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Red cell development in adult humans results in the mean daily production of 2x1011 erythrocytes. Mature hemoglobinized and enucleated erythrocytes develop from multipotent hematopoietic stem/progenitor cells through more committed progenitor cell types such as BFU-E and CFU-E. The studies on the molecular mechanisms of erythropoiesis in the human system require a sufficient number of purified erythroid progenitors of the different stages of erythropoiesis. Primary human erythroid progenitors are difficult to obtain as a homogenous population in sufficiently high cell numbers. Various culture conditions for the in vitro cell culture of primary human erythroid progenitors have been previously described. Mainly, the culture resulted in the generation of rather mature stages of Epo-dependent erythroid progenitors. In this study our efforts were directed towards the isolation and characterization of more early red cell progenitors that are Epo-independent. To identify such progenitors, CD34+ cells were purified from cord blood and cultured under serum free conditions in the presence of the growth factors SCF, IL-3 and hyper-IL-6, referred to as SI2 culture conditions. By immunomagnetic bead selection of E-cadherin (E-cad) positive cells, E-cad+ progenitors were isolated. These Epo-independent E-cad+ progenitors have been amplified under SI2 conditions to large cell numbers. The E-cad+ progenitors were characterized for surface antigen expression by flow cytometry, response to growth factors in proliferation assay and for their differentiation potential into mature red cells. Additionally, the properties of E-cad+ progenitors were compared to those of two other erythroid progenitors: Epo-dependent progenitors described by Panzenböck et al. (referred to as SCF/Epo progenitor), and CD36+ progenitors described by Freyssinier et al. (Panzenböck et al., 1998; Freyssinier et al., 1999). Finally, the gene expression profile of E-cad+ progenitors was compared to the profiles of SCF/Epo progenitors and CD36+ progenitors using the DNA microarray technique. Based on our studies we propose that Epo-independent E-cad+ progenitors are early stage, BFU-E like progenitors. They respond to Epo, despite the fact that they were generated in the absence of Epo, and can completely undergo erythroid differentiation. Furthermore, we demonstrate that the growth properties, the growth factor response and the surface marker expression of E-cad+ progenitors are similar to those of the SCF/Epo progenitors and the CD36+ progenitors. By the comparison of gene profiles, we were also able to demonstrate that the Epo-dependent and Epo-independent red cell progenitors are very similar. Analyzing the molecular differences between E-cad+ and SCF/Epo progenitors revealed several candidate genes such as galectin-3, cyclin D1, AMHR, PDF and IGFBP4, which are potential regulators involved in red cell development. We also demonstrate that the CD36+ progenitors, isolated by immunomagentic bead selection, are a heterogeneous progenitor population containing an E-cad+ and an E-cad- subpopulation. Based on their gene expression profile, CD36+ progenitors seem to exhibit both erythroid and megakaryocytic features. These studies led to a more updated model of erythroid cell development that should pave the way for further studies on molecular mechanisms of erythropoiesis.
In this thesis, I investigated the factors influencing the growth and vertical distribution of planktonic algae in extremely acidic mining lakes (pH 2-3). In the focal study site, Lake 111 (pH 2.7; Lusatia, Germany), the chrysophyte, Ochromonas sp., dominates in the upper water strata and the chlorophyte, Chlamydomonas sp., in the deeper strata, forming a pronounced deep chlorophyll maximum (DCM). Inorganic carbon (IC) limitation influenced the phototrophic growth of Chlamydomonas sp. in the upper water strata. Conversely, in deeper strata, light limited its phototrophic growth. When compared with published data for algae from neutral lakes, Chlamydomonas sp. from Lake 111 exhibited a lower maximum growth rate, an enhanced compensation point and higher dark respiration rates, suggesting higher metabolic costs due to the extreme physico-chemical conditions. The photosynthetic performance of Chlamydomonas sp. decreased in high-light-adapted cells when IC limited. In addition, the minimal phosphorus (P) cell quota was suggestive of a higher P requirement under IC limitation. Subsequently, it was shown that Chlamydomonas sp. was a mixotroph, able to enhance its growth rate by taking up dissolved organic carbon (DOC) via osmotrophy. Therefore, it could survive in deeper water strata where DOC concentrations were higher and light limited. However, neither IC limitation, P availability nor in situ DOC concentrations (bottom-up control) could fully explain the vertical distribution of Chlamydomonas sp. in Lake 111. Conversely, when a novel approach was adopted, the grazing influence of the phagotrophic phototroph, Ochromonas sp., was found to exert top-down control on its prey (Chlamydomonas sp.) reducing prey abundance in the upper water strata. This, coupled with the fact that Chlamydomonas sp. uses DOC for growth, leads to a pronounced accumulation of Chlamydomonas sp. cells at depth; an apparent DCM. Therefore, grazing appears to be the main factor influencing the vertical distribution of algae observed in Lake 111. The knowledge gained from this thesis provides information essential for predicting the effect of strategies to neutralize the acidic mining lakes on the food-web.
Today, analytical chemistry does not longer consist of only the big measuring devices and methods which are time consuming and expensive, which can furthermore only be handled by the qualified staff and in addition the results can also only be evaluated by this qualified staff. Usually, this technique, which shall be described in the following as 'classic analytic measuring technique', requires also rooms equipped especially and often a relative big quantity of the test compounds which should be prepared especially. Beside this classic analytic measuring technique, limited on definite substance groups and requests, a new measuring technique has gained acceptance particularly within the last years, which one can often be used by a layman, too. Often the new measuring technique has very little pieces of equipment. The needed sample volumes are also small and a special sample preparation isn't required. In addition, the new measuring instruments are simple to handle. They are cheap both in their production and in the use and they permit even a continuous measurement recording usually. Numerous of this new measuring instruments base on the research in the field of Biosensorik during the last 40 years. Since Clark and Lyon in the year 1962 were able to measure glucose with a simple oxygen electrode, completed by an enzyme the development of the new measuring technique did not have to be held back any longer. Biosensors, special pickups which consists of a combination from a biological component (permits a specific recognition of the analyte also without purification of the sample previously) and a physical pickup (convert the primary physicochemical effect into an electronically measurable signal), conquered the market. In the context of this thesis different tyrosinasesensors were developed which fulfilling the various requests, depending on origin and features of the used tyrosinase. One of the tyrosinasesensors for example was used for quantification of phenolic compounds in river and sea water and the results could correlated very well with the corresponding DIN-test for the determination of phenolic compounds. An other developed tyrosinasesensor showed a very high sensitiveness for catecholamines, substances which are of special importance in the medical diagnostics. In addition, the investigations of two different tyrosinases, which were carried out also in the context of this thesis, have shown, that a special tyrosinase (tyrosinase from Streptomyces antibioticus) will be the better choice as tyrosinase from Agaricus bisporus, which is used in the area of biosensor research till now, if one wants to develop in future even more sensitive tyrosinasesensors. Furthermore, first successes became reached on a molecular biological field, the production of tyrosinasemutants with special, before well-considered features. These successes can be used to develop a new generation of tyrosinasesensors, tyrosinasesensors in which tyrosinase can be bound directionally both to the corresponding physical pickup or also to another enzyme. From this one expects to achieve ways minimized which the substance to be determined (or whose product) otherwise must cover. Finally, this should result in an clearly visible increase of sensitivity of the Biosensor.