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Contribution of Proteomics and Bioinformatics to The Understanding of The Platelet Secretome

為了解決Blum Hettich的問題，作者LE THAO NGOC NHI 這樣論述：

Background: Outdated allogenic platelet concentrates (PCs) from blood establishments can be used as source material for the preparation of different types of human platelet lysates (HPLs) for clinical applications in cell therapy and regenerative medicine. It is important to understand how the subs

tantial variations in the mode of preparation of HPLs can affect their protein composition and biological function in order to optimize quality and safety as well as clinical applications.Aims: To unveil the proteomes and biological functions of various HPLs to help optimize their clinical applicati

ons.Material and Methods: Outdated PCs were obtained from Taipei Blood Center. The PCs were separated into 3 sub-pools that were subjected to 3 processing methodologies to produce 7 different types of HPLs: (1) Freeze-thaw Platelet Lysate (FTPL): PC was frozen and thawed to release the platelet cont

ent into the plasma compartment; (2) Serum Converted Platelet Lysate (SCPL): PC was supplemented with calcium chloride to convert fibrinogen into fibrin; (3) Heat treated-Serum Converted Platelet Lysate (HSCPL): SCPL was heat-treated (56°C, 30 min); (4) Platelet Pellet Lysate (PPL): isolated platele

ts, depleted of plasma, were lysed by freeze/thaw; (5) Heat-treated-Platelet Pellet Lysate (HPPL): PPL was heat-treated (56°C, 30 min); (6) Micro-filtered-HPPL (HPPL0201): a 0.2-0.1 µm filtration sequence was used to remove large molecules or particles; and (7) Nanofiltered-HPPL (HPPL0201P20): HPPL0

201 was filtered through Planova 20N, a 19-nm virus removal filter. Label-free proteomic was first performed by precipitating proteins by acetone followed by trypsin digestion and LC-MS/MS analysis to obtain a global understanding of the HPL proteomes. For accurate proteome quantification, the HPLs

were depleted of 14 major plasma components using MARS (Multiple Affinity Removal Spin Cartridge Human), followed by trypsin digestion. Peptides were collected, labeled with Tandem Mass Tag (TMT) reagents and analyzed by LC-MS/MS using Orbitrap Fusion Lumos Tribrid Quadrupole-ion trap-Orbitrap mass

spectrometer. Lists of identified and quantified proteins were studied and searched against bioinformatics platforms for pathway enrichment and characterization analysis. Western blotting was then conducted to validate the quantitative results from proteomics.Results: In total, we detected 1441 prot

eins in label-free proteomics, 952 proteins in TMT-labeling experiment (1) of HPLs pairs before and after depletion and 1114 proteins in TMT-labeling experiment (2) of the seven depleted-HPLs. Most of the proteins were from cytoplasm and had catalytic activity. The identified proteomes were previous

ly identified to be associated with platelets. Most of proteins were from cytoplasm and had catalytic activity with the main biological processes involved in the hemostasis and immune system. Different processing steps impacted HPLs composition and their associated canonical pathways. The quantifica

tion achieved in proteomics was compatible with semiquantitative immunoblotting of proteins.Conclusion: Our proteomics data revealed substantial differences in those HPL preparations that may have relevant various impacts on their functionality and application in cell therapy and regenerative medic

ine. Each HPLs has a specific cohort of abundant proteins and distinct proportion of proteins that could be used to define a scientifically-based rationale for optimal clinical use.

競爭公司在抽屜滑軌之專利技術布局研究

為了解決Blum Hettich的問題，作者邱怡翔 這樣論述：

專利布局策略往往要蒐集大量的專利池才能做出最佳的判斷，有計畫的選定ESPACENET和TIPO，再耐心地測試關鍵字，才能有效率的蒐集相關專利，以往沒有做專利檢索的工作，研發的過程就常常出現失敗的風險。專利的命名沒有制式化，雖然有分類號可以篩選，但是專利的分類還是要仰仗人工閱讀，才能做出完整的樹狀圖。既然已經做人工閱讀，也可以順便做出更容易篩選的資料庫，讓未來的研發工作者可以更快速的檢索主要專利申請人的資訊。利用分類好的專利資料庫，便可以進行專利分析，圓餅圖可以看出主要的專利佔比，折線圖可以看出專利趨勢，專利佈局圖可以看出市場操作。本研究的結論是進入隱藏式滑軌的市場首要跨過的門檻是緩歸加拍拍鎖

專利，而要進入騎馬抽的市場就要突破面板結合器的專利，另外要進入伺服器滑軌就要克服前支架扣片的專利。專利佈局策略則是以母國為出發點，利用優先權的方式再向EP或是PCT申請，利用進入國家申請階段再向其他國家做專利申請。如此的操作手法可以在初期達到篩選工作，省下專利申請費用，並且可以掌握重點專利做其他國家的專利家族申請，讓專利保護力最大化。BLUM就是如此操作，他在母國申請的專利數中只有30%會向其他國家申請，而且是利用EP和PCT當跳板，布局的國家常常超過8個。這種操作模式就可以省下次級的專利申請費用，並且可將重點專利做最大化的利用。關鍵字:專利池,分類號,優先權