The research projects of the Chair of Bioanalytics (Prof.
Ralf Hoffmann) have the overall goal to develop new analytical
tools to identify posttranslational modifications as well as to
characterize proteins for their phosphorylation, hydroxylation,
methylation, glycosylation, desamidation and glycation sites.
These mostly reversible but partially irreversible modifications
alter and thereby regulate both the structural and functional
characteristics of proteins in response to cellular or extracellular
signals. Currently we analyze the tau protein, which is a heavily
modified brain specific protein and proposed to carry one or several
modifications peculiar to Alzheimer's disease (PHF-Tau). Despite
world wide research efforts for several decades, the cause of
Alzheimer's disease as well its link to Tau and its medications
is still unclear. Our work aims to identify novel posttranslational
modifications as well as to characterize the phosphoraylation
pattern of Tau, which longest human isoform consists of 441 amino
acids. For its purification we have developed three chromatographic
separations that rely on different protein interactions to the
stationary phase, which most importantly do neither discriminate
the splicing forms nor posttranslational variants. The purified
Tau was quantified relative to each other, their phosphorylation
degree was estimated and the distribution of several known phosphorylation
sites in the different Tau versions was investigated. Furthermore,
we could find a new modification that had not been described for
Tau before, which could regulate the Tau-tubulin interaction as
well as Tau aggregation. To analyze hydroxyllated amino acids
in collagen types, such as hydroxylysine and four hydroxyproline
isomers, we have developed two chromatographic methods that are
also suitable for quantification. These methods were used to characterize
the acid soluble collagen types VI and VIII for their hydroxyamino
acids. Together with a research group from the United States we
identified a new methyl transferase and characterized this enzyme
for its methylation specificity of the guanidino group of arginine
residues in proteins. Recently, we have established solid phase
synthesis of glycated peptides. The synthesized model peptides
will be used to develop the analytical tools to characterize glycated
proteins as well as advanced glycation end products (AGEs).
The Chair of Molecular Biological-biochemical Processing
Technology (Prof. Andrea A. Robitzki) has enlarged the research
and developmental focal points in molecular tissue engineering
as well as in drug discovery and functional protein characterisation
using cell and tissue based multi-electrode arrays by two further
research areas like nanobiotechnology / nanoelectronics and laser
directed manipulation and catapulting of viable biological systems.
One height was e.g. the successful development and proof in principle
of a functional autoimmune antibody-biosensor as a prediagnostic
module of preeclampsia (EPH gestosis) during pregnancy. The principle
of this cardiomyocyte based multielectrode array is the online
and real time monitoring of the contraction rate and arrhythmia
by a multielectrode configuration on an array. The frequency of
the motility of the cardiomyocyte model represents a functional
monitoring of physiological AT1 receptor agonists (detection limit
10-11M) as well as AT1 autoimmune antibodies
in sera. Moreover these electronic bioassays can be applied for
an enlarged clinical diagnosis, for biomedical and biotechnological
research e.g. the detection of cellular events mediated by ligand-receptor
interactions. For the development of further cell and tissue based
microarrays (impedance spectroscopy) ischemic 2D/3D in vitro cardiomyocytes
and a 3D in vitro mammalian retina have been developed and characterised.
Another technology platform combined to sensorics was established
for laser manipulation and catapulting of living embryonic neuronal
precursor cells. The goal of the technology is the establishment
of neuronal regeneration and transplant concepts in the field
of remyelinisation of nerves. For optimizing molecular biochips
and in the course of a miniaturisation for micro-implants and
-prostheses projects were started in the field of molecular transistors
and nanostructuring of implant surfaces for controlled drug delivery
or release.
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-beta" (TGF-beta) family. TGF-beta is a pleiotropic
factor. TGF-beta 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-beta
activity or TGF-beta responsiveness strongly affect tissue homeostasis
and tissue function. It is thus not surprising that modifications
of the TGF-beta 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-beta activity
or TGF-beta responsiveness. A correlation between these modifications
and altered susceptibility towards cancer and immune system malfunction
is being established. For example, loss of TGF-beta responsiveness
in skin, gut and liver quite differently affected tumor incidence
in these tissues and reflected the frequency of respective mutations
in tumors of skin, gut and liver from patients. In addition, we
demonstrated that loss of TGF-beta responsiveness in T-cells augmented
susceptibility towards asthma, hepatitis and arthritis, whereas
increased TGF-beta 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) lie within
the area of regenerative medicine. One focus of the group's interests
is the elucidation of the mechanisms of regeneration following
liver damage. Possible non-haematopoietic functions of peptides
hitherto considered as primarily haematopoietic have been identified
by the group and the investigation of the roles of thrombopoietin
(TPO) and erythropoietin (EPO) in liver regeneration have become
a particular focus of the group's attention. Application of EPO
led to a quantifiable acceleration of liver regeneration following
partial hepatectomy in pigs. Currently, the precise nature of
the effects of "haematopoietic peptides" on liver regeneration
are being investigated in various model systems. Amongst other
techniques, we are studying changes in gene expression with our
own in-house developed oligo microarrays and investigating both
quantitative and qualitative changes in regenerating tissue. The
practical aim of our studies is to help to derive therapies which
support liver regeneration via the application of regeneration-inducing
mediators and extracorporeal liver support.
The research group efforts towards the "development of
a bioreactor circuit for sterile chondrocyte stimulation and bioanalytical
characterization" had the development of a modular bioreactor
system as a major goal. The design and construction of the system
surrounding the stimulation chamber, especially the sensory and
control components, were identified as being of the utmost importance.
Due to the extremely challenging operational requirements, a modular
system consisting of individual bioreactor modules, a control
unit, a user interface and individual system communications ports
was devised.
A single module consists of a bioreactor and associated circuitry
with a nutrition reservoir, a supply pump, a flow sensor and a
temperature sensor. In addition, a secondary circuit for online
analysis including relevant sensors, a dosing pump and a three-way
valve for extracting samples for offline analysis is connected
to the primary circuit. A secondary reservoir can also be attached
to the primary nutrition reservoir. This secondary reservoir can
be used to regulate the supply of high value nutritional components
such as plasma or serum and can also provide "conditioned"
feeding with autologous nutrients in a fed batch mode.
In practise, six cartilage constructs were simultaneously and
identically stimulated and cultivated. From six such constructs,
one could be destined for clinical implantation, whilst the remainder
could be used for biochemical, biophysical and genetic analyses.
The junior research group Applied Molecular Evolution
(PD Dr. Susanne Brakmann) is concerned with directed evolution
techniques for the functional optimization of nucleic acid polymerases
and also, techniques for the realization of single molecule DNA
sequencing. The studies on polymerases focus on the development
of variants of T7 DNA polymerase and HIV Reverse Transcriptase
with altered error rate and on the development of variants of
T7 RNA polymerase with altered substrate tolerance. A major advance
concerns the development of genetic selection approaches for the
identification and isolation of active polymerases from polymerase
mutant libraries which are produced e.g. by error-prone PCR or
by DNA shuffling. Usually, such libraries contain predominatly
inactive variants (more than 90 %). By using certain polymerase-deficient
E. coli strains or strains with limited polymerase activity
we were able to specifically enrich active polymerase variants
that complemented the missing activity. In the case of T7 RNA
polymerase, this complementation failed; however, we were able
to identify and isolate active variants after transcription (and
expression) of a marker protein (GFP). Following the genetic selection,
active variants (DNA and RNA polymerases) were then screened for
specific substrate tolerances using microplate assays (FRET- and
fluorescence detection).
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.
Our research projects are supported by the "Sächsische
Aufbaubank", the "HWP", "EFRE" and the
"DFG". We work in close collaboration with the group
of Dr. Francois Cornélis (University of Evry and University
Paris VII), the group of Jose Cardoso de Menezes (Technical University
of Lisbon, Portugal) as well as with several partners in Germany.
The scientific interests of the junior research group Molecular
Medicine of Infectious Diseases (PD Dr. Reinhard Straubinger)
focus on the molecular mechanisms that pertain to infectious diseases
caused by spirochetes (Lyme borreliosis, Relapsing fever) in man
and animals. In this context, the role of the cytokines Interleukin
(IL)-23 and IL-17 in chronic Lyme arthritis was also investigated
and modifications of the vaccination strategies against Lyme borreliosis,
which may be relevant for clinical use, were evaluated.
During the last months it became evident that two linear plasmids,
which are known to be essential for the infectiousness of Borrelia
burgdorferi, have no impact on the phagocytosis rate displayed
by polymorphonuclear neutrophils (PMNs) and monocytes. This means,
that the host's innate immune system is not affected by proteins
coded by these plasmids in terms of spirochete internalization.
Currently, phagocytosis rates of PMNs and monocytes facing spiral-
or spherical-shaped B. burgdorferi organisms are compared.
This is of special interest, since spiral-shaped organisms are
thought to be important for an active infection with B. burgdorferi,
while spherical-shaped spirochetes show reduced metabolic activity
and therefore might be the cause for persistent infection. Furthermore,
for the first time we succeeded to cultivate an additional borrelia
species, Borrelia persica, in vitro. Due to the
conspicuous biological differences seen among B. burgdorferi
und B. persica - e.g. B. burgdorferi is found in
the host's tissue and causes slowly developing clinical signs,
while B. persica is predominately found in the blood stream
and is the cause of relapsing fever attacks - the direct comparison
of both species offers a solid basis to investigate the molecular
mechanisms of persistent infection in more detail. Chronic inflammatory
responses in joints may result from persistent infections years
later. The underlying pathogenic mechanisms are unknown so far.
In this context the group investigates the inflammation supporting
or even initiating role of IL-23 and IL-17 in the murine animal
model. Finally, the impact of a modified immunization regimen
on the production of vaccine-induced antibodies in the dog was
evaluated quantitatively and qualitatively for five vaccines that
are available against Lyme borreliosis in Europe and the USA.
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 areas 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 libraries
for the in vitro evolution of this enzyme were generated. Using
rational design a new exonuclease variant could be developed,
showing an increase in the optimum incubation temperature by 8
°C and allowing a more efficient production and storage of
the enzyme. In Cooperation with the group of Prof. Sträter
(BBZ) protein crystals could be grown for two thermostable proteins
homologous to exonuclease.
On the other hand a procedure for the screening of large libraries
of protein variants has been enhanced and an adaptation of the
process to new enzyme classes was started. Therefore the recombinant
expression and activity assays for various enzymes (restriction
endonucleases, aldoketo-reductases, dehydrogenases and dioxygenases)
were established. Various libraries were generated for example
enzymes of these classes. The screening of these libraries was
started.
The junior research group Protein-Ligand Interaction by
Ion Cyclotron Resonance Mass Spectrometry (Dr. Andrea Sinz)
employed the Fourier transform ion cyclotron resonance (FTICR)
mass spectrometry to analyze complex protein mixtures. We succeeded
in identifying hundreds of proteins from E. coli cell lysates
by combining several chromatographic separation steps with high-resolution
FTICR mass spectrometry. The strategy based on liquid chromatography
possesses the potential to replace two-dimensional gel electrophoresis,
which is currently the predominately employed separation technique
in proteomics studies.
We continued our research efforts in improving the strategy
to elucidate low-resolution three-dimensional structures of protein
complexes by chemical cross-linking and high-resolution mass spectrometry.
Highly complex mixtures, as they are created by chemical cross-linking
of proteins, are analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry
without further pre-separation steps. In order to facilitate identification
of cross-linking products, we employed isotope-labeled cross-linking
reagents. Based on the distance constraints between the complex
constituting components, information on interfaces between the
binding partners is obtained. As model systems, we employed two
protein complexes between calmodulin and its target peptides melittin
and a C-terminal peptide of the skeletal muscle myosin light chain
kinase. For the complex between calmodulin and the peptide from
the skeletal muscle myosin light chain kinase, the distance constraints
were in agreement with the published NMR structure. For the complex
between calmodulin and melittin, two structural models were created
based on the distance constraints obtained by chemical cross-linking.
Further applications of the strategy to map protein interfaces
by chemical cross-linking and high-resolution FTICR mass spectrometry
currently consist in the identification of interacting regions
between calmodulin and a C-terminal peptide derived from adenylyl
cyclase 8 (cooperation with Prof. Dermot Cooper, Cambridge University,
UK) as well as in the analysis of laminin self-aggregation (cooperation
with Dr. Neil Smyth, University of Cologne).
The junior research group Solid-state NMR Studies of the
Structure of Membrane-associated Proteins (PD Dr. Daniel Huster)
has been dealing with the characterization of the structure and
dynamics of membrane bound molecules such as the ras protein and
the carrier peptide hCT(9-32). One focus of the investigation
was the comprehensive application of 2H NMR methods to determine
motional amplitudes and correlation times of the motion of the
lipid chain modifications of the C-terminus of ras. For hCT(9-32),
a structural model of the membrane associated state was built
on the basis of the structural parameters determined by solid-state
NMR methods.
A second topic of the research was the investigation of natural
and tissue engineered cartilage. By means of solid-state NMR spectroscopy,
the molecular components of the tissue could be individually detected
and their dynamical parameters have been determined. This rational
has first been applied to characterize artificial tissue. The
goal of this research is the quantitative characterization of
the molecular properties of tissue engineered cartilage. Thus,
a quality control / quality assurance analysis will be established
that allows for the optimization of individual production of autologus
cartilage tissue.
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