Every organism contains a characteristic number of chromosomes that have to be segregated equally into two daughter cells during mitosis. Any error during chromosome segregation results in daughter cells that lost or gained a chromosome, a condition known as aneuploidy. Several studies from our laboratory and across the world have previously shown that aneuploidy per se strongly affects cellular physiology. However, these studies were limited mainly to the chromosomal gains due to the availability of several model systems. Strikingly, no systemic study to evaluate the impact of chromosome loss in the human cells has been performed so far. This is mainly due to the lack of model systems, as chromosome loss is incompatible with survival and drastically reduces cellular fitness. During my PhD thesis, for the first time, I used diverse omics and biochemical approaches to investigate the consequences of chromosome losses in human somatic cells. Using isogenic monosomic cells derived from the human cell line RPE1 lacking functional p53, we showed that, similar to the cells with chromosome gains, monosomic cells proliferate slower than the parental cells and exhibit genomic instability. Transcriptome and proteome analysis revealed that the expression of genes encoded on the monosomic chromosomes was reduced, as expected, but the abundance was partially compensated towards diploid levels by both transcriptional and post transcriptional mechanisms. Furthermore, we showed that monosomy induces global gene expression changes that are distinct to changes in response to chromosome gains. The most consistently deregulated pathways among the monosomies were ribosomes and translation, which we validated using polysome profiling and analysis of translation with puromycin incorporation experiments. We showed that these defects could be attributed to the haploinsufficiency of ribosomal protein genes (RPGs) encoded on monosomic chromosomes. Reintroduction of p53 into the monosomic cells uncovered that monosomy is incompatible with p53 expression and that the monosomic cells expressing p53 are either eliminated or outgrown by the p53 negative population. Given the RPG haploinsufficiency and ribosome biogenesis defects caused by monosomy, we show an evidence that the p53 activation in monosomies could be caused by the defects in ribosomes. These findings were further supported by computational analysis of cancer genomes revealing those cancers with monosomic karyotype accumulated frequently p53 pathway mutations and show reduced ribosomal functions. Together, our findings provide a rationale as to why monosomy is embryonically lethal, but frequently occurs in p53 deficient cancers.

Whole genome duplication (WGD) is commonly accepted as an intermediate state between healthy cells and aneuploid cancer cells. Usually, cells after WGD get removed from the replicating pool by p53-dependent cell cycle arrest or apoptosis. Cells, which are able to bypass these mechanisms exhibit chromosomal instability (CIN) and DNA damage, promoting the formation of highly aneuploid karyotypes. In general, WGD favors several detrimental consequences such as increased drug resistance, transformation and metastasis formation. Therefore, it is of special interest to investigate the limiting factors and consequences of tetraploid proliferation as well as the adaptations to WGD. In the past it has been difficult to study the consequences of such large-scale genomic changes and how cells adapt to tetraploidy in order to survive. Our lab established protocols to generate tetraploids as well as isolated post-tetraploid/aneuploid single cells clones derived from euploid parental cell lines after induction of cytokinesis failure. This system enables to study the consequences and adaptations of WGD in newly generated tetraploid cells and evolved post-tetraploid clones in comparison to their isogenic parental cell line. Using newly generated tetraploids from HCT116 cells, we identified USP28 and SPINT2 as novel factors limiting the proliferation after WGD. Using mass spectrometry and immunoprecipitation, we revealed an interaction between USP28 and NuMA1 upon WGD, which affects centrosome coalescence of supernumerary centrosomes, an important process that enhances survival of tetraploids. Furthermore, we validated the occurrence of DNA damage in tetraploid cells and found that USP28 depletion diminished the DNA damage dependent checkpoint activation. SPINT2 influences the proliferation after WGD by regulating the transcription of CDKN1A via histone acetylation. Following proliferating tetraploid cells, we confirmed the activation of the DNA damage response (DDR) by immunoblotting and microscopic approaches. Furthermore, we show that the DDR in the arising post-tetraploid clones is reduced. Further experiments verified the appearance of severe mitotic aberrations, replication stress and accumulation of reactive oxygen species in newly generated tetraploids as well as in the aneuploid cancer cells contributing to the occurrence of DNA damage. Using various drug treatments, we observed an increased dependency on the spindle assembly checkpoint in aneuploid cancer cells compared to their diploid parental cell line. Additionally, siRNA knock down experiments revealed the kinesin motor protein KIF18A as an essential protein in aneuploid cells. Taken together, the results point out cellular consequences of proliferation after tetraploidization as well as the cellular adaptations needed to cope with the increased amount of DNA.

Impaired genome integrity has severe consequences for the viability of any cell. Unrepaired DNA lesions can lead to genomically unstable cells, which will often become predisposed for malignant growth and tumorigenesis, where genomic instability turns into a driving factor through the selection of more aggressive clones. Aneuploidy and polyploidy are both poorly tolerated in somatic cells, but frequently observed hallmarks of cancer. Keeping the genome intact requires the concentrated action of cellular metabolism, cell cycle and DNA damage response. This study presents multi-omics analysis as a versatile tool to understand the various causes and consequences of impaired genome integrity. The possible computational approaches are demonstrated on three different datasets. First, an analysis of a collection of DNA repair experiments is shown, which features the creation of a high-fidelity dataset for the identification and characterization of DNA damage factors. Additionally, a web-application is presented that allows scientists without a computational background to interrogate this dataset. Further, the consequences of chromosome loss in human cells are analyzed by an integrated analysis of TMT labeled mass spectrometry and sequencing data. This analysis revealed heterogeneous cellular responses to chromosome losses that differ from chromosome gains. My analysis further revealed that cells possess both transcriptional and post-transcriptional mechanisms that compensate for the loss of genes encoded on a monosomic chromosome to alleviate the detrimental consequences of reduced gene expression. In my final project, I present a multi-omics analysis of data obtained from SILAC labeled mass spectrometry and dynamic transcriptome analysis of yeast cells of different ploidy, from haploidy to tetraploid. This analysis revealed that unlike cell volume, the proteome of a cell does not scale linearly with increasing ploidy. While the expression of most proteins followed this scaling, several proteins showed ploidy-dependent regulation that could not be explained by transcriptome expression. Hence, this ploidy-dependent regulation occurs mostly on a post-transcriptional level. The analysis uncovered that ribosomal and translation related proteins are downregulated with increasing ploidy, emphasizing a remodeling of the cellular proteome in response to increasing ploidy to ensure survival of cells after whole genome doubling. Altogether this study intends to show how state-of-the-art multi-omics analysis can uncover cellular responses to impaired genome integrity in a highly diverse field of research.

The handling of oxygen sensitive samples and growth of obligate anaerobic organisms requires the stringent exclusion of oxygen, which is omnipresent in our normal atmospheric environment. Anaerobic workstations (aka. Glove boxes) enable the handling of oxygen sensitive samples during complex procedures, or the long-term incubation of anaerobic organisms. Depending on the application requirements, commercial workstations can cost up to 60.000 €. Here we present the complete build instructions for a highly adaptive, Arduino based, anaerobic workstation for microbial cultivation and sample handling, with features normally found only in high cost commercial solutions. This build can automatically regulate humidity, H2 levels (as oxygen reductant), log the environmental data and purge the airlock. It is built as compact as possible to allow it to fit into regular growth chambers for full environmental control. In our experiments, oxygen levels during the continuous growth of oxygen producing cyanobacteria, stayed under 0.03 % for 21 days without needing user intervention. The modular Arduino controller allows for the easy incorporation of additional regulation parameters, such as CO2 concentration or air pressure. This paper provides researchers with a low cost, entry level workstation for anaerobic sample handling with the flexibility to match their specific experimental needs.

Most mitochondrial proteins are synthesized in the cytosol and targeted to the mitochondrial surface in a post-translational manner. The surface of the endoplasmic reticulum (ER) plays an active role in this targeting reaction. ER-associated chaperones interact with certain mitochondrial membrane protein precursors and transfer them onto receptor proteins of the mitochondrial surface in a process termed ER-SURF. ATP-driven proteins in the membranes of mitochondria (Msp1, ATAD1) and the ER (Spf1, P5A-ATPase) serve as extractors for the removal of mislocalized proteins. If the re-routing to mitochondria fails, precursors can be degraded by ER or mitochondria-associated degradation (ERAD or MAD respectively) in a proteasome-mediated reaction. This review summarizes the current knowledge about the cooperation of the ER and mitochondria in the targeting and quality control of mitochondrial precursor proteins.

Amino acids, apart from being building blocks of proteins, serve various cellular and metabolic functions1,2. Changes in amino acid handling have been observed in a wide range of human pathologies, including diabetes and various metabolic disorders (aminoacidopathies)3–5. Saccharomyces cerevisiae is used as a model to investigate how increase in amino acid content (in the form of amino acid dropout mix: AAM) in growth medium influences cell growth. Intriguingly, it was observed that increasing the concentration of AAM in the media (double or triple times; 2 X AAM and 3 X AAM respectively), severely affects the growth of auxotrophic but not of prototrophic yeast strains in presence of glucose as carbon substrate. Increased concentration of Ehrlich amino acids, which are degraded to fusel acidic/alcoholic compounds, induced the observed slow growth phenotype of BY4742. These phenotypes can be rescued by either re-establishing the functional leucine biosynthetic pathway in BY4742 (leucine auxotroph) or increasing leucine in proportion to the increased AAM. Interestingly, the amino acid dependent growth phenotypes are absent when cells grow in media containing non-fermentable carbon sources. Furthermore, the deletion of ILV2 or ILV3 (genes encoding enzymes involved in the leucine biosynthetic pathway) also rescues the growth phenotype of BY4742 on 2 X AAM and 3 X AAM growth media. It was found that Ilv3 is the potential switching point and links cellular growth to redox homeostasis. The possibility of leucine limitation per se or transport competition between different Ehrlich amino acids and leucine, as a cause for the observed phenotypes, is ruled out. Upregulation of the branched-chain amino acid pathway inhibits cell growth of BY4742 on 2 X AAM. Although we could not detect KIV, the α-keto acid intermediate formed by the Ilv3. It is proposed that KIV itself (or its unknown downstream product) leads to the onset of the observed phenotypes. Different studies suggest that oxidative stress (due to accumulation of branched-chain amino acids (BCaa) and their α-keto acids) contributes to the neurological damage of MSUD patients6–9. It was also observed that the trigger of the BCaa bio-synthesis pathway on 2 X AAM growth conditions also contributes to the significant oxidative stress in the cell. In conclusion, we propose that yeast can be used as a suitable model system to study how accumulation of BCaa and their α-keto acids are lead to oxidative stress that is potentially toxic to cells. Further, this knowledge and the underlying molecular mechanisms will enhance our understanding of MSUD in humans.

Signal transduction systems are of great importance for the adaptation of organisms to new conditions. These systems occur most frequently in bacteria and are well-understood thanks to research. It was not until 10 years after the discovery of two-component systems in bacteria that such a system was reported in archaea. This work provides new insights into signal transduction in archaea, through the characterization of a histidine kinase MA4377 and its multi-component system in the methanogenic archaeon Methanosarcina acetivorans. MA4377 is a hybrid kinase of bacterial origin and was probably integrated into M. acetivorans via horizontal gene transfer. Based on the fused receiver domains, MA4377 is classified as a hybrid kinase that regulates a cell response upon the perception of a signal through a multi-component system. These systems consist of four components, the first of which is the kinase. MA4377 has autophosphorylation activity and is phosphorylated at a conserved histidine residue (His497). While the kinase activity is independent of the redox state of the protein, the PAS domain is mandatory for autokinase activity. Using different protein variants, it could be shown that the two fused receiver domains are not involved in the phosphorelay. Rather, the single receiver domain MA4376 serves as the second component of the system. The signal is ultimately transmitted to a transcription factor (MA4375) of the Msr family. Factors of this family are involved in the regulation of methanogenesis, among other things. MA4377 could thus be involved in a multi-component system regulating methanogenesis. The receiver MA4376 plays a central role here since feedback regulation on the kinase was observed. Further investigations will show to what extent cross-regulation with other kinases takes place and in what way the receiver MA4376 plays a key role in this multi-component system.

Iron is an essential nutrient for all life forms, including plants, but of limiting availability in many environments, affecting productivity of both food production and carbon capturing. Iron is essential because of its broad function as a catalyst of redox reactions and processes involving O2 chemistry in the catalytic centers of enzymes. Because of the nature of these reactions, excess amounts of the nutrient can be toxic, requiring a fine tuning of the cellular iron content, to both accommodate the essential demand and avoid detrimental effects simultaneously. A question of this project is how plant metabolism is modified in iron-deficient conditions, for which the green alga Chlamydomonas reinhardtii as a microbe is an excellent reference organism. The metabolic flexibility of C. reinhardtii, specifically the capacity for both heterotrophic (on acetate) and autotrophic (on CO2) growth, offers a unique opportunity to distinguish the impact of iron nutrition on photosynthetic versus respiratory metabolism. During steady-state photoheterotrophic Fe-limited growth, where the cells are provided with light, CO2, and acetate, but lack extracellular iron, cells maintain respiration while decreasing photosynthetic contribution to the energetics of the cell. This thesis analyzes the transition from photoautotrophic (light and CO2) to photoheterotrophic cultures in the context of Fe-nutrition by adding a reduced carbon source to phototrophic cultures and assessing the developing changes to the metabolism time-dependently, in various levels of readouts. Based on the transcriptome analysis, all major cellular processes and pathways respond to the availability of acetate, but Fe-limited cells specifically sacrifice photosynthetic capacity towards respiratory activity in the first 12h after the additional carbon source becomes available, allowing to gain mechanistic insights of transitioning between different ways of life, dependent on the nutritional makeup of the environment. Secondly, exposure to high extracellular iron amounts, its opportunities, and the mechanisms of avoiding deleterious effects as a result from it, had been under-investigated before the beginning of this thesis. Physiological and photosynthetic parameters, elemental analysis, transcriptomics, and a mutant depleted of functional acidic vacuoles, proposed to be involved in the storage for transition metals, were utilized to further the understanding of the processes. Altogether, the results presented in this thesis illustrate how C. reinhardtii can be successfully used as a model organism to study a large variety of aspects of cell and molecular biology, including dynamic acclimations to changing environments.

Glycine constitutes the major neurotransmitter at inhibitory synapses of lower brain regions. A rapid removal of glycine from the synaptic cleft and consequent recycling is crucial for synaptic transmission in systems with high effort on temporal precision. This is mainly achieved by glycine translocation via two glycine transporters (GlyTs), namely GlyT1 and GlyT2. At inhibitory synapses, GlyT2 was found to be specifically expressed by neurons, supplying the presynapse with glycine needed for vesicle filling. In contrast, GlyT1 is attributed to astrocytes and primarily mediates the termination of synaptic transmission by glycine removal from the synaptic cleft. Employing patch-clamp recordings from principal neurons of the lateral superior olive (LSO) in acute brainstem slices of GlyT1b/c knockout (KO) mice and wildtype (WT) littermates at postnatal day 20, I analyzed how postsynaptic responses are changed in a GlyT1-depleted environment. During spontaneous vesicle release I found no change of postsynaptic responses, contradicting my initial hypothesis of prolonged decay times. Electrical stimulation of fibers of the medial nucleus of the trapezoid body (MNTB), which are known to form fast, reliable and highly precise synapses with LSO principal neurons, revealed that GlyT1 is involved in proper synaptic function during sustained, high frequent synaptic transmission. Stimulation with 50 Hz led to a stronger decay time and latency prolongation in GlyT1b/c KO, accelerating to 60% longer decay times and 30% longer latencies. Additionally, a more pronounced frequency-dependent depression and fidelity decrease was observed during stimulation with 200 Hz in GlyT1b/c KO, resulting in 67% smaller amplitudes and only 25% of WT fidelity at the end of the challenge. Basic properties like readily releasable pool, release probability, and quantal size (q) were not altered in GlyT1b/c KO, but interestingly q decreased during 50 Hz and 100 Hz challenges to about 84%, which was not observed in WT. I conclude that stronger accumulation of extracellular glycine due to GlyT1 loss leads to prolonged activation of postsynaptic glycine receptors (GlyRs). As a further consequence, activation of presynaptic GlyRs in the vicinity of the synaptic cleft might be enhanced, accompanied by a stronger occurrence of shunting inhibition. Furthermore, I assume a GlyT1-dependent glycine shuttle, which is absent at GlyT1b/c KO synapses. This could result in a diminished glycine supply to GlyT2 located at more distant sites, causing a disturbed replenishment during periods with excess release of glycine. Conclusively, my study reveals a contribution of astrocytes in fast and reliable synaptic transmission at the MNTB-LSO synapse, which in turn is crucial for proper sound source localization.

Potassium (K) is essential for the processes critical for plant performance, including photosynthesis, carbon assimilation, and response to stress. K also influences translocation of sugars in the phloem and regulates sucrose metabolism. Several plant species synthesize polyols and transport these sugar alcohols from source to sink tissues. Limited knowledge exists about the involvement of K in the above processes in polyol-translocating plants. We, therefore, studied K effects in Plantago major, a species that accumulates the polyol sorbitol to high concentrations. We grew P. major plants on soil substrate adjusted to low-, medium-, or high-potassium conditions. We found that biomass, seed yield, and leaf tissue K contents increased in a soil K-dependent manner. K gradually increased the photosynthetic efficiency and decreased the non-photochemical quenching. Concomitantly, sorbitol levels and sorbitol to sucrose ratio in leaves and phloem sap increased in a K-dependent manner. K supply also fostered plant cold acclimation. High soil K levels mitigated loss of water from leaves in the cold and supported cold-dependent sugar and sorbitol accumulation. We hypothesize that with increased K nutrition, P. major preferentially channels photosynthesis-derived electrons into sorbitol biosynthesis and that this increased sorbitol is supportive for sink development and as a protective solute, during abiotic stress