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siRNA Loaded Lipidoid Nanoparticles and the Immune SystemKasiewicz, Lisa N. 01 May 2018 (has links)
Delivery vehicles are necessary for many therapeutics to overcome the various challenges in their path. It is clear, however, that the relationship between delivery vehicles and the immune system is a complex one. One such delivery vehicle is the lipidoid nanoparticle, which has been shown to be potent in several cell types. This thesis details the first time lipidoids have been used for wound delivery, and demonstrates the successful silencing of an inflammatory protein, TNFα, in the context of diabetic ulcers. Knockdown is seen in an in vitro macrophage-fibroblast coculture model, as well as in nondiabetic and diabetic mice wound models. Lipidoids silence roughly half of the TNFα gene expression in the diabetic wound and have been shown to help the wound close faster than untreated controls. Of course, immune activation can decrease therapeutic efficacy or trigger dangerous reactions in the patient. Learning more about what chemical moieties cause an immune response would allow for the design of a particle that could better resist immune clearance and avoid the creation of a secondary response. This thesis investigated the effect of a lipidoid library on the immune system using a two pronged approach. The lipidoids were first tested against human peripheral blood mononuclear cells and then were injected into mice to probe the in situ immune response. Several types of B cells were examined in this latter case, namely germinal center B cells, plasma cells, and memory B cells. A T cell dependent response occurred, favoring memory B cells for most of the lipidoids tested. There was an increase in free antibody in the blood that reflected this increase in antibody producing cells. Nitrogen rings and carbon tail lengths of eleven and twelve carbons were particularly reactive, though it appears that the amine head group determines immune response more than the tail. Further work will analyze whether these increases in immune cells reflect a loss of therapeutic efficacy, as current ramifications are unclear. An in-depth T cell subset analysis with flow cytometry would also help complete the picture.
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Study on Surface-Medicated Transfer of Small Interfering RNA into Mammalian Cells toward Large-Scale Analysis of Gene Functions / 大規模遺伝子機能解析に向けた表面から哺乳類細胞へのsiRNA導入に関する研究 / ダイキボ イデンシ キノウ カイセキ ニ ムケタ ヒョウメン カラ ホニュウルイ サイボウ エ ノ siRNA ドウニュウ ニ カンスル ケンキュウFujimoto, Hiroyuki 23 January 2009 (has links)
Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第14265号 / 工博第3016号 / 新制||工||1448(附属図書館) / 26592 / UT51-2008-T25 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 木村 俊作, 教授 大嶋 正裕 / 学位規則第4条第1項該当
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Transfekce DNA a siRNA pomocí nanočástic: studium pomocí gene reporter assay. / Reporter gene studies for nanoparticle mediated DNA and siRNA delivery.Kovářová, Barbora January 2017 (has links)
Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Barbora Kovářová Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ. Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: Reporter gene studies for nanoparticle mediated DNA and siRNA delivery Keywords: transfection, plasmid DNA, siRNA, nanoparticles Gene therapy is a promising field offering potential in several currently incurable diseases. Gene therapy is mediated by modulation of gene expression in specific cells by delivering exogenous nucleic acids. One of current tasks of nucleic acid delivery is exploring several synthetic vectors which would have a potential to overcome the disadvantages of commonly used viral vectors. The present study focused on different types of polyethyleneimine-based nanoparticles for plasmid DNA (pDNA) and small interfering RNA (siRNA) delivery. Integration of imaging contrast agents with gene delivery vehicles is advantageous for tracking the gene delivery process both in vivo and in vitro. Gadolinium...
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Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite ExpressionAhmad, Aftab, Zia-Ur-Rehman, Muhammad, Hameed, Usman, Qayyum Rao, Abdul, Ahad, Ammara, Yasmeen, Aneela, Akram, Faheem, Bajwa, Kamran, Scheffler, Jodi, Nasir, Idrees, Shahid, Ahmad, Iqbal, Muhammad, Husnain, Tayyab, Haider, Muhammad, Brown, Judith 14 September 2017 (has links)
Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the beta C1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, V b, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T-1 plants harbored a single copy of the V beta transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/ CLCuMB complex. Among the test plants, plant V beta-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic V beta-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.
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Functionalization of Glucan Dendrimers and Bio-applications / グルカンデンドリマーの機能化とバイオ応用Takeda, Shigeo 25 May 2020 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22660号 / 工博第4744号 / 新制||工||1741(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 秋吉 一成, 教授 大内 誠, 准教授 佐々木 善浩 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM
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Chemically Modified Oligonucleotides Silence Mutant SPTLC1 in an in vitro Model of HSAN1Karnam, Havisha Bindu 05 September 2018 (has links)
Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a monogenic, autosomal dominantly inherited, neurodegenerative disorder resulting in loss of pain and temperature sensation in the distal limbs. HSAN1 is caused by point mutations in a single allele of serine palmitoyltransferase long chain base 1 (SPTLC1), resulting in production of neurotoxic deoxysphingolipids (dSLs). Oligonucleotide therapeutics (ONTs) can be used to downregulate the mutant allele and/or the wild type allele and thus are viable treatment strategies. We investigated the ability of two classes of ONTs, short interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), to downregulate SPTLC1 in an in vitro model of HSAN1 derived from the C133W mouse model overexpressing mutant hamster SPTLC1. We screened a panel of siRNAs and ASOs targeting mutant hamster SPTLC1 and identified four lead compounds. We demonstrated these compounds’ ability to reduce mutant hamster SPLTC1 and/or wild type mouse SPTLC1 mRNA in CHO cells and C57BL/6J embryonic mouse primary cortical neurons. We then showed that these compounds downregulate hamster and mouse SPTLC1 mRNA and protein in embryonic primary cortical neuron cultures derived from C133W mice. These compounds demonstrate therapeutic potential and should be developed further in vivo.
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Novel Roles for B-Raf in Mitosis and CancerBorysova, Meghan E. K 03 April 2009 (has links)
The MAP kinase pathway is well known for its key roles in regulating cell proliferation and cell cycle progression. MAP kinases have also been implicated in mitotic functions, however these functions are less-well understood. Recent studies from our laboratory used Xenopus egg extracts to identify B-Raf as an essential activator of the MAPK cascade during mitosis. Therefore, the first objective of my dissertation research was to determine if B-Raf has functional significance during mitosis in human somatic cells.
Using RNA interference against B-Raf and various immunofluorescence techniques, I show that B-Raf: (1) localizes to and is phosphorylated at a key mitotic structure, (2) is critical for proper mitotic spindle assembly and chromatin congression, (3) is important for the engagement of microtubules with kinetochores during mitosis, and (4) is necessary for activation of the spindle assembly checkpoint.
It has been demonstrated that B-Raf is a prominent oncogene, constitutively activated in the vast majority of melanomas and other cancers. I hypothesized that oncogenic B-Raf expression perturbs mitosis and causes aneuploidy.
First, we show that oncogenic B-Raf expression correlates with mitotic abnormalities in human melanoma cells and that spindle defects are induced when oncogenic B-Raf is ectopically expressed. Further, using FISH and karyotype analysis, I demonstrate that oncogenic B-Raf drives aneuploidy and chromosome instability in primary, immortalized, and tumor cells.
In summary, my dissertation studies elucidate novel roles for B-Raf in mammalian mitosis. In addition, my studies show for the first time that oncogenic B-Raf disrupts mitosis causing chromosomal instability. I propose that oncogenic B-Raf-induced chromosome instability contributes to tumorigenesis.
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Untersuchung der biologischen Aktivität von Nanopartikeln in TumorschnittkulturenMerz, Lea Maria 01 April 2020 (has links)
Die Entdeckung der RNA‐Interferenz (RNA‐i) als ein natürlicher Prozess der Genregulation sorgte auf Grund des möglichen Einsatzes für therapeutische Zwecke für großes Interesse. Probleme beim Transport der RNA‐i‐induzierenden kleinen RNA‐Moleküle (siRNAs) traten vor allem hinsichtlich Ladungsverhältnissen, Instabilität und Molekulargewicht auf. Um diese Probleme zu lösen, wurden zunehmend Transportsysteme aus Nanopartikeln untersucht. Die verwendeten Polyethylenimine (PEIs) sind positiv geladene, verzweigt oder linear aufgebaute Polymere, welche mit RNAs (siRNA, miRNA) Komplexe bilden. Modifikationen der PEI‐Kom‐ plexe können zu einem effektiveren Transport der RNA, zu veränderten pharmakokinetischen Eigenschaften und zu einer verbesserten Biokompatibilität führen. Der Erfolg dieser therapeu‐ tischen Nanopartikel ist insbesondere von der effizienten zellulären Aufnahme, der Fähigkeit der Gewebepenetration und vom Erreichen der Zielzellen abhängig. Der Großteil der bisheri‐ gen Untersuchungen von PEIs wurde an Zellkulturen durchgeführt. Die hier erzielten Ergeb‐ nisse lassen sich jedoch nur schwer auf komplexe in‐vivo‐Systeme übertragen.
Aus diesem Grund wurde in der vorliegenden Arbeit die Anwendung der PEIs an Tumorschnitt‐ kulturen untersucht. Dafür wurde zuerst die Kultivierung von 350 μm durchmessenden Dünn‐ schicht‐Präparaten von Xenotransplantat‐Tumoren aus PC3 und U87 Zelllinien etabliert. Eine erfolgreiche Kultivierung konnte über 14 Tage durchgeführt werden. An bestimmten Zeit‐ punkten nach Kultivierungsbeginn (Tag 1, 3, 5, 7 und 14) wurden die Morphologie und die Vitalität (Apoptose, Proliferation) durch eine HE‐ oder immunhistochemische Färbung be‐ stimmt. Es konnte gezeigt werden, dass die Morphologie der Tumorzellen sowie die Prolifera‐ tion und Apoptose über den gesamten Kultivierungszeitraum erhalten blieben.
Im nächsten Schritt wurde sowohl der Transport der siRNA von nicht‐modifizierten und modi‐ fizierten PEIs als auch der Transport durch deren Lipopolyplex‐Derivate (PEI‐siRNA‐Komplexe mit Liposomenhülle) analysiert. Für die Visualisierung der Komplexe wurde fluoreszenzmar‐ kierte siRNA verwendet. Die PEI‐siRNA‐Komplexe wurden auf die Schnittkulturen gegeben und über 24 Stunden kultiviert. In der mikroskopischen Analyse und ihrer Quantifizierung erfolgte die erste Einschätzung der Gewebepenetration. Es zeigten sich Unterschiede in der Gewebe‐ penetration der verschiedenen Nanopartikel, abhängig von deren Oberflächenladung.
Um die biologische Aktivität der Nanopartikel in lebenden Zellen besser evaluieren zu können, wurde im dritten Schritt der Knockdown eines endogenen Genproduktes untersucht. Zielgen war hier das Onkogen Survivin, welches ein wichtiger endogener Überlebensfaktor der Tu‐ morzellen darstellt. Es gelang der Nachweis eines durch PEI‐siRNA‐Komplexe vermittelten Knockdowns des Survivin‐Gens.
Insgesamt konnten somit Gewebeschnittkulturen als Grundlage für die ex‐vivo Untersuchung von Nanopartikeln etabliert werden. Es konnten nicht nur Aussagen über die Gewebepenet‐ ration der Nanopartikel, sondern auch über ihre biologischen Aktivitäten in einer intakten Ge‐ webestruktur getroffen werden.:Bibliographische Beschreibung 3
1 Einleitung 5
1.1 Der Einsatz der RNA-Interferenz (RNA-i) und von Nanopartikeln in der Medizin 5
1.2 Die RNA-Interferenz 6
1.2.1 Der Prozess der RNA-Interferenz 6
1.2.2 Vor- und Nachteile der RNA-Interferenz 9
1.3 Nanopartikel als Transportsysteme der siRNA: Polyethylenimine 10
1.4 Die Transportfähigkeit von Polyethyleniminen und ihre Optimierung 12
1.5 Anwendung der Polyethylenimine in in-vivo-Systemen 14
1.6 Präklinische Tumormodelle 15
1.6.1 Zellkulturen 15
1.6.2 Xenograft-Modelle und Gewebeschnittkulturen 16
2 Fragestellung 18
3 Literatur 19
4 Publikationen 24
5 Anlagen 32
6 Zusammenfassung und Ausblick 33
7 Erklärung über die eigenständige Abfassung der Arbeit 36
8 Lebenslauf 37
9 Danksagung 40
10 Erklärung über den wissenschaftlichen Beitrag 41
11 Teilnahmebestätigung: Vorlesung zur „Guten Wissenschaftlichen Praxis“ 42
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Local application of Usag-1 siRNA can promote tooth regeneration in Runx2-deficient mice / Usag-1 siRNAの局所投与はRunx2欠損マウスの歯牙再生を促進するMishima, Sayaka 24 January 2022 (has links)
京都大学 / 新制・論文博士 / 博士(医学) / 乙第13462号 / 論医博第2249号 / 新制||医||1055(附属図書館) / (主査)教授 萩原 正敏, 教授 松田 秀一, 教授 齊藤 博英 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Dissection of RNA entry into RNAi using a novel protein-RNA tethering systemCuerda-Gil, Diego January 2021 (has links)
No description available.
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