Im Zuge der Bemühungen um neue, tumorspezifische Therapieansätze für die Myelomerkrankung hat sich der C-X-C-Chemokinrezeptor 4 (CXCR4) aufgrund seiner zentralen Rolle in der Tumorgenese als vielversprechender Angriffspunkt hervorgetan. Im Sinne eines theranostischen Konzepts wird der Rezeptor mithilfe eines radioaktiv markierten Liganden quantifiziert und anschließend von rezeptorspezifischen Radiotherapeutika als Zielstruktur genutzt. Die CXCR4-Expression ist allerdings ein höchst dynamischer Prozess mit großer inter- und intraindividueller Heterogenität, der u.a. durch eine begleitende Chemotherapie beeinflusst werden kann. Ob sich therapieinduzierte Veränderungen der Rezeptorexpression gezielt nutzen lassen, um die CXCR4-Expression zu optimieren und so die Effektivität der CXCR4-gerichteten Strategien zu steigern, wurde bislang nicht untersucht. Vor diesem Hintergrund wurden in der vorliegenden Arbeit verschiedene, in der Myelomtherapie etablierte Substanzen sowohl einzeln als auch in Kombination hinsichtlich ihres Einflusses auf die CXCR4-Expression von MM-Zelllinien und primären MM-Zellen unter in vitro Bedingungen analysiert. In den durchgeführten Experimenten zeigte sich eine hohe Variabilität der CXCR4-Expression der MM-Zellen nach Therapieinduktion, die sich als substanz-, dosis- und zeitabhängig herausstellte. Die Ergebnisse bestätigten das große Potenzial der therapieinduzierten Modulation der CXCR4-Expression. Im weiteren Verlauf sind translationale Forschungsansätze gerechtfertigt, die die Übertragbarkeit der in vitro gewonnenen Ergebnisse auf die komplexen Vorgänge im lebenden Organismus überprüfen. Langfristiges Ziel ist der Entwurf eines patientenzentrierten, multimodalen Therapiekonzepts, welches das CXCR4-gerichtete theranostische Konzept mit einer individuell angepassten, medikamentösen MM-Therapie kombiniert. / In the course of developing new tumor specific therapeutic approaches for non-yet curable myeloma disease C-X-C chemokine receptor 4 (CXCR4) has emerged as a promising target due to its crucial role in myeloma tumorigenesis. Within a theranostic concept CXCR4 is quantified using radioactively labeled ligands and afterwards targeted by receptor-specific radiopharmaceuticals. However, CXCR4 expression is a very dynamic process with a high inter- and intraindividual heterogeneity which can be influenced by concomitant chemotherapy. Whether therapy induced changes in receptor expression can be used to enhance CXCR4 expression and thus to improve efficacy of CXCR4-based theranostics has not been examined so far. In this context the present study evaluated the effect of several anti-myeloma drugs (bortezomib, cyclophosphamide, dexamethasone, doxorubicin, lenalidomide) on CXCR4 expression of different human myeloma cell lines as well as patient-derived CD138+ plasma cells under in vitro conditions. Findings disclosed a high variability of CXCR4 expression on myeloma cells after drug application which turned out to be substance-, dose- and time-dependent. The results confirmed the high potential of therapy-induced modulation of CXCR4 expression. In further course, translational research approaches are justified to verify the transferability of the in vitro findings to the complex macro- and microenvironment in vivo. Long-term goal is the development of a patient-centered, multimodal therapy concept which combines CXCR4 based theranostics with a personalized drug-based therapy.

This diploma thesis deals with the composition of culture media for rooting Vitis vinifera in vitro. Based on the findings of the literary research, various compositions of culture media have been suggested. The success of rooting on individual media has been evaluated.

A Dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, for the Degree of Master of Science (MED). JOHANNESBURG, 1986 / The Literature Review covers the immunosuppressive and immunopotentiating properties of antibiotics on the immune system and the effects these could have on the resolution of an infection. The possible pathogenic mechanisms of C. albicans are also reviewed in this section. The experimental section shows that pre-treatment of mice with erythromycin increases the mean survival time following intraperitoneal inoculation of C. albicans. It was shown that erythromycin enhanced lymphocyte transformation and PMNL migration in both in-vivo and in-vitro situations. These enhanced immunological components probably caused improved survival times in the aforementioned animal experiments. To investigate the effects of oral administration of erythromycin on in-vivo PMNL migration in adult volunteers a new quantitative test which could only be applied to humans was developed and is described in detail. Using this method preliminary data were obtained which show that erythromycin increases PMNL migration in-vivo. / IT2018

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<p> Chick embryos at stages primitive streak to three somites were explanted on the vitelline membrane and cultured by the method of New (1955) or by Gallera's modification of the method (Nicolet and Gallera. 1963). Antisera produced in rabbits against adult chicken brain extract. adult chicken kidney extract. and embryo brain extract were placed on the uppermost side of the embryo preparation. The embryos were recovered after 24 to 36 hours further incubation. Defects of the central nervous system. the posterior trunk. and the extra-embryonic membranes occurred in embryos exposed to adult brain antiserum. Embryos exposed to adult kidney antiserum developed exactly the same kinds of defects. Embryo brain antiserum produced similar abnormalities which included defects of the central nervous system and of the extra-embryonic membranes, and in addition de-. fective somites. However, embryos exposed to gamma globulin solutions containing antibodies against neural-specific antigens and not against common tissue antigens were normal. In control experiments, embryos exposed to saline solution, to normal rabbit serum, and to normal rabbit serum gamma globulins developed normally. </p> <p> Histological examination of ~epresentative antisera treated embryos revealed that there were extensive areas of disorganization and necrosis of neural tissue. In embryos with short trunks, the caudal proliferation centre was necrotic. Embryos exposed to gamma globulins of absorbed adult brain antiserum were histologically normal. </p> <p> The antisera used in these experiments were characterized by double diffusion in agar gel. It was demonstrated by this method that the antisera contained antibodies against common tissue antigens as well as against tissue-specific antigens. It was also shown that certain antigens common to all adult organs were present in the embryo and in the extra-embryonic membranes during the time that the embryos were exposed to the antisera. </p> <p> Embryos which had been exposed to various of the tissue antisera for 8.5, 21.5 or 32 hours were sectioned. The sections were stained with FITC-labelled goat anti-rabbit gamma globulins in order to localize distribution of the antibodies. Fluorescence was located on the ectoderm of the embryos and of their extra-embryonic membranes, in the lumen of the neural tube and in the cavity of the otic vesicles. This demonstrated that, under the conditions of the experiment, the antibodies were available to the embryo. </p> <p> Antisera which affected the embryo and the extra-embryonic membranes were shown to contain antibodies against antigens actually present in the embryos and in the extra-embryonic membranes during the time of exposure. The only antiserum which had no adverse effects on the embryos was the one which contained only antibodies against antigens not demonstrated to be present in the embryo during the time of exposure. </p> / Thesis / Master of Science (MSc)

𝘐𝘯 𝘷𝘪𝘵𝘳𝘰 selection has been widely used to isolate single-stranded DNA molecules from large random-sequence pools that are able to perform a desired catalytic reaction, or bind to a target molecule. Using this technique, we have created a DNA enzyme, named DET22-18, which has a uniquely linked chemical catalysis/real-time signaling capability. It is a true enzyme with a 𝘬cₐₜ of~7 min⁻¹ -the second fastest rate ever reported for a DNA enzyme. The DNA enzyme cleaves a substrate containing a single ribonucleotide linkage embedded in a DNA chain and sandwiched between a fluorophore-labeled deoxyribonucleotide and a quencher-modified deoxyribonucleotide. The ability of DET22-18 to generate a large fluorescence signal provides a useful tool to engineer potential allosteric deoxyribozyme biosensors for real-time detection of important biological targets. To provide a proof of concept that a reporter system can be built from the above signaling DNA enzyme, the cis-acting version of this enzyme, DEC22-18A, was engineered into an allosterically regulated deoxyribozyme biosensor that can report ATP. A preliminary investigation was also conducted to determine a possible secondary structure of the DNA enzyme. This study lays a foundation for pursuing novel signaling DNA enzymes for biological detection directed applications. / Thesis / Master of Science (MSc)

Sequences at the 5' end of the nascent RNA are known to be important as signals for encapsidation of the genome of vesicular stomatitis virus. In order to define the specific sequences involved in this process and to develop an in vitro encapsidation system, in vitro transcription from SP64-based plasmids was used to synthesize RNA molecules corresponding to various portions of the viral 5' plus strand sequence. Some of these RNAs were tested for their ability to bind the capsid N protein in vitro. N protein in this assay was provided either from VSV mRNA programmed reticulocyte lysates or from infected cell extracts or, in collaboration with Dr. Sue Moyer (Gainesville, Florida), purified from viral nucleocapsids. This thesis describes the construction of the SP64-based plasmids and the use of their RNA transcription products in the experiments described above. I also constructed a series of plasmids that could direct the synthesis of RNA molecules which have many of the features of VSV defective particle genomes. Two of the constructs generate a defective-like RNA carrying a reporter gene capable of expressing the bacterial lac Z protein. These RNAs have the potential, after in vitro encapsidation and transfection into mammalian cells, of producing readily detectable helper-dependent virions. / Thesis / Master of Science (MS)

In vitro liver models that closely mimic the in vivo microenvironment are central for understanding hepatic functions and intercellular communication processes. Bottom-up shotgun proteomic analysis of the hepatic cells can lend insight into such processes. This technique employs liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of protein abundances by measuring intensities of their corresponding peptides. Organotypic 3D liver models have been developed in our laboratory that consist of hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM), which serves as a mimic for the Space of Disse. Each component within these models is easily separable allowing for systematic evaluation of the cells and PEMs. In this study, proteomes of hepatocytes from PEM containing models, cultured with and without LSECs, were compared to those from monolayers. Changes in core metabolism were evaluated among all culture conditions. Overall, all cultures were ketogenic and performed gluconeogenesis. The presence of the PEM led to increases in proteins associated with mitochondrial-based β-oxidation and peroxisomal proteins. The PEMs also limited production of structural proteins, which are linked to dedifferentiation of hepatocytes, suggesting that cell-ECM interactions are essential for maintenance of their liver-like state. The presence of LSECs increased levels of carboxylesterases and other phase I and phase II detoxification enzymes suggesting that intercellular signaling mediates enzyme abundance. Taken together, these results suggest that the cell-cell (from the LSECs) and cell-ECM (from the PEMs) interactions exert different, yet crucial effects, and both are required for the preservation of metabolic liver functions and differentiated phenotypes. Changes in the PEMs as a result of cell culture were also evaluated but exhibited minimal differences at this time point. Proteomes of LSECs monolayers were also characterized. Enzymes related to the metabolism of amino acids, lipids, oxidative phosphorylation and phase I and phase II detoxification processes were all identified in LSECs monolayers highlighting their role in these processes. Characterization of 3DHL LSECs was not possible due to ion suppression resulting from the presence of excess contaminant proteins. Nonetheless, this study provides a foundation in which LSECs from 3D liver models can be compared against in future studies. / Ph. D.