Thermodynamics and Heat Transport of doped Spin-Ice Materials

In this thesis the low-temperature magnetism of the spin-ice systems Dy2Ti2O7 and Ho2Ti2O7 is investigated. In general, a clear experimental evidence for a sizable magnetic contribution kappa_{mag} to the low-temperature, zero-field heat transport of both spin-ice materials is observed. This kappa_{mag} can be attributed to the magnetic monopole excitations, which are highly mobile in zero field and are suppressed by a rather small external field resulting in a drop of kappa(H). Towards higher magnetic fields, significant field dependencies of the phononic heat conductivities kappa_{ph}(H) of Ho2Ti2O7 and Dy2Ti2O7 are found, which are, however, of opposite signs, as it is also found for the highly dilute reference materials (Ho0.5Y0.5)2Ti2O7 and (Dy0.5Y0.5)2Ti2O7. The dominant effect in the Ho-based materials is the scattering of phonons by spin flips which appears to be significantly stronger than in the Dy-based materials. Here, the thermal conductivity is suppressed due to enhanced lattice distortions observed in the magnetostriction. Furthermore, the thermal conductivity of Dy2Ti2O7 has been investigated concerning strong hysteresis effects and slow-relaxation processes towards equilibrium states in the low-temperature and low-field regime. The thermal conductivity in the hysteretic regions slowly relaxes towards larger values suggesting that there is an additional suppression of the heat transport by disorder in the non-equilibrium states. The equilibration can even be governed by the heat current for particular configurations. A special focus was put on the dilution series Dy2Ti2O7x. From specific heat measurements, it was found that the ultra-slow thermal equilibration in pure spin ice Dy2Ti2O7 is rapidly suppressed upon dilution with non-magnetic yttrium and vanishes completely for x>=0.2 down to the lowest accessible temperatures. In general, the low-temperature entropy of (Dy1-xYx)2Ti2O7, considerably decreases with increasing x, whereas its temperature-dependence drastically increases. Thus, it could be clarified that there is no experimental evidence for a finite zero-temperature entropy in (Dy1-xYx)2Ti2O7 above x>=0.2, in clear contrast to the finite residual entropy S_{P}(x) expected from a generalized Pauling approximation. A similar discrepancy is also present between S_{P}(x) and the low-temperature entropy obtained by Monte Carlo simulations, which reproduce the experimental data from 25 K down to 0.7 K, whereas the data at 0.4 K are overestimated. A straightforward description of the field-dependence kappa(H) of the dilution series with qualitative models justifies the extraction of kappa_{mag}. It was observed that kappa_{mag} systematically scales with the degree of dilution and its low-field decrease is related to the monopole excitation energy. The diffusion coefficient D_{mag} for the monopole excitations was calculated by means of c_{mag} and kappa_{mag}. It exhibits a broad maximum around 1.6 K and is suppressed for T<=0.5 K, indicating a non-degenerate ground state in the long-time limit, and in the high-temperature range for T>=4 K where spin-ice physics is eliminated. A mean-free path of 0.3 mum is obtained for Dy2Ti2O7 at about 1 K within the kinetic gas theory.

Improving safety and establishing episomal maintenance of Adeno-associated viral vectors

Adeno-associated viral (AAV) vectors are among the most widely used gene transfer systems in basic and pre-clinical research and have been employed in more than 160 clinical trials. AAV vectors are commonly produced in producer cell lines like HEK293 by co-transfection with a so-called vector plasmid and one (in this work) or two so-called helper plasmids. The vector plasmid contains the transgene cassette of interest (TEC) flanked by AAV’s inverted terminal repeats (ITRs) which serve as packaging signals, whereas the helper plasmid provides the required AAV and helper virus functions in trans. A pivotal aspect of AAV vectorology is the manufacturing of AAV vectors free from impurities arising during the production process. These impurities include AAV vector preparations that contain capsids containing prokaryotic sequences, e.g. antibiotic resistance genes originating from the producer plasmids. In the first part of the thesis we aimed at improving the safety of AAV vectors. As we found that encapsidated prokaryotic sequences (using the ampicillin resistance gene as indicator) cannot be re-moved by standard purification methods we investigated whether the producer plasmids could be replaced by Minicircles (MCs). MCs are circular DNA constructs which contain no functional or coding prokaryotic sequences; they only consist of the TEC and a short sequence required for production and purification. MC counterparts of a vector plasmid encoding for enhanced green fluorescent (eGFP) protein and a helper plasmid encoding for AAV serotype 2 (AAV2) and helper Adenovirus (Ad) genes were designed and produced by PlasmidFactory (Bielefeld, Germany). Using all four possible combinations of plasmid and MCs, single-stranded AAV2 vectors (ssAAV) and self-complementary AAV vectors (scAAV) were produced and characterized for vector quantity, quality and functionality. The analyses showed that plasmids can be replaced by MCs without decreasing the efficiency of vector production and vector quality. MC-derived scAAV vector preparations even exceeded plasmid-derived preparations, as they displayed up to 30-fold improved transduction efficiencies. Using MCs as tools, we found that the vector plasmid is the main source of encapsidated prokaryotic sequences. Remarkably, we found that plasmid-derived scAAV vector preparations contained a much higher relative amount of prokaryotic sequences (up to 26.1 %, relative to TEC) compared to ssAAV vector preparations (up to 2.9 %). By replacing both plasmids by MCs the amount of functional prokaryotic sequences could be decreased to below the limit of quantification. Additional analyses for DNA impurities other than prokaryotic sequences showed that scAAV vectors generally contained a higher amount of non-vector DNA (e.g. adenoviral sequences) than ssAAV vectors. For both, ssAAV and scAAV vector preparations, MC-derived vectors tended to contain lower amounts of foreign DNA. None of the vectors tested could be shown to induce immunogenicity. In summary we could demonstrate that the quality of AAV vector preparations could be significantly improved by replacing producer plasmids by MCs. Upon transduction of a target tissue, AAV vector genomes predominantly remain in an episomal state, as duplex DNA circles or concatemers. These episomal forms mediate long-term transgene expression in terminally differentiated cells, but are lost in proliferating cells due to cell division. Therefore, in the second part of the thesis, in cooperation with Claudia Hagedorn and Hans J. Lipps (University Witten/Herdecke) an AAV vector genome was equipped with an autonomous replication element (Scaffold/matrix attachment region (S/MAR)). AAV-S/MAR encoding for eGFP and a blasticidin resistance gene and a control vector with the same TEC but lacking the S/MAR element (AAV-ΔS/MAR) were produced and transduced into highly proliferative HeLa cells. Antibiotic pressure was employed to select for cells stably maintaining the vector genome. AAV-S/MAR transduced cells yielded a higher number of colonies than AAV-ΔS/MAR-transduced cells. Colonies derived from each vector transduction were picked and cultured further. They remained eGFP-positive (up to 70 days, maximum cultivation period) even in the absence of antibiotic selection pressure. Interestingly, the mitotic stability of both AAV-S/MAR and control vector AAV-ΔS/MAR was found to be a result of episomal maintenance of the vector genome. This finding indicates that, under specific conditions such as the mild selection pressure we employed, “common” AAV vectors persist episomally. Thus, the S/MAR element increases the establishment frequency of stable episomes, but is not a prerequisite.