@phdthesis{Šustr2020,
  author    = {Šustr, David},
  title     = {Molecular diffusion in polyelectrolyte multilayers},
  doi       = {10.25932/publishup-48903},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-489038},
  school      = {Universit{\"a}t Potsdam},
  pages     = {106},
  year      = {2020},
  abstract  = {Research on novel and advanced biomaterials is an indispensable step towards their applications in desirable fields such as tissue engineering, regenerative medicine, cell culture, or biotechnology. The work presented here focuses on such a promising material: polyelectrolyte multilayer (PEM) composed of hyaluronic acid (HA) and poly(L-lysine) (PLL). This gel-like polymer surface coating is able to accumulate (bio-)molecules such as proteins or drugs and release them in a controlled manner. It serves as a mimic of the extracellular matrix (ECM) in composition and intrinsic properties. These qualities make the HA/PLL multilayers a promising candidate for multiple bio-applications such as those mentioned above. The work presented aims at the development of a straightforward approach for assessment of multi-fractional diffusion in multilayers (first part) and at control of local molecular transport into or from the multilayers by laser light trigger (second part). The mechanism of the loading and release is governed by the interaction of bioactives with the multilayer constituents and by the diffusion phenomenon overall. The diffusion of a molecule in HA/PLL multilayers shows multiple fractions of different diffusion rate. Approaches, that are able to assess the mobility of molecules in such a complex system, are limited. This shortcoming motivated the design of a novel evaluation tool presented here. The tool employs a simulation-based approach for evaluation of the data acquired by fluorescence recovery after photobleaching (FRAP) method. In this approach, possible fluorescence recovery scenarios are primarily simulated and afterwards compared with the data acquired while optimizing parameters of a model until a sufficient match is achieved. Fluorescent latex particles of different sizes and fluorescein in an aqueous medium are utilized as test samples validating the analysis results. The diffusion of protein cytochrome c in HA/PLL multilayers is evaluated as well. This tool significantly broadens the possibilities of analysis of spatiotemporal FRAP data, which originate from multi-fractional diffusion, while striving to be widely applicable. This tool has the potential to elucidate the mechanisms of molecular transport and empower rational engineering of the drug release systems. The second part of the work focuses on the fabrication of such a spatiotemporarily-controlled drug release system employing the HA/PLL multilayer. This release system comprises different layers of various functionalities that together form a sandwich structure. The bottom layer, which serves as a reservoir, is formed by HA/PLL PEM deposited on a planar glass substrate. On top of the PEM, a layer of so-called hybrids is deposited. The hybrids consist of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) -based hydrogel microparticles with surface-attached gold nanorods. The layer of hybrids is intended to serve as a gate that controls the local molecular transport through the PEM-solution-interface. The possibility of stimulating the molecular transport by near-infrared (NIR) laser irradiation is being explored. From several tested approaches for the deposition of hybrids onto the PEM surface, the drying-based approach was identified as optimal. Experiments, that examine the functionality of the fabricated sandwich at elevated temperature, document the reversible volume phase transition of the PEM-attached hybrids while sustaining the sandwich stability. Further, the gold nanorods were shown to effectively absorb light radiation in the tissue- and cell-friendly NIR spectral region while transducing the energy of light into heat. The rapid and reversible shrinkage of the PEM-attached hybrids was thereby achieved. Finally, dextran was employed as a model transport molecule. It loads into the PEM reservoir in a few seconds with the partition constant of 2.4, while it spontaneously releases in a slower, sustained manner. The local laser irradiation of the sandwich, which contains the fluorescein isothiocyanate tagged dextran, leads to a gradual reduction of fluorescence intensity in the irradiated region. The release system fabricated employs renowned photoresponsivity of the hybrids in an innovative setting. The results of the research are a step towards a spatially-controlled on-demand drug release system that paves the way to spatiotemporally controlled drug release. The approaches developed in this work have the potential to elucidate the molecular dynamics in ECM and to foster engineering of multilayers with properties tuned to mimic the ECM. The work aims at spatiotemporal control over the diffusion of bioactives and their presentation to the cells.},
  language  = {en}
}
@phdthesis{Ziege2022,
  author    = {Ziege, Ricardo},
  title     = {Growth dynamics and mechanical properties of E. coli biofilms},
  doi       = {10.25932/publishup-55986},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-559869},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xi, 123},
  year      = {2022},
  abstract  = {Biofilms are complex living materials that form as bacteria get embedded in a matrix of self-produced protein and polysaccharide fibres. The formation of a network of extracellular biopolymer fibres contributes to the cohesion of the biofilm by promoting cell-cell attachment and by mediating biofilm-substrate interactions. This sessile mode of bacteria growth has been well studied by microbiologists to prevent the detrimental effects of biofilms in medical and industrial settings. Indeed, biofilms are associated with increased antibiotic resistance in bacterial infections, and they can also cause clogging of pipelines or promote bio-corrosion. However, biofilms also gained interest from biophysics due to their ability to form complex morphological patterns during growth. Recently, the emerging field of engineered living materials investigates biofilm mechanical properties at multiple length scales and leverages the tools of synthetic biology to tune the functions of their constitutive biopolymers. This doctoral thesis aims at clarifying how the morphogenesis of Escherichia coli (E. coli) biofilms is influenced by their growth dynamics and mechanical properties. To address this question, I used methods from cell mechanics and materials science. I first studied how biological activity in biofilms gives rise to non-uniform growth patterns. In a second study, I investigated how E. coli biofilm morphogenesis and its mechanical properties adapt to an environmental stimulus, namely the water content of their substrate. Finally, I estimated how the mechanical properties of E. coli biofilms are altered when the bacteria express different extracellular biopolymers. On nutritive hydrogels, micron-sized E. coli cells can build centimetre-large biofilms. During this process, bacterial proliferation and matrix production introduce mechanical stresses in the biofilm, which release through the formation of macroscopic wrinkles and delaminated buckles. To relate these biological and mechanical phenomena, I used time-lapse fluorescence imaging to track cell and matrix surface densities through the early and late stages of E. coli biofilm growth. Colocalization of high cell and matrix densities at the periphery precede the onset of mechanical instabilities at this annular region. Early growth is detected at this outer annulus, which was analysed by adding fluorescent microspheres to the bacterial inoculum. But only when high rates of matrix production are present in the biofilm centre, does overall biofilm spreading initiate along the solid-air interface. By tracking larger fluorescent particles for a long time, I could distinguish several kinematic stages of E. coli biofilm expansion and observed a transition from non-linear to linear velocity profiles, which precedes the emergence of wrinkles at the biofilm periphery. Decomposing particle velocities to their radial and circumferential components revealed a last kinematic stage, where biofilm movement is mostly directed towards the radial delaminated buckles, which verticalize. The resulting compressive strains computed in these regions were observed to substantially deform the underlying agar substrates. The co-localization of higher cell and matrix densities towards an annular region and the succession of several kinematic stages are thus expected to promote the emergence of mechanical instabilities at the biofilm periphery. These experimental findings are predicted to advance future modelling approaches of biofilm morphogenesis. E. coli biofilm morphogenesis is further anticipated to depend on external stimuli from the environment. To clarify how the water could be used to tune biofilm material properties, we quantified E. coli biofilm growth, wrinkling dynamics and rigidity as a function of the water content of the nutritive substrates. Time-lapse microscopy and computational image analysis revealed that substrates with high water content promote biofilm spreading kinetics, while substrates with low water content promote biofilm wrinkling. The wrinkles observed on biofilm cross-sections appeared more bent on substrates with high water content, while they tended to be more vertical on substrates with low water content. Both wet and dry biomass, accumulated over 4 days of culture, were larger in biofilms cultured on substrates with high water content, despite extra porosity within the matrix layer. Finally, the micro-indentation analysis revealed that substrates with low water content supported the formation of stiffer biofilms. This study shows that E. coli biofilms respond to the water content of their substrate, which might be used for tuning their material properties in view of further applications. Biofilm material properties further depend on the composition and structure of the matrix of extracellular proteins and polysaccharides. In particular, E. coli biofilms were suggested to present tissue-like elasticity due to a dense fibre network consisting of amyloid curli and phosphoethanolamine-modified cellulose. To understand the contribution of these components to the emergent mechanical properties of E. coli biofilms, we performed micro-indentation on biofilms grown from bacteria of several strains. Besides showing higher dry masses, larger spreading diameters and slightly reduced water contents, biofilms expressing both main matrix components also presented high rigidities in the range of several hundred kPa, similar to biofilms containing only curli fibres. In contrast, a lack of amyloid curli fibres provides much higher adhesive energies and more viscoelastic fluid-like material behaviour. Therefore, the combination of amyloid curli and phosphoethanolamine-modified cellulose fibres implies the formation of a composite material whereby the amyloid curli fibres provide rigidity to E. coli biofilms, whereas the phosphoethanolamine-modified cellulose rather acts as a glue. These findings motivate further studies involving purified versions of these protein and polysaccharide components to better understand how their interactions benefit biofilm functions. All three studies depict different aspects of biofilm morphogenesis, which are interrelated. The first work reveals the correlation between non-uniform biological activities and the emergence of mechanical instabilities in the biofilm. The second work acknowledges the adaptive nature of E. coli biofilm morphogenesis and its mechanical properties to an environmental stimulus, namely water. Finally, the last study reveals the complementary role of the individual matrix components in the formation of a stable biofilm material, which not only forms complex morphologies but also functions as a protective shield for the bacteria it contains. Our experimental findings on E. coli biofilm morphogenesis and their mechanical properties can have further implications for fundamental and applied biofilm research fields.},
  language  = {en}
}
@phdthesis{Zhang2020,
  author    = {Zhang, Jianrui},
  title     = {Completely water-based emulsions as compartmentalized systems via pickering stabilization},
  doi       = {10.25932/publishup-47654},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-476542},
  school      = {Universit{\"a}t Potsdam},
  pages     = {II, 119},
  year      = {2020},
  abstract  = {Completely water-based systems are of interest for the development of novel material for various reasons: On one hand, they provide benign environment for biological systems and on the other hand they facilitate effective molecular transport in a membrane-free environment. In order to investigate the general potential of aqueous two-phase systems (ATPSs) for biomaterials and compartmentalized systems, various solid particles were applied to stabilize all-aqueous emulsion droplets. The target ATPS to be investigated should be prepared via mixing of two aqueous solutions of water-soluble polymers, which turn biphasic when exceeding a critical polymer concentration. Hydrophilic polymers with a wide range of molar mass such as dextran/poly(ethylene glycol) (PEG) can therefore be applied. Solid particles adsorbed at the interfaces can be exceptionally efficient stabilizers forming so-called Pickering emulsions, and nanoparticles can bridge the correlation length of polymer solutions and are thereby the best option for water-in-water emulsions. The first approach towards the investigation of ATPS was conducted with all aqueous dextran-PEG emulsions in the presence of poly(dopamine) particles (PDP) in Chapter 4. The water-in-water emulsions were formed with a PEG/dextran system via utilizing PDP as stabilizers. Studies of the formed emulsions were performed via laser scanning confocal microscope (CLSM), optical microscope (OM), cryo-scanning electron microscope (SEM) and tensiometry. The stable emulsions (at least 16 weeks) were demulsified easily via dilution or surfactant addition. Furthermore, the solid PDP at the water-water interface were crosslinked in order to inhibit demulsification of the Pickering emulsion. Transmission electron microscope (TEM) and scanning electron microscope (SEM) were used to visualize the morphology of PDP before and after crosslinking. PDP stabilized water-in-water emulsions were utilized in the following Chapter 5 to form supramolecular compartmentalized hydrogels. Here, hydrogels were prepared in pre-formed water-in-water emulsions and gelled via α-cyclodextrin-PEG (α-CD-PEG) inclusion complex formation. Studies of the formed complexes were performed via X-ray powder diffraction (XRD) and the mechanical properties of the hydrogels were measured with oscillatory shear rheology. In order to verify the compartmentalized state and its triggered decomposition, hydrogels and emulsions were assessed via OM, SEM and CLSM. The last chapter broadens the investigations from the previous two systems by utilizing various carbon nitrides (CN) as different stabilizers in ATPS. CN introduces another way to trigger demulsification, namely irradiation with visible light. Therefore, emulsification and demulsification with various triggers were probed. The investigated all aqueous multi-phase systems will act as model for future fabrication of biocompatible materials, cell micropatterning as well as separation of compartmentalized systems.},
  language  = {en}
}
@phdthesis{Zhang2005,
  author    = {Zhang, Baichen},
  title     = {Dissection of phloem transport in cucurbitaceae by metabolomic analysis},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-6644},
  school      = {Universit{\"a}t Potsdam},
  year      = {2005},
  abstract  = {This thesis aimed to investigate several fundamental and perplexing questions relating to the phloem loading and transport mechanisms of Cucurbita maxima, by combining metabolomic analysis with cell biological techniques. This putative symplastic loading species has long been used for experiments on phloem anatomy, phloem biochemistry, phloem transport physiology and phloem signalling. Symplastic loading species have been proposed to use a polymer trapping mechanism to accumulate RFO (raffinose family oligosaccharides) sugars to build up high osmotic pressure in minor veins which sustains a concentration gradient that drives mass flow. However, extensive evidence indicating a low sugar concentration in their phloem exudates is a long-known problem that conflicts with this hypothesis. Previous metabolomic analysis shows the concentration of many small molecules in phloem exudates is higher than that of leaf tissues, which indicates an active apoplastic loading step. Therefore, in the view of the phloem metabolome, a symplastic loading mechanism cannot explain how small molecules other than RFO sugars are loaded into phloem. Most studies of phloem physiology using cucurbits have neglected the possible functions of vascular architecture in phloem transport. It is well known that there are two phloem systems in cucurbits with distinctly different anatomical features: central phloem and extrafascicular phloem. However, mistaken conclusions on sources of cucurbit phloem exudation from previous reports have hindered consideration of the idea that there may be important differences between these two phloem systems. The major results are summarized as below: 1) O-linked glycans in C.maxima were structurally identified as beta-1,3 linked glucose polymers, and the composition of glycans in cucurbits was found to be species-specific. Inter-species grafting experiments proved that these glycans are phloem mobile and transported uni-directionally from scion to stock. 2) As indicated by stable isotopic labelling experiments, a considerable amount of carbon is incorporated into small metabolites in phloem exudates. However, the incorporation of carbon into RFO sugars is much faster than for other metabolites. 3) Both CO2 labelling experiments and comparative metabolomic analysis of phloem exudates and leaf tissues indicated that metabolic processes other than RFO sugar metabolism play an important role in cucurbit phloem physiology. 4) The underlying assumption that the central phloem of cucurbits continuously releases exudates after physical incision was proved wrong by rigorous experiments including direct observation by normal microscopy and combined multiple-microscopic methods. Errors in previous experimental confirmation of phloem exudation in cucurbits are critically discussed. 5) Extrafascicular phloem was proved to be functional, as indicated by phloem-mobile carboxyfluorescein tracer studies. Commissural sieve tubes interconnect phloem bundles into a complete super-symplastic network. 6) Extrafascicular phloem represents the main source of exudates following physical incision. The major transported metabolites by these extrafacicular phloem are non-sugar compounds including amino acids, O-glycans, amines. 7) Central phloem contains almost exclusively RFO sugars, the estimated amount of which is up to 1 to 2 molar. The major RFO sugar present in central phloem is stachyose. 8) Cucurbits utilize two structurally different phloem systems for transporting different group of metabolites (RFO sugars and non-RFO sugar compounds). This implies that cucurbits may use spatially separated loading mechanisms (apoplastic loading for extrafascicular phloem and symplastic loading for central phloem) for supply of nutrients to sinks. 9) Along the transport systems, RFO sugars were mainly distributed within central phloem tissues. There were only small amounts of RFO sugars present in xylem tissues (millimolar range) and trace amounts of RFO sugars in cortex and pith. The composition of small molecules in external central phloem is very different from that in internal central phloem. 10) Aggregated P-proteins were manually dissected from central phloem and analysed by both SDS-PAGE and mass spectrometry. Partial sequences of peptides were obtained by QTOF de novo sequencing from trypsin digests of three SDS-PAGE bands. None of these partial sequences shows significant homology to known cucurbit phloem proteins or other plant proteins. This proves that these central phloem proteins are a completely new group of proteins different from those in extrafascicular phloem. The extensively analysed P-proteins reported in literature to date are therefore now shown to arise from extrafascicular phloem and not central phloem, and therefore do not appear to be involved in the occlusion processes in central phloem.},
  subject      = {phloem},
  language  = {en}
}
@phdthesis{Zemella2019,
  author    = {Zemella, Anne},
  title     = {Fluoreszenzmarkierung und Modifizierung von komplexen Proteinen in eukaryotischen zellfreien Systemen durch die Etablierung von orthogonalen tRNA/Aminoacyl-tRNA-Synthetase-Paaren},
  doi       = {10.25932/publishup-44236},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-442361},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XI, 141},
  year      = {2019},
  abstract  = {Die funktionelle Charakterisierung von therapeutisch relevanten Proteinen kann bereits durch die Bereitstellung des Zielproteins in ad{\"a}quaten Mengen limitierend sein. Dies trifft besonders auf Membranproteine zu, die aufgrund von zytotoxischen Effekten auf die Produktionszelllinie und der Tendenz Aggregate zu bilden, in niedrigen Ausbeuten an aktivem Protein resultieren k{\"o}nnen. Der lebende Organismus kann durch die Verwendung von translationsaktiven Zelllysaten umgangen werden- die Grundlage der zellfreien Proteinsynthese. Zu Beginn der Arbeit wurde die ATP-abh{\"a}ngige Translation eines Lysates auf der Basis von kultivierten Insektenzellen (Sf21) analysiert. F{\"u}r diesen Zweck wurde ein ATP-bindendes Aptamer eingesetzt, durch welches die Translation der Nanoluziferase reguliert werden konnte. Durch die dargestellte Applizierung von Aptameren, k{\"o}nnten diese zuk{\"u}nftig in zellfreien Systemen f{\"u}r die Visualisierung der Transkription und Translation eingesetzt werden, wodurch zum Beispiel komplexe Prozesse validiert werden k{\"o}nnen. Neben der reinen Proteinherstellung k{\"o}nnen Faktoren wie posttranslationale Modifikationen sowie eine Integration in eine lipidische Membran essentiell f{\"u}r die Funktionalit{\"a}t des Membranproteins sein. Im zweiten Abschnitt konnte, im zellfreien Sf21-System, f{\"u}r den G-Protein-gekoppelten Rezeptor Endothelin B sowohl eine Integration in die endogen vorhandenen Endoplasmatisch Retikulum-basierten Membranstrukturen als auch Glykosylierungen, identifiziert werden. Auf der Grundlage der erfolgreichen Synthese des ET-B-Rezeptors wurden verschiedene Methoden zur Fluoreszenzmarkierung des Adenosin-Rezeptors A2a (Adora2a) angewandt und optimiert. Im dritten Abschnitt wurde der Adora2a mit Hilfe einer vorbeladenen tRNA, welche an eine fluoreszierende Aminos{\"a}ure gekoppelt war, im zellfreien Chinesischen Zwerghamster Ovarien (CHO)-System markiert. Zus{\"a}tzlich konnte durch den Einsatz eines modifizierten tRNA/Aminoacyl-tRNA-Synthetase-Paares eine nicht-kanonische Aminos{\"a}ure an Position eines integrierten Amber-Stopcodon in die Polypeptidkette eingebaut und die funktionelle Gruppe im Anschluss an einen Fluoreszenzfarbstoff gekoppelt werden. Aufgrund des offenen Charakters eignen sich zellfreie Proteinsynthesesysteme besonders f{\"u}r eine Integration von exogenen Komponenten in den Translationsprozess. Mit Hilfe der Fluoreszenzmarkierung wurde eine ligandvermittelte Konformations{\"a}nderung im Adora2a {\"u}ber einen Biolumineszenz-Resonanzenergietransfer detektiert. Durch die Etablierung der Amber-Suppression wurde dar{\"u}ber hinaus das Hormon Erythropoetin pegyliert, wodurch Eigenschaften wie Stabilit{\"a}t und Halbwertszeit des Proteins ver{\"a}ndert wurden. Zu guter Letzt wurde ein neues tRNA/Aminoacyl-tRNA-Synthetase-Paar auf Basis der Methanosarcina mazei Pyrrolysin-Synthetase etabliert, um das Repertoire an nicht-kanonischen Aminos{\"a}uren und den damit verbundenen Kopplungsreaktionen zu erweitern. Zusammenfassend wurden die Potenziale zellfreier Systeme in Bezug auf der Herstellung von komplexen Membranproteinen und der Charakterisierung dieser durch die Einbringung einer positionsspezifischen Fluoreszenzmarkierung verdeutlicht, wodurch neue M{\"o}glichkeiten f{\"u}r die Analyse und Funktionalisierung von komplexen Proteinen geschaffen wurden.},
  language  = {de}
}
@phdthesis{Zaupa2010,
  author    = {Zaupa, Alessandro},
  title     = {Physical crosslinking of gelatin : a supramolecular approach to biomaterials},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-52888},
  school      = {Universit{\"a}t Potsdam},
  year      = {2010},
  abstract  = {This work describes the realization of physically crosslinked networks based on gelatin by the introduction of functional groups enabling specific supramolecular interactions. Molecular models were developed in order to predict the material properties and permit to establish a knowledge-based approach to material design. The effect of additional supramolecular interactions with hydroxyapaptite was then studied in composite materials. The calculated properties are compared to experimental results to validate the models. The models are then further used for the study of physically crosslinked networks. Gelatin was functionalized with desaminotyrosine (DAT) and desaminotyrosyl-tyrosine (DATT) side groups, derived from the natural amino acid tyrosine. These group can potentially undergo to π-π and hydrogen bonding interactions also under physiological conditions. Molecular dynamics (MD) simulations were performed on models with 0.8 wt.-\% or 25 wt.-\% water content, using the second generation forcefield CFF91. The validation of the models was obtained by the comparison with specific experimental data such as, density, peptide conformational angles and X-ray scattering spectra. The models were then used to predict the supramolecular organization of the polymer chain, analyze the formation of physical netpoints and calculate the mechanical properties. An important finding of simulation was that with the increase of aromatic groups also the number of observed physical netpoints increased. The number of relatively stable physical netpoints, on average zero 0 for natural gelatin, increased to 1 and 6 for DAT and DATT functionalized gelatins respectively. A comparison with the Flory-Rehner model suggested reduced equilibrium swelling by factor 6 of the DATT-functionalized materials in water. The functionalized gelatins could be synthesized by chemoselective coupling of the free carboxylic acid groups of DAT and DATT to the free amino groups of gelatin. At 25 wt.-\% water content, the simulated and experimentally determined elastic mechanical properties (e.g. Young Modulus) were both in the order of GPa and were not influenced by the degree of aromatic modification. The experimental equilibrium degree of swelling in water decreased with increasing the number of inserted aromatic functions (from 2800 vol.-\% for pure gelatin to 300 vol.-\% for the DATT modified gelatin), at the same time, Young's modulus, elongation at break, and maximum tensile strength increased. It could be show that the functionalization with DAT and DATT influences the chain organization of gelatin based materials together with a controlled drying condition. Functionalization with DAT and DATT lead to a drastic reduction of helical renaturation, that could be more finely controlled by the applied drying conditions. The properties of the materials could then be influenced by application of two independent methods. Composite materials of DAT and DATT functionalized gelatins with hydroxyapatite (HAp) show a drastic reduction of swelling degree. In tensile tests and rheological measurements, the composites equilibrated in water had increased Young's moduli (from 200 kPa up to 2 MPa) and tensile strength (from 57 kPa up to 1.1 MPa) compared to the natural polymer matrix without affecting the elongation at break. Furthermore, an increased thermal stability from 40 °C to 85 °C of the networks could be demonstrated. The differences of the behaviour of the functionalized gelatins to pure gelatin as matrix suggested an additional stabilizing bond between the incorporated aromatic groups to the hydroxyapatite.},
  language  = {en}
}
@phdthesis{Yildiz2023,
  author    = {Yildiz, Tugba},
  title     = {Dissecting the role of the TusA protein for cell functionality and FtsZ ring assembly in Escherichia coli},
  doi       = {10.25932/publishup-61713},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-617135},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XI, 171},
  year      = {2023},
  abstract  = {In this work, the role of the TusA protein was investigated for the cell functionality and FtsZ ring assembly in Escherichia coli. TusA is the tRNA-2-thiouridine synthase that acts as a sulfur transferase in tRNA thiolation for the formation of 2-thiouridine at the position 34 (wobble base) of tRNALys, tRNAGlu and tRNAGln. It binds the persulfide form of sulfur and transfers it to further proteins during mnm5s2U tRNA modification at wobble position and for Moco biosynthesis. With this thiomodification of tRNA, the ribosome binding is more efficient and frameshifting is averted during the protein translation. Previous studies have revealed an essential role of TusA in bacterial cell physiology since deletion of the tusA gene resulted in retarded growth and filamentous cells during the exponential growth phase in a rich medium which suddenly disappeared during the stationary phase. This indicates a problem in the cell division process. Therefore the focus of this work was to investigate the role of TusA for cell functionality and FtsZ ring formation and thus the cell separation. The reason behind the filamentous growth of the tusA mutant strain was investigated by growth and morphological analyses. ΔtusA cells showed a retarded growth during the exponential phase compared to the WT strain. Also, morphological analysis of ΔtusA cells confirmed the filamentous cell shape. The growth and cell division defects in ΔtusA indicated a defect in FtsZ protein as a key player of cell division. The microscopic investigation revealed that filamentous ΔtusA cells possessed multiple DNA parts arranged next to each other. This suggested that although the DNA replication occurred correctly, there was a defect in the step where FtsZ should act; probably FtsZ is unable to assemble to the ring structure or the assembled ring is not able to constrict. All tested mutant strains (ΔtusD, ΔtusE and ΔmnmA) involved in the mnm5s2U34 tRNA modification pathway shared the similar retarded growth and filamentous cell shape like ΔtusA strain. Thus, the cell division defect arises from a defect in mnm5s2U34 tRNA thiolation. Since the FtsZ ring formation was supposed to be defective in filaments, a possible intracellular interaction of TusA and FtsZ was examined by fluorescent (EGFP and mCherry) fusion proteins expression and FRET. FtsZ expressing tusA mutant (DE3) cells showed a red mCherry signal at the cell poles, indicating that FtsZ is still in the assembling phase. Interestingly, the cellular region of EGFP-TusA fusion protein expressed in ΔtusA (DE3) was conspicuous; the EGFP signal was spread throughout the whole cell and, in addition, a slight accumulation of the EGFP-TusA fluorescence was detectable at the cell poles, the same part of the cell as for mCherry-FtsZ. Thus, this strongly suggested an interaction of TusA and FtsZ. Furthermore, the cellular FtsZ and Fis concentrations, and their change during different growth phases were determined via immunoblotting. All tested deletion strains of mnm5s2U34 tRNA modification show high cellular FtsZ and Fis levels in the exponential phase, shifting to the later growth phases. This shift reflects the retarded growth, whereby the deletion strains reach later the exponential phase. Conclusively, the growth and cell division defect, and thus the formation of filaments, is most likely caused by changes in the cellular FtsZ and Fis concentrations. Finally, the translation efficiencies of certain proteins (RpoS, Fur, Fis and mFis) in tusA mutant and in additional gene deletion strains were studied whether they were affected by using unmodified U34 tRNAs of Lys, Glu and Gln. The translation efficiency is decreased in mnm5s2U34 tRNA modification-impaired strains in addition to their existing growth and cell division defect due to the elimination of these three amino acids. Finally, these results confirm and reinforce the importance of Lys, Glu and Gln and the mnm5s2U34 tRNA thiolation for efficient protein translation. Thus, these findings verify that the translation of fur, fis and rpoS is regulated by mnm5s2U34 tRNA modifications, which is growth phase-dependent. In total, this work showed the importance of the role of TusA for bacterial cell functionality and physiology. The deletion of the tusA gene disrupted a complex regulatory network within the cell, that most influenced by the decreased translation of Fis and RpoS, caused by the absence of mnm5s2U34 tRNA modifications. The disruption of RpoS and Fis cellular network influences in turn the cellular FtsZ level in the early exponential phase. Finally, the reduced FtsZ concentration leads to elongated, filamentous E. coli cells, which are unable to divide.},
  language  = {en}
}
@phdthesis{Wunderlich2014,
  author    = {Wunderlich, Kai},
  title     = {Entwicklung einer parallelen Mehrkomponentenanalyse von Antigen-Antik{\"o}rper-Reaktionen in der Dopinganalyse},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-76869},
  school      = {Universit{\"a}t Potsdam},
  pages     = {VIII, 130},
  year      = {2014},
  abstract  = {Weltweit streben Anti-Doping Institute danach jene Sportler zu {\"u}berf{\"u}hren, welche sich unerlaubter Mittel oder Methoden bedienen. Die hierf{\"u}r notwendigen Testsysteme werden kontinuierlich weiterentwickelt und neue Methoden aufgrund neuer Wirkstoffe der Pharmaindustrie etabliert. Gegenstand dieser Arbeit war es, eine parallele Mehrkomponentenanalyse auf Basis von Antigen-Antik{\"o}rper Reaktionen zu entwickeln, bei dem es prim{\"a}r um Verringerung des ben{\"o}tigten Probevolumens und der Versuchszeit im Vergleich zu einem Standard Nachweis-Verfahren ging. Neben der Verwendung eines Multiplex Ansatzes und der Mikroarraytechnologie stellten ebenfalls die Genauigkeit aller Messparameter, die Stabilit{\"a}t des Versuchsaufbaus sowie die Performance {\"u}ber einen Einfach-Blind-Ansatz Herausforderungen dar. Die Anforderung an den Multiplex Ansatz, keine falschen Signale trotz {\"a}hnlicher Strukturen zu messen, konnte durch die gezielte Kombination von spezifischen Antik{\"o}rpern realisiert werden. Hierf{\"u}r wurden neben Kreuzreaktivit{\"a}tstests auf dem Mikroarray parallel erfolgreich Western Blot Versuche durchgef{\"u}hrt. Jene Antik{\"o}rper, welche in diesen Versuchen die gesetzten Anforderungen erf{\"u}llten, wurden f{\"u}r das Ermitteln der kleinsten nachweisbaren Konzentration verwendet. {\"U}ber das Optimieren der Versuchsbedingungen konnte unter Verwendung von Tween in der Waschl{\"o}sung sowohl auf Glas als auch auf Kunststoff die Hintergrundfluoreszenz reduziert und somit eine Steigerung des Signal/Hintergrundverh{\"a}ltnisses erreicht werden. In den Versuchen zu Ermittlung der Bestimmungsgrenze wurde f{\"u}r das humane Choriongonadotropin (hCG-i) eine Konzentration von 10 mU/ml, f{\"u}r dessen beta-Untereinheit (hCG-beta) eine Konzentration von 3,6 mU/ml und f{\"u}r das luteinisierende Hormon (LH) eine Konzentration von 10 mU/ml bestimmt. Den ermittelten Wert im Serum f{\"u}r das hCG-i entspricht dem von der Welt-Anti-Dopin-Agentur (WADA) geforderten Wert in Urin von 5 mU/ml. Neben der Ermittlung von Bestimmungsgrenzen wurden diese hinsichtlich auftretender Matrixeffekte in Serum und Blut gemessen. Wie aus den Versuchen zur Ermittlung von Kreuzreaktivit{\"a}ten auf dem Mikroarray zu entnehmen ist, lassen sich das LH, das hCG-i und hCG-β ebenfalls in Serum und Blut messen. Die Durchf{\"u}hrung einer Performance-Analyse {\"u}ber einem Einfach-Blind-Ansatz mit 130 Serum Proben, wurde ebenfalls {\"u}ber dieses System realisiert. Die ausgewerteten Proben wurden anschließend {\"u}ber eine Grenzwertoptimierungskurve analysiert und die diagnostische Spezifit{\"a}t ermittelt. F{\"u}r die Messungen des LH konnte eine Sensitivit{\"a}t und Spezifit{\"a}t von 100\% erreicht werden. Demnach wurden alle negativen und positiven Proben eindeutig interpretiert. F{\"u}r das hCG-β konnte ebenfalls eine Spezifit{\"a}t von 100\% und eine Sensitivit{\"a}t von 97\% erreicht werden. Die hCG-i Proben wurden mit einer Spezifit{\"a}t von 100\% und eine Sensitivit{\"a}t von 97,5\% gemessen. Um den Nachweis zu erbringen, dass dieser Versuchsaufbau {\"u}ber mehrere Wochen stabile Signale bei Vermessen von identischen Proben liefert, wurde ein {\"u}ber zw{\"o}lf Wochen angesetzter Stabilit{\"a}tstest f{\"u}r alle Parameter erfolgreich in Serum und Blut durchgef{\"u}hrt. Zusammenfassend konnte in dieser Arbeit erfolgreich eine Mehrkomponentenanalyse als Multiplex Ansatz auf einem Mikroarray entwickelt werden. Die Durchf{\"u}hrung der Performance-Analyse und des Stabilit{\"a}tstests zeigen bereits die m{\"o}gliche Einsatzf{\"a}higkeit dieses Tests im Kontext einer Dopinganalyse.},
  language  = {de}
}
@phdthesis{Woehlecke2021,
  author    = {Woehlecke, Sandra},
  title     = {Das erweiterte Fachwissen f{\"u}r den schulischen Kontext als Leitlinie f{\"u}r eine additive fachliche Lehrveranstaltung im Lehramtsstudium Biologie},
  doi       = {10.25932/publishup-52120},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-521209},
  school      = {Universit{\"a}t Potsdam},
  pages     = {304},
  year      = {2021},
  abstract  = {Das Fachwissen von Lehrkr{\"a}ften weist f{\"u}r die Auspr{\"a}gung fachdidaktischer Expertise eine hohe Bedeutung auf. Welche Merkmale universit{\"a}re Lehrveranstaltungen aufweisen sollten, um Lehramtsstudierenden ein berufsspezifisches Fachwissen zu vermitteln, ist jedoch {\"u}berwiegend noch unklar. Innerhalb des Projekts PSI-Potsdam wurde auf theoretischer Grundlage das fach{\"u}bergreifende Modell des erweiterten Fachwissens f{\"u}r den schulischen Kontext entwickelt. Als Ansatz zur Verbesserung des Biologie-Lehramtsstudiums diente dieses Modell als Konzeptionsgrundlage f{\"u}r eine additive Lehrveranstaltung. Hierbei werden Lerngelegenheiten geboten, um das universit{\"a}r erworbene Fachwissen {\"u}ber zellbiologische Inhalte auf schulische Kontexte anzuwenden, z.B. durch die Dekonstruktion und anschließende Rekonstruktion von schulischen Lerntexten. Die Wirkung des Seminars wurde in mehreren Zyklen im Forschungsformat der Fachdidaktischen Entwicklungsforschung beforscht. Eine der zentralen Forschungsfragen lautet dabei: Wie kann eine Lerngelegenheit f{\"u}r Lehramtsstudierende der Biologie gestaltet sein, um ein erweitertes Fachwissen f{\"u}r den schulischen Kontext f{\"u}r den zellbiologischen Themenbereich „Struktur und Funktion der Biomembran" zu f{\"o}rdern? Anhand fall{\"u}bergreifender Analysen (n = 29) wird im empirischen Teil aufgezeigt, welche Einstellungen zum Lehramtsstudium in der Stichprobe bestehen. Als ein wichtiges Ergebnis kann hierbei herausgestellt werden, dass sich das Fachinteresse hinsichtlich schulisch und universit{\"a}r vermittelter Inhalte bei den untersuchten Studierenden auffallend unterscheidet, wobei dem Schulwissen ein deutlich h{\"o}heres Interesse entgegengebracht wird. Die Berufsrelevanz fachlicher Inhalte wird seitens der Studierenden h{\"a}ufig am Schulwissen festgemacht. Innerhalb konkreter Einzelfallanalysen (n = 6) wird anhand von Lernpfaden dargestellt, wie sich {\"u}ber mehrere Design-Experimente hinweg fachliche Konzepte entwickelt haben. Bei der Beschreibung wird vor allem auf Schl{\"u}sselstellen und H{\"u}rden im Lernprozess fokussiert. Aus diesen Ergebnissen folgend werden vorgenommene Iterationen f{\"u}r die einzelnen Zyklen beschrieben, die ebenfalls anhand der iterativen Entwicklung der Design-Prinzipien dargelegt werden. Es konnte gezeigt werden, dass die Schl{\"u}sselstellen sehr individuell aufgrund der subjektiv fokussierten Inhalte zu Tage treten. Meist treten sie jedoch im Zusammenhang mit der Verkn{\"u}pfung verschiedener fachlicher Konzepte oder durch kooperative Aufschl{\"u}sselungen von Konzepten auf. Fachliche H{\"u}rden konnten hingegen in Form von fachlich unangemessenen Vorstellungen fall{\"u}bergreifend identifiziert werden. Dies betrifft unter anderem die Vorstellung der Biomembran als Wand, die mit den Vorstellungen einer Schutzfunktion und einer formgebenden Funktion der Biomembran einhergeht. Weiterhin wird beleuchtet, wie das erweiterte Fachwissen f{\"u}r den schulischen Kontext zur Bearbeitung der Lernaufgaben angewendet wurde. Es hat sich gezeigt, dass sich bestimmte Lerngelegenheiten eigenen, um bestimmte Facetten des erweiterten Fachwissens zu f{\"o}rdern. Insgesamt scheint das Modell des erweiterten Fachwissens f{\"u}r den schulischen Kontext {\"a}ußerst geeignet zu sein, um anhand der Facetten und deren Beschreibungen Lerngelegenheiten oder Gestaltungsprinzipien f{\"u}r diese zu konzipieren. F{\"u}r das untersuchte Lehr-Lernarrangement haben sich kleinere Adaptationen des Modells als sinnvoll erwiesen. Hinsichtlich der Methodologie konnten Ableitungen f{\"u}r die Anwendung der fachdidaktischen Entwicklungsforschung f{\"u}r additive fachliche Lehrveranstaltungen dieser Art herausgestellt werden. Um den Professionsbezug der fachwissenschaftlichen Anteile im Lehramtsstudium zu verbessern, ist der weitere Einbezug des erweiterten Fachwissens f{\"u}r den schulischen Kontext in die fachwissenschaftlichen Studienanteile {\"u}beraus w{\"u}nschenswert.},
  language  = {de}
}
@phdthesis{Witte2009,
  author    = {Witte, Jeannine},
  title     = {Rhabdomerorganisation und -morphogenese im Komplexauge von Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-41847},
  school      = {Universit{\"a}t Potsdam},
  year      = {2009},
  abstract  = {Sehzellen von Insekten sind epitheliale Zellen mit einer charakteristischen, hochpolaren Morphologie und Organisation. Die molekularen Komponenten der Sehkaskade befinden sich im Rhabdomer, einem Saum dicht gepackter Mikrovilli entlang der Sehzelle. Bereits in den 70er Jahren des letzten Jahrhunderts wurde beschrieben, dass die Mikrovilli entlang einer Sehzelle eine unterschiedliche Ausrichtung besitzen, oder in anderen Worten, die Rhabdomere entlang der Sehzell-L{\"a}ngsachse verdreht sind. So sind in den Sehzellen R1-R6 bei dipteren Fliegen (Calliphora, Drosophila) die Mikrovilli im distalen und proximalen Bereich eines Rhabdomers etwa rechtwinkelig zueinander angeordnet. Dieses Ph{\"a}nomen wird in der Fachliteratur als rhabdomere twisting bezeichnet und reduziert die Empfindlichkeit f{\"u}r polarisiertes Licht. Es wurde f{\"u}r das Drosophila-Auge gezeigt, dass diese strukturelle Asymmetrie der Sehzellen mit einer molekularen Asymmetrie in der Verteilung phosphotyrosinierter Proteine an die Stielmembran (einem nicht-mikrovill{\"a}ren Bereich der apikalen Plasmamembran) einhergeht. Zudem wurde gezeigt, dass die immuncytochemische Markierung mit anti-Phosphotyrosin (anti-PY) als lichtmikroskopischer Marker f{\"u}r das rhabdomere twisting verwendet werden kann. Bisher wurde haupts{\"a}chlich die physiologische Bedeutung der Rhabdomerverdrehung untersucht. Es ist wenig {\"u}ber die entwicklungs- und zellbiologischen Grundlagen bekannt. Ziel der vorliegenden Arbeit war es, die Identit{\"a}t der phosphotyrosinierten Proteine an der Stielmembran zu kl{\"a}ren und ihre funktionelle Bedeutung f{\"u}r die Entwicklung des rhabdomere twisting zu analysieren. Zudem sollte untersucht werden, welchen Einfluss die inneren Sehzellen R7 und R8 auf die Verdrehung der Rhabdomere von R1-R6 haben. F{\"u}r die zwei Proteinkinasen Rolled (ERK) und Basket (JNK) vom Typ der Mitogen-aktivierten Proteinkinasen (MAPK) konnte ich zeigen, dass sie in ihrer aktivierten (= phosphorylierten) Form (pERK bzw. pJNK) eine asymmetrische Verteilung an der Stielmembran aufweisen vergleichbar der Markierung mit anti-PY. Weiterhin wurde diese asymmetrische Verteilung von pERK und pJNK ebenso wie die von PY erst kurz vor Schlupf der Fliegen (bei ca. 90\% pupaler Entwicklung) etabliert. Durch Pr{\"a}inkubationsexperimente mit anti-PY wurde die Markierung mit anti-pERK bzw. anti-pJNK unterbunden. Diese Ergebnisse sprechen daf{\"u}r, dass pERK und pJNK zu den Proteinen geh{\"o}ren, die von anti-PY an der Stielmembran erkannt werden. Da es sich bei ERK und JNK um Kinasen handelt, ist es naheliegend, dass diese an der Entwicklung des rhabdomere twisting beteiligt sein k{\"o}nnten. Diese Hypothese wurde durch die Analyse von hypermorphen (rl SEM)und hypomorphen (rl 1/rl 10a) Rolled-Mutanten {\"u}berpr{\"u}ft. In der rl SEM-Mutante mit erh{\"o}hter Aktivit{\"a}t der Proteinkinase erfolgte die asymmetrische Positionierung von pERK an der Stielmembran sowie die Mikrovillikippung schon zu einem fr{\"u}heren Zeitpunkt in der pupalen Entwicklung. Im adulten Auge war die anti-PY-Markierung im distalen Bereich der Sehzellen intensiver sowie der Kippwinkel vergr{\"o}ßert. In der rl 1/rl 10a-Mutanten mit reduzierter Kinaseaktivit{\"a}t waren die anti-PY-Markierung und der Kippwinkel im proximalen Bereich der Sehzellen verringert. Die Proteinkinase ERK hat somit einen Einfluss auf die zeitliche Etablierung des rhabdomere twisting wie auch auf dessen Auspr{\"a}gung im Adulttier. Die Rhabdomerverdrehung sowie die {\"A}nderung im anti-PY-Markierungsmuster erfolgen an den Sehzellen R1-R6 relativ abrupt auf halber Ommatidienl{\"a}nge, dort wo das Rhabdomer von R7 endet und das von R8 beginnt. Es stellte sich deshalb die Frage, ob die Rhabdomerverdrehung an R1-R6 durch die Sehzelle R7 und/oder R8 beeinflusst wird. Um dieser Frage nachzugehen wurden Mutanten analysiert, denen die R7- oder die R8-Photorezeptoren bzw. R7 und R8 fehlten. Das wichtigste Ergebnis dieser Untersuchungen war, dass bei Fehlen von R8 die Rhabdomerverdrehung bei R1-R6 nach keinen erkennbaren Regeln erfolgt. R8 ist somit Voraussetzung f{\"u}r die Etablierung der Rhabdomerverdrehung in R1-R6. Folgendes Modell wurde auf Grundlage dieses und weiterer Ergebnisse erarbeitet: Im dritten Larvenstadium rekrutiert R8 die Sehzellpaare R2/R5, R3/R4 und R1/R6. Dabei werden R1-R6 durch den Kontakt zu R8 „polarisiert". Abschließend wird R7 durch R8 rekrutiert. Dies f{\"u}hrt zu einer Fixierung der Polarit{\"a}t von R1-R6 durch R7. Die Ausf{\"u}hrung der Mikrovillikippung anhand der festgelegten Polarit{\"a}t erfolgt in der sp{\"a}ten Puppenphase. Die Proteinkinase ERK ist an diesem letzten Morphogeneseprozess beteiligt.},
  language  = {de}
}