Participants and Projects
Name:             Michael Matt

University:     Dartmouth College

Supervisor:   Prof. Dr. Oliver Fackler

Workgroup:   Virology

Project:          Unc119: An acyl chaperone protein with important functions in T cell receptor signaling

Summer

School Call:   2013

Description:  Nef, a protein important for the pathogenicity of the HIV virus, co-opts T cell receptor signaling upon receptor stimulation, interfering with pathways that block virus replication and promoting signaling events that boost viral replication. Nef achieves this effect partly through the intracellular accumulation of the important Src-type tyrosine kinase Lck. Recently, Unc119, a protein that aids in the trafficking of myristoylated proteins, has been shown to release intracellular accumulation of Lck and restore important T cell functions. This project aimed to test the possibility of Unc-mediated release of kinase accumulation for other Src-type kinases, as well as uncover the specific residues in Unc119 that mediate its interaction with Lck. Using site-directed mutagenesis, I introduced mutations into an Unc119.RFP plasmid and observed the effect of those mutations on the subcellular localization of Lck using immunofluorescence and microscopy techniques. 

 

 

Name:             Berrak Albostan

University:     Bilkent University

Supervisor:   Prof.Dr. Michael Boutros

Workgroup:   Division of Signalling and Functional Genomics

Project:          Tumor-stroma Interactions in the Context of Prostate Cancer Metastasis to Bone

Summer

School Call:   2014

Description:  Some types of cancer such as breast and prostate tend to metastasize to mesenchymal stromal cells, tissues originated from bone. To find what is the reason of these cancer types’ metastasis to bone, mesenchymal stem cells being found also in bone from human donors were used as a model system of the interaction between MSC and cancer cells. By applying in vitro laboratory methods included cell culture of multiple cell lines, RNAi technology, functional assays such as Transwell Migration Assays, QPCR for gene expression analysis as well as microscopy, I investigated extracellular matrix proteins like integrins and collagen and their effect on migration.

 

 

Name:             Alessandro Enrico

University:     Institut polytechnique de Grenoble, France

Supervisor:   Prof. Dr. Joachim Spatz

Workgroup:   Cell Adhesion and Signaling  group

Project:          Fabrication of fibronectin fibrillar network  for cancer cell migration studies

Summer

School Call:   2014

Description:  After seeding 3T3 fibroblast cells on appropriate substrate they were cultured to confluence for 4 days. Micro-patterned substrates were also used in order to check possible top-guided alignment of protein fibers. Then cells have been lysed and the underlying matrix has been fixed and labelled using different techniques. Assessment of fibronectin fibril density and geometry as well as characterization of network thickness and physical properties were performed through AFM analyses and immunofluorescence imaging. Cell migration essay of HT1080 cells on fibrillar FN were conducted by time-lapse video microscopy,  in order to analyse the dynamics of cancer cells on this kind of substrates (more resembling a real tissue than the 2D FN coating) and the degradation of fibrillar fibronectin matrices caused by cell migration.

 

 

Name:             Anastasiia Gainullina

University:     St.Petersburg State Polytechnical University

Supervisor:    Prof. Dr. Joachim Wittbrodt

Workgroup:   Developmental Biology/Physiology

Project:          Investigating Optic Fissure Closure

Summer

School Call:   2014

Description:  Incorrect optic fissure closure causing congenital disease coloboma might lead to childhood blindness. Gene network involved into the optic fissure closure regulation was deeply studied but precise cellular  and molecular mechanisms are still unclear and require more careful approach. The coloboma phenotype was shown on a murine model but switching to the fish model allows to observe eye embryogenesis with the help of time-lapse imaging choosing new gene candidates regulating optic fissure closure. Currently the adjustment of the murine model to the fish model is performed. 2 genes determined by an array of being upregulated in respect of the fissure closure were chosen. They were cloned for further investigation of the localization, amount and function of their transcripts and products by ISH, IHC and gene modification.  

 

 

Name:             Ankita Garg

University:     National Institute of Technology, Jalandhar, India.

Supervisor:    Dr. Sylvia Erhardt

Workgroup:   Chromatin & centromere biology

Project:          Regulation of Chromosome Segregation by tRNA Methyltransferase Dnmt2

Summer

School Call:   2014

Description:  Our work was primarily based on Dnmt2, which is known to be a Cytosine-5 tRNA Methyltranferase. Its localization has been found to be sensitive to RNase and heat shock that we analyzed by Drosophila larval neuroblasts immunostaining where we also identified the recovery time optimum for the cells to start division again after heat shock. We also observed that Dmnt2 depletion causes mitotic defects such as formation of anaphase bridges, lagging chromosomes and DNA fragments in neuroblasts. An interesting correlation was found between the mitotic defects in wild type and Dnmt2 mutant flies corresponding to various heat shock treatments. In addition to this, I got hands on experience in tissue culture, fly genetics, and learnt techniques like deconvolution microscopy and immunostaining.

 

 

Name:             Sophie Guillaume

University:     National Graduate School of Chemistry of Montpellier, France

Supervisor:    Dr. Julien Béthune

Workgroup:   Posttranscriptional regulation of mRNA expression and localization 

Project:          Is Coatomer a translational regulator: characterisation of an unexpected mRNA/protein interaction:

Summer

School Call:   2014

Description:  In the 90s, Coatomer (the proteinous coat of COPI vesicles) has been described to form a coat around budding vesicles. Few years ago, an interaction between Coatomer and the mRNA coding for asialoglycoprotein receptor (a protein receptor found at the plasma membrane of liver cells) has been described (Luis A. de la Vega and Richard J. Stockert, 1999). The project was to confirm this interaction between the 5’-UTR of ASGR mRNA and Coatomer. After showing this interaction, it would have been interesting to identify the RNA sequence on the ASGR mRNA which is responsible for this interaction and to identify the part of the Coatomer complex which is responsible for the interaction.

 

 

Name:             Vladislav Kulbatski

University:     Belarusian State University

Supervisor:    Prof. Dr. Jan-Erik Siemens

Workgroup:   AG Siemens, Institute of Pharmacology

Project:          Role of Reactive Oxygen Species in hypothalamic thermoregulation

Summer

School Call:   2014

Description:  The molecular mechanism underlying thermoregulation orchestrated by warm-sensitive neurons are largely unknown. Warm sensitive neurons: sparsely distributes in the POA (10%). TRPM2 is expressed at high levels in the brain of mammalians. TRPM2 can be activated by exposure to warm temperatures (>35 degrees C) apparently via direct heat-evoked channel gating. NAD(+)- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. TRPM2 gating by ADPR is facilitated by hydrogen (H2O2), cyclic ADPR (cADPR) and Ca2+. Endogenously H2O2 is generated through oxidative phosporylation in mitochondria and exogenous generation is induced in concept with other reactive oxygen species (ROS). Project dialled with ROS levels in anterior-preoptic and posterior areas of hypothalamus in mice during LPS-induced fever.

 

 

Name:             Oleg Magnes

University:     KAIST

Supervisor:    Prof. Dr. Roland Eils

Workgroup:   Computational Oncology

Project:          Attempts to identify new driver mutations and significant pathways in various types of lymphoma

Summer

School Call:   2014

Description:  In our project we analyzed somatic mutation data obtained from whole genome sequencing of 115 lymphomas. We used several approaches, such as network-assisted approaches and search for mutual exclusivity patterns. Network-assisted approach were based on protein-protein interaction networks, and allowed us to use a wide range of graph algorithms on the genetic data. Mutual exclusivity means that certain mutations in genes from significant cancer pathways do not co-occur (or co-occur very rarely) across all samples, because they would destroy the same functional module (e.g. pathway), so there is no selective advantage of having both mutations. For example, such effect can be shown on PI(3)K, p53, and Rb pathways. Thus, search of mutual exclusivity patterns can point out some new important oncogenes. This project aimed to find some new genes and pathways that might be drivers of lymphoma genesis. During the project we obtained results, which are in correspondence with current literature data. Also we found several candidate genes, which are interesting candidates for future cancer research.

 

 

Name:             Matthew Neale

University:     Northeastern University

Supervisor:    Prof. Dr. Freddy Frischknecht

Workgroup:   Parasitology

Project:          Screening and Identification of Sporozoite Motility Inhibitors          

Summer

School Call:   2014

Description:  Malaria is a devastating tropical disease caused by the parasite Plasmodium.  To cause a new infection, parasites in the mosquito saliva (called sporozoites) are inoculated into the skin when the mosquito probes for a blood meal.  Once in the skin, sporozoites rely on active movement to find and enter vasculature.  Sporozoite motility is therefore an essential feature of parasite infection in vertebrates. The inhibition of sporozoite motility at this skin step could therefore provide an additional mode of prophylaxis against malaria by preventing sporozoite entry into vasculature and subsequent infection.  The Medicines for Malaria Venture (MMV) Malaria Box is a set of 400 diverse compounds known to have antimalarial activity against the asexual blood stage of Plasmodium falciparum.  The goal of this project was to perform an in vitro screen of this compound library against the sporozoite stage of the parasite life cycle to identify potential inhibitors of parasite motility.   

 

 

Name:             Katharina Ross

University:     Massachusetts Institute of Technology

Supervisor:    Prof. Dr. Christof von Kalle

Workgroup:   Applied Stem Cell Biology

Project:          

Summer

School Call:   2014

Description:  

 

 

Name:             Ziliya Usmanova

University:     Moscow Institute of Physics and Technology

Supervisor:    Prof. Dr. Matthias Mayer

Workgroup:   Allosteric regulation of protein conformation

Project:          Characterisation of Hsp90 and Aha1 interaction via cross-linking experiments

Summer

School Call:   2014

Description:  Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. It has 3 main domains : N-domain  (Nucleotide binding domain), M-domain (Middle domain) and C-domain (dimerization domain ). Biological function of Hsp90 is absolutely dependent on its ability to both bind and hydrolyze ATP. Aha1 has an activating  effect on the ATPase cycle of Hsp90. Despite functions of every domain were revealed, nobody clearly explained mechanisms of binding reactions. In my Summer Internship project I was asked to characterise interaction of Hsp90 and Aha1 using well known cross-linking technique.  In addition to this I've learned methods to purify proteins which are extremely important in Biochemistry.

 

 

Name:             Victoria Zeuner

University:     Northeastern University, Boston

Supervisor:    Dr. Dirk Grimm

Workgroup:   Virus-Host Interactions

Project:          Evaluating the Effectiveness of Different Cas9s

Summer

School Call:   2014

Description:  The CRISPER system is the adaptive immune system of bacteria and integrates short fragments of foreign DNA into a repeat locus in the host cell genome. The CRISPER Technology uses Cas9, which are RNA-guided nucleases, to cleave the foreign DNA once it re-infects the cells. The system works by creating a DNA Double Strand Break (DSB) at the site in the genome that is wished to be modified. This break then signals the organism to repair it. This step allows researchers to insert or delete sequences into the genome. The project is to evaluate different Cas9’s effectiveness in modifying the genomic sequence and gene expression. The Cas9s that are being worked with are:

• S. pyogenes (Sp),

• S. thermophilus (St),

• N. meningitidis (Nm) and,

• S. aureus (Sa).

The end goal of the project will be to look at the effectiveness of the different Cas9s to delete HIV and HPV from the human genome.

 

 

Name:             Angela Zhu

University:     Massachusetts Institute of Technology

Supervisor:    Dr. Björn Tews

Workgroup:   Molecular Mechanisms of Tumor Invasion

Project:          Elucidating the mechanisms of the peroxidase and chaperone functions of Peroxiredoxin-1 in     

                        aiding glioblastoma invasion        

Summer

School Call:   2014

Description:  Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumors and linked to radio- and chemotherapy response as well as longer survival. The Tews Lab has studied the peroxiredoxin 1 (PRDX1) gene at 1p34.1 for promoter methylation and expression in primary gliomas and investigated its role in radio- and chemosensitivity of glioma cells in vitro. Using in vitro methods such as site-directed mutagenesis, I conducted invasion assays to test which function of PRDX1- its peroxidase and/or its chaperone function- was primarily responsible for increased invasion in glioblastoma cells. I also investigated the potential link between oxidative stress from treating cells with temozolomide and adenanthin, common chemotherapy treatments, and increased levels of peroxiredoxin protein by treating culturing glioblastoma cells that contained overexpressed and knocked-down levels of PRDX1 with these chemotherapy agents.

 

 

Name:             Shelby E. Hutchins

University:     Northeastern University

Supervisor:    Prof. Martina Muckenthaler

Workgroup:   Molecular Medicine MMPU Group

Project:          -

Summer

School Call:   2015

Description:  -

 

 

Name:             Irena Kuzma

University:     Northeastern University

Supervisor:    Prof. Christof von Kalle

Workgroup:   Aoplied Stem Cell Biology

Project:          -                        

Summer

School Call:   2015

Description:  -

 

 

Name:             Kseniia Lysakovskaia

University:     Lomonosov Moscow State University

Supervisor:    Prof. Roland Eils

Workgroup:   Cellular Mechanics Group

Project:          Vimentin silencing: influence on phenotypic properties of MDA-MB-231 breast cancer cell line

Summer

School Call:   2015

Description:  Vimentin is the intermediate filament protein and the major cytoskeleton component of mesenchymal cells. Vimentin has been widely recognized as a canonical marker for epithelial-to-mesenchymal transition (EMT). EMT is a multi-step process during which epithelial cells lose their cell-cell contacts and acquire motile and invasive phenotype. Cancer cells are able to exploit EMT program for their tumorigenic advantage. It was reported that vimentin is overexpressed in various epithelial-originated cancers and its overexpression correlates with accelerated tumor growth, invasion and poor prognosis.

The aim of my project was to examine influence of vimentin knock-down on phenotypic responses of cancer cells. We used highly aggressive and invasive MDA-MB-231 breast cancer cell line and its subclones with knock-down of vimentin by different small hairpin RNAs (shRNAs). As a first part of the project I validated the knock-down efficiency utilizing different techniques: quantitative immunobloting, qRT-PCR and immunofluorescence. Afterwards I used microfluidic optical stretcher as well as time-lapse microscopy to assess how vimentin knock-down affects cell mechanical and migratory/invasive properties of the cancer cells.

 

 

Name:             Matias Porras Paniagua

University:     Massachusetts Institute of Technology

Supervisor:    Prof. Hans-Georg Kräusslich

Workgroup:   Reserch Group Kräusslich

Project:          -

Summer

School Call:   2015

Description:  -

 

 

Name:             Itamar Shapira

University:     Vanderbildt University

Supervisor:    Prof. Felix Wieland

Workgroup:   Vesicular Transport, Protein-Lipid Interactions

Project:          Investigation of Palmitoylating DHHC Class Enzyme Role in Membranous Morphology

Summer

School Call:   2015

Description:  GP2, a protein found in Ebola, causes significant morphological changes in affected cells, resulting in significant membrane branching. The transmembrane domain of GP2 contains two cysteine residues which are palmitoylated by DHHC class enzymes. It is hypothesized that the palmitoylation step is important to the aforementioned morphological changes. By creating and utilizing mutant GP2 lacking these palmitoylated residues, the team will be able to observe any changes in the membrane morphology, thus shedding light on the role of palmitoylation in membrane structure. This summer, progress was made on creating in vivo and in vitro Drosophila models by cloning the constructs necessary for micro-injection into Drosophila embryos and transfection of Drosophila cell cultures using the Gal-4/UAS Drosophila expression system.

 

 

Name:             Andrés Tapia del Fierro

University:     National Graduate School of Chemistry of Montpellier

Supervisor:    Dr. Julien Béthune

Workgroup:   Posttranscriptional regulation of mRNA expression and localization

Project:          Study of the role of COPI vesicles in neuronal differentiation

Summer

School Call:   2015

Description:  Nearly all proteins made by a cell need to be localized at defined places to fulfill their functions. Specific localization can be achieved either by localized translation (for example by transporting mRNAs to specific places) or protein transport. About a third of eukaryotic proteins rely on the secretory pathway to reach their final localizations. These proteins are made at the endoplasmic reticulum (ER) and then travel within small coated vesicles from one organelle to the next. A class of these transport carriers, the COPI vesicles, transport proteins between the Golgi apparatus and the ER, as well as within the Golgi stacks.  Recent evidence suggests that Golgi-derived vesicles play a critical role in axonal growth and polarization of neuronal cells. Establishment and maintenance of cellular polarity is an essential process that underlies cellular fate and identity by creating unique cellular compartments and cell/cell interfaces. Hence, it is not surprising that cellular polarization and specific protein localization are two interwoven processes. The precise role and function of COPI vesicles in the highly polarized neuronal cells has however, to date, been hardly studied. Yet, recent data indicates that, in neurons, COPI vesicles travel on long distances within the axon, regulate neurite outgrowth and may associate with localized mRNAs. Altogether this suggests that COPI vesicles play a yet overlooked important role in neuronal differentiation that we plan to characterize in a mouse cellular model for neuronal differentiation.

 

 

Name:             Sergei Timofeev

University:     St. Petersburg State University

Supervisor:    Prof. Michael Lanzer

Workgroup:   Malaria 1

Project:          Protection against severe Malaria by hemoglobins S and C

Summer

School Call:   2015

Description:  The structural human hemoglobinopathies HbS and HbC are the most common genetic disorders that confer a significant protection against severe Malaria caused by Plasmodium falciparum. However, the mechanism of its protection remains unclear. It has been reported that parasitized erythrocytes containing hemoglobins S and C present a reduced cytoadherence to the microvasculature, that result to reduced vascular obstruction, endothelial inflammation and damage.

The aim of my project was to investigate interactions of infected red blood cells carrying HbS and normal hemoglobin (HbA) with endothelial cells (EC) in vitro. Using cultures of EC and P. falciparum in red blood cells with HbA and HbS we have investigated cytoadhesion behavior of infected erythrocytes and response of EC in flow and static conditions by FACS and fluorescent microscopy. 

 

 

Name:             Tania Bishola Tshitenge

University:     University of Nairobi International Centre of Insect Physiology and Ecology (ICIPE)

Supervisor:     Prof. Dr. Christine Clayton

Workgroup:    mRNA turnover in trypanosomes

Project:          Regulation of RBP 10 Expression in Trypanosoma brucei Investigation of biological role of 4E-interacting protein (4EIP) in T. brucei

Summer

School Call:   2016

Description:  Trypanosoma brucei is an extracellular flagellated parasite that causes sleeping sickness in humans (Human African Trypanosomiasis) and Nagana in cattle (Animal African Trypanosomiasis). The parasite is transmitted to mammalian hosts by the bite of tsetse fly. Differentiation of the parasite from the mammalian bloodstream forms to the procyclic forms that develop in tsetse fly as well as its adaptation to different energy sources require an adequate regulation of gene expression. To control expression of genes, trypanosome relies largely on post-transcriptional mechanisms entailing changes in mRNA stability and translation. These mechanisms are largely determined by sequences in the 3’-untranslated regions (UTRs) of mRNAs and RNA-binding proteins (RBPs) that bind to these regions have been reported as responsible for the regulation. One of these molecules, RBP10, has been recently found to be specific to bloodstream-form trypanosomes (BSF) where it is essential for expression of BSF-specific mRNAs. Recent studies also suggested that RBP10 might repress translation of procyclic-form-specific mRNAs in bloodstream forms. My summer school project aimed at investigating the role of 3'UTR of RBP10 in its regulation during the life cycle of the parasite. This was achieved through in vitro differentiation of the parasite together with reporter gene assays (CAT assays) and northern blotting analysis. I also investigated the biological role of another protein, 4E-Interacting protein (4EIP) in Trypanosoma brucei by using genetic reverse studies.

 

 

Name:             Carla Garcia Cabau

University:     Universitat Ramon Llull, IQS School of Engineering, Barcelona

Supervisor:    Dr. rer. nat. Richard Wombacher

Workgroup:   AK Wombacher, Institute of Pharmacy and Molecular Biotechnology

Project:          Novel chemical protecting groups for cell-type specific- and conditional control of drugs

Summer

School Call:   2016

Description:  Targeting of only specific cell-types in a whole organism with drugs is a challenging task, especially when the target of the drug (often a protein) is ubiquitous and located intracellularly. In this project, the basic approach in order to specifically target molecular interactions in particular cell types was the use of protecting groups that react to a specific stimulus (presence of a protein, special metabolic state). For this, special protecting group precursors were synthesized. As a model system, a fluorophore (fluorescein) was converted chemically into a non-fluorescent profluorophore using the protecting groups. The conditions required to liberate the free fluorophore were analyzed in vitro and inside living cells using fluorescence spectroscopy and microscopy. Once the properties of these protecting groups have been fine-tuned, they can be used to cage molecular effectors like drugs. Ideally these molecules would only be active when the desired stimulus is present (conditional deprotection). This would open up new ways for specialized therapies in medicine, as well as interesting sensors or effectors for synthetic biology and chemical biology.

 

 

Name:             Amy Foster

University:     

Supervisor:    Dr. Sylvia Erhardt

Workgroup:   

Project:          

Summer

School Call:   2016

Description:  

 

 

Name:             Robert Freeman

University:     Northeastern University

Supervisor:    Prof. Dr. Thomas Holstein

Workgroup:   Center for Organismal Studies, Department of Molecular Evolution and Genomics

Project:          A Reconsideration of the Role of Apoptosis in Hydra Regeneration

Summer

School Call:   2016

Description:  Hydra have astonishing regenerative abilities, allowing for head and/or body column regrowth following a wound. Previous research suggests that head regeneration following a mid-gastric amputation of a hydra begins with a release of Wnt3, caused by the apoptosis of interstitial-stem cells. A second wave of Wnt3 releases after surrounding epithelial cells engulf these apoptotic cells. Surprisingly, this apoptosis induced regeneration was not observed during foot regeneration, despite the similarity of tissue and wound stimuli. In order to further explore the relationship between apoptosis and regeneration, a variety of assays were established to detect apoptotic events within hydra. A method using Acridine Orange was developed, in order to stain vacuoles containing apoptotic cells. The TUNEL Assay was adapted and optimized to end-label and fluoresce apoptotic nuclei. Macerations helped to quantify the number of cells and cell types within the hydra. Both Acridine Orange and TUNEL staining proved to be robust and versatile methods to detect apoptotic events in hydra, exhibited by various control experiments. Utilizing both of these methods, we demonstrated that in both foot and head regeneration apoptotic events occur, contrary to previous studies. Further investigations will be necessary to support alternative hypotheses. Assays employing caspase activity, DNA Laddering, and Dead-or-Alive Cell Staining will help to bolster our current foundation.

 

 

Name:             Kateryna Nesteruk

University:     Taras Shevchenko National University of Kyiv, Ukraine

Supervisor:    Dr. Carmen Ruiz de Almodóvar

Workgroup:   Molecular and Cellular Mechanisms of the Neurovascular link

Project:          The role of caspase-8 in the adult vascular system

Summer

School Call:   2016

Description:  Caspase-8 is a cysteine protease involved in regulating cellular apoptosis and necroptosis. Caspase-8 deficiency leads to embryonic lethality in mice due to defects in endothelial tissues., indicating that Caspase-8 is a pro-survival factor for the vasculature during embryonic development. Nevertheless it is not known, whether Caspase-8 is also needed to maintain vascular homeostasis in later developmental stages and adulthood. To study the function of caspase-8 in the adult vascular system we use a tamoxifen inducible Cre/LoxP system to specifically delete Caspase-8 in the endothelium of adult mice. During my summer internship, my task was to analyse the recombination efficiency of these mice. To do so, I isolated lung endothelial cells from these mice after tamoxifen treatment and performed qPCRs to show the specific downregulation of Caspase-8. In addition, I used mTmG, double-fluorescent reporter mice mated to our endothelial specific Cre-driver line to analyse recombination efficiency in the endothelium by immunofluorensce.

 

 

Name:             Nadezda Podvalnaya

University:     D.Mendeleev University of Chemical Technology of Russia

Supervisor:    Dr. Tamas Fischer

Workgroup:   Epigenetics and Genomic Stability

Project:          Investigation the interaction between poly-A polymerase (Pla1) and MTREC complex in Schizosaccharomyces pombe.

Summer

School Call:   2016

Description:  Degradation of cryptic unstable transcripts (CUTs) in yeasts is a complicated process, which requires the participation of a great number of different proteins. In Schizosaccharomyces pombe, was shown that the MTREC (Mtl1-Red1 core) complex has an important role in targeting of CUTs to the nuclear exosome for degradation. The poly-A polymerase, Pla1 interacts with the MTREC complex and take part in the polyadenylation of the CUTs, which is necessary for their degradation. The aim of the current project is to understand the interaction of the Pla1 and the MTREC complex in details and to find out if Pla1 is recruited to the CUTs by the MTREC complex? Using yeast - two hybrid assay and in vitro expression of recombinant proteins we found out the minimal protein regions involved in the interaction between Pla1 and the MTREC complex. Further mutant variants, which will abolish the interaction between MTREC and Pla1 will be used to assess the in vivo effect.

 

 

Name:            Welda Eleanor Haryanto Tjhoi

University:     Zhejiang University, School of Medicine, Hangzhou, China

Supervisor:    Prof. Dr. Michael Lanzer

Workgroup:   AG Lanzer

Project:          Analysing the Role of Phosphorylation in the Drug Resistance Function of PfCRT

Summer

School Call:   2016

Description:  Post Translational Modifications (PTMs) are enzymatic modifications of proteins during or after protein biosynthesis. Phosphorylation is the most common PTM. It can either activate or inhibit the activity of transporters and also regulate their cellular localization. In Plasmodium falciparum, the localization of the 'Chloroquine Resistance Transporters' (PfCRT) to the food vacuolar membrane is regulated by phosphorylation (T416). However, the role of the phosphorylation sites at position 33 and 411 still remain to be investigated. The CRISPR-Cas9 system was previously used in the lab to mutate the residue 33 from serine to alanine. The aim of this practical is to investigate whether phosphorylation regulates the PfCRT function as a drug carrier. The IC50 assay will be used to measure the sensitivity in the mutant strain towards several antimalarial drugs.

 

 

Name:             Cynthia Amisigo

University:     University of Ghana, Ghana, Africa

Supervisor:    Prof. Dr. Christine Clayton

Workgroup:   mRNA turnover in trypanosomes

Project:          Effect on Deferoxamine on Trypanosoa Brucei

Summer

School Call:   2017

Description:  Trypanosoma brucei is the causative agent of sleeping sickness in human and nagana in cattle. Iron is very crucial to the parasites’ growth and survival. Treatment of this disease is still a challenge hence the need to come up with more therapeutic interventions. Iron is very crucial to the parasites’ growth and survival. Developing treatment options that deprives the parasite of iron could be lethal. The aim of this project was to determine the effect of iron chelator deferoxamine on the transcriptome and growth of Trypanosoma brucei.

 

 

Name:             Nataliia Gnyliukh

University:     Ivan Franko National University of Lviv, Ukraine

Supervisor:    Prof. Dr. Matthias Mayer

Workgroup:   Allosteric regulation of protein conformation

Project:          Characterization of the Hsp70-JDP interaction

Summer

School Call:   2017

Description:  Heat shock protein 70 (Hsp70) is a molecular chaperone which plays a key role in large variety of protein folding processes in the cell. Biological functions of Hsp70 are regulated in the ATP-dependent manner. Switching of the Hsp70 between the ATP and ADP bound states results in the changes of affinity of the Hsp70 for the substrates, which allows efficient binding and release of the substrates. JDPs (J-domain containing proteins) are co-chaperones that stimulate the ATPase activity of Hsp70, leading to the efficient entrapment of the substrates. Examples of human JDPs include Hdj1 and auxilin. However, a molecular basis which underlies the Hsp70-JDP interaction was not well understood. Based on structural data, four amino acids were defined in possibly playing a role in the interaction between Hsp70 and JDPs. FIAsH-Tag is widely used in enzyme activity studies and protein localization, turnover, and trafficking. Another part of the project was to design DnaJ (E. coli Hsp40) with FIAsH-Tag for further studies of its interaction with Hsp70.

 

 

Name:             Anna Goncharova

University:     Moscow Institute of Physics and Technology, State University, Russia

Supervisor:    PD Dr. Karl Rohr

Workgroup:   Biomedical Computer Vision Group (BMCV)

Project:           Automatic analysis of telomeres in microscopy images

Summer

School Call:   2017

Description:  Telomeres are found at the termini of chromosomes and play an essentially important role in DNA damage protection. The length of a telomere is strongly connected with cell aging: during the time, it is becoming shorter and without telomeres, a cell will die. Mammalian telomeres form large duplex loops (telomere loops) which are visible in microscopy images. In my project, I performed image analysis to investigate telomere properties and compare them with previous studies.

 

 

Name:             Liliia Ibneeva

University:     Lomonosov Moscow State University, Russia

Supervisor:    Prof. Michael Knop

Workgroup:   Systems Biology, Meiosis and Signal Transduction

Project:          High-throughput genome-wide screening for novel methionine aminopeptidases and auxiliary proteins

Summer

School Call:   2017

Description:  Selective protein degradation is an important part of normal protein turnover maintaining proteome homeostasis. So-called N-end rule pathways play a key role in the recognition of unnecessary or defective proteins, which are marked with specific degradation signals comprised of well-defined N-terminal residues and/or post-translational modifications, termed N-degrons. Two major branches of the N-end rule target proteins carrying distinct N-degrons for proteasomal degradation: the Ac/N-end rule pathway, which targets proteins carrying N-terminally acetylated residue, and the Arg/N-end rule pathway, which targets proteins with unacetylated residues at the N-terminus. While the Arg/N-end rule pathway has been extensively characterized using artificial model substrates, our knowledge of endogenous substrates is limited to cases of endoproteolytic cleavage. Here we applied high-throughput genome-wide screening in Saccharomyces cerevisiae based on synthetic genetic array technology combined with tandem fluorescent timer reporter to identify hereto unknown factors contributing to generation of Arg/N-end rule substrates by unconventional initiator methionine cleavage modulated by N-terminal acetylation.

 

 

Name:             Svetlana Maltseva

University:     Novosibirsk State University, Russia

Supervisor:    Prof. Karsten Rippe

Workgroup:   Division Chromatin Networks, B066

Project:          Using CRISPR/Cas9 for epigenetic perturbations and effector recruitment to chromatin in mammalian cells

Summer

School Call:   2017

Description:  CRISPR/Cas9 is a promising system to target endogenous loci in living cells. The endonuclease deficient version of the Cas9 protein (dCas9) was previously used to recruit effector proteins or RNA to target DNA sequences in mammalian cells. This can be achieved by fusing effector proteins directly to dCas9 or by introducing natural or synthetic RNA sequences into the single guide RNA (sgRNA) which themselves can bind desired effector proteins. In this project we validated this method by microscopic imaging of a fluorescently-tagged dCas9 protein recruited to telomeres of human U2OS cells. We used both a modified sgRNA with 2xMS2 loops and unmodified one. We show that this system can be successfully used to target telomeres as well as to recruit effector proteins through the MS2-loop containing sgRNA. Furthermore, we characterized the effects of recruiting transcriptional activator proteins to a reporter gene locus either as direct dCas9 fusion or as MS2 coat protein (MCP) fusion recruited through sgRNA component. Our results show that CRISPR/Cas9 approach provides a powerful tool to perform complex perturbations on chromatin of living cells and study regulative non-coding RNAs and their potential role in cancer.

 

 

Name:            Yuliia Nikonishyna

University:     Taras Shevchenko National University of Kyiv, Ukraine

Supervisor:    Prof. Hilmar Bading

Workgroup:   Neurobiology group, Interdisciplinary Center for Neurosciences

Project:          Effects of excitotoxicity on neurons and astrocytes

Summer

School Call:   2017

Description:  Excitotoxic cell death contributes to the development of slowly progressing neurodegenerative disorders (Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis) as well as acute brain and spinal cord injuries (stroke, head traumas). Excitotoxicity is defined as a pathological process that results in neuronal cell death because of excessive glutamate receptor activation. The most important attribute of excitotoxicity is an activation of extrasynaptic N-methyl-D-aspartate (NMDA) receptors which were shown to initiate cell death cascades in neurons. However, details of all molecular pathways involved in the cell death induced by extrasynaptic NMDA receptors remain elusive and need to be investigated. The task of my project was to study the distribution of nuclear proteins, including nuclear actin, in hippocampal neurons and astrocytes after excitotoxicity induction by introducing three different DNA constructs. Methods I used during internship included primary neuronal cell culture, lipofectamine transfection, immunohistochemistry, fluorescent microscopy and confocal live imaging.

 

 

Name:             Angélica Maria Estrada Pacheco        

University:     University of Havana, Cuba

Supervisor:    Katja Machemer-Noonan in cooperation with MCTP  

Workgroup:   COS   

Project:          R2R3-MYB transcription factors       

Summer

School Call::   2017/2018

Description:  R2R3-MYB transcription factors (TFs) belong to a large and functionally diverse protein superfamily in plants. They are involved in regulating stomata aperture (VviMYB60), controlling stamen development (VviMYB80), and regulating the phenylpropanoid pathway. These include activators of the anthocyanin, flavonoid, proanthocyanidin (PA) and stilbene pathways, and also members of the C2-repressor motif clade. (Wong et al., 2016) During this period, we explored the ability of two transcription factors, VviMYB14 and VviMYB15, to activate transcription of the pGT4, pGT5 and pLac promoters via transient expression in plant protoplasts via electroporation followed by Dual Luciferase Reporter Assay (DLR Assay). We applied additional methods such as transformation of E. coli, DNA purification, check digest technology and PCR, GATEWAY ™ Cloning Technology.

 

 

Name:             Anna Caridys Ramírez Suárez          

University:     University of Havana/Center for Genetic Engineering and Biotechnology, Havana, Cuba.

Supervisor:    Prof. Dr. Barbara Mueller   

Workgroup:   Dr. Barbara Mueller´s group, Department for Infectology, Virology, CIID 

Project:          Investigating HIV inhibition and effect on early and late steps of HIV replication of a vimentin targeting peptide        

Summer

School Call:   2017/2018

Description:  Cytoskeleton proteins have been reported to play a role in HIV infection1. Vimentin, an intermediate filament of the cytoskeleton has been identified as an attractive cellular target for inhibiting HIV replication by our lab in Havana and a vimentin targeting peptide capable of modulating the supramolecular structure of this protein is being studied2. The present project aims to further characterize the HIV-1 inhibition observed for this peptide and to comprehend the mechanisms by which the vimentin targeting peptide inhibits HIV infection. Particularly, experiments to evaluate the early or late stages of HIV replication in presence of this antiviral peptide were performed, based on the long-standing expertise in the field of Barbara Mueller´s group. Cell culture, immunostaining, confocal fluorescence microscopy, quantitative western blotting, sequence alignment, FACS analysis, plasmid characterization, cytotoxicity assay and infectivity assays were applied, and I got more in familiar with these methods during this singular CellNetworks Lab Training Program.

1Sasaki, H. et al. (1995).Myosin-actin interaction plays an important role in human immunodeficiency virus type 1 release from host cells. Proc Natl Acad Sci USA 92, 2026–2030.

 2Fernández-Ortega C et  al (2016). Identification of vimentin as a potential therapeutic target against HIV infection. Viruses 8:98. doi:10.3390/v8060098.

 

 

Name:             Karla Zechinelli Pérez          

University:     Universidad Popular Autónoma del Estado de Puebla, Puebla, Mexico.    

Supervisor:    Dr. Carla Galmozzi 

Workgroup:   AG Bukau. Biogenesis & quality control of proteins 

Project:          Exploring the co-translational action of the Escherichia coli molecular chaperone GroEL         

Summer

School Call:   2017/2018 

Description:  In Escherichia coli protein folding mainly relays in a chaperone network that comprise: Trigger factor (TF), DnaK and GroEL. The co-translational folding is facilitated by TF and Dnak that associates with nascent polypeptide chains in a different manner. On the other hand GroEL is a large barrel-shape protein complex which provides an isolated environment and assistance in the folding of proteins between 20 - 60 KDa  (Ewalt et al., 1997; Saibil et al., 2013; Castanié-Cornet et al., 2014). It is assumed to act strictly post-translational, guiding final folding steps of a subgroups of proteins. However, in vitro evidences show that can also associate to translating ribosomes (Ying et al., 2006) suggesting a possible co-translational role in vivo. In this study we used Selective Ribosome Profiling (SeRP) approach in order to study the translational action of the GroEL chaperone. The SeRP was performed for GroEL in different background strains such as: wild type, single TF and DnaK deletions strain (Δtig, ΔDnaK) and the double mutant (ΔtigΔdnak) with the purpose to understand the GroEL action on nascent chain and the interplay between the three main chaperones in E. coli. Firstly, the project focused on the characterization of the strain lacking TF and DnaK. The double mutant growing behavior was described, in addition, since cells compensate the lack of both chaperones by increasing the expression levels of GroEL (Genevaux et al., 2004) the overexpression level of GroEL was quantified. Finally, two different SeRP protocols were use in order to compare the GroEL interactome stabilizing the ribosome-nascent chain-GroEL complex in presence or absence of crosslinking reagents.

 

 

 

Name:             Sucel Palomares

University:     University Centro de ingeniería Genética y Biotecnología Universidad de la Habana

Supervisor:    Rub Russell

Workgroup:   Russell Lab

Project:          Analysis of the potentialities of the Mechismo program. Case study CK2. Analysis of the impact of post-translational modifications in leukemia and the identification and analysis of available public databases.

Summer

School Call:   2017/2018

Description:  The acute myeloid leukemia (AML) is a type of cancer in which the bone marrow makes abnormal myeloblasts (a type of white blood cell), red blood cells, or platelets. This diseases has a five-year overall survival rate of 26 percent and is the most common acute leukemia type in adults. Protein kinase CK2 sustains acute myeloid leukemia cell growth, but its role in leukemia stem cells is largely unknown. We studied the potentialities of the Mechismo program to analyze the effect of protein modifications, using the CK2 protein as a model. The impact of post-translational modifications in leukemia was also analyzed, identifying public databases available for analysis in patients and cell lines, including the effect of drugs on these biological systems.

 

 

Name:             Karla Lisette Andrade López

University:     Autonomous University of Morelos State

Supervisor:   PhD student Manu Goyal

Workgroup:   Posttranscriptional regulation of mRNA expression and membrane-associated RNA-binding proteins

Project:          Validate apoptosis markers and neuronal markers in P19 Wt and Copg1 KO cells

Summer

School Call:   2017/2018 

Description:  In eukaryotes there are three major different types of vesicles involved in the secretory pathway. (I) Clathrin coated vesicles that transport proteins in late secretory pathway, (II) COPII vesicles that export proteins from ER to Golgi, (III) COPI vesicles that transport proteins from Golgi to ER. Béthune's lab is interested in studying paralogue specific role of COPI pathway during neuronal differentiation.

My goal was to study the role of membrane proteins, those could be involved in regulation of the neuronal differentiation in P19 cells. I used several molecular biology techniques, for example western blotting and immunostaining to monitor protein expression. I made cloning of membrane signaling proteins to study the secretory pathway, and I differentiated P19 cells into neurons using Retinoic acid.

 

 

Name:             Elizabeth López Sánchez

University:     Institute of Biotechnology, UNAM Mexico

Supervisor:   Ricardo Carvalho

Workgroup:   Lab Sabine Strahl

Project:          Expression of the O-mannosyl transferase POMT 1 tagged with the reporter protein mCherry.

Summer

School Call:   2017/2018

Description:  Protein glycosylation is divided into N-linked glycosylation and O-linked glycosylation, all beginning in the endoplasmic reticulum (ER). The biosynthesis of O-mannosyltranferase proteins called POMT1 and POMT2 in mammals and PMT 1-6 in fungi. At the luminal site of the ER membrane, PMTs catalyse the transfer of the mannose residue from dolichol phosphate β-D-mannose (Dol-P-Man) to the Ser or Thr amino acids of nascent polypeptides.It is believed that PMTs in yeast and the POMT complex in mammals have highly similar biochemical characteristics and substrate requirements. However, in contrast to baker´s yeast, in mammals only two PMTs have been identified. POMT1 (family PMT4) and POMT2 (family 2) act as a heteromerical complex that is only functional when both proteins are present. The manosylation by the POMT1-POMT2 complex is essential for the glycosylation and functionality of α-dystroglycan (α-DG), the extracellular subunit of the dystroglycan complex that mediates the binding between the cell surface and the extracellular matrix.It was reported that in humans, a heterogeneous group of congenital muscular dystrophies, called secondary α-dystroglycanopathies, are related to a reduction of O-mannosylglycans of α-DG. Therefore, to understand what causes α-dystroglycanopathies, it is necessary to indentify the physical interactions of the human POMT1-POMT2 complex and the machinery involved in O-mannosylation in the mammalian ER. We plan to use reporter fluorescent proteins such as EGFP and mCherry to tag the N-terminal or C-terminal of POMT1 and POMT2 to study the colocalisation of POMT1 and POMT2 as well as the cellular localization of the complex. The plasmid POMT1-mCherry was the only construct obtained in the timeframe of the study. After the plasmid sequence was confirmed it was co-transfected with provided FLAG-HA-POMT2 for over expression in HEK293T cells lacking POMT1 and POMT2 (∆POMT1/2). Subsequently, tagged POMT1 expression was analysed by inmunnoflurescence and inmunoblot. To access if the ectopically expressed POMT1 was functional we performed a West Germ Agglutinin pull-down to enrich for N-Acetylglucosamine rich proteins, and therefore glycosylated α-DG. We observed that the tagged POMT1 was able to rescue α-DG glycosylation defect of ∆POMT1/2 HEK293T cells. Furthermore, POMT1 localised in the ER in HEK 293 cells. The results of this study represent a significant advance in the understanding of the functioning of the machinery involved in the protein O-mannosylation in humans.

 

 

Name:             Santa Mariela Olivera Ugarte

University:     University of Havana and Center for Genetic Engineering and Biotechnology, Cuba

Supervisor:   Dr. Volker Lohmann

Workgroup:   Molecular Virology Department, Dr. Lohmann’s Group

Project:          Toll-like receptor 3 signaling pathway screening project

Summer

School Call:   2017/2018 

Description:  Toll-like receptor 3 (TLR3) recognizes double stranded RNA (dsRNA) that constitutes an essential replication intermediate for many RNA viruses, as Hepatitis C Virus (HCV) (Alexopoulou L. et al., 2001). The activation of transcription factors like IRF3 and NFκB involved in TLR3 signaling pathway triggers induction of a large set of interferon-stimulated genes (ISGs) and interferons (type I and III), which then initiate the antiviral response (Schroder M. and Bowie A.G., 2005). Studies on dsRNA sensors including the TLR3 signaling pathway, which led to the identification of all key players and several dozens of additional regulating factors, have mainly been conducted on easily available and easy-to-handle human cell lines like HEK293 or HeLa (Zhang S.Y. et al., 2013). Few if any studies included hepatocyte-derived cell lines, which leaves us with little knowledge about potential specific regulators involved in hepatic TLR3 responses. To determine whether so far undescribed specific regulators play a role in the TLR3 response of hepatocytes, a genome-wide CRISPR/Cas9-mediated knockout screen in hepatocytes-derived cell lines was previously carried out in Dr. Lohmann’s lab. A total of 55 candidates for TLR3 signaling pathway regulators were identified. The aim of the current project is to evaluate the contribution of these putative candidates to the modulation of TLR3 response. The methodologies employed for this task consisted on: siRNA transfection, qPCR, electroporation of hepatocyte-derived cell lines with VCV replicons, Luciferase Assay, Plasmid Purification, in vitro transcription and total RNA extraction. A deeper knowledge about how the innate immune response against HCV is developed in hepatocytes could take us closer to understanding the persistence of hepatitis C infection.

 

 

Name:             Scarlett Overhage

University:     Universidad Autónoma de Nuevo León, Mexico

Supervisor:   Prof. Dr. Michael Lanzer

Workgroup:   AG Lanzer Parasitology

Project:          Adhesion Assays of Plasmodium falciparum mutants on CSA coating

Summer

School Call:   2017/2018 

Description:  Malaria is a disease caused by protozoan parasite named Plasmodium transmitted through female Anopheles mosquito. Malaria is so lethal mainly because Plasmodium causes modifications on the surface of host erythrocytes making them rigid and inflexible by exporting specific proteins to the RBCs (red blood cells) membrane during the intraerythrocytic stages. This leads to binding of infected-RBC with specific adhesion receptors on endothelial cells causing blockage of microvasculature. CSA is the major adhesion receptor mediating cytoadhesion on endothelial cells during pregnancy. The aim of this study was to compare the binding capacity of different Plasmodium falciparum mutants to CSA.

 

Name:             Ana Mariel Zúñiga Pereira

University:     Universidad de Costa Rica

Supervisor:   Dr. Holger Lorenz

Workgroup:   Imaging Facility

Project:          

Summer

School Call:   2017/2018 

Description:  Fluorescent protein (e.g. GFP) technology has been used, among other applications, for the study of the localization, topology and function of proteins inside mammalian cells. Especially, an endoplasmic reticulum (ER) type 1 membrane protein has been employed for the study of atypical ER degradation pathways. To address numerous open research questions of the protein's degradation path, several microscopy techniques were applied: brightfield, fluorescence, widefield and confocal microscopy; Total Internal Reflection Fluorescence (TIRF) and long-term live cell imaging (6D microscopy). Other techniques like Fluorescence Recovery after Photobleaching (FRAP) and the Fluorescence Protease Protection (FPP) assay were also applied.

 

Name:             Kyle Weise

University:     Northeastern University

Supervisor:   Rob Russell

Workgroup:   Protein Evolution Group

Project:          Frontend software development for protein interaction visualization

Summer

School Call:   2018 

Description:  Protein interactions make up incredibly complex networks. It is important to not only to analyze these networks, but to visualize them as well. Our lab developed a pipeline that takes in a list of proteins or proteins pairs, along with optional mutation sites, and displays the interactions in an easy to comprehend network graph. The pipeline is written in the Python programming language, and the visualization is handled by the popular JavaScript graphing library Cytoscape.js. I redesigned the layout for the website that interfaces the pipeline, added some additional functionality, and made it overall more interactive for end users.