The overall goal of research of the Chair of Bioanalytics
(Prof. Ralf Hoffmann) is to understand the mechanisms of protein
regulation by posttranslational modifications. These enzymatic
modifications are typically reversible but are sometimes irreversible
chemical modifications of specific positions within a protein
chain. Important and well investigated modifications are phosphorylations
on the hydroxyl groups of serine, threonine and tyrosine residues
(phosphoric acid esters) and glycosylation on the side chains
of serine, threonine and asparagine residues. Such modified proteins
will be analyzed with state of the art equipment including mass
spectrometry, HPLC and two-dimensional gel electrophoresis. Thus,
methods will be optimized or developed to analyze these or other
less investigated protein modifications.
We are currently analysing the so-called Tau-protein, which
is highly phosphorylated in the brains of Alzheimer`s disease
(AD) patients and carries most likely disease-specific modifications
(PHF-Tau). Despite worldwide efforts of many research groups for
several decades, the causes of Alzheimer´s disease are still
unknown. One possible hint towards the disease is the Tau-protein,
which consists mainly of a 441 amino acid residues long splicing
form. Research is focused on disease-related alterations of this
protein in the brains of AD patients. In collaboration with two
research groups in the U.S.A. new modifications in PHF-Tau have
been identified. Analyses of the complex phosphorylation patterns
of bovine and human Tau samples using mass spectrometry and two-dimensional
gel electrophoresis indicated that the phosphorylation patterns
of bovine Tau and PHF-Tau (from AD patients) are very similar.
The Chair of Molecular Biological-biochemical Processing
Technology (Prof. Andrea A. Robitzki) has established successfully
the research and developmental focal points in the area of molecular
tissue engineering as well as in the field of drug discovery
using cell and tissue based multi-electrode microarrays and nanobiotechnology.
The main topic of the projects was for the first time the discovery
and characterisation of the spatial and temporal gene expression
pattern of the GDNF family receptor α4 in the embryonic
retina. These results were important and represented the precondition
for the establishment of a novel ligand-receptor test system in
3D in vitro retina models combinated with a bioelectronic
reading out system. A further trend-setting clinical diagnostic
module for preeclampsia and transplant rejection could be developed
by a novel cardiomyocyte-based biosensor for a sensitive, ultra
fast monitoring of angiotensin receptor 1 (AT1)-autoimmune-antibodies
in human sera. Further research projects were successfully launched
in the fields of laser-directed force sensors for a quantitative
biomechanical detection of cell forces, collagen matrix encapsulation
of active cardiomyocytes, novel 3D in vitro mammalian retina models
for drug screening as well as surface modified polymers for micro-laser
manipulation and biosensor technology.
The Chair of Molecular Pathogenesis (Prof. Manfred Blessing)
investigates the function of growth factors and cytokines. The
research is focused on members of the "Transforming Growth
Factor-ß" (TGF-ß) family. TGF-ß is a pleiotropic
factor. TGF-ß is a central regulator of the immune system
promotes proliferation and differentiation of mesenchymal cells
but strongly inhibits epithelial cell proliferation and modulates
angiogenesis and apoptosis. Because of this broad spectrum of
activities which may be quite opposite in different cell types,
changes in TGF-ß activity or TGF-ß responsiveness
strongly affect tissue homeostasis and tissue function. It is
thus not surprising that modifications of the TGF-ß system
are found in a variety of diseases including cancer, immune system
malfunction and impaired regeneration. For functional analysis
we generated a series of transgenic mice with local and cell-type
specific modifications in TGF-ß activity or TGF-ß
responsiveness. A correlation between these modifications and
altered susceptibility towards cancer and immune system malfunction
is being established. For example, loss of TGF-ß responsiveness
in skin and liver quite differently affected tumor incidence in
these tissues and reflected the frequency of respective mutations
in skin- and livertumors from patients. In addition, we demonstrated
that loss of TGF-ß responsiveness in T-cells augmented susceptibility
towards asthma, hepatitis and arthritis, whereas increased TGF-ß
secretion by T-cells rendered resistance towards asthma. At the
same time cell-type specific target genes of this central regulator
are being identified in these models using high density DNA-chip
arrays. From these analyses we expect new candidate genes which
due to their cell-type specificity lead to the development of
new diagnostic, prognostic and therapeutic tools.
The main research interest of the Chair of Structural Analysis
of Biopolymers (Prof. Norbert Sträter) is the study of
the relationship between the three-dimensional structure of proteins
and their function by using biochemical and biophysical methods,
mainly protein crystallography. Here, currently enzymes are being
studied, mostly metalloenzymes, but also other proteins of medical
or biotechnological relevance. Aside from the study of the structure
of the native protein, a prime interest is the characterization
of the catalytic mechanism or function of a protein on a molecular
basis. Current projects focus on metallohydrolases, pharmacologically
relevant extracellular receptor proteins and proteins involved
in signal transduction. The place of the group's research is at
the interface between chemistry and life sciences: Prominent examples
are the analysis of the chemical catalytic mechanisms of enzymes
or the molecular mode of action of synthetic drugs on the biological
targets by structural analysis of the complex structures.
The research interests of the Chair of Cell Techniques and
Applied Stem Cell Biology (Prof. Augustinus Bader) encompass
the field of Regenerative Medicine. Novel patented bioreactors
have been developed and applied to cultivate vital three-dimensional
cartilage grafts. The exacting requirements for the cultivation
of cartilage cell (chondrocyte) constructs demands GMP conditions
for construction and operation of the minibioreactor (Imbiotor)
and associated supply flowpath. Porcine and human articular chondrocytes
are cultivated in several biocompatible matrices with distinct
compositions and mechanical characteristics and are stimulated
to form cartilaginous structures. Interactions of the cells with
the supporting matrix are characterized biochemically and by DNA
microarray gene expression analysis. Bioreactor structure and
culture condition development have yielded promising results in
terms of cell proliferation, differentiation, and resistance to
perfusion-induced shear effects. The two-step stimulation reactor
provides integrated pressure application and will be equipped
with a complex biosensor system in cooperation with other groups
in the BBZ.
A further field of bioreactor development concerns the configuration
and control of modular bioreactors for hepatocytes distinguished
by high cellular production efficiency. Overexpression of telomerase
should allow the proliferation of human hepatocytes with maintaining
redifferentiation potential.
The development of a skin substitute from both dermal and epidermal
precursors on synthetic biocompatible membranes should produce
preliminary results using the new bioreactor system.
Finally, the search for solutions for the high rejection rate,
thrombogenicity, lack of antimicrobial efficacy, and poor biocompatibility
of currently applied arterial protheses comprise the Medimplant
project.
The junior research group Applied Molecular Evolution
(Dr. Susanne Brakmann) employs the method repertoire of directed
evolution to the functional optimization of nucleic acid polymerases
and exonucleases. Predominant results of the past year comprise
the soluble expression of three polymerases, the establishment
of a system for the selection of DNA and RNA polymerase activity
in Escherichia coli, as well as the implementation of a FRET-based
assay for the detection and assessment of RNA polymerase activity.
The junior research group Molecular Diagnostics - Microarray
Techniques (Dr. Peter Ahnert) analyses interindividual diversity
in the etiology and pathogenesis of autoimmune diseases, especially
rheumatoid arthritis. Autoimmune diseases like rheumatoid arthritis
(RA) are complex diseases with various causative factors. It is
well accepted that genetic predisposition plays a role in disease
susceptibility and disease progression. Most likely, not just
the well known HLA locus is involved. However, to date it is unclear
which other genes and their variants may be important. The study
of candidate genes and their polymorphisms appears to be a promising
approach to elucidate this problem. Likely, combinations or patterns
of low penetrance gene variants are important here and for complex
diseases in general. This points in the direction of systems biology.
The goal of this project is to contribute to the elucidation of
the genetic component in the etiology and pathogenesis of RA and
to further the development of methods for investigating the genetics
of complex diseases in general.
We apply statistical methods to the analysis of biological
networks for the selection of candidate genes. SNPs are selected
from public databases. For genotyping we use the GENOLINK system
which is based on primer extension and mass spectrometry. We apply
methods from statistical genetics and machine learning to the
analysis of genotype-phenotype associations.
The junior research group Molecular Medicine of Contagious
Diseases (Dr. Reinhard Straubinger) deals with vaccination
strategies in dogs against Lyme borreliosis, with molecular mechanisms
of persistent infection in Borrelia burgdorferi and with
the roles of IL-23 and IL-17 in the chronic Lyme arthritis. Lyme
disease is a tick-transmitted infectious disease of man and animals
that is caused by spiral-formed, mobile bacteria that belong to
spirochetal family (Borrelia burgdorferi sensu lato). Borrelia
can survive in host tissue despite a strong humoral and cellular
immune response by the host and despite antibiotic therapy. Interestingly,
Borrelia burgdorferi organisms can not only be found in
their typical spiral-formed shape, but also as a spherical entity
("cyst") that may represent a so far unknown survival
form. Clinically, Lyme borreliosis in humans is characterized
by three phases. Days to weeks after the infection with the infectious
agent Borrelia burgdorferi, the skin around the site of
initial infection may show discoloration, that can be noticed
as a circular reddening known as erythema migrans. Weeks to months
later, acute inflammatory responses in joints, nervous system
and in the heart may dominate the clinical picture. A few patients
will develop chronic inflammations in joints years after the infection.
The mechanisms that cause these changes are unknown. In this context,
the group investigates the pathogenesis-supporting or even initiating
roles of the cytokines interleukin (IL)-23 and IL-17 support in
detail in the mouse model. Furthermore, the issue whether the
cyst is the origin for persistent infection is investigated. Finally,
the humoral immune response in dogs after vaccination with commercial
vaccines against Lyme borreliosis, which are available in the
USA and Europe, is studied by quantitative and qualitative methods.
The research field of the junior research group Protein
Engineering (Dr. Thomas Greiner-Stöffele) is the application
and development of methods for the rational protein design and
for in vitro evolution of proteins. Two projects were in
the focus of the group. On the one hand the improved stabilization
of exonuclease III of E. coli has been continued. For this
purpose further rational mutations were introduced into the protein
and new assay systems for the in vitro evolution of this
enzyme were developed. An additional protein homologous to exonuclease
III was isolated from a thermophilic organism (Methanothermobacter
thermautotrophicus), recombinant produced and characterized.
The enzyme shows strong DNA binding properties combined with a
weak DNase activity. On the other hand a new procedure for the
screening of large libraries of protein variants has been developed.
Using the model enzyme RNase T1 the method could be established.
RNase T1 cleaves single-stranded RNA with a high preference after
guanosine residues. Applying the new method an RNase T1 variant
could be isolated for the first time, which cleaves RNA better
after adenosine residues. This corresponds to a shift of the enzyme
specificity by a factor of 1,000,000. The detailed characterization
of this variant has been started.
The junior research group Protein-Ligand Interaction by
Ion Cyclotron Resonance Mass Spectrometry (Dr. Andrea Sinz)
developed methods and protocols to apply the technique of Fourier
transform ion cyclotron resonance (FTICR) mass spectrometry for
analyzing complex protein mixtures. Several chromatographic separation
steps using nano-HPLC (high performance liquid chromatography)
coupled to nano-ESI (electrospray ionization) FTICR mass spectrometry
will be applied. We expect to identify hundreds of proteins from
complex protein mixtures, such as E.coli cell lysates.
Chemical cross-linking combined with high-resolution mass spectrometry
represents a powerful technique to determine low-resolution three-dimensional
structures of protein complexes. After the cross-linking reaction,
reaction mixtures are analyzed by nano-HPLC / nano-ESI-FTICR mass
spectrometry. Low-resolution three-dimensional structure models
of the protein complexes are created on the basis of the obtained
distance constraints.
Non-covalent peptide-metal complexes are characterized by electrospray
ionization Fourier transform ion cyclotron resonance (ESI-FTICR)
mass spectrometry. Metal binding properties of a peptide derived
from the sea urchin protein bindin as well as mutants, in which
histidine residues are replaced by serines, are investigated by
ESI-FTICRMS. Both stoichiometry and binding affinity of the peptide/metal
complexes are determined.
The junior research group Solid-state NMR Studies of the
Structure of Membrane-associated Proteins (Dr. Daniel Huster)
has been dealing with the characterization of the structure and
dynamics of membrane bound molecules such as the Ras protein,
Bacteriorhodopsin, and the fusogenic peptide B18. In particular,
structural data was acquired for the C-terminus of the Ras protein.
Further, structural changes of B18 in the course of the peptide
mediated fusion event have been observed. Detailed investigations
of the molecular motions of Bacteriorhodopsin revealed a very
heterogeneously distributed dynamics of the fast motions in the
backbone of the molecule. In additional studies, the group has
investigated the influence of fluorescence and spin probes as
well as cholesterol analogs on membrane morphology. The use of
paramagnetic relaxation rates for the determination of membrane
protein topology has been successfully demonstrated. Finally,
in detailed studies the structure and dynamics of the macromolecules
in articular cartilage has been described on an atomic lengths
scale.
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