Class of 2013 Abstract / Specific Aims: To elucidate the effect of the use of three different inlet configurations, percent ethanol in formulation, and propellant used on the percent respirable drug and MMAD of aerosolized particles from MDIs that contained beclomethasone dipropionate (BDP). Methods: The inlet configurations assessed in this study were the United States Pharmacopeia (USP) throat, the Alberta idealized mouth-throat replica (biological throat), and a large volume spacer (globe). ACI analyses were conducted on four different MDI formulations with regards to each of the three inlet configurations in quadruplicate. The two hydrofluoroalkane propellants assessed were HFA-134 and HFA-227. All four solution formulations contained 0.3% (w/w) beclomethasone dipropionate (BDP), two of which contained 8% (w/w) ethanol (one each with HFA-134a and HFA-227) and two contained 20% (w/w) ethanol (one each with HFA-134a and HFA-227). All experiments were conducted at a flow rate of 28.3L/min using an actuator with an orifice diameter of 0.29mm and a 50μL metered-valve. After each ACI test, the drug collected on each stage of the impactor was rinsed with known volumes of diluent and quantified by high performance liquid chromatography (HPLC). The MMAD was determined by using DistFit to lognormally fit the ACI data. The resiprable fraction was calculated as the mass of the drug collected on stages 3 through filter of the ACI divided by the total mass of the drug aerosolized. The two-sided student's t-test was the statistical test utilized, with an a priori alpha-value of 0.05. Main Results: The USP and biological throats had significantly lower percent respirable drug compared to the globe regardless of concentration of ethanol or propellant (p<0.05). The MMADs were significantly lower for configurations with the USP and biological throats as compared to the globe (p<0.05). The only formulation with a significant percent respirable drug difference between the USP and biological throats regarding was the 20% ethanol/HFA-227 formulation (20.9+/-0.15 and 16.8+/-1.3 respectively, p=0.005), with the USP throat having the significantly greater percent respirable drug. The USP throat had significantly larger MMADs compared to the biological throat regardless of formulation (p<0.05). For both propellants, the 8% ethanol formulation had significantly greater percent respirable drug compared to the 20% formulation for all three inlets (p<0.05). The 20% ethanol formulations had significantly higher MMADs compared to the 8% ethanol formulations in both the USP throat and globe and with both propellants (p<0.05). Only the 20% ethanol formulations demonstrated a significant difference when varying propellant while keeping all else constant, with the HFA-134a formulations having higher percent respirable drug with all inlets as compared to HFA-227 (p<0.05). When propellant used was varied with all else kept constant, the HFA-227 formulations had significantly higher MMADs compared to the HFA-134a formulations (p<0.05). Conclusion: It was found that significant differences in percent respirable drug and particle size (MMAD) resulting from varying inlet configurations was a function of formulation parameters, most notably, ethanol concentration. The differences may be attributed to factors that increased the time necessary for the evaporation of atomized particles prior to deposition in the impactor, the initial atomized droplet diameter, and/or the likelihood of particle impaction with regards to the mouth-throat inlet utilized. Further assessment is needed to evaluate the correlation of this data with in vivo analyses.

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)