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Anwendungsorientierte Grundlagenforschung und produktorientierte Anwendungsforschung als Spekulationsgeschäft ein Anwendungsfall für die Optionspreistheorie /Schultheiss, Rainer. January 2001 (has links) (PDF)
Stuttgart, Universiẗat, Diss., 2001.
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Isolierte humane Hepatozyten : Stellenwert für die Grundlagenforschung und klinische Praxis /Katenz, Ekaterina. Unknown Date (has links)
Berlin, Charité, University-Med., Diss., 2005.
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Molecular mechanisms underlying Woodhouse-Sakati syndrome: characterization of DCAF17 with specific, polyclonal antibodies / Molekulare Grundlagen des Woodhouse-Sakati Syndroms: Charakterisierung des DCAF 17 mit spezifischen, polyklonalen AntikörpernBlocka, Joanna January 2019 (has links) (PDF)
Woodhouse-Sakati syndrome (WSS) is a rare multisystemic, autosomal recessive disease. The underlying cause of WSS are mutations of C2orf37 gene, which result in a truncated protein. Little is known about the function of C2orf37 (DDB1-CUL4A-associated factor 17, DCAF17) apart from it being part of the DDB1-CUL4-ROC1 E3 ubiquitin ligase complex, specifically binding directly to DDB1 and serving as a substrate recruiter for E3. There are two major isoforms of DCAF17: beta (65 kDa, 520 amino acids) and alpha (27 kDa, 240 amino acids), which is a C-terminal part of beta. The intracellular localization of the WSS protein is thought to be primarily the nucleolus. A murine ortholog protein was found to be expressed in all tissues with a relatively higher expression in the brain, liver, and skin.The aim of this work was to investigate DCAF17 in HeLa cells in more detail, in particular the redistribution of both WSS isoforms on the subcellular and -nuclear level as well as their chemical features. For these experiments, I developed, through recombinant expression and affinity purification, a specific polyclonal antibody against a WSS-epitope 493-520. Furthermore, three other specific polyclonal antibodies were obtained through affinity purification with help of commercially produced high-affinity epitope peptides.By means of these antibodies, I determined- through immunofluorescence and subcellular protein fractionation- that, apart from the redistribution of the WSS protein within the non-soluble = chromatin-bound nuclear fraction, a significant amount of both WSS isoforms is present in the soluble nuclear fraction. Indeed, treatment of purified nuclear envelopes with an increasing concentration of NaCl as well as urea confirmed a non-covalent binding of the WSS protein to the nuclear envelope with the detachment ofbeta-WSS at a lower NaCl concentration than alpha-WSS. In regard to the chromatin-bound WSS protein, I performed hydrolysis of nuclear and nucleolar extract with DNase and RNase. The results indicate that the WSS protein is bound to DNA but not RNA, with alpha-WSS being possibly located more abundantly in the nucleolus, whereas beta-WSS within other subnuclear departments. Furthermore, in all the above-mentioned experiments, a presence of an 80-kDa protein, which specifically reacted with the polyclonal high-affinity antibodies and showed similar redistribution and chemical features as alpha- and beta-WSS, was observed. In order to investigate whether this protein is a posttranslationally modified WSS isoform, I performed deglycosylation and dephosphorylation of nuclear extract, which showed no disappearance or change in abundance of the 80-kDa band on Western blot. While other ways of poststranslational modification cannot be excluded as the cause of occurrence of the 80-kDa protein, an existence of a third, yet undescribed, major isoform is also conceivable. Summarizing, this work contributed to a deeper characterization of the WSS protein, which can help future investigators in developing new experimental ideas to better understand the pathology of WSS. / Woodhouse-Sakati Syndrom (WSS) ist eine seltene, autosomal rezessive Multisystemerkrankung, deren Ursache Mutationen im C2orf37 Gen, resultierend in einem trunkierten Protein, sind. Die Funktion des C2orf37 (DDB1-CUL4A-associated factor 17, DCAF 17) ist weitgehend unbekannt. Das Protein ist Teil des DDB1-CUL4-ROC1 E3-Ubiquitin-Ligase-Komplexes, wo es direkt an DDB1 bindet und Substrate für E3 rekrutiert. Zwei Isoformen des DCAF17: beta (65 kDa, 520 Aminosäuren) und alpha (27 kDa, 240 Aminosäuren), die ein C-terminaler Teil der beta-Isoform ist, sind heutzutage bekannt. Man geht von einer primär nukleolären Lokalisation des WSS-Proteins in den Zellen aus. Untersuchungen des murinen C2orf37-Ortholog-Proteins ergaben eine Expression in allen Zellen mit einer erhöhten Expression im Gehirn, in der Leber und in der Haut. Das Ziel dieser Arbeit war es, DCAF17 in HeLa-Zellen zu untersuchen, insbesondere die Lokalisation beider WSS-Isoformen auf dem subzellulären und -nukleären Niveau sowie deren chemische Eigenschaften. Durch rekombinante Expression und Affinitätsreinigung entwickelte ich spezifische polyklonale Antikörper gegen das WSS-Epitop 493-530. Zudem reinigte ich drei weitere spezifische polyklonale Antikörper mithilfe kommerziell produzierter hochaffiner Epitop-Peptide auf. Mithilfe dieser Antikörper konnte ich- durch Immunfluoreszenz und subzelluläre Proteinfraktionierug- eine Lokalisation des WSS-Proteins in der löslichen Kernfraktion, zusätzlich zu der bereits bekannten chromatingebundenen Kernfraktion, nachweisen. Die Behandlung reiner Kernhüllen mit steigernden NaCl-Konzentrationen und Harnstoff zeigte eine nicht-kovalente Bindung des DCAF17 an die Kernhülle mit einer Ablösung der beta-Isoform von der Kernhülle bereits bei niedrigeren NaCl-Konzentrationen als im Falle der alpha-Isoform. Um das chromatingebundene DCAF17 genauer zu untersuchen, führte ich eine Hydrolyse des Ganzkern- und Nukleolusextraktes mit DNase und RNase durch. Diese ergab eine Bindung des WSS-Proteins an die DNA, jedoch nicht an die RNA, mit der möglichen Hauptlokalisation der alpha-Isoform im Nukleolus und der beta-Isoform in anderen subnukleären Kompartimenten. Des Weiteren wurde in den oben beschriebenen Experimenten ein 80-kDa Protein nachgewiesen, das eine spezifische Reaktion mit den polyklonalen hochaffinen Antikörpern sowie eine dem WSS-Protein ähnliche subzelluläre / -nukleäre Lokalisation und chemische Eigenschaften zeigte. Um zu untersuchen, ob es sich um ein posttranslational modifiziertes DCAF17 handelt, führte ich Deglycosylierung und Dephosphorylierung des Ganzkernextraktes durch. Diese zeigten weder ein Verschwinden noch eine Änderung des 80-kDa-Signals auf Immunoblots. Obwohl eine andere Art einer posttranslationalen Proteinmodifizierung ist nicht ausgeschlossen, entspricht dieses Protein möglicherweise einer dritten, bisher nicht beschriebenen, Hauptisoform des DCAF17.
Zusammenfassend trug diese Arbeit zur genaueren Charakterisierung des WSS-Proteins bei. Dies kann zukünftigen Forschern helfen, die Pathologie des WSS besser zu verstehen.
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Moralischer Realismus und Antirealismus : zur neueren Debatte um Grundlagenfragen der Ethik /Tarkian, Tatjana. Unknown Date (has links)
Göttingen, Universiẗat, Diss., 2002.
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Anwendungsorientierte Grundlagenforschung und produktorientierte Anwendungsforschung als Spekulationsgeschäft ein Anwendungsfall für die Optionspreistheorie /Schultheiß, Rainer. January 2001 (has links)
Stuttgart, Univ., Diss., 2000.
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Cell culture models of Chorea Acanthocytosis and their evaluationGlaß, Hannes 29 May 2018 (has links) (PDF)
Chorea Acanthocytosis (ChAc) is an autosomal recessive inherited disease caused by loss- of-function mutation in the VPS13A gene which encodes CHOREIN protein. This study used induced pluripotent stem cells (iPSCs) as well as neural progenitor cells (NPCs) to generate medium spiny neurons (MSN) as well as midbrain dopaminergic neurons (mDAN).
The first objective of this thesis was to generate and characterize a stem cell based disease model of ChAc. The second objective was to establish two different differentiation protocols that yield different neuronal sub types that are affected in ChAc, and compare whether they harbor similar phenotypes and whether the faster protocol can be used to model the disease accurately.
The generated iPSCs were characterized using AP staining as an early marker for reprogramming, qPCR for analysis of residual expression of exogenous transcription factors, immunocytochemistry (ICC) for staining of pluripotency markers as well as markers for mesoderm, ectoderm and endoderm formation upon three germ layer formation. Karyotyping was conducted to exclude aberrant clones. Western blot using CHOREIN antibody revealed that the cell lines retained their disease identity.
There were no differences observed between wild type and ChAc lines in stem cell and neuron populations in either protocol. qPCR analysis, investigating the expression of previously described markers for characterization, revealed no significant clustering between wild type and ChAc lines in either protocol. A disturbed ratio of globular and filamentous actin is causative for the aberrant shape of ChAc erythrocytes. Investigation of the ratio in mature neurons revealed a significant reduction of this ratio in MSN but no difference in mDAN cultures. When the ratio of cytosolic and filamentous tubulin and the acetylation of tubulin were investigated, no differences were found between wild type and ChAc lines.
Mature neurons of both differentiation protocols were subjected to treatment with the proteotoxic stress inducer L-canavanine and the unfolded protein response (UPR) inducer tunicamycin. Survival was analyzed with the PrestoBlue assay as well as lactate dehydroxylase (LDH) release assay. Both cultures of mature neurons showed an increased susceptibility to the respective drugs. Furthermore the data suggests that MSN cultures are more vulnerable against proteotoxic stress (L-canavanine). Kinetics of tunicamycin poisoning were not different within MSN cultures but indicated a late cell death of ChAc lines under mDAN differentiation conditions. DNA damage plays a major role in the progression of neurodegenerative diseases. The amount of double strand breaks (DSB) was assessed in mature cultures of MSN and mDAN differentiations. There was no difference in basal level of DSB. When etoposide was applied to induce DNA damage, increased susceptibility of ChAc lines was observed. Albeit significant, the effect size was very small.
Seahorse was used to characterize energy metabolism. Glycolysis was not impaired in ChAc lines in either protocol. Furthermore, MSN differentiation showed no difference in any parameter related to oxidative phosphorylation, while under mDAN conditions, coupling efficiency and spare respiratory capacity was increased for ChAc lines. The non-respiratory oxygen consumption was increased in ChAc lines in MSN cultures but decreased in mDAN cultures.
The yeast homolog of VPS13A interacts with vesicle and mitochondrial membranes. Therefore, this study focuses on vesicle and mitochondria homeostasis. Live cell imaging of mature neurons of MSN differentiations revealed a decreased amount and reduced motility of mitochondria. Even though mitochondria were normally shaped their size was reduced. mDAN differentiations harbored a reduced amount and shortened mitochondria. These mitochondria, however, showed an increased motility. When analyzing aligned mature neurons in microfluidic chambers (MFCs), a strong phenotype was already observed in proximal regions, which resembled the distal parts of the channels. Hence, the dysregulation, that occurs distal in healthy controls, happens closer to the soma in diseased cells. The mitochondria potential marker JC-1 showed a hyperpolarization of mitochondria in MSN culture and a depolarization in mDAN cultures.
When investigated in MFCs of mDAN cultures, there was a significant increase in potential observed at the distal position of ChAc lines, while wild type cultures showed no difference. Experiments conducted on the lysosomal compartments showed a decrease in proximal parts of ChAc MSN cultures when compared to wild type. Their shape was altered as well. mDAN cultures featured no significant morphological changes. Trafficking analysis revealed an increase in motility in MSN cultures but a decrease in mDAN cultures. When lysosomes were analyzed in MFCs only mDAN cultures showed an increase in retrograde transport.
In order to investigate whether the in vitro phenotypes of Huntington (Htt) and ChAc are similar, some of the previous experiments were conducted in MSN differentiations of one Htt line. Cells from Htt behaved similar to ChAc lines when DNA damage response was investigated. Analysis of mitochondrial parameters showed no difference as well. However, the non-respiratory oxygen consumption was not increased and resembled wild type.
When Htt neurons were investigated during live cell imaging, shortened mitochondria were found. Their number was not reduced significantly. However, a trend for reduction was observed. Mitochondria of Htt cells were more motile than ChAc or wild type lines. Mitochondrial potential was increased in Htt and comparable to ChAc. Lysosomal count showed a reduction and the area of Htt lysosomes was significantly smaller than wild type or ChAc. Lysosomes of Htt cells were more motile than their wild type or ChAc counterparts.
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Academic enterprise : a new mission for universities or a contradiction in term? : four papers on the long-term implications of increasing industry involevement and commercialization in academia /Larsen, Maria Theresa. January 2007 (has links) (PDF)
Diss.--Copenhagen Business School, 2007. / Enth. 4 Beitr.
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Public research funding and private innovation : the case of the pharmaceutical industry /Book, Robert Aaron. January 2002 (has links)
Thesis (Ph. D.)--University of Chicago, Graduate School of Business, August 2002. / Includes bibliographical references. Also available on the Internet.
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Cell culture models of Chorea Acanthocytosis and their evaluationGlaß, Hannes 17 April 2018 (has links)
Chorea Acanthocytosis (ChAc) is an autosomal recessive inherited disease caused by loss- of-function mutation in the VPS13A gene which encodes CHOREIN protein. This study used induced pluripotent stem cells (iPSCs) as well as neural progenitor cells (NPCs) to generate medium spiny neurons (MSN) as well as midbrain dopaminergic neurons (mDAN).
The first objective of this thesis was to generate and characterize a stem cell based disease model of ChAc. The second objective was to establish two different differentiation protocols that yield different neuronal sub types that are affected in ChAc, and compare whether they harbor similar phenotypes and whether the faster protocol can be used to model the disease accurately.
The generated iPSCs were characterized using AP staining as an early marker for reprogramming, qPCR for analysis of residual expression of exogenous transcription factors, immunocytochemistry (ICC) for staining of pluripotency markers as well as markers for mesoderm, ectoderm and endoderm formation upon three germ layer formation. Karyotyping was conducted to exclude aberrant clones. Western blot using CHOREIN antibody revealed that the cell lines retained their disease identity.
There were no differences observed between wild type and ChAc lines in stem cell and neuron populations in either protocol. qPCR analysis, investigating the expression of previously described markers for characterization, revealed no significant clustering between wild type and ChAc lines in either protocol. A disturbed ratio of globular and filamentous actin is causative for the aberrant shape of ChAc erythrocytes. Investigation of the ratio in mature neurons revealed a significant reduction of this ratio in MSN but no difference in mDAN cultures. When the ratio of cytosolic and filamentous tubulin and the acetylation of tubulin were investigated, no differences were found between wild type and ChAc lines.
Mature neurons of both differentiation protocols were subjected to treatment with the proteotoxic stress inducer L-canavanine and the unfolded protein response (UPR) inducer tunicamycin. Survival was analyzed with the PrestoBlue assay as well as lactate dehydroxylase (LDH) release assay. Both cultures of mature neurons showed an increased susceptibility to the respective drugs. Furthermore the data suggests that MSN cultures are more vulnerable against proteotoxic stress (L-canavanine). Kinetics of tunicamycin poisoning were not different within MSN cultures but indicated a late cell death of ChAc lines under mDAN differentiation conditions. DNA damage plays a major role in the progression of neurodegenerative diseases. The amount of double strand breaks (DSB) was assessed in mature cultures of MSN and mDAN differentiations. There was no difference in basal level of DSB. When etoposide was applied to induce DNA damage, increased susceptibility of ChAc lines was observed. Albeit significant, the effect size was very small.
Seahorse was used to characterize energy metabolism. Glycolysis was not impaired in ChAc lines in either protocol. Furthermore, MSN differentiation showed no difference in any parameter related to oxidative phosphorylation, while under mDAN conditions, coupling efficiency and spare respiratory capacity was increased for ChAc lines. The non-respiratory oxygen consumption was increased in ChAc lines in MSN cultures but decreased in mDAN cultures.
The yeast homolog of VPS13A interacts with vesicle and mitochondrial membranes. Therefore, this study focuses on vesicle and mitochondria homeostasis. Live cell imaging of mature neurons of MSN differentiations revealed a decreased amount and reduced motility of mitochondria. Even though mitochondria were normally shaped their size was reduced. mDAN differentiations harbored a reduced amount and shortened mitochondria. These mitochondria, however, showed an increased motility. When analyzing aligned mature neurons in microfluidic chambers (MFCs), a strong phenotype was already observed in proximal regions, which resembled the distal parts of the channels. Hence, the dysregulation, that occurs distal in healthy controls, happens closer to the soma in diseased cells. The mitochondria potential marker JC-1 showed a hyperpolarization of mitochondria in MSN culture and a depolarization in mDAN cultures.
When investigated in MFCs of mDAN cultures, there was a significant increase in potential observed at the distal position of ChAc lines, while wild type cultures showed no difference. Experiments conducted on the lysosomal compartments showed a decrease in proximal parts of ChAc MSN cultures when compared to wild type. Their shape was altered as well. mDAN cultures featured no significant morphological changes. Trafficking analysis revealed an increase in motility in MSN cultures but a decrease in mDAN cultures. When lysosomes were analyzed in MFCs only mDAN cultures showed an increase in retrograde transport.
In order to investigate whether the in vitro phenotypes of Huntington (Htt) and ChAc are similar, some of the previous experiments were conducted in MSN differentiations of one Htt line. Cells from Htt behaved similar to ChAc lines when DNA damage response was investigated. Analysis of mitochondrial parameters showed no difference as well. However, the non-respiratory oxygen consumption was not increased and resembled wild type.
When Htt neurons were investigated during live cell imaging, shortened mitochondria were found. Their number was not reduced significantly. However, a trend for reduction was observed. Mitochondria of Htt cells were more motile than ChAc or wild type lines. Mitochondrial potential was increased in Htt and comparable to ChAc. Lysosomal count showed a reduction and the area of Htt lysosomes was significantly smaller than wild type or ChAc. Lysosomes of Htt cells were more motile than their wild type or ChAc counterparts.:List of abbreviations
Introduction
1. Neurodegenerative diseases
1.1. Chorea-acanthocytosis – a clinical overview
1.2. Chorea-Acanthocytosis – genetic considerations
2. Disease modelling
2.1. Human disease models
2.2. Induced pluripotent stem cells
2.3. Multipotent neuronal progenitor cells
3. Objectives of this thesis
Materials & Methods
1. Cell culture procedures
1.1. Coating
1.2. Matrigel
1.3. PLO/laminin
1.4. Gelatin coating
1.5. Mouse embryonic fibroblast isolation
1.6. Generation of feeder cells
1.7. Human fibroblast culture
1.8. Reprogramming
1.9. iPSC culture
1.10. Culture of small molecule neuronal precursor cells (smNPC)
1.11. MSN differentiation
1.12. mDAN differentiation
2. Nucleic acid biochemistry
2.1. mRNA isolation
2.2. cDNA generation
2.3. Polymerase chain reaction (PCR)
2.4. Agarose gel electrophoresis
3. Cell survival analysis
3.1. PrestoBlue cell viability assay
3.2. Cytotoxicity detection kit:
3.3. DNA damage analysis
4. Metabolic characterization
5. Protein biochemistry
5.1. Alkaline phosphatase staining
5.2. Preparation of immunocytochemistry samples
5.3. Isolation of globular and filamentous actin
5.4. Whole cell protein Isolation
5.5. Cytosolic protein isolation
5.6. Protein concentration measurement
5.7. Western blot
6. Live cell imaging
7. Statistics
Results
1. Generation of induced pluripotent stem cells
1.1. Silencing of exogenous transcription factors
1.2. Karyotyping of iPSC clones
1.3. Evaluation of pluripotency
1.4. Alkaline phosphatase staining
1.5. Staining of pluripotency markers
1.6. Three germ layer formation
1.7. Confirmation of ChAc phenotype by CHOREIN western blot
2. Characterization of differentiation potential
2.1. Differentiation efficiency
2.2. Characterization by qPCR
2.3. Ratio of polymerized and unpolymerized cytoskeleton proteins
2.4. Cell survival upon stress induction
2.5. DNA damage in mature neurons
2.6. Characterization of metabolism
3. Live cell imaging
3.1. Mitochondrial dynamics
3.1.1. Morphological analysis
3.1.1.1. Undirected neurons (96 well plate format)
3.1.1.2. Microfluidic chambers
3.1.2. Trafficking analysis
3.1.2.1. 96 well
3.1.2.2. Microfluidic chambers
3.1.3. JC-1
3.1.3.1. 96 well
3.1.3.2. Microfluidic chambers
3.2. Lysosomal dynamics
3.2.1. Morphological analysis
3.2.1.1. 96 well
3.2.1.2. Microfluidic chambers
3.2.2. Trafficking
3.2.2.1. 96 well
3.2.2.2. Microfluidic chambers
4. Comparison with Huntington’s disease
4.1. DNA damage
4.2. Characterization of metabolism
4.3. Live cell imaging
4.3.1. Mitochondria
4.3.1.1. Morphological analysis
4.3.1.2. Trafficking
4.3.1.3. JC-1
4.3.2. Lysosomes
4.3.2.1. Morphological analysis
4.3.2.2. Trafficking
Discussion
1. Characterization of ChAc lines
1.1. ChAc stem cell lines show no impaired differentiation potential
1.2. Neurons from MSN differentiation have an altered G/F actin ratio
1.3. Mature neurons from ChAc lines are susceptible to UPR, proteotoxicity and DNA damage
1.4. ChAc neurons are not susceptible to DNA damage
1.5. Energy dynamics in ChAc and Huntington lines feature a shift to glycolysis
2. Live cell imaging of ChAc lines
2.1. Video analysis is reproducible and sensitive
2.2. ChAc lines have altered mitochondria shape and trafficking
2.3. Treatments are not selective on ChAc lines mitochondria
2.4. Mitochondrial potential is altered in ChAc lines
2.5. ChAc lysosomes feature normal morphology but altered trafficking
2.6. Lysosomes of MSN cultures respond poorly to treatments
3. MSN and mDAN differentiation highlight different aspects of the disease
References
List of figures
List of tables
Acknowledgments
Appendix
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The Bark Project - Material Research in the Natural Science and DesignWenig, Charlett 29 June 2022 (has links)
The Bark Project is a practice-based research venture that uses scientific tools to characterize materials and design methods to explore materials that lead to longer lifecycle applications for tree bark. This project is part of the Matters of Activity excellence cluster research group Adaptive Fibrous Materials, which is aimed at a deeper understanding of biological material, their activity within their environment and adaptation for future scenarios.
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