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Chemical pollution is one of the main contributors to the degradation of lotic ecosystems and their biodiversity. Among chemicals driving lotic biodiversity decline are anthropogenic organic micropollutants (AOM), which affect the survival and functioning of freshwater organisms. Continuous exposure of freshwater organisms to AOM leads to adverse effects that sometimes cannot be traced with standard toxicity methods such as standard toxicity testing or biodiversity indices. Among these effects of AOM are selective or mutagenic effects that cause impaired species genetic diversity. Thus, the correlation between different levels of AOM and genetic diversity of species is still poorly understood. However, it can be explored by applying population genetics screening.
In Chapter 1 of this thesis, background information on environmental pollution, genetic screening, and the detection of evolutionary-relevant AOM effects in freshwater organisms are described and the thesis goals are identified. The main goal of the thesis is to study whether AOM exposure occurring in European rivers causes a significant evolutionary footprint in freshwater species and leads to a selection of more tolerant geno-and phenotypes. Therefore, population genetics indices together with high-resolution chemical exposure screening of a widespread indicator invertebrate species, Gammarus pulex (Linnaeus, 1758), living in polluted and pristine European rivers were investigated.
In Chapter 2, the development of a genetic screening method for G. pulex (microsatellites) is described. Due to genetic differentiation and the presence of morphologically cryptic lineages, the available sets of target loci do not enable a reliable population genetic characterization of G. pulex from central Germany. Thus, a novel set of microsatellite loci for a high-precision assessment of population genetic diversity was here applied. Eleven loci were first identified and thereafter amplified in G. pulex from three rivers. The new loci reliably amplified and indicated polymorphisms in the studied amphipods. The amplification resulted in the successful identification of genetically distinct populations of G. pulex from the analyzed rivers. Moreover, the microsatellite loci were amplified in other genetic lineages of G. pulex and another Gammarus species, G. fossarum, promising a broader applicability of the loci in related amphipod species.
In Chapter 3, the effects of AOM on species genetic differentiation and sensitivity to toxic chemicals in a typical central European river with pristine and AOM-polluted sections was investigated. The river’s site-specific concentrations of AOM were assessed by chemical analysis of G. pulex tissue and water samples. To test, whether different levels of AOM in the river select for pollution-dependent genotypes, the genetic structure of G. pulex from the river was analyzed. Finally, the toxicokinetics of and sensitivity to the commonly used insecticide imidacloprid were determined for amphipods sampled at pristine and polluted sections to assess whether various levels of AOM in the river influence sensitivity of G. pulex to imidacloprid. The results indicated that different levels of AOM did not drive genetic divergence of G. pulex within the river but led to an increased sensitivity of exposed amphipods to imidacloprid. The amphipods living in polluted river sections were more sensitive to the insecticide due to chronic exposure to toxic levels of AOM.
In Chapter 4, the relationship between site-specific pollution levels of AOM and genetic diversity parameters of G. pulex was analyzed at the regional scale within six rivers in central Germany. The genetic structure of G. pulex in the studied area was tested for relatedness to the waterway distance between sites. Gammarus pulex genetic diversity parameters, including allelic richness and inbreeding rate, were tested against environmental pollution parameters using linear mixed-effect- and structural-equation models. According to the results, G. pulex genetic diversity parameters were significantly associated with the detected AOM levels. At sites with high concentrations of AOM and toxicity potential G. pulex showed reduced genetic diversity and increased rates of inbreeding. These results suggest that AOM play a major role in shaping the genetic diversity of G. pulex in rivers.
According to the findings presented here, the applied microsatellites can be used to successfully detect changes in genetic patterns in freshwater amphipods facing increased levels of AOM. The findings indicate that levels of AOM representative for European rivers do not lead to the separation of genotypes among G. pulex as the connectivity between sites majorly contributes to species’ genetic structure. However, the chronic exposure to increased levels of toxic AOM leads to a reduction of species genetic diversity and increases the sensitivity of G. pulex to the toxic chemical effects.
Standard biorelevant media reflect the average gastrointestinal (GI) physiology in healthy volunteers. The use of biorelevant media in in vitro experiments has become an important strategy to predict drug behaviour in vivo and is often combined with in silico tools in order to simulate drug plasma profiles over time. In addition to the healthy population, the effects of disease state or co-administration of other drugs on plasma profiles must be considered to assure drug efficacy and safety. Thus, there is a need for a more accurate representation of the human GI physiology when it is altered by disease or co-administered drugs in in vitro dissolution experiments.
This thesis focused on the development of biorelevant media and dissolution tests reflecting GI physiology in circumstances where the gastric pH is elevated. Diseases linked to an elevated gastric pH are hypochlorhydria and achlorhydria, but these days treatment with acid-reducing agents (ARAs) is the single greatest cause of elevation in gastric pH. pH-dependent drug-drug interactions (DDIs) with ARAs are frequent, as the ARAs are used in a number of diseases using a variety of drugs. As the drugs currently on the market are often poorly soluble and ionisable, their dissolution is highly dependent on the pH of the GI tract, especially the gastric pH.
The thesis research consisted of several steps. In the first step, physiological changes in the human GI tract during the therapy with ARAs were identified. Parameters of the standard biorelevant gastric medium FaSSGF were adjusted to the identified changes to reflect the impact of ARA co-administration on the gastric physiology. The media aim to assess the potential extent of the ARA impact on gastric physiology by introducing biorelevant media pairs, ARA pH 4 and pH 6 media, of which one reflects a lesser, and the other a stronger impact of ARAs.
In the second step these ARA media were implemented in in vitro dissolution set-ups.
The dissolution of poorly soluble ionisable drugs was assessed using one-stage, two-stage and transfer model set-ups, as well as using a more evolved in vitro system TIM-1. Comparison of results from dissolution set-ups using the standard, low pH, gastric biorelevant medium FaSSGF (pH 1.6 or 2), and the same set-ups using ARA pH 4 and pH 6 media, shows a decrease in dissolution rate and extent for weakly basic compounds PSWB 001 and dipyridamole, and an increase in rate and extent of dissolution for the weakly acidic compound raltegravir potassium, when the gastric pH is elevated. Due to different physicochemical properties, the extent of the impact of physiological changes during ARA therapy (when either ARA pH 4 or pH 6 medium is selected) on dissolution varied among the model drugs. Thus, the bracketing approach, which considers a range of the possible ARA co-administration impact on drug dissolution, was confirmed to be best practice in assessing the impact of ARAs.
In the third step, dissolution data from in vitro experiments with ARA media was implemented into in silico models. The predictions using various in silico model approaches in Simcyp™ Simulator (minimal and full PBPK model, dissolution input using DRM and DLM) successfully bracketed in vivo data on drug administration during ARA therapy and correctly predicted an overall decrease in plasma concentration for the two model weakly basic compounds and an increase in plasma concertation for the model weakly acidic compound.
In all assessed scenarios, the ARA methods proved to be an essential part of evaluating and predicting the impact of ARAs on drug pharmacokinetics, and appropriately predicted the extent of a possible impact of ARAs on the drug plasma profiles. Thus, the ARA biorelevant media and dissolution tests were demonstrated to be valuable tools reflecting administration of drugs when the gastric pH is elevated and able to predict the impact of ARA therapy on drug administration.
The ability to evaluate the impact of human (patho) physioloy on drug behaviour in the gastrointestinal tract is of great importance, as the GI conditions play a significant role in drug release and absorption. Thus, there is great interest on the part of the pharmaceutical industry and regulatory agencies to develop best practices in this field, especially for pH-dependent DDIs. The media and dissolution tests developed in this thesis are biorelevant methods appropriate for evaluation of the impact of elevated gastric pH on drug efficacy and safety. Such methods, used as a risk assessment tool, in connection with evaluation of the efficacy window and potential toxicity, may help to increase confidence about decisions as to whether a pH-effect will occur and whether it is relevant or not, prior to conducting clinical studies. They may also enable changes in inclusion/exclusion criteria during recruiting for large-scale efficacy trials. In fact, the biopharmaceutic approach to drug development is becoming standard practice on a number of fronts, including metabolic DDIs, renal and hepatic insufficiency, powering decision-making process and possibly even waiving certain types of clinical studies.
...
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative movement disorder caused by expansion of CAG repeats in the ATXN2 gene beyond 33 units, while healthy individuals carry 22-23 repeats. First symptoms of SCA2 include uncoordinated movement, ataxic gait and slowing of the saccadic eye movements in line with the early pronounced atrophy of cerebellum, spinal cord and brainstem. Cerebellar Purkinje cells and spinal cord motor neurons are the most affected cells from ATXN2 expansions. Later on, patients manifest distal amyotrophy, problems in breathing and swallowing, depression and cognitive decline caused by widespread degeneration throughout the brain. The striking loss of mass in the brain, due to severe myelin fat atrophy, is accompanied by a similar reduction in the peripheral fat stores. After the devastating progression of disease, the severity and duration of which depends on the CAG repeat size, genetic background and environmental factors, patients succumb to SCA2 mostly because of respiratory failure at the terminal stage. Larger repeat sizes lead to an earlier manifestation of the disease and a more rapid progression. Aside from SCA2, intermediate-length and short pathogenic CAG expansions in ATXN2 between 26-39 repeats significantly increase the risk of developing other neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), fronto-temporal lobar dementia (FTLD) or Parkinson plus tauopathies like progressive supranuclear palsy (PSP) in various cohorts across the world.
Ataxin-2 (ATXN2) is a ubiquitously expressed cytosolic protein most famous for its involvement in neurodegenerative disease caused by the expanded poly-glutamine (polyQ) domain corresponding to a genomic (CAG)n tract. This N-terminal polyQ domain has no known function, other than increasing the aggregation propensity of mutant ATXN2 and facilitating interaction with other polyQ containing proteins, leading to their sequestration. The progressive accumulation of ATXN2 into cytosolic foci, and also that of its interaction partners over time, underlies the molecular pathomechanism. Next to polyQ domain, ATXN2 also contains a Like-Sm domain (Lsm), an Lsm-associated domain (LsmAD), multiple proline-rich domains (PRD) and a Poly(A)-Binding-Protein (PABP)-interacting motif (PAM2).
Through its Lsm/LsmAD domains, ATXN2 directly binds to a large number of transcripts, regulating their quality and translation rate. In a similar fashion, through its direct interaction with PABP via PAM2 motif, ATXN2 indirectly modifies the fate of even larger number of transcripts and global translation. Several PRDs scattered across the protein help ATXN2 associate with growth factor receptors and other endocytosis factors, modulating nutrient uptake and downstream signaling.
ATXN2 is a stress response factor. Therefore, its involvement in nutrient uptake plays a crucial part in cell’s capability to overcome non-permissive conditions. Upon nutrient deprivation, oxidative stress, proteotoxicity, heat stress or Ca2+ imbalance, ATXN2 relocalizes into cytosolic ribonucleoprotein particles known as stress granules (SGs), together with PABP, several eukaryotic translation initiation factors, many other RNA-binding proteins (RBP) with their target transcripts and the small ribosomal subunit. Collectively, they modulate the stability of the trapped transcripts, favoring the maturation and translation of IRES-dependent stress response proteins instead, according to the specific need. Many RBPs interact either directly or in an RNA-dependent manner in the SGs, and due to the large number of ALS-causing mutations identified in them (such as TDP-43, FUS, TIA-1, hnRNPA2/B1), SGs became a hot topic in neuropathology. Acute SGs serve to halt translation and growth, and to spend energy only for survival until stress disappears. However, chronic SG assembly eventually activates apoptotis leading to cell death. While the polyQ expansions in ATXN2 enhance SG stability, reduce their dissociation rate after stress, and lead to aberrant post-translational modifications of other SG components like TDP-43, complete loss of ATXN2 delays SG formation and results in easily dissolvable foci.
Most of the stressors that induce SG formation eventually converge on energetic deficit. Therefore, it is logical that the ultimate task of SGs is to stop further growth when it cannot be afforded. In yeast, the molecular mechanism underlying this growth arrest was explained as sequestration of the master growth regulator complex, Target-of-Rapamycin Complex 1 (TORC1), into SGs in an ATXN2-dependent manner. The repressor effect of ATXN2 on mammalian TORC1 (mTORC1) and global protein translation had already been documented in earlier studies; complete loss of ATXN2 function in knock-out mouse (Atxn2-KO) resulted in mTORC1 hyperactivity and transcriptional upregulation of multiple ribosomal subunits indicating an increased need for these machines. ...
Resistant microbes are a growing concern. It was estimated that about 33,000 of people die because of the infections caused by multidrug resistant bacteria each year in Europe (ECDC, 2018, https://www.ecdc.europa.eu/). Bacteria can acquire resistance against toxic compounds via different mechanisms and intrinsic active efflux is one of the first mechanisms deployed by bacterial cells. The membrane-localized efflux pumps catalysing this reaction, extract toxic compounds from the interior of the cell and transport these to the outside, thereby maintaining sub-lethal toxin levels in the cytoplasm, periplasm and membranes. Gram-negative three-component efflux pumps, analysed in this study, are composed of an inner membrane protein, a member of the Resistance-Nodulation cell Division (RND) superfamily, an Outer Membrane Factor (OMF) protein and a Membrane Fusion Protein (MFP) that connects the two afore mentioned components into an active efflux pump. The pumps described in this work, AcrAB-TolC and EmrAB-TolC, are drug efflux pumps belonging to the RND and MFS superfamilies, respectively, while CusCBA is an efflux pump that belongs to the RND heavy metal efflux family. Another efflux pump that was used as a model for the design of an in vitro assay for the silver ion transport studies, CopA, belongs to the P-type ATPase superfamily. All pumps analysed in this study are part of the resistance system of Escherichia coli, which is a highly clinically relevant pathogen.
In order to examine the AcrAB-TolC, CopA and CusA efflux pumps, the individual components were separately produced in E. coli, purified to monodispersity and reconstituted in large unilamellar vesicles, LUVs. Means for the optimized production and adequate conditions for efficient reconstitution were presented in this study. The activity of AcrB in LUVs was detected using fluorescence quenching of the dye 8-hydroxy-1,3,6 pyrenetrisulfonate (pyranine), which is incorporated inside the proteoliposomes and is sensitive to the pH changes in its surrounding. The inactive AcrB variant with a substitution in the proton relay network, D407N, showed no activity in proteoliposomes, which correlates with the measurements done in empty liposomes. When AcrA was co-reconstituted with AcrB D407N proteoliposomes it did not restore protein activity. To test the assembly of the AcrAB-TolC pump out of its single components, an in vitro assay was established where the complex assembly was tested with AcrAB- and TolC-containing liposomes. These experiments showed putative AcrAB-TolC formation in the presence or absence of a pump substrate, taurocholate, as well as in the presence of the pump inhibitor, MBX3132. The assembly appeared stable over time and results were invariant in the presence or absence of a pH gradient across the AcrAB-containing membrane.
After determination of the ATPase activity of the P-type ATPase, CopA, in detergent micelles, the protein was reconstituted in LUVs. Quenching of the Ag+-sensitive dye Phen Green SK (PGSK), present on the inside of the CopA-containing proteoliposomes, was observed in presence of ATP and Ag+. Under the same conditions, but in absence of Ag+-ions, quenching was reduced by 80 % after 300 seconds. No PGSK-quenching was observed in control liposomes in the presence of ATP and Ag+. The additional presence of sodium azide led to minimal reduction of the PGSK-quenching as expected since sodium azide is not an inhibitor of P-type ATPases, but the quenching rate was similar to that of the same experimental condition with control liposomes.
The RND superfamily member CusA, as part of the tripartite CusCBA efflux pump, has been proposed to sequester Ag+ or Cu+ from either the cytoplasmic or periplasmic side of the inner membrane. The periplasmic transport of silver ions was implied from an in vitro assay where the quenching of a pH sensitive dye, 9-amino-6-chloro-2-methoxyacridine (ACMA), indicates acidification of the lumen of the proteoliposomes containing CusA when an inwardly directed pH was imposed. The same experiment with the CusA D405N variant, which was previously reported to be an inactive variant, also led to ACMA quenching, although at a slightly lower rate. Under application of an inwardly directed pH and a (negative inside), CusA-containing proteoliposomes showed a strong quenching of the incorporated PGSK dye, suggesting strong Ag+ influx.
The Major Facilitator Superfamily-(MFS-) type EmrAB-TolC pump has an analogous structural setup as the RND-type AcrAB-TolC pump. To examine the efflux of one of its substrates, carbonyl - cyanide m-chlorophenylhydrazone (CCCP), a plate-based susceptibility assay was used. The presence of the EmrAB-TolC pump confers lower susceptibility levels towards CCCP in E. coli, compared to cells not expressing the pump or cells expressing only the MFS component, indicating that EmrAB-TolC extrudes CCCP.
The work done in this study opens up a path towards investigation of drug and metal resistance in vitro. The methodologies to obtain proteoliposomal samples of multicomponent efflux pumps and subsequent measurements of drug/metal ion and H+ fluxes, as well as the determination of pump assembly are crucial for the future research on pump catalysis and transport kinetics. The in vivo drug-plate assays done in this work provide initial insights for future investigations of the drug susceptibility of E. coli expressing the MFS-type tripartite efflux pumps.
Terahertz (THz) technology is an emerging field that considers the radiation between microwave and far-infrared regions where the electronic and photonic technologies merge. THz generation and THz sensing technologies should fill the gap between photonics and electronics which is defined as a region where THz generation power and THz sensing capabilities are at a low technology readiness level (TRL). As one of the options for THz detection technology, field-effect transistors with integrated antennae were suggested to be used as THz detectors in the 1990s by M. Dyakonov and M. Shur from where the development of field-effect transistor-based detector began. In this work, various FET technologies are presented, such as CMOS, AlGaN/GaN, and graphene-based material systems and their further sensitivity enhancement in order to reach the performance of well-developed Schottky diode-based THz sensing technology. Here presented FET-based detectors were explored in a wide frequency range from 0.1 THz up to 5 THz in narrowband and broadband configurations.
For proper implementation of THz detectors, the well-defined characterization is of high importance. Therefore, this work overviews the characterization methods, establishes various definitions of detector parameters, and summarizes the state-of-the-art THz detectors. The electrical, optical, and cryogenic characterization techniques are also presented here, as well as the best results obtained by the development of the characterization methods, namely graphene FET stabilization, low-power THz source characterization for detector calibration, and technology development for cryogenic detection.
Following the discussion about the detector characterization, a wide range of THz applications, which were tested during the last four years of Ph.D. and conducted under the ITN CELTA project from HORIZON2020 program, are presented in this work. The studies began with spectroscopy applications and imaging and later developed towards hyperspectral imaging and even passive imaging of human body THz radiation. As various options for THz applications, single-pixel detectors as well as multi-pixel arrays are also covered in this work.
The conducted research shows that FET-based detectors can be used for spectroscopy applications or be easily adapted for the relevant frequency range. State-of-the-art detectors considered in this work reach the resonant performance below 20 pW/√Hz at 0.3 THz and 0.5 THz, as well as 404 pW/√Hz cross-sectional NEP at 4.75 THz. The broadband detectors show NEP as low as 25 pW/√Hz at around 0.6 THz for the best AlGaN/GaN design and 25 pW/√Hz around 1 THz for the best CMOS design. As one of the most promising applications, metamaterial characterization was tested using the most sensitive devices. Furthermore, one of the single-pixel devices and a multi-pixel array were tested as an engineering solution for a radio astronomy system called GREAT in a stratosphere observatory named SOFIA. The exploration of the autocorrelation technique using FET-based devices shows the opportunity to employ such detectors for direct detection of THz pulses without an interferometric measurement setup.
This work also considers imaging applications, which include near-field and far-field visualization solutions. A considerable milestone for the theory of FET technology was achieved when scanning near-field microscopy led to the visualization of plasma (or carrier density) waves in a graphene FET channel. Whereas another important milestone for the THz technology was achieved when a 3D scan of a mobile phone was performed under the far-field imaging mode. Even though the imaging was done through the phone’s plastic cover, the image displayed high accuracy and good feature recognition of the smartphone, inching the FET-based detector technology ever so close to practical security applications. In parallel, the multi-pixel array testing was carried out on 6x7 pixel arrays that have been implemented in configurable-size aperture and imaging configurations. The configurable aperture size allowed the easier detector focusing procedure and a better fit for the beam size of the incident radiation. The imaging has been tested on various THz sources and compared to the TeraSense 16x16 pixel array. The experimental results show the big advantage of the developed multi-pixel array against the used commercial technology.
Furthermore, two ultra-low-power applications have been successfully tested. The application on hyper-frequency THz imaging tested in the specially developed dual frequency comb and our detector system for 300 GHz radiation with 9 spectral lines led to outstanding imaging results on various materials. The passive imaging of human body radiation was conducted using the most sensitive broadband CMOS detector with a log-spiral antenna working in the 0.1 – 1.5 THz range and reaching the optical NEP of 42 pW/√Hz. The NETD of this device reaches 2.1 K and overcomes the performance limit of passive room-temperature imaging of the human body radiation, which was less than 10 K above the room temperature. This experiment opened a completely new field that was explored before only by the multiplier chain-based or thermal detectors.
...
The dissertation studied reused Roman coins (AD 100 – 400) that were found in medieval cemeteries (AD 400 – 1400) in the territory of Serbia. The evaluation process was traced through three different periods and cultural contexts: (1) in the period of Roman domination in the central Balkans (AD 1 – 400), i.e. the “primary context” of their use and circulation; (2) in the time of transition from the late antiquity to early medieval period (AD 400 – 700); and (3) in the high and late Middle Ages (AD 900 – 1400), where the last two were considered to be a “secondary context” in which the Roman coins were no longer a valid currency.
It was observed that the reused Roman coins, as a distinctive category of archaeological finds, impose a necessity for reconsideration of the relationship between the disciplines of archaeology and numismatics; encouraging a greater cooperation and discussion between the two. Considering the use and evaluation of Roman coins in their “primary context”, it is possible to presume that the strength of the political Roman system was the crucial factor in the formation and maintaining the stability of the value of Roman coins. The act of reuse should not be automatically equalized with recycling; implying only to use value, but at the same time it was not possible to assume that the value was formed only on a purely symbolical level. The (re)use of Roman coins in the funeral practices from c. AD 400 to 700 was considered to be a part of wider and occasional practice of incorporating older Roman issues in the coin pool by the “barbarian” or Byzantine authorities. It could be then concluded that the value of Roman coins was understood more as a potential attribute than as a fixed category; enabling one to simultaneously “overvalue “ and “undervalue” these objects. In the period from c. AD 900 to 1400, the reuse of Roman coins was detected only within the cemeteries of the peasantry and in a context of gradual increase of general coin use in the central Balkan communities of the Middle Ages. This was understood as an indicator that the Roman coins were not perceived as particularly valuable per se, but since the were recognized as category of objects that became more important in defining social relationships they were then incorporated in the funeral rituals and reinterpreted by the medieval population.
FPP und GGPP sind Intermediate des Mevalonat-Weges und fungieren als post-translationale Modifikation kleiner GTPasen. Die Prenylierung kleiner GTPasen erfolgt katalysiert von spezifischen Prenyltransferasen und ist notwendig um die kleinen GTPasen in Membranen zu verankern, wo ihre Aktivierung stattfindet. Zu den intrazellulären Funktionen der GTPasen gehören unter anderem der Aufbau des Cytoskeletts, das neuronale Zellwachstum, die Leitung und Ausläuferbildung von Axonen, das Dendritenwachstum, die Synapsenformation, die synaptische Plastizität und die Apoptose. Diese Funktionen spielen in der Gehirnalterung sowie in neurodegenerativen Erkrankungen wie der Alzheimer Demenz (AD) und auch bei der Glioblastoma multiforme (GBM) eine wichtige Rolle.
Im Zuge einer in vivo Studie an C57BL/6 Mäusen konnten in der vorliegenden Arbeit altersbedingte Veränderungen der Lokalisation verschiedener Rho- und Rab-GTPasen in Membran- und Cytosol-Präparationen sowie der GGTase-I in Gehirnen gealterter Tiere gezeigt werden. Die zelluläre Lokalisation der Rho GTPasen Rac1, RhoA und Cdc42 verschiebt sich im Alter zu reduzierten Membran-gebundenen und erhöhten cytosolischen Gehalten. Dies ist mit einer Reduktion der Protein- und mRNA- Gehalte des Enzyms GGTase-Iβ assoziiert, der Untereinheit der GGTase-I, die die Bindung des Isoprenoids GGPP an die Rho-GTPasen reguliert. Diese wiederum korrelieren direkt mit der altersbedingten Reduktion der relativen GGTase-Aktivität. Die in vitro Inhibition der GGTase-I mittels GGTI-2133 an SH-SY5Y Zellen erwies sich als Modell, welches die gleichen Effekte wie die gealterten Gehirne in vivo zeigt.
7, 8-Dihydroxyflavon (7, 8-DHF) ist ein natürlich vorkommendes Flavon, welches als hoch affiner selektiver TrkB-Rezeptor-Agonist fungiert und hierdurch wie das Neurotrophin BDNF das Überleben von Neuronen, deren Differenzierung, synaptische Plastizität und Neurogenese vermittelt. In vivo verursacht die orale Gabe von 7, 8-Dihydroxyflavon in Gehirnen alter Tiere eine Abnahme des Isoprenoids GGPP, die Zunahme der prenylierten Membran-gebundenen GTPase Rac1 und eine Reduktion des Gehaltes an Membran-gebundenem Rab3A auf das Niveau der Gehalte in den Gehirnen der jungen Kontroll-Tiere. Das Neurotrophin BDNF interagiert mit dem TrkB-Rezeptor und ist in der Lage direkt an den Rac1-spezifischen GEF Tiam1 zu binden, wodurch dieser aktiviert wird und Veränderungen der zellulären Morphologie der betroffenen Neurone induziert. Während das Alter und die orale Gabe von 7, 8-Dihydroxyflavon in vivo keine Effekte auf die Proteingehalte von BDNF und TrkB in der Tierstudie aufzeigten, konnte eine alterbedingte Reduktion von Tiam1 im Hirngewebe detektiert werden, die wiederum durch 7, 8-Dihydroxyflavon aufgehoben werden konnte.
Die Isoprenoide FPP und GGPP, sowie die Regulation kleiner GTPasen spielen auch eine wichtige Rolle im Zusammenhang mit Veränderungen der APP-Prozessierung in der molekularen Pathogenese der AD. Bei der APP-Prozessierung sind die beiden Sekretasen β- und γ-Sekretase für die Bildung des β-Amyloid-Peptids verantwortlich. In vitro Studien mit dem β-Sekretase-Inhibitor IV und dem γ-Sekretase-Inhibitor DAPT an untransfizierten und APP-transfizierten HEK293 Zellen (HEK293-APP695wt und HEK293-APPsw Zellen) konnten zeigen, dass sowohl die β- als auch die γ-Sekretase an der Regulation der Isoprenoide FPP und GGPP beteiligt sind. FPP und GGPP liegen in APP-transfizierten HEK293 Zellen erhöht vor. Die Inhibition der β-Sekretase führt zur Reduktion von FPP und GGPP. Durch die Inhibition der γ-Sekretase wird ausschließlich FPP reduziert. Weiterhin liegen in APP-transfizierten HEK293 Zellen die Membran-gebundenen prenylierten Rho-GTPasen Rac1, Cdc42 und RhoA erhöht vor. Das Membran-gebundene prenylierte H-Ras kommt jedoch in APP-transfizierten Zellen im Vergleich zu untransfizierten HEK293 Zellen in deutlich niedrigeren Mengen vor. Die Inhibition der β-Sekretase bedingt die Reduktion von Membran-gebundenem prenylierten Rac1 und auch von Membran-gebundenem H-Ras in HEK293-APPsw Zellen.
Veränderungen von Signaltransduktionswegen, die durch kleine GTPasen vermittelt werden, haben sich auch bei der GBM als zentraler Teil der molekularen Pathogenese herausgestellt. Hierbei ist die Prenylierung durch FPP und GGPP die Voraussetzung für die Membran-Insertion und onkogenen Funktion der Ras- und Rho-Proteine über die Stimulierung des Ras-Raf-MEK-ERK Signalweges. In dieser Arbeit konnte gezeigt werden, dass der HMG-CoA-Reduktase Inhibitor Lovastatin die Bildung der beiden Isoprenoide FPP und GGPP in U87 und U343 Glioblastoma Zellen verringert und hierdurch die Isoprenylierung von H-Ras und Rac1 reduziert. Das natürlich vorkommende Monoterpen Perrilylalkohol hingegen inhibiert die Prenyltransferasen FTase und GGTase und verändert dadurch die post-translationale Prenylierung der GTPasen Rac1 und H-Ras in U87 und U343 Zellen ohne die Isoprenoide FPP und GGPP signifikant zu beeinflussen. Jedoch bewirkt Perillylalkohol in U343 Zellen eine Erhöhung des GGPPs. Beide Substanzen bewirkten die Reduktion der ERK-Phosphorylierung und der Migration, Invasion und Proliferation der untersuchten U87 und U343 Glioblastoma Zellen.
Die HAART hat einen Durchbruch in der Therapie der HIV-Infektion bewirkt und so zu einer drastischen Senkung der Mortalität und Morbidität geführt. Um diesen Ansprüchen weiterhin gerecht zu werden und sie bestenfalls zu übertreffen, erfordert eine ständige Weiterentwicklung der HAART mit neuen und ausgefeilteren Alternativen. Ein weiterer Schritt in diese Richtung ist die Entwicklung einer neuen Formulierung des Kombinationspräparates LPV/r (Kaletra®) von der „lipophilen Kapselform“ zur „hydrophilen Tablettenform“, aus der Wirkstoffgruppe der Proteasehemmer. Lopinavir (LPV) ist ein HIV-Proteasehemmer der mit Ritonavir (r oder RTV) als fixe Kombination (LPV/r) hergestellt wird. Der Proteasehemmer Ritonavir wird dabei in subtherapeutischer Dosierung als Booster verwendet, dadurch wird eine Verbesserung der pharmakokinetischen Eigenschaften erzielt. Der Vorteil hierbei sind die höheren Lopinavir-Plasmaspiegel die erreicht werden. Diese Kombination wird als Kaletra® (LPV/r) vermarktet.
LPV/r ist erhältlich als lipophile Kapselform (133,3/33,3mg) oder in Flüssigform (80/20mg pro ml). Beide erfordern eine kühle Lagerung und müssen mit einer fettreichen Mahlzeit eingenommen werden, um optimale Lopinavir Plasmaspiegel zu erzielen.
Durch das „Melt Extrusion (Meltrex)“ Produktionsverfahren gelang die Herstellung einer „hydrophilen Tabletteform“ (200/50mg und 100/25mg) mit verbesserter Bioverfügbarkeit. Dadurch reduzierte sich die einzunehmende Anzahl von 6 Kapseln pro Tag auf 4 Tabletten pro Tag. Zudem bedarf die LPV/r Tablette keiner Kühlung und kann nahrungsunabhängig eingenommen werden.
Ziel dieser Untersuchung war es zu prüfen, welche LPV/r (Kaletra®) Darreichungsform, Kapsel oder Tablette, in einer HAART von HIV-Patienten bevorzugt wird. Es sollte ermittelt werden, ob bei gleichbleibender Wirksamkeit kombiniert mit einer verbesserten Verträglichkeit und Handhabung (weniger Tabletten, nahrungsunabhängige Einnahme und keine Kühlung), die überwiegende Mehrzahl der HIV-Patienten sich zugunsten der LPV/r Tablette, im Sinne einer verbesserten Lebensqualität bzw. Gemütszustandes, entscheiden werden.
Dies geschah anhand einer prospektiven, nicht randomisierten Studie mit 238 HIV-infizierten Patienten, die über mindesten 16 Wochen oder länger eine LPV/r Kapsel haltige antiretrovirale Kombinationstherapie einnahmen und am Tag 0 auf LPV/r Tabletten umgestellt wurden, ohne weitere Änderungen in ihrer bisherigen HAART vorzunehmen. Der darauffolgende Beobachtungszeitraum betrug 32 Wochen. Es wurden Vorher-, Nachher-Fragebogen ausgefüllt und die Patienten unterzogen sich einer Vorher-, Nachher-Laboruntersuchung (CD4 und HI-Viruslast). Zudem wurde nach der subjektiven Präferenz gegenüber beiden Darreichungsformen (Kapsel oder Tablette) gefragt.
Zusammenfassend lässt sich sagen, dass unter der LPV/r Tablette bei gleichbleibender antiretroviraler Wirksamkeit, signifikant weniger intestinale Nebenwirkungen auftraten und daran geknüpft signifikant weniger Medikamente gegen intestinale Beschwerden eingenommen wurden. Was bei den Patienten zu einer deutlichen Präferenz der LPV/r-Tablette (71,2 %) gegenüber der LPV/r Kapsel (3,0 %) führte. Die Ergebnisse zu Lebensqualität zeigten zwar eine tendenzielle Besserung aber zusammen mit den Gemütszuständen ergaben sich hier keine signifikanten Unterschiede.
Nach der vorliegenden Untersuchung muss die LPV/r Tablette im Vergleich zur LPV/r-Kapsel, als die überlegene antiretrovirale Therapieoption in Betracht gezogen werden. Angesichts zahlreicher Einschränkungen durch die Infektion und die Notwendigkeit einer lebenslangen Therapie, kann dies, ein bedeutender Beitrag zur Therapietreue sein und dadurch den Erfolg einer HIV-Therapie wesentlich mitbestimmen.
Das Forschungsziel dieser Arbeit war die Abklärung der Frage, ob sich in implantierten Intraokularlinsen der Marke Acrysof®, die in Deutschland kurz nach deren Markteinführung erworben wurden, Mikrovakuolen im Optikkörper befinden. Diese sogenannten Glistenings, die der Hersteller auf eine neuartige Verpackung zurückführte, welche ausschließlich in den Vereinigten Staaten Verwendung fand, führten in den USA zu Explantationen und zum Rückruf so verpackter Kunstlinsen dieses Typs. Unser Arbeitsprogramm umfaßte hauptsächlich die Untersuchung von Patienten, bei denen eine Acrysof®-Intraokularlinse implantiert wurde, an der Spaltlampe und anschließende Dokumentation mittels Fotospaltlampe. Zusätzlich wurden Sehschärfebestimmungen durchgeführt. Analog wurden Untersuchungen und Dokumentationen bei Patienten, denen im gleichen Zeitraum und vom gleichen Operateur eine herkömmliche PMMA-Kunstlinse implantiert wurde, durchgeführt. In allen untersuchten Intraokularlinsen des neuen Modells aus Acryl konnten Mikrovakuolen festgestellt und fotografisch dokumentiert werden. Diese Veränderungen konnten in keiner der PMMA-Kunstlinsen beobachtet werden. In unseren Ergebnissen fanden sich sonst durchgehend Hinweise auf die mittlerweile von anderen gewonnenen Erkenntnisse über die neue Linse. Es konnten keine Hinweise auf eine Visusminderung durch die Glistenings festgestellt werden. Die Analyse der Literatur ergab, daß nach dem heutigem Stand der Erkenntnisse über die Glistenings, von einer Flüssigkeitsaufnahme in den Optikkörper der IOL ausgegangen wird. Die Flüssigkeit sammelt sich in einige mikrometer großen Räumen in der Polymerstruktur der Kunstlinse. Diese Mikrovakuolen werden durch die Brechkraftdifferenz zwischen deren Inhalt und dem IOL-Material optisch sichtbar und erscheinen bei der Spaltlampenuntersuchung als multiple Glitzerpunkte. Experimentelle Arbeiten haben gezeigt, daß die Ausbildung der Mikrovakuolen durch Temperaturschwankungen und durch die Anwesenheit von Serum begünstigt wird. Ein verstärktes Vorkommen der Glistenings bei Patienten, bei denen sich durch Beeinträchtigung der Blut-Kammerwasser-Schranke oder der Blut-Retina-Schranke, Serumbestandteile im Kammerwasser befinden können, hier sind besonders Diabetiker zu nennen, wurde von einigen Autoren berichtet. Auch wir konnten bei einer nachträglichen Auswertung unserer Unterlagen Hinweise darauf finden, daß es bei solchen Patienten zur verstärkten Glisteningbildung kommt. Die Aussage der Herstellerfirma, die Glistenings hingen mit der neuartigen Verpackung und Sterilisation der IOLs in dem AcryPak-System, welches nur in den USA zum Einsatz kam und 1995 vom Markt genommen wurde, zusammen, wurde durch unsere Ergebnisse widerlegt. Besonders die frühere englischsprachige Literatur spiegelt einen Konsens mit der vom Hersteller vertretenen Auffassung wider. Neuere Arbeiten, die unsere Ergebnisse bestätigen weisen auf ein Poster auf einem Kongress 1999 in Seattle als erstes bekanntwerden von Glistenings in herkömmlich verpackten AcrySof®-IOL hin, obwohl schon frühere Berichte von Autoren aus Europa, auch von unserer Arbeitsgruppe, vorlagen. Die Mikrovakuolen haben nur in sehr seltenen Einzelfällen, bei denen es zu massenhaftem Vorkommen gekommen war, zu Beeinträchtigungen der Sehfunktionen geführt und eine Explantation erforderlich gemacht. Trotzdem sind weitere Forschungen, für die wir einige Ansätze vorschlagen, notwendig. Auch das Vorkommen von schwerwiegenden Trübungen bei anderen Kunstlinsentypen zeigt die Notwendigkeit für verstärkte Forschungen und gesetzliche Regulierungen auf diesem Gebiet. Insbesondere die hervorragend niedrigen Nachstarraten, die mit der Implantation von AcrySof®-Kunstlinsen einher gehen, berechtigen jedoch zu ihrer Weiterverwendung. Während es unwahrscheinlich scheint, daß die Glistenings noch schwerwiegende Probleme bereiten werden, so ist jedoch gesichert, daß durch die Verwendung von AcrySof®Kunstlinsen weniger YAG-Laser-Kapsulotomien notwendig sind. Daß sich damit auch die mit diesem Eingriff verbundenen Kosten und Komplikationen reduzieren ist selbstredend. Der Verlauf der Forschungen zu diesem Thema wird dargelegt. Die Notwendigkeit präziserer Studienaufbauten und besserer technischer Ausstattung wird deutlich. Durch den fortgeführten Einsatz der AcrySof®-IOL wurde die ophthalmologische Sichtweise der IOL verändert. Die IOL wird heute als Implantat betrachtet, welches die Aphakie korrigiert aber auch den Nachstar verhüten soll. Im kleinen Rahmen der Entwicklungsgeschichte der IOLs selbst, könnte dies den Beginn einer neuen IOL-Generation bedeuten.