMSCs培養在這兩種不同的基質上，加入藥劑誘導向脂肪細胞分化七天。透過細胞計數、形態學觀察和成像分析，我們系統地比較了細胞在兩種環境下的形態變化、脂大小 和分化進程。再透過RNA-seq分析，我們找出了三維及二維環境培養的細胞的差異表達基因，並做出火山圖與熱圖。 本研究建立一種新型三維球形微孔的培養環境、，成地地 hMSCs提供更接近體內條件的微環境。進一步觀察hMSCs在三維環境中生長與分化，期許 組織工程和再生醫學領域提供新的見解和潛在的應用方向。