Rancière, autonomía como emancipación intelectual 23

5mC - 5-methyl cytosine

CAF - chromatin assembly factor

CENP - centromere binding protein

CPP - cell penetrating peptide

DFC - dense fibrillar component

DNA - deoxyribonucleic acid

DRAQ5 - deep red fluorescing anthraquinone Nr. 5

EM - electron microscopy

ESI - electron spectroscopic imaging

ER - endoplasmatic reticulum

FC - fibrillar center

FITC - fluoresceinisothiocyanate

GC - granular component

GFP - green fluorescent protein

HIV - human immunodeficiency virus

HMGN - high-mobility group nucleosome binding protein

hnRNP - heterogeneous nuclear ribonucleoprotein particles

HP1 - heterochromatin protein 1

mRFP - monovalent red fluorescent protein

mRNA - messenger RNA

MBD - methyl cytosine binding domain proteins

MeCP2 - methyl cytosine binding protein 2

MW - molecular weight

NLS - nuclear localization sequence

TAT - HIV transactivator of transcription

PBD - PCNA binding domain

pc - phase contrast

PCNA - proliferating cell nuclear antigen

PI - propidium iodide

PML - promyeolytic leukemia

S - Swedberg sedimentation unit

SMT - single molecule tracking

Martin, R. M., Leonhardt, H., and Cardoso, M. C. (2005). DNA labeling in living cells. Cytometry A 67, 45-52.

Walther W., Minow T., Martin R., Fichtner I., Schlag P.M., Stein U. (2006). Uptake, biodistribution, and time course of naked plasmid DNA trafficking after intratumoral in vivo jet injection. Human Gene Therapy 17:1–14

Tunnemann, G., Martin, R. M., Haupt, S., Patsch, C., Edenhofer, F., and Cardoso, M. C. (2006). Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells. FASEB J 20, 1775-1784.

Martin, R. M., Tunnemann, G., Leonhardt, H., and Cardoso, M. C. (2007). Nucleolar marker for living cells. Histochem Cell Biol 127, 243-251.

Martin, R. M., Gorisch, S. M., Leonhardt, H., and Cardoso, M. C. (2007). An unexpected link between energy metabolism, calcium, chromatin condensation and cell cycle. Cell Cycle 6-19, 2422-24.

Tunnemann, G., Ter-Avetisyan, G., Martin, R. M., Stockl, M., Herrmann, A., and Cardoso, M. C. (2007). Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. J Pept Sci. 14:469-76.

Grunwald, D., Martin, R. M., Buschmann, V., Bazett-Jones, D. P., Leonhardt, H., Kubitscheck, U., and Cardoso, M. C. (2008). Probing intranuclear

environments at the single molecule level. Biophys J. 94:2847-58

Martin R.M., Cardoso M.C. (2008).

Chromatin condensation modulates access and binding of nuclear proteins Manuscript submitted at JMB

Nov 2004 Oral presentation at the Max-Delbrück-Center for Molecular Medicine (MDC) Cardiovascular Retreat, Gross Dölln

Sept 2005 Oral presentation at the Max-Delbrück-Center for Molecular Medicine (MDC) & Leibniz-Institut für Molekulare Pharmakologie (FMP) PhD-Retreat, Straussberg

Nov 2005 Oral presentation “DNA Labeling in Living Cells”, CenTech, Münster

Dec 2005 Oral & poster presentation at Graduate biophysics program; Workshop on Cell Biology and Microscopy, Altleiningen

July 2006 Poster presentation, International symposium “Optical analysis of biomolecular machines”, Berlin

Sept 2006 Oral presentation at the Max-Delbrück-Center for Molecular Medicine (MDC) & Leibniz-Institut für Molekulare Pharmakologie (FMP) PhD-Retreat, Motzen

Dec 2006 Poster presentation at “Chromatin structure and function” conference, Punta Cana, Dom. Rep.

Aug 2007 Oral presentation at Wilhelm Bernhard Workshop on the Cell

Acknowledgments

The projects of this thesis would not have been possible without the help of many people. Most of them are listed as coauthors in the publications above. Here I would like to express my grateful thanks especially to M. Cristina Cardoso for all the guidance and support over the years with enthusiasm and patience in scientific working as well as for fruitful and inspiring discussions. Together with Cristina I have to thank here Heinrich Leonhardt for the supervision of the thesis in all steps and his help and support during all the experimental work and preparation of figures and manuscripts. Both of them as single persons and as a team have encouraged me to appreciate and invest in my work and enjoy all about science.

The nice atmosphere in the Cardoso Lab has encouraged me to a motivated work and in this way I wanted to thank and appreciate all former and present members of this lab. Some of them I wanted to mention explicitly for their support in the daily live in the lab, namely the irreplaceable Petra, Marion and Ingrid. I also thank Anje for introducing me to confocal microscopy and being there in case of problems, questions and discussions.

Special thanks I owe to the people I have worked with in close collaboration during the different projects. First of all it is David Grünwald whom I thank for the excellent cooperation, very inspiring discussions and making my visits in Münster a delight. I would like to thank Volker Buschmann for his advice and cooperation as a colleague while joining the Cardoso Lab and beyond for nice times outside of the lab. I greatly appreciate the team in Munich and especially Kourosh and Oliver for creating a cheerful and pleasant atmosphere during the ‘Illuminati’ meetings as well as in workshops and meetings

Finally I would like to thank my Family who accompanied me in all the years with support, help and patience in all aspects of live. Additional thanks go out to Lars and Olaf for having nice times and support on hard days, the exchange of ideas and just being real friends.

Chapter 3.1 DNA labeling in living cells

This project was started already 2002 during my diploma thesis in the group of M. Cristina Cardoso at the Max-Delbrück-Center in Berlin, Germany. The comparison of DNA dyes in Fig. 1 and the linescan analysis of DRAQ5 with histone GFP labeled chromatin in Fig. 2 are part of the Diploma thesis. All following experiments were performed, designed, analyzed and evaluated by me, except for FACS experiments at the core facility at the MDC-Berlin, performed by Hans-Peter Rahn. The manuscript including the figures was written and designed by me with the help and advice of M. Cristina Cardoso and Heinrich Leonhardt.

Chapter 3.2 Nucleolar marker for living cells

The project to develop and use cell penetrating peptides as nucleolar marker for living cells was initiated by me. The experiments for this project were designed, performed and analyzed by me, excluding the uptake of the nucleolar marker into live rat cardiomyocytes performed by Gisela Tünnemann. The manuscript was written and prepared by me with the helpful advice of M. Cristina Cardoso and proofreading by G. Tünnemann.

Chapter 3.3 Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells.

For this project I performed together with Gisela Tünnemann the cell cycle progression assay and contributed with the design and performance of the FRAP and transduction experiments. Simone Haupt, Cristoph Patsch and Frank Edenhofer provided the reporter cell line (3T3-FDR1.2 cells) and fluorescently labeled TAT-Cre protein as well as performed the respective experiments. The manuscript and figures were prepared by Gisela Tünnemann and me, supported by M. Cristina Cardoso with scientific advice and discussion of the results.

Chapter 3.4 Live cell analysis of cell penetration ability and toxicity of oligo-arginines This project was initiated by M. Cristina Cardoso, Gisela Tünnemann and me after discussing our results on cell penetrating peptides and the nucleolar marker. Gohar Ter-Avetisyan performed, evaluated and illustrated all transduction experiments. I supported the initial experiments with supervision and help during the performance and contributed unpublished experimental data. Martin Stöckl analyzed the transduction frequency of NBD-labeled peptides in large unilamellar vesicles (LUV) by spectrophotometry. Andreas Herrman provided expertise and equipment for the LUV experiments and M. Cristina Cardoso accompanied all stages of the experimental part and the manuscript preparation with scientific and practical advice. Gisela Tünnemann performed evaluated and illustrated experiments in Fig. 2/Fig. 3. and wrote the manuscript. My part during the manuscript preparation was the help in analyzing and displaying the data, preparing the supplementary movie and scientific proofreading of the manuscript.

condensation and cell cycle

The concept of the project layout was done together with M. Cristina Cardoso and Sabine M. Görisch. The experiments were designed and performed by me, including the data analysis. Further I wrote the paper manuscript and prepared the figures with supportive advice and fruitful discussions with M. Cristina Cardoso and Heinrich Leonhardt. Tony J. Collins (McMaster University, Hamilton, Canada) made helpful comments on the manuscript. Chapter 3.6 Chromatin condensation modulates access and binding of nuclear proteins

All experiments in this manuscript were developed, performed and analyzed by me including the writing of the manuscript and preparation of the figures with support by M. Cristina Cardoso.

Chapter 3.7 Probing intranuclear environments at the single molecule level.

My contribution to this project was the design and performance of the experimental live cell work together with David Grünwald, who has built the single molecule microscope setup.

In addition my part was the design and performance of the FRAP and FLIP control experiments as well as the testing of plasmid constructs, microinjection and confocal microscopy localization studies. The evaluation of the FRAP and FLIP experiments was supported by Volker Buschmann, who also contributed importantly to the analysis of the single molecule data. During the work for the manuscript my part was the preparation of figures, contribution to the text, discussion of the results and editing of the final manuscript with the help and advice of M. Cristina Cardoso.

Fakultät Biologie”

Betreuung: Hiermit erkläre ich, dass die vorgelegte Arbeit an der LMU von Herrn Prof. Dr. Heinrich Leonhardt betreut wurde.

Anfertigung: Hiermit versichere ich ehrenwörtlich, dass die Dissertation von mir selbstständig und ohne unerlaubte Hilfsmittel angefertigt wurde. Über Beiträge, die im Rahmen der kumulativen Dissertation in Form von Manuskripten und Publikationen in der Dissertation enthalten sind, wurde im Kapitel 6.4 Rechenschaft abgelegt und die eigenen Leistungen wurden aufgelistet.

Prüfung: Hiermit erkläre ich, dass die Dissertation weder als ganzes noch in Teilen an einem anderen Ort einer Prüfungskommission vorgelegt wurde. Weiterhin habe ich weder an einem anderen Ort eine Promotion angestrebt oder angemeldet oder versucht eine Doktorprüfung abzulegen.

München, den__________________________________ (Robert Martin)

6.5 Curriculum Vitæ