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Doctoral Projects 2016

Doctoral projects offered by LSM faculty members to applicants for 2016:

Please note:

  • You have to apply with our online application tool.
  • If the available doctoral projects don’t match your interest, you are free to add a proposal matching the research of one of the LSM faculty groups to our online application tool. (http://www.lsm.bio.lmu.de/faculty/).
  • Please click on the name of the supervisor to get redirected automatically to further information on the research group and the scientific contents. 

Supervisor: PD Dr. Bettina BOELTER / PD Dr. Serena SCHWENKERT (Plant Sciences, Biochemistry, Molecular Biology)

Title: Regulation of protein import into chloroplasts.

 

Chloroplasts originated from an ancient cyanobacterial ancestor, which was engulfed by a eukaryotic cell and subsequently integrated into the host organism as an endosymbiont. Substantial horizontal gene transfer to the nuclear genome resulted in the chloroplast developing into a semiautonomous organelle that critically depends on importing the majority of its proteins from the cytosol. This posttranslational process includes specific targeting in the cytosol as well as transport of chloroplast (pre)proteins across the double envelope membrane.
Short-term acclimation of chloroplasts to various environmental triggers requires drastic changes in its proteome composition. The amount of photosynthetic complexes needs to be adapted in response to varying light intensities or temperature to ensure optimal performance. This in turn requires a fast response in adaptation of the import efficiency.
The regulation of import capacity can occur on several levels. Firstly, this involves tightly regulated posttranslational modifications, i.e. the phosphorylation of preproteins in the cytosol, which is mediated by STY kinases. How this phosphorylation and/or dephosphorylation changes in response to environmental influences and how this affects the import capacity can be studied in this project.
In the outer and inner envelope membrane of chloroplasts, complex translocation machineries named TOC (Translocon at the outer envelope of chloroplasts) and TIC (Translocon at the inner envelope of chloroplasts), respectively, mediate import of plastid proteins. The Tic complex consists of seven or eight components, including putative translocation channels, chaperones and regulatory subunits. The exact molecular function and coordination of subunits as well as regulative redox-related features in acclimation processes of the Toc and Tic complexes are just beginning to emerge and will be addressed in this project.

*Dependent on funding, the 2 doctoral positions will be available by March 2016.

Reference: Redox meets protein trafficking. Bölter B, Soll J, Schwenkert S. Biochim Biophys Acta. 2015 Sep;1847(9):949-56.

Supervisor: Prof Michael BOSHART (Genetics, Biochemistry and Cell Biology)

Title 1: A novel cAMP signaling pathway in pathogenic parasites.

The cyclic AMP signaling pathway is highly conserved in most eukaryotes, but very unconventional in phylogentically distant kinetoplastid protozoa that include important pathogens causing neglected tropical diseases, like Trypanosoma and Leishmania. By genome-wide RNAi screening several components of a novel pathway have been identified in trypanosomes and reverse genetic analysis suggests a role in the orientation and migration of the parasite in its host. The pathway acts downstream of a large family of receptor type adenylate cyclases that are differentially expressed and intracellularly targeted during development of the parasite in its insect vector. The doctoral project will investigate the role of this signaling pathway by reverse genetic manipulations in a culture model of stage differentiation for trypanosomes. We want to understand how individual signaling proteins e.g CARP1 and CARP3 work in this pathway and how they interact. Methods will include proteomics, biochemistry on recombinant proteins and interaction analysis with the BioID method. We expect insight into the regulation of the vector-parasite interaction that might be exploitable for transmission blocking strategies and disease prevention of neglected tropical diseases.

Gould MK, Bachmaier S, et al. (2013) Antimicrob Agents Chemother 57: 4882-4893
Salmon D, Vanwalleghem G, et al. (2012) Science 337: 463-466
Salmon D, Bachmaier S, et al. (2012) Mol Microbiol 84: 225-242

Title 2: Metabolic adaptation and differentiation of Trypanosoma.

Metabolic pathways and fluxes change in the different life cycle stages of the protozoan parasite Trypanosoma that adapts to different mammalian and fly host environments in its infectious cycle. The project will investigate the interplay between stage differentiation, (iso-)citrate metabolism and gluconeogenesis and the regulation of these processes. A large collection of knock-out mutants is available and collaborations established for metabolite and flux analysis by MS and stable isotope labeling. New mutants will be generated and phenotypically analysed in a culture development system for trypanosomes. Specific developmental stages will be purified by fluorescence activated cell sorting (FACS) from these cultures and subjected to metabolomic and proteomic analysis. The project is embedded in a collaborative network with teams in France and Japan. The ultimate goal is to develop innovative strategies to disrupt the infectious cycle of the deadly parasite causing sleeping sickness in man, a neglected tropical disease.

Allmann, S. et al. (2013) J Biol Chem 288, 18494-18505
Kolev, N.G. et al. (2012) Science 338, 1352-1353
Riviere, L. et al. (2009) Proc Natl Acad Sci U S A 106, 12694-12699

*For both projects the candidate is expected to apply for a fellowship.

Supervisor: Dr Anne-Kathrin CLASSEN (Cell Biology and Epigenetics)

  • Title 1: Cellular signaling and epigenetic plasticity in response to tissue stress.
  • Title 2: Cell biology and biomechanics of epithelial morphogenesis.

Please visit our website for more information:
http://www.bioimaging.bio.lmu.de/research/junior_research_group_classen/index.html

Supervisor: Prof. Kirsten JUNG (Microbiology, Cell Biology, Biochemistry)

Title 1: Posttranslational modification of translation elongation factor P.


Translation, the assembly of proteins by the ribosome machinery, is an essential cellular process. Our recent findings indicate that ribosomes stall during translation of three or more consecutive proline residues (PPP) or specific XPPX sequences, and that translation elongation factor P (EF-P) and the eukaryotic ortholog eIF-5A, respectively, relieve this translational arrest (Ude et al., 2013; Peil et al., 2013; Gutierrez et al., 2013). The functionality of EF-P and eIF-5A to stimulate peptide bond formation of stalled ribosomes strictly depends on the posttranslational modification of the proteins. While hypusinylation of eIF-5A is observed in all eukaryotes tested thus far, 30 % of all bacteria have EF-P that is ß-lysinylated. It will be the aim of the project to identify and characterize novel modification pathways of EF-P in bacteria belonging to the Firmicutes (e.g., Staphylococcus and Enterococcus) and test the functionality of these EF-P proteins in vitro and in vivo.


Ude, S., Lassak, J., Starosta, A. L., Kraxenberger, T., Wilson, D.N., Jung, K. (2013) Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches. Science 339:82-85.
Peil, L., Starosta, A.L., Lassak, J., Atkinson, G., Virumäe, K., Spitzer, M., Tenson, T., Jung, K., Remme, J., Wilson, D.N. (2013)Distinct XPPX-motifs induce ribosome stalling, which are rescued by the translation elongation factor
Gutierrez, E., Shin, B.S., Woolstenhulme, C.J., Kim, J.R., Saini, P., Buskirk, A.R. Dever, T.E. (2013) eIF5A promotes translation of polyproline motifs. Mol. Cell 51:35-45.

Title 2: The physiological role of a novel nutrient-sensing histidine kinase/response regulator network during host colonization.


When carbon sources become limiting for growth, bacteria must choose which of the remaining nutrients should be used first. We have identified a nutrient-sensing signaling network in Escherichia coli that is activated at the transition to stationary phase (Behr et al., 2014). The network is composed of two histidine kinase/response regulator systems and two transport proteins. The peptide/amino acid-responsive YehU/YehT system was found to have a negative effect on expression of the target gene yhjX (encoding a transporter) of the pyruvate-responsive YpdA/YpdB system, while the YpdA/YpdB system stimulates expression of yjiY (encoding a CstA-like tranporter), the target of the YehU/YehT system. The project focuses on the role of this network during host colonization of UPEC strains using a combination of in vivo reporter assays, single cell studies, and biochemical approaches.

Behr, S., Fried, L., Jung, K. (2014) Identification of a novel nutrient-sensing histidine kinase/response regulator signaling network in Escherichia coli, J. Bacteriol. 96:2023-2029.

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Supervisor: Dr. Macarena Marín (Cell biology, Biochemistry, Genetics)

Title: Identification of rhizobia effectors involved in internalization and intracellular persistence.

Before the onset of Nitrogen fixation, symbiotic rhizobia must cross the epidermis and colonize root cortical cells. Two mechanisms describe how they overcome the epidermal barrier: root hair and crack-entry infection. In both cases, when bacteria reach the inner cortex, they invade cortical cells by a process resembling endocytosis (1). The mechanisms controlling rhizobia internalization and persistence in host cortical cells are unknown, although cortical compatibility and infection are of key importance, because they determine nodule occupancy, and thus Nitrogen fixation and legume yield. The aim of this project is to address the role of rhizobia effector proteins in internalization and persistence. Effectors are proteins secreted to the apoplast or directly into the host cytoplasm, where they subvert host signaling cascades to promote susceptibility, for example by undermining the plant immune system (2). We will use molecular, cell-biological and biochemical methods to identify candidate effectors involved in this process and their host targets.

This project will be co-supervised by Prof. Parniske.

1. Oldroyd, G. E., et al (2011). "The rules of engagement in the legume-rhizobial symbiosis." Annu Rev Genet 45: 119-144.
2. Marín, M. and Ott, T.(2013). “Intrinsic disorder in pathogen effectors: Protein flexibility as an evolutionary hallmark in a molecular arms race.” Plant Cell 25:3153-7

Supervisor: PD Dr. Stylianos MICHALAKIS (Gene Therapy, Neuroscience, Pharmacology, Epigenetics)

Title: Epigenetic mechanisms in neuronal plasticity and neurodegeneration.


Neuronal networks show a remarkable degree of plasticity during physiological and pathophysiological processes. This plasticity goes along or is even mediated by major adjustments in the expression of key genes. Unfortunately, the mechanisms controlling gene expression and neuronal plasticity are poorly understood, but it is suggested that epigenetic mechanisms crucially contribute to these biological processes. The aim of this project is to investigate how epigenetic mechanisms at the level of DNA and chromatin contribute to gene regulation, neuronal plasticity and neurodegeneration. The methodology will include state-of-the-art in vitro and in vivo biochemical, genetic, cell biological and viral gene transfer methods in relevant mouse models.

 

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Supervisor: Prof. Joerg NICKELSEN (Plant Sciences, Biochemistry, Genetics)

Title: Regulation of chloroplast gene expression in Chlamydomonas.

Due to the endosymbiotic evolutionary history of chloroplasts, most of the multimeric plastid protein complexes are formed by subunits which are encoded by either the nuclear or the chloroplast genome. This intrinsic complicated situation has led to the development of a coordination system for gene expression in both cellular compartments as well as a chloroplast biogenesis machinery that ensures the ordered step-wise assembly of photosynthetic complexes within the organelle. Moreover, both processes appear to be interlinked via feed-back control mechanisms that involve the sensing of non-assembled complex subunits. Accumulating evidence  indicate that numerous factors are involved in these regulatory processes. Some of which share characteristic motifs like e.g. TPR, PPR and other repeat domains or possess classical RNA binding domains and are typically organized in high-molecular weight complexes [Schwarz et al. (2007) Plant Cell 19: 3627-3639; Johnson et al. (2010) Plant Cell 22: 234-248]. This project aims at elucidating the molecular function of nucleus-encoded proteins potentially being involved in the biogenesis of thylakoid membranes in the model system Chlamydomonas reinhardtii.

Dependent on funding, the doctoral position will be available by summer 2016.

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Supervisor: Prof. Martin PARNISKE (Genetics, Biochemistry, Cell Biology)

Title: Cell-specific reprogramming of legume roots for endosymbiotic infection.
The vast majority of land plants can establish arbuscular mycorrhiza (AM), an agronomically important symbiosis with fungi to improve mineral and water supply. Legumes use an ancient signal transduction program, co-opted from the ancestral AM, to establish intracellular (endosymbiotic) interactions with nitrogen-fixing rhizobia, in the root nodule symbiosis. Successful symbiotic infection relies on an ancient cellular differentiation program for the intracellular accommodation of the microsymbionts. Although a number of important symbiosis genes essential for the infection process have been identified through genetic approaches, the precise cellular and molecular events underlying bacterial colonization of the root are poorly understood. The project will focus on nuclear signalling events and associated transcriptional reprogramming that take place in host cells engaged in rhizobial infection. Calcium-spiking is a specific nuclear response associated with the early stages of infection. This project aims to decipher the spatio-temporal interconnection between calcium-spiking and the dynamics of transcription factor accumulation and complex conformation preceding host cell transcriptional reprogramming for infection. We will focus on the transcription factor CYCLOPS which acts in a complex with CCaMK, an important early integrator of calcium signalling. We have recently shown that CYCLOPS phosphorylation is essential for its transcriptional activity, and is associated with structural alterations of the CYCLOPS dimer (Singh et al., 2014). FRET-based strategies will be used to follow CYCLOPS complex formation in living cells during infection and how this correlates with calcium signalling. The CYCLOPS promoter appears to be critical for successful symbiotic signalling. We will identify the cis- and trans-acting factors that determine the complex expression pattern of CYCLOPS. We will employ the INTACT (Isolation of Nuclei TAgged in specific Cell Types) technology (Deal and Henikoff 2011) to specifically target the small subpopulation of cells undergoing reprogramming and couple it with RNA sequencing to access gene transcription networks specifically associated with early stages of rhizobial infection. The comparison of cell-specific transcriptomes of wild type and infection-deficient mutants will aid in the identification of genes directly related to endosymbiotic entry. The project will thus provide new insights into the cellular reprogramming during endosymbiotic root colonization.
Singh S, Katzer K, Lambert J, Cerri M, Parniske M (2014) CYCLOPS, A DNA-binding transcriptional activator, orchestrates symbiotic root nodule development. Cell Host&Microbe. 15(2):139–52

Deal RB, Henikoff S (2011) The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nature Protoc 1: 56-68

Supervisor: PD Dr. Katrin PHILIPPAR (Plant Biochemistry, Physiology)

Philippar

Title: Molecular analysis of fatty acids export from plastids.

Fatty acids (FAs) build the majority of cellular lipids, which are essential not only for membrane function, but also for growth and development of organisms. Since in plants FA synthesis occurs in plastids, transport to the ER for further lipid assembly is required. The mode of plastid FA-export, however, was an open question until only recently we described FAX1 (fatty acid export 1). FAX1 is a novel membrane protein in the plastid inner envelope and mediates FA-export (see Fig. 1; Li et al., 2015). In the model plant Arabidopsis thaliana FAX1 is crucial for biomass production, male fertility, and synthesis of FA-derived compounds such as lipids, ketone waxes or pollen cell walls. FAX1 belongs to a family of seven proteins: four are predicted to be in plastids, three most likely integrate into membranes of the secretory pathway. In vertebrates, FAX relatives are mitochondrial membrane 'TMEM14' proteins with unknown biological function. Thus, this protein family represents a powerful tool not only to increase lipid/biofuel production in the green lineage, but also to explore novel transport systems in vertebrates.
The project aims to characterise FAX proteins in Arabidopsis, crop plants and other model systems (e.g. green microalgae). Research should clarify their impact of FAX proteins on cellular metabolism, in particular on FAs, lipids and FA/lipid-derived compounds as well as on carbon energy metabolites. A spotlight will be on synthesis of storage oils in seeds, i.e. the significance of FAX in oil synthesis. In the long run, overproduction of FAX proteins in combination with enhanced expression of FA/lipid biosynthesis enzymes should lead to development of strategies for biofuel production. The structure/function correlation of FAX1 is completely novel. Thus, research will enable us to study plant-specific but also general aspects of a new intracellular FA-transport pathway. Evaluation of FAX topology, structure, and FA/lipid binding, functional characteristics as well as identification of interaction partners will help to describe the plastid FA-export pathway. Research covers techniques of modern Plant Molecular Biology and Physiology (mutant generation, ultrastructural analysis, transcriptomics, metabolomics, proteomics), Cell Biology (in vivo GFP-targeting, protein-protein interaction) as well as Biochemistry and functional analysis of membrane proteins (purification of protein complexes, functional assays).

Reference:
Li, N., Gügel, I, Giavalisco, P., Zeisler, V., Schreiber, L., Soll, J., and Philippar, K. (2015). FAX1, a novel membrane protein mediating plastid fatty acid export. PLoS Biology 13(2): e1002053. doi:10.1371/journal.pbio.1002053.
- Roberts RG (2015) Flagging the Fatty Acid Ferryman. Synopsis on Li et al. (2015), doi:10.1371/journal.pbio.100205
- TAIR feature: 1st Paper of the Month [March 18, 2015], https://www.arabidopsis.org: "This month we feature FAX1, a Novel Membrane Protein Mediating Plastid Fatty Acid Export, by Li, et.al."

 

Supervisor: PD Dr. Serena SCHWENKERT (Plant Sciences, Biochemistry, Molecular Biology)

Title: Chaperones and TPR proteins in organellar protein import and chloroplast biogenesis.

In the eukaryotic cell most organellar proteins are synthesized as preproteins in the cytosol and are either post- or co-translationally translocated into chloroplasts, mitochondria or the endoplasmic reticulum. During posttranslational protein transport the preproteins have to be kept in an import competent state and molecular crowding has to be prevented, since this can easily lead to misfolding or aggregation. This process is assisted by the molecular chaperones HSP90 and HSP70, which also mediate interaction of preproteins with the membrane surfaces. This interaction is conferred by tetratricopeptide repeat (TPR) proteins, which are associated with the translocon complexes. The aim of this project is to characterize the molecular and regulatory function of these TPR docking proteins during posttranslational protein transport. The functional and structural analyses of these docking proteins will not only provide insight into the import mechanism of preproteins but also elucidate the role of chaperones and the respective receptors in specific targeting of preproteins. Moreover, not only the cytosolic, but also the chloroplast resident TPR proteins as well as HSP90 are indispensable for chloroplast biogenesis. We will therefore investigate the composition and mode of action of the chloroplast HSP90 machinery, with special focus on its conformational dynamics in comparison with the cytosolic localized chaperone setup. To this end we will apply a variety of biochemical and molecular biological techniques, such as protein purification, co-immunoprecipitation and expression of fluorescent proteins.

*Please note that this project is pending on funding acceptance.


For further information see: http://www.en.botanik.bio.lmu.de/research/soll/subgroups/schwenkert/index.html

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