Research Report Laboratory Prof. Dr. A. E. Sippel

Universitt Freiburg
Institut fr Biologie III
Schnzlestrasse 1
D-79104 Freiburg i. Br.

Molecular Mechanisms of Gene Regulation and Cell Differentiation in the Blood Forming System of Vertebrates

Team: Elly Bretschger*, Ruth Camargo Vassao*, Thorsten Ebel*, Nicole Faust, Manfred Fliegauf*, Thomas Frischmann*, Thomas Grewal*, Tim Grewe*, Albert Grnder*, Lorenz Hgele*, Dieter Hartz*, Christoph Hoefer*, Anita Imm*, Susanne Knall*, Susanne Koenig*, Joyce v. Natzmer*, Feng Quian*, Richard Quinn*, Harald Saueressig*, Gabriele Schfer*, Maria Shnyreva*, Diana Winter*, Andr Zimmermann*
*part of the time



Stability of trees against wind loads

Blood contains many different types of cells. These cells are relativelyshort-lived and have to be replaced continuously throughout life. Even thoughmature blood cells have very different functions, they originally are derivedfrom the same pluripotent stem cells. They develop in diverging cell lineages,directed by specific external growth and differentiation factors alongendogenously determined cell differentiation programs (hematopoiesis).

Our interest in the hematopoietic system of vertebrates concentrates onmolecular events in the nucleus of precursor cells which control decisions forcell lineage commitment and cell differentiation. Our research is guided by theassumption that the molecular mechanisms of transcriptional regulation ofcell-type and cell-stage specific marker genes identify the key molecules incontrol of cell growth and differentiation. By using an in vitro celldifferentiation system for myelopoiesis, in which embryonal stem cells developinto macrophages, we have studied the specific transcriptional activation ofthe myeloid specificly expressed marker gene for lysozyme. We have identifiedDNA-sequences in cis and transcription factors in trans which define thecorrect locus control function of this gene. By altering in a controlled waythe expression of members of one myeloid specific transcription factor familywe can deregulate normal cell differentiation at different levels ofmyelopoietic differentiation. The study of regulatory nuclear proteins andtheir interactions at the level of specific chromatin sites in the genome isimportant for our understanding of oncogenesis in the hematopoietic system andfor development of gene therapy strategies in tumor therapy.

Numbers in parentheses refer to publications.

1. In vitro differentiation of embryonal stem cells: a model for hematopoiesis

Faust, Saueressig, Hartz in cooperation with M. Wiles, Basel and C. Bonifer, Freiburg (7,20,29,30)

In an effort to study basic principles of marker gene activation duringmyeloid lineage development, we established an in vitro differentiation systembased on mouse embryonic stem (ES) cells. Under the influence of definedcytokines, ES cells give rise either to an intensified transient erythropoiesisor to a cell population consisting predominantly of macrophages. We can showthat the expression of the endogenous mouse -globin and lysozyme M geneare faithful internal standards indicating the proportion of transienterythroid or final macrophage cells respectively in the differentiationculture. This controlled in vitro differentiation system was used fortitrations of endogenous transcription factor levels during myeloid celldevelopment and for quantitative studies on transgene activation.Undifferentiated ES-cells were stably transfected with the chicken lysozymegene locus, which had been shown previously by interspecies transfer to expresslysozyme RNA cell type specifically in macrophages of transgenic mice. Inundifferentiated transfected ES cell clones, the transgene was consistentlyinactive. Upon in vitro differentiation, the transgene was activatedexclusively in macrophages. The in vitro cell differentiation system could thenbe used to study the molecular determinants of myeloid-specific gene activationand the influence of hematopoietic regulators on myelopoiesis through theireffect on transfected marker gene expression. Top

2. Mechanisms to overcome deregulation of transgenes by chromosomal position effects and their practical use

Saueressig, Faust, Winter, Schfer, Hoefer in cooperation with C. Bonifer, Freiburg, and F. Grosveld, Rotterdam (1,5,6,8,9,14,15,17,18,19,21,28,29,30,35)

In recent years tremendous progress has been made in understanding themechanisms of regulated transcription on the molecular level. However, ourknowledge about gene regulation must be evaluated with respect to our abilityto transplant genes back into the genome of cells and organisms withpredictable correct transcriptional activity. Genes randomly inserted into hostgenomes are most often inactive or incorrectly regulated, due to thederegulating influcence of the neighboring chromatin at their site ofchromosomal insertion (chromosomal position effect). We could show intransgenic mouse lines, as well as in individual transgenic cell clones in theES cell derived in vitro differentiation system, that a construct containingthe DNA of the entire chromatin domain of the chicken lysozyme locus from -11.7kb to +9,5 kb mediates consistent position independent high level cell-typespecific transgene activity. The stunning result of position independentexpression of the complete transgene locus offered the opportunity to map thecis requirements for the locus control function by deletion analysis. By againusing the clonal position effect assay in transgenic mice and the ES cellderived in vitro cell differentiation system for macrophages, we could showthat the transgene loses its position independency as soon as we delete one ofthe transcriptional enhancer regions of the gene. These results stronglysuggest that the locus control function does not map to a particular regulatoryelement in the gene domain, but rather is a feature of the cooperative actionof most likely all regulatory elements of the gene locus, to assemble stableregulatory super complexes in the chromatin of the transgene as outlined inFigure 1. The 'concerted action model' for eukaryotic gene activation washighlighted in a meaningful stable transfection experiment in chickenpromacrophage cells. When we transferred the lysozyme locus with a smalldeletion in the promoter region, several enhancer protein complexes could notassemble in the chromatin of the transgene locus, whereas they are present onthe endogenous wild type locus in the same cell.

In the active chicken lysozyme locus, the border regions of the domain oflosely packaged chromatin on both sides co-locate with so-called nuclear matrixattachment regions (MARs). We could show in stable transfection experiments incells in culture that the lysozyme domain border regions interfere withenhancer promoter interaction when placed inbetween them and that they insulatetransgenes from chromosomal position effects when placed at both ends of thetransgene construct.

With these experiments we have detected a total of two mechanisms forinsulation of transgenes from chromosomal position effects. Regulatory unitsof naturally evolved genes appear to assemble stable regulatory transcriptionfactor complexes, which are inert towards positional stress and the influenceof foreign regulatory elements. This feature self-isolates gene loci. Inaddition, at least some genes, as exemplified by the chicken lysozyme gene, dohave insulator elements which help to block interference from neighboringchromatin by a different mechanism.

These results from basic research on the mechanisms counteracting positioneffects have produced valuable insights for gene technology. Our resultssuggest that it is possible to design transgene vectors for the predictable andcorrect expression of randomly inserted transgenes. In fact the chickenlysozyme 5' domain border region was used to suppress transcriptionalvariability of transgenes in plants caused by position effects. The entirelysozyme gene domain was used as a vector for a macrophage expressed foreigngene in transgenic rabbits. We will now explore these tooles for application ingene therapy.

Picture available here

Figure 1. The 'concerted action model' for eukaryotic gene regulation.

The schematic diagram shows multifactorial regulatory complex formation forfour different transgene situations. a. Stable large transcription factorcomplex of the active wild type locus; b. ectopic expression and positiondependent variable transcription of a partially deleted regulatory complexaffected by a neighbouring enhancer; c. low level position dependent variableexpression due to an instable, incomplete regulatory complex; d. inactivetransgene due to promoter deletion; no stable transcription factor assembly onenhancers is possible. Same shading depict transcription factor proteins of thesame regulatory element (E1, E2, two lysozyme enhancers; Ex, enhancer of aneighbouring gene). Thin line represents loops of nucleosomal chromatin betweenregulatory elements. Black triangles mark positions of DNA deletions in theotherwise complete gene domain construct. Top

3. The chromosomal position effect resistent transgene leads the way tothe definition of a new cell type and to a transcription factor familyresponsible for cell-type and cell-stage specific gene expression inmacrophages

Faust in cooperation with C. Bonifer, Freiburg (9,13,15,17,18,19,21,30)

A transgene which is independent in its transcriptional level and cell-typespecific expression of its site of genomic integration is a rare and mostvaluable tool. Only in the case of position effect resistance it is possible touse randomly inserted transgene constructs as an analytical probe for cell-typespecificity.

Up to now, cell types were mostly characterized by microscopical phenotypeand by molecular marker proteins at the cell membrane. Due to lack of surfacemarkers it was previously not possible to distinguish between embryonal andadult macrophages in vertebrates. We could show that the endogenous lysozymegene in embryonal macrophages is 30 fold lower expressed than in adultmacrophages of chicken. When the chicken lysozyme gene activity was measured inmouse ES-cell derived macrophages the same lower activity was detected ascompared with the interspecies transgene in adult macrophages of transgenicmice. Thus by using the lysozyme gene as a probe we could show that twodifferent macrophage populations develop from embryonic and adult hematopoietictissues in vertebrates. In fact the chicken lysozyme transgene was regulated inthe mouse exactly as it is in its natural host, the chicken. By using the wellcharacterized transgene as probe to distinguish between embryonal and adultmacrophages, we were able, on a molecular level, to analyze differences innuclear transcription factor activities in the two cell types. Chromatinmappings and transcription factor DNA-binding studies clearly showed thatmembers of the CCAAT-enhancer binding protein (=C/EBP) family of transcriptionfactors, by acting at the -2.7 kb enhancer, are the key regulators for lysozymegene activity in both myeloid cell types. Top

4. Protein interactions of the members of the transcription factor C/EBP family

Zimmermann, Grewal, Frischmann, Shnyreva, in cooperation with R. Morrigl, Freiburg (26,33,37)

There are a number of reasons why we chose to focus on the transcriptionfactor C/EBP. It is myeloid specificly expressed in the hematopoietic system.Its DNA-binding activity in nuclear extracts of macrophages strictly followslysozyme gene activity, as it was described in the previous chapter. Mutationof C/EBP-binding sites in lysozyme gene enhancers eliminates their in vivoactivity. Finally, C/EBP binding sites are present in each one of the three 5'flanking enhancers plus the promoter proximal region of the lysozyme gene.

The function of the large transcription factor complex, consisting of theenhancer and the promoter protein complexes and its dynamic changes duringmacrophage development, can only be understood by a detailed description of theprotein components and their molecular interactions. It is most likely thatC/EBPs contribute to the protein-protein interactions between the individualregulatory elements, as indicated schematically in Fig. 2 between the -6.1 kbenhancer and the promoter. Therefore we have started to study protein contactsof the various isoforms of this transcription factor family in the yeasttwo-hybrid system. Three levels of protein surface interactions are analyzedfor the transactivating C/EBPalpha, and delta proteins andthe dominant negative forms of C/EBPgamma and CHOP (= C/EBP homologprotein, can be a chimeric oncogene product in malignant liposarcoma). A,Intramolecular interactions between different protein domains; B,intermolecular interactions between the various isoforms, and C, interactionsbetween C/EBPs and other transcription factors. For each of the threeinteraction levels one particular case was detected and is now studied in moredetail. In some C/EBPs repressing interactions exist between thetransactivation and the DNA-binding domain which are reversed upon unfolding.CHOP shows a particular strong interaction with some of the C/EBP homodimers,which could be indicative for dynamic changes in multifactorial transcriptionfactor DNA complexes. Protein interactions between C/EBPs and specific membersof the STAT transcription factor family point to cross-talking betweendifferent cytokine signal transduction pathways in macrophages.

Picture available here

Figure 2. A model for the molecular interaction of transcriptionfactor complexes at the -6.1 kb enhancer and the promoter of the chicken lysozyme gene.
C/EBP, NFI and some of the basic transcription factors are shown bound tothe DNA motifs T1, T2, D and E of the enhancer and the promoter proximal C/EBP-and the TATA-motif of the promoter region. Protein-protein and protein-DNAinteractions previously studied by us and others are indicated by red. Thoseinteractions currently studied in our group are marked black. Top

5. Towards a functional analysis for transcription factor Nuclear Factor I (NFI)

Ebel, Qian, Grnder, Bretschger, in cooperation with P. Lichter, Heidelberg, U. Kruse, La Jolla and E. Stavnezer, Cincinnati (3,4,10,12,22,25,26,27,31,40)

The transcription factor NFI (TGGCA-protein, CTF) was 1980 independentlydiscovered by Hurwitz's group in New York and our group as cellular replicationprotein for Adenovirus and central DNA-binding protein in the -6.1 kb enhancerof the chicken lysozyme gene respectively. We later found that NFI is not aunique transcription factor protein but consists of a whole family of relatedproteins with identical DNA-binding activities. The large number of isoforms isderived from isoform amplifications on the DNA, RNA and protein level.

We could show that there are 4 seperate genes for NFI in the chicken, mouseand human genome. We mapped the NFIA gene to the human chromosomal position1p31.2, the NFIB gene to 9p24.1, and the two genes NFIC and X to 19p13.3. Thechicken NFIB gene was shown to contain 12 exons stretched over more than 150 kbof genomic DNA. Each gene produces transcripts which alternatively splice intodifferent mRNAs. Splice variations are mostly in the C-terminal half of theopen reading frames, containing the transactivation domains of the proteins.Corresponding splice variants are seen between paralogous genes in one speciesand the orthologous genes in different vertebrate species. Paralogous proteinhomologies are mostly restricted to the N-terminal DNA-binding domain,orthologous homologies are over 90%, extending through all domains of theproteins. The number of dimeric NFI transcription factor variants isdramatically increased on the protein level by free heterodimerization ofsubunit monomers.

NFI DNA-binding activity is ubiquitously found in all tested vertebratecells, which had contributed to a relative lack of interest in functionalstudies. However, using our specific cDNA variants as tools, we observedistinct differential but overlapping expression patterns in different celltypes and have thus initiated the analysis of NFI function an two levels.

On the level of protein we study their intra- and intermolecular proteininteractions in the yeast two-hybrid system and a partial enhancerreconstitution assay. Again, as in the case of transcription factor C/EBPproteins, we find for NFIB of the mouse intramolecular repression betweenDNA-binding and transactivation domains and composit type of transactivationfunction in the C-terminal half of the protein. As an example for transcriptionfactor interaction, with NFI we have analyzed the specific cooperativeDNA-binding and transcriptional activation function of the Ski oncoproteinmediated by NFI variants.

In order to approach functional studies on the level of the organisms, wehave isolated parts of the mouse NfiA and B genes, have constructed casettevectors for "knock-out/knock-in" transgenic mice strains via homologousrecombination in ES cells. Top

6. Genetic modification of C/EBP expression in the ES cell deriveddifferentiation system and the effects on development of hematopoietic cells

Knig, Hgele, Grewe, Faust and Hartz, Fliegauf in cooperation with M. Reth, Freiburg and L. Larue, Paris (24,36,38,39)

For our studies of mechanisms of regulation of cell growth anddifferentiation in the hematopoietic system we again used our ES-cell derivedin vitro cell differentiation system. This true stem cell system offers theadvantage of relative easy genetic manipulations. To test whether the myeloidspecific transcription factor family, which we detected as crucial formacrophage specific gene activation, is a general regulator of myelopoiesis, wemodified the expression of C/EBPs by genetic gain of function and loss offunction experiments.

Transgene mediated constitutive overexpression of C/EBPalpha and CHOPproved to be lethal for ES-cell growth. In order to eliminate the growthregulatory function of the trans dominant wild type CHOP protein we used adeletion in the basic DNA-binding region of the protein. We could show thataccording to higher or lower levels of expression of the CHOP-br-mutant protein cell differentiation was inhibited either already on the levelof ES-cells or later in the development of hematopoietic cells. It becameclear, that in order to study later events in hematopoiesis, as for examplelysozyme gene activation in mature macrophages under the influence of C/EBPoverexpression, undisturbed by earlier deregulated processes, it is necessaryto develop genetic systems for inducible C/EBP expression.

A first type of inducible gain of function experiment was done with thegene for a C/EBPalpha fusion protein containing the estrogen receptorligand binding domain. We saw that in opposite to the expression of wild typeC/EBPalpha, which had directly killed transfected ES-cell clones, estrogeninduction of C/EBPalpha activity at later differentiation states lead tovarious differentiation phenotypes according to C/EBPalpha ER expressionlevel and time point of induction. Currently we are working on a second type ofinducible gain of function system for the overexpression of C/EBP variants. Weconstruct ES-cell clones with inducible Cre-recombinase and C/EBP genevariants, which will be activated after deletion of a blocking DNA sequenceelement by site specific recombination.

In the in vitro differentiation system quantitative PCR titrations showedan increase of C/EBPalpha expression to parallel macrophagedifferentiation and to trail C/EBP increase by several days in thecourse of ES-cell derived myelopoiesis. We were therefore surprised to find apronounced developmental phenotype in an ES cell C/EBPalpha loss offunction experiment. C/EBPalpha antisense-RNA expression inundifferentiated ES cells inhibited the development of normal embroid bodiesand caused differentiation of parietal endoderm cells. The apparent massivetransition of early embryonic cell differentiation in vitro indicates theimportance of low level C/EBPalpha expression for the control of theearliest steps of cell differentiation in blastocyst inner cell mass. Top

7. Replication timing of the lysozyme gene domain

Faust, Zimmermann, in cooperation with H. Cedar, Jerusalem

The chicken lysozyme is one of best characterized eukaryotic gene domains.Most important is the fact that the transgene locus belongs to the very fewcases which are transcriptionally activ independent of the site of chromosomalintegration. We have therefore chosen this gene as a model system for aninvestigation of the programmed process governing gene replication.

For many genes a good correlation exists between early S-phase replicationand gene transcription. In order to better understand the role of replicationtiming we have designed a series of experiments to dissect the cis and transrequirements for the coupled regulation of both basic processes in chickencells and in the transgenic in vitro cell differentiation system derived frommouse ES-cells. Top

8. Public acceptance of clinical somatic gene therapy and animal transgenesis

v. Natzmer, Sippel (2,8,16,17,21,32)

Because of the strong criticism of gene technology in our country, it isnecessary to know more about reservations and the degree of popular acceptanceof specific aspects of the biological high-tech sector. We have performed astudy on the knowledge and acceptance of somatic gene therapy by medicalpractitioners and biology teachers. In addition, we have dealt on many publicoccasions with ethical, legal and social issues of gene therapy and animaltransgenesis. Top


Ongoing research from our group is supported by grants from the DeutscheForschungsgemeinschaft (SFB 364 and SFB 388), the Fonds der ChemischenIndustrie, and the German Israeli Foundation of Scientific Research and Development.

Publications 1993-6/1997

  1. Sippel, A.E., Schfer, G. Faust, N., Saueressig, H., Hecht, A. and Bonifer, C. (1993)
    Chromatin domains constitute regulatory units for the control of eukaryotic genes; Cold Spring Harbor Symposia on Quant. Biol. 58, 37-44

  2. Sippel, A.E. (1993)
    Das Genom und die Individualitt seines Trgers; BIUZ Jg. 23 (Biologen in unserer Zeit) 4/93, 60-61

  3. Kruse, U. and Sippel, A.E. (1994)
    The genes for transcription factor Nuclear Factor I give rise to  corresponding splice variants between vertebrate species; J Mol. Biol., 238, 860-865

  4. Kruse, U. and Sippel, A.E. (1994)
    Transcription factor Nuclear Factor I proteins form stable homo- and heterodimers; FEBS Letters 348, 46-50

  5. Bonifer, C., Yannoutsos, N., Krger, G., Grosveld, F. and Sippel, A.E. (1994)
    Dissection of the locus control function located on the chicken lysozyme gene domain in transgenic mice; Nucl. Acids Res. 22, 4202-4210

  6. Huber, M.C., Bosch, F.X., Sippel, A.E. and Bonifer, C. (1994)
    Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNaseI hypersensitive site formation; Nucl. Acids Res. 22, 4195-4201

  7. Faust, N., Bonifer, C., Wiles, M.V. and Sippel, A.E. (1994)
    An in vitro differentiation system for the examination of transgene activation in mouse macrophages; DNA and Cell Biol. 13, 901-907

  8. Sippel, A.E., Saueressig, H., Faust, N., Schfer, G. and Bonifer, C. (1994)
    Control of expression of the chicken lysozyme gene and its potential application in transgenic birds; in Proceedings of Genetics Applied to Livestock Production (ed. E.B. Burnside) Vol. 21, 307-313

  9. Bonifer, C., Bosch, F.X., Faust, N., Schuhmann, A. and Sippel, A.E. (1994)
    Evolution of gene regulation as revealed by differential regulation of the chicken lysozyme transgene and the endogenous mouse lysozyme gene in mouse macrophages; Europ. J. Biochem. 226, 227-235a

  10. Qian, F., Kruse, U., Lichter, P. and Sippel, A.E. (1995)
    Chromosomal localization of the four genes for the human transcription factor Nuclear Factor I (NFIA, B, C and X) by FISH; Genomics 28, 66-73

  11. Huber, M., Graf, T., Sippel, A.E. and Bonifer, C. (1995)
    Dynamic changes in the chromatin of the chicken lysozyme gene domain during macrophage differentiation; DNA and Cell Biol. 4, 397-402

  12. Ebel, T.T. and Sippel, A.E. (1995)
    A rapid method to deplete endogenous DNA-binding proteins from reticulocyte lysate translation systems; Nucl. Acids Res. 23, 2076-2077

  13. Sippel, A.E., Saueressig, H., Huber, M.C., Stief, A., Borgmeyer, U. and Bonifer, C. (1996)
    Identification of cis-acting elements as DNase hypersensitive sites in lysozyme gene chromatin; Methods in Enzymology 274, 233-246

  14. Bonifer, C., Huber, M.C., Faust, N. and Sippel, A.E. (1996)
    Regulation of the chicken lysozyme locus in transgenic mice; Critical Reviews in Eukaryotic Gene Expression 6, 285-297

  15. Bonifer, C., Huber, M.C., Jgle, U. and Sippel, A.E. (1996)
    Prerequisites for tissue specific and position independent expression of a gene locus in transgenic mice; J. Mol. Med. 74, 663-671

  16. Sippel, A.E. (1996)
    Gene Therapy - a new medical technique and points to consider; in "Annual Review of Law and Ethics". Eds. Byrd, B.S., Hruschka, J. and Joerden, J.C. Verlag Dunker and Humblot, Berlin, Vol. 4, 35-47

  17. Sippel, A.E., Saueressig, H., Huber, M.C., Faust, N. and Bonifer, C. (1996)
    Insulation of transgenes from chromosomal position effects; in "Transgenic animals: Generation and Use". PartIII/B/41 p.1-9; Ed. Houdebine, L.-M.Harwood Academic Publishers

  18. Bonifer, C., Faust, N., Huber, M.C., Saueressig, H. and Sippel, A.E. (1997)
    The chicken lysozyme chromatin domain; in "Nuclear Organization, Chromatin Structure and Gene Expression". Eds. van Driel, R. and Otte, A. Oxford University Press; in press

  19. Faust, N., Huber, M.C., Sippel, A.E., Bonifer, C. (1997)
    Different macrophage populations develop from embryonic/ fetal and adult hematopoietic tissues; Experimental Hematology; in press

  20. Faust, N., Knig, S, Bonifer C. and Sippel, A.E. (1997)
    Transgene analysis in mouse embryonic stem cells differentiating in vitro; in "Human Genome Methods". Ed. Adolph, K.W. CRC-Press; in press

  21. Saueressig, H., Faust, N. and Sippel, A.E. (1997)
    Mechanisms to overcome deregulation of transgenes by chromosomal position effects; in "Animal Biotechnology: Molecular Breeding and Transgenics"; Eds. Li, N., Schook, L.B., Wu, C. and Chen, Y.International Academic Publishers; in press

  22. Kruse, U., Ebel, T.T. and Sippel, A.E. (1997)
    Generation of transcription factors in rabbit reticulocyte lysate depleted of endogenous DNA-binding proteins; in "Methods in Molecular Biology", ed M.J. Tymms, Humana Press; in press

  23. Faust, N., Bonifer, C. and Sippel, A.E. (1997)
    A major role for CCAAT/enhancer binding proteins in transcriptional stimulation of the chicken lysozyme gene during macrophage activation by bacterial lipopolysaccharide; submitted

  24. Hartz, D.G., Fliegauf, M., Saueressig, H., Khler, F., Larue, L. and Sippel, A.E. (1997)
    Antisense expression of transcription factor C/EBPalpha induces parietal endoderm differentiation in murine embryonic stem cells; in preparation

  25. Richmond, C., Tarapore, P., Zheng, G., Heyman, H.C., Cohen, S., Kelder, B., Kopchick, J., Kruse, U., Sippel, A.E., Colmenares, C., and Stavnezer, E. (1997)
    DNA binding and transcriptional activation by the Ski oncoprotein mediated by interaction with NF1; in preparation

  26. and electronic publications in the EMBL/GenBank database: Y07685-Y07693 (9 MM NFI cDNA sequences)


Doctoral Theses 1993-6/1997


Diploma Theses 1993-6/1997


Running Diploma and Doctoral Theses