Kaiserslautern - Fachbereich Biologie
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One of the main tasks of molecular biology is understanding the mechanisms of molecular biological processes. This brings the problem of creating regulatory networks and therefore finding key regulators. In order to do it, it is important to have such representation of the data that can reveal the distinct patterns within the big groups. On one side, there are numerous experimentally determined kinetic information about the alteration of molecular presence in the observed system. On the other side, there are documented throughout the years evidences of the involvement of molecules in different biological processes. Both sources of the information have their drawbacks: experimental data reflect only a fleeting molecular state of each individual organism and therefore are often high-variant and noisy; functional groups were determined as generalization of known roles of molecules in biological processes and therefore can be not complete and only partially relevant to certain experimental conditions and individual organisms. Our goal is to get the overview of the experimentally observed molecules and extract the knowledge from both sources, avoiding constrains of noise distractions and generalization bias. The resulted optimal representation of the experimental data then would help to pinpoint potential regulators.
The proposed method is called the Signature Topology (ST) approach, as it uses the functional topology as the prior knowledge source and creates a specific signature for the given experimental data. The ST approach is based on knowledge-and-data-driven machine learning algorithm, that is implemented via a dynamic programming approach. Based on both prior knowledge and learning from the data, the proposed approach represents a combination of supervised and unsupervised machine learning. The resulting network structure deals with data abundance and avoids an over-detailed description that may lead to misinterpretation and is able to pick out elements with minor behavior patterns.
The method is tested with artificial data and applied to real-world mass-spectrometry proteome data and NGS-transcriptome data of Chlamydomonas reinhardtii. The proposed approach helps with identification of the potential regulatory genes, whose roles are not explicitly provided in the used functional ontology. Moreover, it shows a successful reduction in data complexity while preserving all individual molecular information reported in the literature and stored in the functional ontology. If the proposed approach analyzes different experimental data with the same ontology, the resulting networks are uniform and therefore can be compared. That gives an opportunity to compare between a great variety of experimental conditions, from different organisms to different
system levels.
Drought is a significant environmental factor that can impair plant growth and development, leading to reduced crop productivity or even plant death. Maintaining sugar distribution from source to sink is crucial for increasing crop production under water limitation conditions. Numerous studies have suggested that nutrition fertilization, especially potassium (K), can enhance plant growth and yield production. To investigate the mechanism of K in sugar long-distance transportation under drought stress, we established a soil-grow system and a hydroponic-grow system with varying amounts of potassium supplementation and analyzed the biochemical and molecular responses in Arabidopsis and potato plants under drought stress conditions. Our findings showed that excess potassium fertilization limited sucrose metabolism, leading to lower drought tolerance in Arabidopsis in both grow systems. However, higher potassium supplementation altered sugar relocation and potassium movement, resulting in an increase in starch yield production in both potato plants with different sink strength capacities. We also proposed that a low amount of sodium increases Arabidopsis drought tolerance under low potassium conditions since a low amount of sodium can improve the control of osmotic potential, leading to more water being retained in plant cells.
Silicon (Si) has received considerable attention recently for its potential in mitigating drought stress, although the effects vary among different plant species. To investigate the mechanism of Si in drought stress tolerance, we applied monosilicic acid in hydroponic media and then applied PEG8000 to simulate drought stress. Our findings revealed that Si-dependent drought mitigation occurred more in the shoot than in the root of Arabidopsis, and we observed silicon accumulation in the shoot of Arabidopsis. In Si-treated plants, more glucose was accumulated in the vacuole, leading to better osmotic potential control under drought stress. RNA sequencing analysis showed that Si altered the activity of sugar transporters and the sugar metabolism process, and increased photosynthesis. However, Si-dependent regulation in sugar transporter showed different responses in potato. Understanding the mechanism of Si in potato requires further studies. Overall, our dissertation provides important information for clarifying the mechanism of Si in drought stress, which forms the basis for further investigation.
Phycobilisomes (PBS) are the major light-harvesting complexes for the majority of cyanobacteria
and allow these organisms to absorb in the so-called green gap. They consist of smaller units called
phycobiliproteins (PBPs), which are composed of an α- and a β-subunit with covalently bound
linear tetrapyrroles (phycobilins). The latter are attached to the apo-PBPs by phycobiliprotein
lyases. Interestingly, cyanobacteria of the genus Prochlorococcus lack complete PBS and instead
use prochlorophyte chlorophyll-binding proteins (Pcbs), which effectively utilize the energy of the
blue light region. The low-light-adapted (LL) strain Prochlorococcus marinus SS120 has a single
PBP, phycoerythrin-III (PE-III). It has been postulated that PE-III is chromophorylated with the
phycobilins phycourobilin (PUB) and phycoerythrobilin (PEB) in a 3:1 ratio. Thereby, the function
of PE-III remains unclear so far, so that light-gathering function and also photoreceptor function
are discussed.
The main goal of this work was to characterize the assembly of PE-III and thus the function of the
six putative phycobiliprotein lyases of P. marinus SS120. Previous work found that the individual
lyases could not be produced in soluble form, so we switched to a dual pDuet™ plasmid system in
E. coli, which was successfully established. Investigation of the binding of PEB to Apo-PE
revealed that the CpeS lyase specifically chromophorylated Cys82 with 3Z-PEB. Unfortunately,
additional chromophorylation could not be observed using the pDuet system. Therefore, in a
second part of the work, the entire PE gene cluster from P. marinus SS120 was to be introduced
into E. coli and expressed. Although the gene cluster was successfully transcribed within E. coli,
no translation was observed, possibly due to incompatible translation initiation between
Prochlorococcus and E. coli. The introduction of a mini PE cluster (CpeAB) into the
cyanobacterium Synechococcus sp. PCC 7002 was also successfully performed, in which case
production of CpeB but not CpeA from Prochlorococcus was detected. Recombinant CpeB was
also detected together with intrinsic PBP in Synechococcussp. 7002, indicating structural similarity
and incorporation into PBS in Synechococcus sp. 7002. Overall, the obtained results suggest that a
cyanobacterial host is a good option for the studies on the assembly of PE-III from P. marinus and,
based on this, future work could aim at generating an artificial operon using synthetic biology to
achieve efficient translation of all genes.
Regulation of sucrose transport between source and sink tissues is critical for plant development and properties. In cells, the dynamic vacuolar sugar homeostasis is maintained by the controlled regulation of the activities of sugar importers and exporters residing in the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 protein, being the closest homolog to the proton/glucose symporter ERDL6, resides in the vacuolar membrane. We raised both, molecular expression and data deriving from non-aqueous fractionation studies indicating that ERDL4 was involved in glucose and fructose allocation across the tonoplast. Surprisingly, overexpression of ERDL4 increased total sugar levels in leaves, which is due to a concomitantly induced stimulation of TST2 expression, coding for the major vacuolar sugar loader. This conclusion is supported by the notion that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. That ERDL4 activity contributes to the coordination of cellular sugar homeostasis is further indicated by two observations. Firstly, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, secondly, the ERDL4 gene is markedly expressed during cold acclimation representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger size of rosettes and roots, a delayed flowering and increased total seed yield. In summary, we identified a novel factor influencing source to sink transfer of sucrose and by this governing plant organ development.
Synapses are the fundamental structures that regulate the functionality of the neural circuit. The ability of the synapse to modulate its structure and function at a fast rate due to various sensory inputs provides the strength to the nervous system to incorporate new adaptations and behaviors in the animal. The synapses are very dynamic throughout the life of the animal starting from early development. Continuous events of formation and elimination of synapse, activation and inhibition of synaptic function are observed in almost all synapses. These processes occur at a high speed and require controlled cellular mechanisms. Imbalance in these processes results in defective nervous system and has been reported in many neurological disorders. Thus, it is important to understand the mechanisms that regulate process of synapse development maintenance and function.
Kinases and phosphatases are the key regulators of cellular mechanisms. Understanding the function of these molecules in the neuron will shed light on the molecular mechanisms of synaptic plasticity. Using Drosophila melanogaster larval neuromuscular junction as a model, Bulat et al. (2014) performed a large RNAi based screen targeting kinome and phosphatome of Drosophila to identify the essential kinases and phosphatases and found Myeloid leukemia factor-1 adaptor molecule (Madm) and Protein phosphatase 4 (PP4) as novel regulators of synapse development and maintenance. The function of these molecules in the nervous system has not been reported and hence I investigated on the role of Madm and PP4 in the regulation of synapse development, maintenance and function.
Myeloid leukemia factor-1 adaptor molecule (Madm), a ubiquitously expressing psuedokinase essentially functions to regulate synaptic growth, stability and function. Using a combination of genetic and high throughput imaging, I could demonstrate that Madm functions to regulate the synaptic growth and stability from the presynapse and synaptic organization form the postsynapse. Also, I could demonstrate that Madm functions in association with mTOR pathway to regulate synapse growth acting downstream of 4E-BP. In addition, using electrophysiology, we could demonstrate that Madm is essential for the basic synaptic transmission with an additive function of retrograde synaptic potentiation. In summary, I could demonstrate that Madm is a novel regulator of synaptic development, maintenance and function.
Protein phosphatase 4 (PP4), a ubiquitously expressing protein phosphatase is involved in the regulation of multiple aspects of the nervous system. I could demonstrate that PP4 is essential for the development of nervous system and the metamorphosis. Using genetics and imaging analysis, I could demonstrate that loss of PP4 results in the abnormal morphology of cell organelles. In addition, I could show that loss of PP4 results in defective brain development with poorly developed structures.
Altogether, in this study, I could demonstrate the importance of novel molecules, a pesudokinase Madm and protein phosphatases PP4 in the nervous system to regulate distinct aspects of the neuron.
Chromosomal aberrations are manifold changes in the configuration of the DNA. Each cell in a tumor
may accumulate different karyotype changes, making it challenging to determine the causes and
consequences of this instability. Therefore, model systems have been developed in the past to
generate and study specific genome alterations. In this thesis, I present the results of my studies on
three types of chromosomal aberrations, all of which may contribute to tumor development or
progression.
Chromothripsis is a phenomenon that describes a one-off massive chromosomal disruption and
reassembly, perhaps arising via DNA damage micronuclei (MN). MN are small DNA-packed nuclear
envelopes. I tested potential causes of DNA damage in MN and found that the rupture of the MN
envelope and the entry of cytosolic fractions increase DNA damage in MN. Furthermore, I addressed
the question of what physiological consequences cell lines with an additional rearranged chromosome
have compared to those with an intact extra chromosome. Strikingly, the cells with more
rearrangements showed a functional advantage resulting in an improved fitness potential.
However, the engineering of polysomic cell lines with fully intact additional chromosomes increases
various cellular stress responses and reduces the proliferation capacity. To investigate how cancer cells
overcome the detrimental consequences of aneuploidy, I explored physiological adaptations of model
cells with a defined additional chromosome that underwent in vivo and in vitro evolution. Interestingly,
unfavorable phenotypes of aneuploid cells, such as the replication stress, were mitigated upon
evolution. Furthermore, I examined the replication on single molecule resolution, showing alteration
after evolution that might underlie the replication stress bypass or tolerance.
In contrast to these unbalanced forms of genomic aberrations, whole genome doubling (WGD) leads
to a full doubled chromosome set, which was shown to evolve into aneuploid karyotypes by
chromosomal instability (CIN), frequently by losing chromosomes. Cells that underwent WGD
accumulate DNA damage in the S phase. I performed a single molecule analysis on the DNA during the
first cell cycle after WGD to elucidate how the DNA damage arises and found that the number of active
origins is not sufficient to replicate the doubled amount of DNA in the first S phase after WGD faithfully.
This starts a genome-destabilizing cascade that eventually promotes tumorigenesis, metastasis, and
poor patient outcome.
Taken together, these studies provide insights into the causes and consequences of three types of
genomic aberrations: chromothripsis, polysomy, and WGD. However different these phenomena may
be, they share one common feature – they contribute to tumor development and progression.
Therefore, elucidating the aberrant cell functions caused by genomic aberrations contributes to a
better understanding of a cancer cell's nature and will perhaps help to find new cancer therapy targets.
Botrytis cinerea is a major plant pathogen infecting more than 1400 plant species. During invasion, the fungus rapidly kills host cells, which is believed to be supported by induction of programmed plant cell death. To comprehensively evaluate the contributions of most of the currently known plant cell death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas9 protocol was established which allowed to perform serial marker-free mutagenesis to generate multiple deletion mutants lacking up to 12 CDIPs. Whole genome sequencing of a 6x and 12x deletion mutant revealed a low number of off-target mutations which were unrelated to Cas9-mediated cleavage. Secretome analyses confirmed the loss of secreted proteins encoded by the deleted genes. Infection tests with the mutants revealed a successive decrease in virulence with increasing numbers of mutated genes, and varying effects of the knockouts on different host plants. Comparative analysis of mutants confirmed significant roles of two polygalacturonases (PG1, PG2) and the phytotoxic metabolites botrydial and botcinins for infection, but revealed no or only weak effects of deletion of the other CDIPs. Nicotiana benthamiana plants with mutated or silenced coreceptors of pattern recognition receptors, SOBIR1 and BAK1, showed similar susceptibility as control plants to infection by B. cinerea wild type and a 12x deletion mutant. These results raise doubts about a major role of manipulation of these plant defence regulators for B. cinerea infection. Despite the loss of most of the known phytotoxic compounds, the on planta secretomes of the multiple mutants retained substantial phytotoxic activity, proving that further, as yet unknown CDIPs contribute to necrosis and virulence. Our study has addressed for the first time systematically the functional redundancy of fungal virulence factors, and demonstrates that B. cinerea releases a highly redundant cocktail of proteins to achieve necrotrophic infection of a wide variety of host plants.
The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1,273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to higher eukaryotes. Here we report the production of full-length SARS-CoV-2 spike protein – lacking the C-terminal membrane anchor – as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.
Towards standardized operating procedures for eDNA-based monitoring of marine coastal ecosystems
(2022)
Marine coastal ecosystems are exposed to a variety of anthropogenic impacts, which
often manifest themselves in the pollution of the surrounding ecosystem. Especially on
densely populated coasts or in regions heavily used for aquaculture, changes in the natural
marine habitat can be observed. In order to protect nature and thus its ecosystem services
for humans, more and more environmental protection laws are coming into force.
Exemplary, operators of facilities known to contribute to pollution are obliged to regularly
monitor the condition of the surrounding environment. The purpose of such so-called
compliance monitoring is to determine whether the prescribed regulations are being
followed. The traditional routine involves sampling by ship, during which sediment
samples are taken from the seabed below the aquaculture cages and all macrofauna
organisms found, such as mussels or worms, are taxonomically determined and quantified
by experts. Based on the community of organisms the ecological status of the sample can
then be inferred. Since this method is very labor- and time-consuming, a reorientation of
the scientific community towards alternative monitoring methods is currently taking place.
A bacteria-based eDNA (environmental DNA) metabarcoding system in particular has
proven to be a suitable monitoring tool. With this molecular method, the composition of
the benthic bacterial community is determined using high-throughput sequencing. The
great advantage of this method is that bacteria, due to their short generation times, react
rapidly to various environmental influences. The composition of this community can then
be used to infer the ecological status of the sample under investigation via sequencing
without the need for laborious enumeration and identification of organisms. Additionally,
sequencing costs are more and more decreasing, proposing eDNA metabarcoding-based
monitoring as a faster and cheaper alternative to traditional monitoring. In order to
implement the method in legislation in the long term, standard protocols need to be
developed. Once these are sufficiently validated, the novel methodology can be
incorporated into regulations to support or even replace traditional monitoring. However,
some steps of the eDNA metabarcoding method, from sampling to ecosystem assessment,
are not yet sufficiently standardized, which is why the development of this work was
necessary. Since there is no consensus in the scientific community on (i) the preservation of
environmental samples during transport, (ii) the reproducibility of ecosystem assessment
among different laboratories, (iii) the most appropriate bioinformatic method for ecosystem
assessment, and (iv) the minimum sequencing depth required to determine ecosystem
status, these sub-steps were investigated. It was found that the most common methods
currently used to preserve samples during transport had no discernible effect on the final
ecosystem assessment. Furthermore, sample processing in independent laboratories
allowed the same ecological interpretations based on the bacterial community, which
resulted in concordant ecosystem assessments among laboratories. This indicates the
overall reproducibility of the eDNA metabarcoding-based method, thus enabling its
implementation in standard protocols. Furthermore, it was shown that corresponding
ecosystem assessments can be obtained with the currently used methods for determining
ecological status based on eDNA data. Critical to predictive accuracy is not the method
itself, but a sufficient number of samples that accounts for the natural spatial and temporal
variability of bacterial communities. It was demonstrated that a very shallow sequencing
depth per sample can be sufficient to use machine learning to prediction the ecological
status of the environmental sample. The quality of this classifications did not depend on
the sequencing depth as assumed but was determined by the separability of individual
categories. The results and recommendations of this work contribute directly to the
standardization of ecological assessment of nearshore marine ecosystems. By establishing
these standard protocols, it will be possible to integrate the eDNA metabarcoding-based
method for monitoring compliance of coastal marine ecosystems into legislative
regulations in the future.
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.