Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06 Archiv

Das größte Problem für HIV-infizierte Personen ist die Tatsache, dass die Infektion nicht „geheilt“ werden kann und die Betroffenen ihr Leben lang infiziert bleiben. Deshalb erfordert die HIV Infektion die dauerhafte Anwendung von Therapien, die das Virus an der Replikation hindern und damit die „Viruslast“ im Körper möglichst gering halten. Die optimale Bekämpfung der HIV-Infektion wären Wirkstoffkombinationen die sowohl die Produktion des Virus durch persistent infizierte Reservoirs als auch die Neuinfektion von HIV-Zielzellen unterbinden. Interessanterweise sind einige zelluläre Faktoren bekannt, die in die HIV-Replikation eingreifen und der HIV-Produktion entgegenwirken können. Ein Beispiel für solche HIV-Restriktionsfaktoren sind die Risp/ Fam21 Proteine, die mit dem HIV Rev Protein interagieren und so vermutlich seine regulatorische Funktion hemmen können. Frühere Untersuchungen an persistent HIV-infizierten Astrozyten belegten einen Zusammenhang zwischen der Expressionsstärke von Risp/ Fam21 und der Hemmung der HIV Produktion in diesen Zellen. In dieser Arbeit wurde mit Hilfe der quantitativen PCR Analyse gezeigt, dass risp/ fam21 Gene in unterschiedlichen Stärken in menschlichen Zellen exprimiert werden. Zur Modulation der Risp/ Fam21 Expression in diesen als auch anderen für HIV relevanten Zellen wurde ein lentivirales Vektorsystem etabliert. In akut infizierten T-Zellen wurde kein Einfluss der Risp/ Fam21-Modulation auf die HIV-Infektion gefunden, was die Theorie nahelegt, dass Risp/ Fam21 Proteine nur in persistent infizierten Zellen wie den o.g. Astrozyten eine Aktivität zeigen könnten. Um neue Inhibitoren der akuten HIV-Infektion gesunder Zellen zu identifizieren, wurde die medizinische Heilpflanze Cistus incanus (Ci) im Hinblick auf ihre anti-HIV Aktivität getestet. Bei dieser Pflanze handelt es sich um eine sehr polyphenolreiche Pflanze und Polyphenole stellen eine interessante Klasse an HIV-Inhibitoren dar. In der vorliegenden Arbeit wurde gezeigt, dass Präparate aus Ci die Infektion von Zellen hemmen, indem sie spezifisch an die Virusoberfläche binden und die Anheftung der Viren an die Zielzellen verhindern. Präparate aus Ci inhibieren ein sehr breites Spektrum an verschiedenen HIV-Laborstämmen und –Patientenisolaten.

Diese Arbeit beschäftigt sich mit der Expression des astrozytenspezifischen Enzyms Glutaminsynthetase in Ergänzung zum gliaspezifischen Marker Repo, um Gliazellen, die mit der embryonalen Entwicklung des Zentralkomplexes in Schistocerca gregaria assoziiert sind, zellulär und molekular zu charakterisieren. Der Zentralkomplex ist ein modulares System neuropiler Strukturen im Mittelhirn aller Insekten, und ist in vielen Verhaltensvorgängen wie Laufen, Fliegen, Stridulation und Ernährung involviert. In der Heuschrecke entwickeln sich die Neuropile des Zentralkomplexes im Laufe der Embryogenese und sind zum Zeitpunkt des Schlüpfens funktionsfähig. Trotz großer Kenntnisse neuronaler Aspekte über die Entwicklung des Zentralkomplexes verbleibt die Funktion der Gliazellen unklar. In dieser Arbeit wurde das Expressionsmuster des astrozytenspezifischen Enzyms Glutaminsynthetase (GS) und des gliaspezifischen Homöobox Gens reversed polarity (repo) in Kombination mit der negativen Expression des neuron-spezifischen Markers Meerrettich Peroxidase (HRP) zur Identifizierung glialer Zellen benutzt. Doppelfärbungen zeigen, dass alle GS-positiven Zellen, die mit dem Zentralkomplex assoziiert sind, gleichzeitig Repo-positiv sind. Zum ersten Mal konnte ich durch diese Kombination nicht nur Zellkörper, sondern auch Projektionen (Gliapodien) der Gliazellen sichtbar machen. Während der Embryogenese, also noch vor der Entwicklung des Zentralkomplexes, formen Gliazellen eine zusammenhängende Population, die aus der Pars intercerebralis in die Region der Faserbündel einwandert. Anschließend verteilen sich die Gliazellen neu und umhüllen jedes der einzelnen Module des Zentralkomplexes. Innerhalb der einzelnen Neuropile des Zentralkomplexes sind keine glialen Zellkörper zu finden. Rekonstruktionen einzelner Zellen zeigen Populationen von Gliazellen, die ausgedehnte umhüllende Projektionen um die Neuropile des Zentralkomplexes, wie den Zentralkörper, senden, während eine andere Population von Gliazellen säulenartige Verzweigungen in den Zentralkörper hinein projiziert. Solche Verzweigungen in den Modulen des Zentralkomplexes sind erst nach Fertigstellung der Neuroarchitektur zu erkennen. Daher kann man annehmen, dass diese Verzweigungen auf ein zuvor entstandenes Gerüst von Neuronen oder Tracheen projizieren. Höchstwahrscheinlich sind diese Gliaprojektionen in die Transmitterregulation innerhalb des Neuropils involviert. Da Gliazellen weitreichende Projektionen (Gliapodien) in und um die Mittelhirnneuropile senden, wurden in gefrorenen Hirnschnitten intrazelluläre Injektionen durchgeführt um zu erforschen, ob diese Gliazellen ein zelluläres Netzwerk via Zellkopplung im Verlauf der Embryogenese bilden. Färbungen individueller Zellen, die an vier unterschiedlichen Injektionsstellen um den Zentralkörper lokalisiert sind, zeigen eine Population gekoppelter Zellen, deren Anzahl und räumliche Verteilung stereotypisch für jeden der Injektionspunkte ist. Darüber hinaus sind sie sowohl bei 70%igem wie auch bei einem embryonalen Entwicklungsstand von 100% miteinander vergleichbar. Anschließende immunhistochemische Experimente bestätigen, dass es sich bei den gekoppelten Zellen um astrozytenähnliche Gliazellen handelt. Durch Hinzufügen von n-Heptanol in das Puffermedium wurde die Zellkopplung verhindert. Da die Zellkopplung auch ohne direkten intersomalen Kontakt auftritt, könnten die erheblichen Verzweigungen der Gliapodien, die sich im Laufe der Embryogenese ausbreiten, involviert sein. Durch die Datenerhebung aller Injektionspunkte kann darauf geschlossen werden, dass die Gliazellen, welche den Zentralkörper umrunden, ein Netzwerk gekoppelter Gliazellen bilden, das als Positionierungssystem der sich entwickelnden Neuropile des Zentralkomplexes dient.

The CHO-K1 cell line is the most common expression system for therapeutic proteins in the pharmaceutical industry. Due to the nature of economics, the cell lines and the vector design are subject to constant change to increase product quality and quantity. During the cultivation, the production cell lines are susceptible to decreasing productivity over time. Often the loss of production can be associated with a reduction of copy number and the silencing of transgenes. During cell line development, the most promising cell lines are cultivated in large batch culture. Consequently, the loss of a stable production cell line can be very cost-intensive. For this reason I developed different strategies to avoid a reduced productivity. Instability of production cell lines can be predicted by the degree of CpG methylation of the driving promoter. Considering that the DNA methylation is at the end of an epigenetic cascade and associated with the maintenance of the repressive state, I investigated the upstream signals of histone modifications with the assumption to obtain a higher predictive power of production instability. For this reason I performed a chromatin immunoprecipitation of the histone modifications H3K9me3 and H3K27me3 as repressive signals and H3ac as well as H3K4me3 as active marks. The accumulations of those signals were measured close to the hCMV-MIE at the beginning of the cultivation and were then compared with the loss of productivity over two month. I found that the degree of the H3 acetylation (H3ac) correlated best with the production stability. Furthermore I was able to identify an H3ac threshold to exclude most of the unstable producers. In the second project I aimed to improve the vector design by considering epigenetic mechanisms. To this end I designed on the one hand a target-oriented histone acetyltransferase to enforce an open and active chromatin status at the transgene. On the other hand I point-mutated methylation-susceptible CpGs within the hCMV-MIE to impede the maintenance of inactive heterochromatin formation. Remarkably, the C to G mutation located 179 bp upstream of transcription start site resulted in very stable antibody producing cell lines. In addition, the examination of cell pools expressing eGFP showed that G-179 promoter variants were less prone to a general methylation and gene amplification, which illustrates the dominating effect in epigenetic mechanisms of one single CpG. The last project was performed to localize stable integration sites within the CHO-K1 genome. In so doing I could show that the transfection leads predominantly to integration into inactive regions. Furthermore I identified promising integration sites with a high potential to induce stable expression. However, those results are preliminary and must be viewed with caution. Further examination needs to be done to confirm these results. Considering the results of all three projects, I propose that the interplay of metabolic burden and selection pressure at an early time point of cultivation plays an important role in cell line development. Small alterations of selection pressure can lead to a decisive change of cell properties. Therefore, stable cells are less susceptible than weak producers. The increase of selection pressure leads to compensatory effect by gene amplification in the instable cell lines. The resulting adjustment of productivity masks the truly stable cells, which precludes the selection of the right cell lines. For this reason the selection pressure, the copy number as well as the growth rate should be considered to minimize repressive effects.

Magnetotactic bacteria (MTB) contain nanometer-sized crystals of a magnetic iron mineral enabling directed swimming along geomagnetic field lines. However, although this unique behavior was discovered already 40 years ago, it still has remained poorly understood at the cellular level and the molecular mechanisms responsible for sensing environmental stimuli and transducing signals to the flagellar motors have been unknown. Therefore, the major goal of this thesis was to investigate the swimming behavior of Magnetospirillum gryphiswaldense both at the behavioral and molecular level. Individual motors of tethered M. gryphiswaldense cells were found to rotate both clockwise and counterclockwise with equal speed. Cells swam at speeds of up to 60 µm s-1 and commonly displayed runs of several hundred µm in length. In striking contrast to E. coli, which reorients the cell body between run intervals at random angles, motor switching events caused swimming reversals with reorientation angles close to 180°. The sensory repertoire of M. gryphiswaldense was analyzed by classical macroscopic chemotaxis assays, and aerotaxis was found to be the dominant behavior. In addition to the strong microaerophilic response in oxygen gradients, I observed tactic bands also under anoxic conditions within gradients of the alternative electron acceptor nitrate, suggesting that aerotaxis is part of a general redox or energy taxis mechanism. The aerotactic response of M. gryphiswaldense was furthermore analyzed by recording and tracking single cells under controlled atmospheric conditions in a gas perfusion chamber. Compared to other well-studied bacteria, M. gryphiswaldense displayed unusually low swimming reversal rates (

In order to react to changes within their environment, plants developed a specific signaling network that enables the cells to convert external stimuli including light, abiotic and biotic stress as well as hormones into cellular signals. One example is the influx of calcium, a second messenger stored in apoplasts or internal reservoirs, into the cytosol. This causes changes in the calcium-ion-concentration within the cell that are recognized by specific sensors including Calmodulin and lead to the induction of a cellular signal response. Calcium signals do not only occur in the cytosol, but also appear within the nucleus, chloroplasts, mitochondria as well as peroxisoms (Bachs et al. 1992, Chigri et al. 2005, Kuhn et al. 2009, Dolze et al. 2013). The import of nuclear encoded proteins into the mitochondria is regulated by calcium and Calmodulin at level of the TIM23- and TIM22-complex. This study identified atTim23.2, the pore-forming component of the TIM23-complex, as a Calmodulin-binding protein. Pull-down-assays using Calmodulin-agarose revealed a specific and calcium-dependent binding. Furthermore, in silico analysis identified two potential Calmodulin-binding domains (CaMBD). Topology studies of atTim23.2 demonstrated that the proposed N-terminal CaMBD is located within the intermembrane space, the binding region within the first loops is located in the matrix of the mitochondria. Moreover, a topology of four transmembran domains of the protein could be shown. The recently in the mitochondria identified Calmodulin-like protein CML30 appeared to be a potential binding partner for atTim23.2. CML30 could be indeed detected in the intermembrane space of the mitochondria, but a direct interaction of the two proteins could not have been detected so far. Furthermore, using the split-ubiquitin system proved the ability of atTim23.2 to dimerize which might be responsible for the regulation of opening and closing of the importpore as it was already shown in S.cerevisiae. However, a correlation between the two functions of atTim23.2 to bind Calmodulin as well as to dimerize could not have been confirmed, yet. Nevertheless, the regulation of the pore via the calcium/Calmodulin signaling network could connect the import process of matrix proteins with the stress regulation of the cell.

The genetic basis underlying adaptive evolution is still largely unknown. Adaptive evolution is facilitated by natural selection that acts on the genetic variation present in a population. Favoring some genetic variants over others, natural selection eventually produces adaptations that allow populations to survive in changing or new environments. Populations colonizing new habitats that differ from their original habitat are often confronted with a multitude of novel ecological constraints to which they need to adapt. A well-annotated genome and a diverse genetic toolkit make the fruit fly Drosophila melanogaster an ideal model system for studying the genetics underlying adaptation. As a cosmopolitan species, D. melanogaster has adapted to a wide range of thermal environments. Despite having a tropical origin in southern-central Africa, it has successfully settled in temperate environments around the world. Thermal adaptations that have helped to deal with the greater range and variability in temperature as well as low-temperature extremes have been required to prosper in temperate environments. Chromatin-based gene regulation is known to be disrupted by varying temperatures. Variation in the temperature, at which flies live, result in varying expression levels of Polycomb group (PcG) regulated genes with higher expression at lower temperatures. Chapter 1 and 2 of this thesis aim to answer the question whether this thermosensitivity of PcG regulation has been detrimental for colonizing temperate environments and thus needed to be buffered by natural selection. Thermosensitivity of PcG regulation was observed in different natural populations of D. melanogaster. A lower degree of thermosensitive expression was consistently found for populations from temperate climates when compared to those from the tropics. In Chapter 1, evidence is presented for positive selection acting on the polyhomeotic (ph) gene region to reduce thermosensitivity of PcG regulation in temperate populations from Europe. The targets of selection appear to be single nucleotide polymorphisms (SNPs) in a relatively small cis-regulatory region between the two PcG target genes polyhomeotic proximal (ph-p) and CG3835 that are highly differentiated between European and African populations. Using reporter gene assays, it was demonstrated that these SNPs influence gene expression and that the European alleles confer reduced thermosensitivity of expression in contrast to the African alleles. In Chapter 2, thermosensitivity of another PcG target gene, vestigial (vg), was investigated in six natural populations including four temperate populations from high-altitude Africa and central to high-latitude Europe, and two tropical populations from the ancestral species range. All four temperate populations exhibited a lower degree of thermosensitive expression than the two tropical populations. The underlying mechanisms of increased buffering, however, seem to differ between these temperate populations. Thermal adaptation to temperate environments also includes dealing with low-temperature extremes. Severe cold stress is a main limiting factor imposed on D. melanogaster by temperate climates. Increased cold tolerance in temperate populations is thought to have evolved by natural selection. Cold tolerance is a quantitative trait that appears to be highly polygenic and has been mapped to different quantitative trait loci (QTL) in the genome. In Chapter 3, such a QTL region was fine-mapped to localize causal genes for increased cold tolerance in temperate flies. As a result, brinker (brk) was identified as a new candidate gene putatively involved in cold stress adaptation.

At least four phylogenetically distinct groups of bacteria encode repeat proteins with the common ability to bind specific DNA sequences with a unique but conserved code. Each repeat binds a single DNA base, and specificity is determined by the amino acid residue at position 13 of each repeat. Repeats are typically 33-35 amino acids long. Comparing repeat sequences across all groups reveals that only three positions are hyper-conserved. Repeats are in most cases functionally compatible such that they can be assembled together into a single chimeric array. This functional conformity and inter-compatibility is a result of structural conservation. Repeat arrays of these proteins have been demonstrated or predicted to form almost identical tertiary structures: a right-handed super helix that wraps around the DNA double strand with the base specifying residue of each repeat positioned in the major groove next to its cognate target base. The mechanism of DNA binding is conserved. The first discovered group, providing the name for the rest, are the Transcription Activator Like Effectors (TALEs) of plant-pathogenic Xanthomonas bacteria. The eukaryotic transactivation domain, which lends this group their name, allows them to activate specifically targeted host genes for the benefit of the bacterial invader. The other groups, discovered after the TALEs, are the RipTALs of Ralstonia solanacearum, the Bats of Burkholderia rhizoxinica, and MOrTL1 and MOrTL2 of unknown marine bacteria. Together they are designated TALE-likes. Each designation contains some allusion to the TALEs. The term RipTAL stands for Ralstonia injected proteins TALE-like, the Bats are Burkholderia TALE likes, and the MOrTLs Marine Organism TALE-likes. This unity of terminology belies disunity in the lifestyles of these different bacteria, and the biological roles fulfilled by these proteins. The TALEs have already been researched extensively. The code that describes the relationship between the base specifying residues and their cognate bases is often referred to as the TALE code. This code was deciphered by two groups independently and published in 2009, a year before I began my doctoral work. Since then research into TALEs has not slowed and a great deal has been learnt both about the native biology and biotechnological uses of TALEs. My work has been focused on the other TALE-like groups, none of which had been previously characterized in terms of DNA recognition properties, before I began my work. RipTALs are effector proteins delivered during bacterial wilt disease caused by R. solanacearum strains. This devastating disease affects numerous crop species worldwide. Characterizing the molecular properties of the RipTALs provides a first step towards uncovering their role in the disease. The Bats and MOrTLs are primarily of interest as comparison groups to the TALEs and RipTALs and as sources of sequence diversity for future efforts into TALE repeat engineering. In the introduction of this dissertation, which explores TALE biology, a particular focus will be placed on the DNA binding properties of TALEs and how this can be put to use in TALE technology. After this the RipTALs, Bats and MOrTLs are each introduced, explaining what is known about their provenance and sequence features. The aims of my doctoral work are then listed and expounded in turn. The proximal goal of my doctoral work was to carry out a comparative molecular characterization of each group of non-TALE TALE-likes. In doing so we hoped to gain insights into the principles of TALE-like DNA-binding properties, evolutionary history of the different groups and their potential uses in biotechnology. In the case of the RipTALs this work should begin to unravel the role these proteins play in bacterial wilt disease, as a means to fight this devastating pathogen. The articles I have worked on covering the molecular characterizations of RipTALs, Bats and MOrTLs are then presented in turn. Working together with others I was able to show that repeats from each group of TALE-likes mediate sequence specific DNA binding, revealing a conserved code in each case. This code links position 13 of any TALE-like repeat to a specific DNA base preference in a reliable fashion. I will argue that the TALE-likes represent a fascinating case of conserved structure and function in a diverse sequence space. In addition the TALEs and RipTALs may simply represent one face of the TALE-likes, a protein family mediating as yet unknown biological roles as bacterial DNA binding proteins.

Dendritic integration is a fundamental element of neuronal information processing. So far, few studies have provided a detailed picture of this process, describing the properties of local dendritic activity and its subcellular organization. Here, I used 2-photon calcium imaging in optic flow processing neurons of the blowfly Calliphora vicina to determine the preferred location and direction of local motion cues for small branchlets throughout the entire dendrite. I found a pronounced retinotopic mapping on both the subcellular and the cell population level. In addition, dendritic branchlets residing in different layers of the neuropil were tuned to distinct directions of motion. Within one layer, local preferred directions varied according to the deflections of the ommatidial lattice. Summing the local receptive fields of all dendritic branchlets reproduced the characteristic properties of these neurons’ axonal output receptive fields. These results corroborate the notion that the dendritic morphology of vertical system cells allows them to selectively collect local motion inputs with particular directional preferences from a spatially organized input repertoire, thus forming filters that match global patterns of optic flow. These data illustrate a highly structured circuit organization as an efficient way to hard-wire a complex sensory task.

Physical changes in neuronal connections, dictated by the neuronal network activity, are believed to be essential for learning and memory. Long-term potentiation (LTP) of synaptic transmission has emerged as a model to study activity-driven plasticity. The majority of excitatory contacts between neurons, called synapses, are found on spines, small dendritic protrusions. LTP is known to trigger the formation and stabilization of new dendritic spines in vitro. Similarly, experience-dependent plasticity in vivo is associated with changes in the number and stability of spines. However, to date, the contribution of excitatory synaptogenesis to the enhanced synaptic transmission after LTP remains elusive. Do new spines form functional synapses with the inputs stimulated during LTP induction and thereby follow Hebbian co-activation rules, or do they connect with random partners? Furthermore, at which time-point are de novo spines functionally integrated into the network? I developed an optical approach to stably and exclusively stimulate the axons of a defined channelrhodopsin-2 (ChR2)-transduced subset of CA3 cell in mature hippocampal slice culture over extended periods of time (up to 24h). I continuously monitored synaptic activation and synaptic structure of CA1 cells dendrites using two-photon imaging. To control the dendritic location where LTP and associated spinogenesis were allowed to take place, I globally blocked Na+-dependent action potential firing and directly evoke neurotransmitter release by local light-evoked depolarization of ChR2-expressing presynaptic boutons (in TTX, 4-AP). I induced optical LTP specifically at this location by combining optogenetic activation with chemical pairing (in low [Mg2+]o, high [Ca2+]o, forskolin, and rolipram). Taking advantage of the NMDA-receptor mediated calcium influx during synaptic activation I assessed the formation of functional synapses using the genetically encoded calcium indicator GCaMP6s. I find that optical LTP led to the generation of new spines, decreased the stability of preexisting spines and increased the stability of new spines. Under optical LTP conditions, a fraction of new spines responded to optical presynaptic stimulation within hours after formation. However, the occurrence of the first synaptic calcium response in de novo spines varied considerably, ranging from 8.5 min to 25 h. Most new spines became responsive within 4 h (1.2 ± 0.9 h, mean ± S.D., n = 16 out of 20), whereas the remainder showed their first response only on the second experimental day (18.2 ± 3.7 h). Importantly, new spines generated under optical LTP were more likely to build functional synapses with light-activated, ChR2-expressing axons than spontaneously formed spines (new responsive spines under optical LTP: 64 ± 4 %; control 1: 0%; control 2: 13 ± 4 %; control 3: 11 ± 4 %). Furthermore, new spines that were responsive to optical presynaptic stimulation were less prone to be eliminated after overnight incubation than new spines that failed to respond (% overnight spine survival; 81 ± 3 % new responsive spines; 58 ± 4 % of new unresponsive spines). In summary, the results from my thesis demonstrate that synapses can form rapidly in an input-specific manner.