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MIC values are expressed as the lowest concentration that caused visible growth inhibition.
Interestingly, the hBD3 behaviour was neither similar to the rapid membranolytic action of
polymyxin B nor to the activity of cell wall inhibitors (ampicillin, penicillin G; Figure 23) which
cause killing over the course of one bacterial generation. J Gen Microbiol 96, 277-81. Jenssen, H.,
Hamill, P., and Hancock, R. E. (2006): Peptide antimicrobial agents. It was demonstrated that the
peptide permeabilise the outer membrane by binding to LPS and that the composition of the LPS
core region plays an important role in this interaction. Interestingly, similarities in the mode of action
between some eukaryotic defensins and bacterially-produced lantibiotics could be revealed. Future
Microbiol 5, 1553-79. Brotz, H. (1997): Wirkungsmechanismus des Lantibiotikums Mersacidin:
Neuartige Interaktion mit einer bakteriellen Zellwandvorstufe. Functionally, antibacterial (primarily
active against Gram-positive bacteria) and antifungal peptides can be distinguished which differ
from vertebrate defensins by their disulphide bridging patterns (C1-C4, C2-C5, C3-C6 for peptides
containing six cysteines). Moreover, it is likely that hBD3 interferes with various membrane-bound
processes in E. Potassium efflux experiments and membrane potential measurements demonstrated
that the membrane impairment by RTDs strongly depends on the energisation of the membrane. They
are activated by cells involved in innate immunity and realize highly antigen-specific immunity. PCC
correlates fluorescence intensities; 1 means perfect relation, while 0 means no relation of the
fluorescence intensities. Initially, the defensin has to cross the outer membrane barrier of Gram-
negative bacteria to gain access to the periplasmic space. MHC II molecules are thought to be
continuously recycled from the plasma membrane to early endosomes and back to the membrane27.
Here, the 15 most abundant patterns are shown (refer to Table S 9-4). Haftungsausschluss In
Englisch ???????? (for international students). Subsequently, cell supernatants were stained with two
different anti-TNF-. Thus, the deficiency of ?-defensins in ileal CD may cause an imbalance of the
microbial composition which in turn triggers infection and inflammation (Salzman et al., 2010). The
lack of intestinal ?-defensins has been linked to a diminished expression of the transcription factor
Tcf-4 of the Wnt Page 23. Figure 25 Transmission electron microscopy of E.coli BW25113 treated
with 10x MIC hBD3. The AM and GL are each fused to repeat units that are responsible for
targeting the enzymes to the cross wall (Baba and Schneewind, 1998; Komatsuzawa et al., 1997). It
has been reported that in S. Thus, the antimicrobial activity may depend on the overall conformation
and charge distribution rather than on their primary sequence. The WaaP and WaaY kinases are
involved in the phosphorylation of the first and second heptose residue in the inner core (Yethon et
al., 1998). Thus, a deletion of one of the genes reduces the net negative charge of the cell envelope
which correlates with a decreased susceptibility towards cationic peptides, as observed here. In
comparison to other carrier molecules like antibodies or viruses, aptamers are described to be low or
non-immunogenic78,119. Genomics 81, 175-83. Rosa, R. D., Santini, A., Fievet, J., Bulet, P.,
Destoumieux-Garzon, D., and Bachere, E. (2011): Big defensins, a diverse family of antimicrobial
peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas.
Clin Exp Immunol 137, 379-85. Falord, M., Karimova, G., Hiron, A., and Msadek, T. (2012):
GraXSR proteins interact with the VraFG ABC transporter to form a five-component system
required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus. The lipid
II concentration was determined by the phosphate content (section 2.5.7). 2.5.7 Phosphate
determination Phosphate determination was performed according to the method of Rouser (1970).
The aim of this thesis was to investigate the potential applicability of aptamers as a novel class of
DC-targeting carriers for immunotherapeutic applications. Under non-inflammatory steady-state
conditions DCs reside as immature cells in different tissues, i.e. they lack co- stimulatory molecules.
Target molecules which are difficult to isolate from the cell surface can be addressed by this
selection strategy100. The coating of the lipid layers gave a response in the range of 6,000 to 6,500
RU. RTDs were added in concentrations corresponding to 10x MIC.
Biochem J 374, 707-14. Brogden, K. A. (2005): Antimicrobial peptides: pore formers or metabolic
inhibitors in bacteria. In contrast, antifungal invertebrate and plant defensins interact with specific
sphingolipids, thereby leading to fungal cell death (compare section 1.3) (Aerts et al., 2009;
Thevissen et al., 2004). Thus, evidence is increasing that invertebrate defensins, and more generally
CS??-type defensins, are rather specific inhibitors of microbial biosynthesis or signalling pathways
than mere membrane active agents. Thus, the deficiency of ?-defensins in ileal CD may cause an
imbalance of the microbial composition which in turn triggers infection and inflammation (Salzman
et al., 2010). The lack of intestinal ?-defensins has been linked to a diminished expression of the
transcription factor Tcf-4 of the Wnt Page 23. To date, only invertebrate defensins with antifungal
activity had been characterised in terms of cellular targets (Thevissen et al., 2004). The biosynthesis
of bacterial cell wall peptidoglycan is a multistep process involving reactions in the cytoplasm and
both sides of the cytoplasmic membrane (for details see Figure 27). One of these countermeasures is
based on the covalent modification of anionic cell envelope molecules, thus decreasing their net
negative charge and their attraction to HDPs. The sensorgram depicts the interaction of oyster
defensins, nisin (positive control) and tachyplesin (negative control) with immobilised DOPC
vesicles containing 0.8 mol% lipid II. All peptides were added at 250 nM. Figure 4 A: NMR-based
modelling of the plectasin-lipid II complex. First of all, the libraries of the 6th and 11th selection
cycles bound weakly to Fc-FN (Figure 4.1.2B). Second, on the single sequence level no similarities
within the DNA sequences were found (Table S 9-1). Here, to increase stability, CpG ODN 1826
has a phosphorothioate backbone. After reattachment the cells were washed twice with wash buffer.
This is consistent with the fact that some defensins completely lack direct antibiotic activities such
as the human enteric ?-defensin HD6. Protein-containing fractions were pooled and dialysed using
Slide-A-Lyzer dialysis cassettes (Pierce; Thermo Fisher Scientific, Schwerte, Germany). MHC I
molecules preferentially bind peptides being 8-9 amino acids in length and having hydrophobic or
basic residues at ?1?2 ?3 ?2-microglobulin peptide binding groove DC ?1 ?2 ?1 ?2 peptide binding
groove DC A) B) Page 19. Finally, the dye is retained within the cell through interactions of the
succinimidyl mioety with primary amines and is equally distributed among daughter cells upon
divisions151. The gel was pre-run for 30 minutes at 370 V and 15 W. J Biol Chem 273, 26310-6.
Yeung, A. T., Gellatly, S. L., and Hancock, R. E. (2011): Multifunctional cationic host defence
peptides and their clinical applications. This quantification is based on the reduction of copper (II)
ions that form a violet-coloured complex in alkaline solution. Added adaptors enable the
immobilization and processing of the sample by the Sequencing instrument. Mean and standard
deviation values were calculated with Microsoft Office Excel 2007. Page 106. To achieve that, the
respective target is either immobilized on a matrix or the non-binders are removed by centrifugation,
electrophoresis or flow cytometry85-88. B: The fungal defensin interacts with the pyrophosphate
moiety of lipid II by forming four hydrogen bonds (involving residues F2, G3, C4 and C37). Neither
activated cell wall sugars (UDP-MurNAc-pp and UDP-GlcNAc) nor the lipid carrier C55-P had
similar antagonistic effects. Surprisingly, the MFI of the labeled control sequence also rose with
increasing concentrations, albeit to a lesser extend (Figure 4.2.1A). This fact is probably caused by
the ability of BM-DCs to continuously internalize surrounding fluids by macropinocytosis30. J Biol
Chem 276, 1772-9. Wong, J. H., and Ng, T. B. (2005): Sesquin, a potent defensin-like antimicrobial
peptide from ground beans with inhibitory activities toward tumor cells and HIV-1 reverse
transcriptase. Biochemistry 43, 9800-12. Bulet, P., and Stocklin, R. (2005): Insect antimicrobial
peptides: structures, properties and gene regulation. For further analysis, selected aptamers are
obtained by solid phase synthesis. 3.5.2 Cell-binding aptamers Aptamers can be developed for a
plethora of target structures, ranging from small molecules to complex organisms91,94-100. In
addition, Pep5 formes pores in energised cells leading to rapid efflux of small molecules (Sahl, 1985;
Sahl and Brandis, 1983). Lipid II was incubated in the presence of Cg-Defh2 at molar ratios ranging
from 1:0.1 to 1:1. The stable complex remained at the application spot, whereas free lipid II migrated
up the TLC plate. RNA Biol 6, 316-320 (2009). 115 Wengerter, B. C. et al. Aptamer-targeted antigen
delivery. Tolerogenic DCs are deficient in adequate signaling for T cell activation or they only deliver
co-inhibitory signals18.
At this point, the selection against Fc-FN was not further investigated. MVBs subsequently fuse
with the peptide-containing vesicle, where the peptide is loaded on the MHC II molecule (5).
Selective delivery of antigens to DCs is only warranted if the aptamers keep their binding ability
upon conjugation. On the other hand, HDPs can dampen harmful proinflammatory immune
responses. Antimicrob Agents Chemother 55, 5325-30. Yamada, S., Sugai, M., Komatsuzawa, H.,
Nakashima, S., Oshida, T., Matsumoto, A., and Suginaka, H. (1996): An autolysin ring associated
with cell separation of Staphylococcus aureus. Heilmann, C., Hussain, M., Peters, G., and Gotz, F.
(1997): Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a
polystyrene surface. The defensin adopts a cysteine-stabled ?-helix ?-sheet structure (CS??) in
solution; the four disulphide bridges are marked in yellow (Schmitt et al., 2012b). Page 48. For
analysis of the cytoplasmic peptidoglycan precursor pool, S. DCs express a variety of endocytic
receptors and prominent examples among them are the C-type lectin receptors31,32. Biophys J 81,
1475-85. Yeaman, M. R., and Yount, N. Y. (2007): Unifying themes in host defence effector
polypeptides. The coating of the lipid layers gave a response in the range of 6,000 to 6,500 RU. For
example, the MR is described to recognize glycan residues of various microorganisms such as
Candida albicans and Mycobacterium tuberculosis35. The cells of the bone marrow were cultivated
in DC-medium or macrophage-medium for 7 days. A clathrin-coated vesicle is formed and fuse
subsequently with early endosomes for enabling cross-presentation on MHC I molecules.
Additionally, professional APCs share some common cell surface structures for antigen recognition,
e.g. Fc receptors for IgG27. Isolation from the lepidopteran Heliothis virescens of a novel insect
defensin with potent antifungal activity. These mice develop either CD8 T cells recognizing MHC I
bound OVA257-264 or CD4 T cells specific for MHC II bound OVA323- 339 recognition. Thus, the
deficiency of ?-defensins in ileal CD may cause an imbalance of the microbial composition which in
turn triggers infection and inflammation (Salzman et al., 2010). The lack of intestinal ?-defensins has
been linked to a diminished expression of the transcription factor Tcf-4 of the Wnt Page 23. Figure 3
The common ancestor hypothesis of disulphide-stabilised host defence effector molecules. Nat
Biotechnol 25, 465-72. Scott, M. G., Vreugdenhil, A. C., Buurman, W. A., Hancock, R. E., and
Gold, M. R. (2000): Cutting edge: cationic antimicrobial peptides block the binding of
lipopolysaccharide (LPS) to LPS binding protein. In particular, we were interested in answering the
following questions. Ouellette, A. J., and Selsted, M. E. (2012): Immunology. HD6 defensin
nanonets. In cell-SELEX, the specific target structure is Page 32. Indeed, addition of LTA inhibited
the antimicrobial activity of RTDs when used at 1:4 molar ratio, but did not antagonise the antibiotic
action of Pep5 (Table 12). The AM and GL are each fused to repeat units that are responsible for
targeting the enzymes to the cross wall (Baba and Schneewind, 1998; Komatsuzawa et al., 1997). It
has been reported that in S. Co-localization, as indicated by shades of yellow, was observed in some
punctate structures. Afterwards, BM-DCs were co-cultured for 72 h with CFSE-labeled OVA-
dependent CD4 T cells and the CFSE profile was measured by flow cytometry. The following
pipetting scheme was used for one PCR reaction (Table 7-11; PCR program see Table 7-3). Page 95.
DCs link the unspecific innate immunity to the antigen-specific adaptive immunity by priming T
cells. RP-HPLC and mass spectrometry analysis of bacterial cell extracts showed that the three oyster
defensins caused the intracellular accumulation of UDP-MurNAc-pp in a similar manner to
vancomycin, which is known to form a complex with the cell wall precursor lipid II (Figure 8).
Blood 109, 2928-35. Gadsby, D. C., Vergani, P., and Csanady, L. (2006): The ABC protein turned
chloride channel whose failure causes cystic fibrosis. Cell Microbiol 7, 1387-97. Froy, O., and
Gurevitz, M. (2003): Arthropod and mollusk defensins--evolution by exon-shuffling. Tolerogenic
DCs are deficient in adequate signaling for T cell activation or they only deliver co-inhibitory
signals18. The samples were centrifuged for 5 minutes at 200 x g and the supernatant discarded.
Autologous DCs are loaded ex vivo with antigens and reinfused into the patient. Target molecules
which are difficult to isolate from the cell surface can be addressed by this selection strategy100. The
antagonisation of the oyster defensins antibacterial activity by lipid II occurred at a 1:1 molar ratio
(Table 9), as also observed for plectasin (Schneider et al., 2010) and for hBD3. All oyster defensins
were active at low micromolar concentrations against the Gram-positive bacteria tested (Table 8), but
did not show significant antimicrobial activity against Gram-negative bacteria, including the oyster
pathogens V. The peptides are subsequently shuttled into the endoplasmic reticulum (ER) and finally
trimmed by endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP).
The most potent peptides kill at low micromolar concentrations and some also exert activity against
multidrug resistant microbes. To further confirm that the peptides interact with LTA, an
antagonisation assay was performed. Pep5 also forms transient pores in energised membranes
leading to rapid efflux of low molecular weight substances from the cytoplasm (Poppinga, 2007;
Sahl, 1985; Sahl and Brandis, 1983). Several factors influence the outcome of SELEX, including
structural characteristics of targets, size and complexity of the starting library, choice of partitioning
and elution methods and concentrations of targets and competitors82,156. In brief, immortalized
murine embryonic stem cell-derived macrophages were treated with increasing concentrations of
oligonucleotides for 24 hours. The DNA libraries derived from the 6th selection cycle targeting Fc-
CTL (Figure 4.1.3A) or Fc-FN (Figure 4.1.3B) bound to both Fc-CTL and Fc-FN proteins. Biochem
J 374, 707-14. Brogden, K. A. (2005): Antimicrobial peptides: pore formers or metabolic inhibitors in
bacteria. We are in the process of investigating aptamer-based silencing of miR-125a in human
macrophages associated with granulomatous diseases184. The pore-forming peptide nisin (10x MIC)
was used as a control. Subsequently, BM-DCs were co-cultured for 72 h with CFSE-labeled OVA-
dependent CD4 T cells and the proliferation profile indicated by changes of CFSE signals was
measured by flow cytometry. Cg- Defm and Cg-Defh2, the more active forms, exhibit a positively
charged residue at position 16 (K and R, respectively; Figure 6) instead of an uncharged glutamine
in Cg- Defh1. MIC determinations were carried out in 96-well polypropylene microtiter plates
(NuncTM; Thermo Fisher Scientific, Schwerte, Germany) by standard broth microdilution using half-
Page 32. Finally, the amount of alive and dead target and control cells was measured in flow
cytometry by using Hoechst as viability marker and quantified as percentage of cytotoxicity. This
may occur by a mechanism known as self- promoted uptake which is based on the interaction of the
cationic peptides with the negatively charged LPS molecules of the outer membrane (compare 1.3)
(Hancock, 1997). In fact, hBD3 rapidly disrupted the outer membrane of E. Incubation of S.
simulans 22 with oyster defensins resulted in the accumulation of the last soluble cell wall precursor
UDP-MurNAc-pp in the cytoplasm, comparable to classical inhibitors of the membrane-bound steps
of cell wall biosynthesis (Figure 8). To date, only invertebrate defensins with antifungal activity had
been characterised in terms of cellular targets (Thevissen et al., 2004). The biosynthesis of bacterial
cell wall peptidoglycan is a multistep process involving reactions in the cytoplasm and both sides of
the cytoplasmic membrane (for details see Figure 27). From the second selection cycle, counter
selection steps were introduced, i.e. DNA was pre-incubated with Fc-FN in SELEX targeting Fc-
CTL and vice versa. B: Inner membrane permeabilisation was determined by following the cleavage
of the extracellular substrate ONPG by the constitutively expressed ?-galactosidase of E. Environ
Microbiol 12, 951-63. Durr, U. H., Sudheendra, U. S., and Ramamoorthy, A. (2006): LL-37, the only
human member of the cathelicidin family of antimicrobial peptides. Secretion of different cytokines
is characteristic for each effector subset, for example, Th2 cells produce high levels of IL-42. Thus, a
high membrane potential - as it can be observed in presence of glucose - seems to be essential for the
membrane-disrupting activity of RTDs.
In general, CpG ODNs are composed of unmethylated CpG motif (cytosine - phosphodiester or
phosphorothioate - guanosine) flanked by 5’ purines and 3’ pyrimidines149. Effective targeting of
antigens to DCs results in activation of T cell-mediated immunity. These cells, including dendritic
cells, macrophages and B cells, are termed professional antigen-presenting cells (APCs). In
accordance with the classical pathway, the antigen is degraded (1d) and the peptide-containing
vesicle fuse with MVBs. 3.3.2 Internalization mechanisms DCs feature various mechanisms to
internalize pathogens; they practice phagocytosis, macropinocytosis and receptor-mediated clathrin-
dependent endocytosis13,27. Eight sequences were grouped into two motif-sharing sequence
families (Figure 4.1.8). The remaining sequences were unique (Table S9-3). Figure 4.1.8: DNA
sequences share sequence similarities According to their composition, some DNA sequences obtained
from cell-SELEX were grouped into sequence family 1 and 2. Binding was assessed by adding anti-
hIgG1 antibody Page 99. Staphylococcus carnosus TM300 harbouring the pAH-PepI vector was
grown in presence of 1% xylose to induce pepI expression. In this work, the mode of action of
defensins from diverse origins was investigated to gain further insights into the molecular
mechanisms of defensin-mediated killing. Consequently, T cells undergo clonal expansion and
differentiation into highly antigen-specific CD4 or CD8 effector T cells. Finally, the peptide-MHC I
complex is transported to the cell membrane (Figure 3.3.3). In addition to the classical MHC I
pathway, DCs are able to load exogenous antigens on MHC I molecules by a mechanism termed
cross-presentation16. The incubation times were chosen in accordance to the incubation times used in
the SELEX approaches. Finally, the radioactivity of the fractions was measured by liquid
scintillation and the percentage of bound DNA calculated by using the depicted formula. After 60
min exposure to RTD-2, cells showed degradation of the cell wall, particularly in the septum area
between two daughter cells (Figure 18 D, E). The coating of the lipid layers gave a response in the
range of 6,000 to 6,500 RU. Opening times Mon. - Sun.: 8 am to midnight Borrowing and returning
items Mon. - Sun.: 10 am to midnight. New lipid layers were prepared for every injection to avoid
the need for a regeneration step. Recombinant production, folding, solution structure, antimicrobial
activities, and gene expression. The bifunctional protein MprF (multiple peptide resistance factor) of
S. Finally, target and control cells will be isolated from the spleen and analyzed by flow cytometry.
This quantification is based on the reduction of copper (II) ions that form a violet-coloured complex
in alkaline solution. CDX-1307 consists of a human anti-MR monoclonal antibody fused to the
human chorionic gonadotropin beta-chain, a tumor antigen frequently expressed by epithelial
tumors59. The T7 RNA-polymerase mutant Y639F was used to enable the introduction of 2’F-
pyrimidines. ATCC 25992: full-length LPS, BW25113: lacks the O antigen, ?waaG: lacks the outer
core region, ?waaY: lacks one phosphate group in the inner core region, ?waaP: deficient in core
phosphate. In the present work, no CD4 T cell cytotoxicity was detectable in in vitro cytotoxicity
assays (Figure 4.3.5). This was not unexpected; in fact, there is no general Page 76. Immunogenetics
59, 949-54. Zhu, S. (2008): Discovery of six families of fungal defensin-like peptides provides
insights into origin and evolution of the CSalphabeta defensins. The cells migrate to peripheral
lymphoid tissues to enable the recognition of the peptide-MHC complex by rare T cell clones
expressing the TCR specific for that particular peptide (signal 1). As expected, the membrane
potential increased by Page 60. LPS molecules typically consist of three distinct regions: a
hydrophobic membrane anchor (lipid A), a short sugar chain substituted with phosphate groups (core)
and a polymer composed of oligosaccharide repeats (O antigen). To further investigate aptamer
targeted CD8 T cell activation in vivo, a cytotoxicity assay in mice will be performed. Moreover,
Pep5 and ?-defensins do not only disrupt the membranes barrier function, but they also release the
major autolysin Atl in staphylococci probably by replacing the enzymes from LTA.
Since then, much work has been carried out to identify the mechanisms of the immune system and
how these are applicable for therapeutic treatments. 3.1 The immune system The mammalian immune
system is a complex network of organs, cells and proteins. In general, CpG ODNs are composed of
unmethylated CpG motif (cytosine - phosphodiester or phosphorothioate - guanosine) flanked by 5’
purines and 3’ pyrimidines149. For example, it is proven to be difficult to sufficiently recapitulate
DC maturation ex vivo52 and ex vivo induced tolerogenicity of DCs was observed to be rapidly
inverted into an activating phenotype after reinfusion into the patient53. For the extraction solution 6
M glacial acetic acid was adjusted to pH 4.2 by addition of pyridine and mixed with two volumes of
n-butanol. However, the latter one was also antagonised by C55-P and negatively charged
phospholipids when added in up to a 5-fold molar excess (Sass et al., 2010). The 1:1 stoichiometry
was also confirmed here by TLC, as indicated by the absence of free lipid II on the plate at this
molar ratio (Figure 10). Nevertheless, aptamers could be identified for targets that enable
presentation to T cells and that are not easy to isolate from the membrane. Biophys J 75, 641-5.
Yang, L., Harroun, T. A., Weiss, T. M., Ding, L., and Huang, H. W. (2001): Barrel-stave model or
toroidal model. Aptamers are nucleic acids ligands with a defined shape that bind with high affinity
and specificity to their particular targets. Then, the pellets were washed with 40 ml 0.1 M sodium
citrate buffer (pH 4.7), centrifuged as described above and finally resuspended in 20 ml sodium
citrate buffer (pH 4.7). The suspension was mixed with an equal volume of 1-butanol and incubated
at RT for 30 min under stirring. This result was not expected, because Fc-FN was used in the counter
selection step in Fc-CTL-SELEX and vice versa. Hypnosis dissertation guide monitor institutional
spending. Altogether, these data indicate that at lethal concentrations, oyster defensins do not
compromise the membrane integrity of S. aureus. Figure 7 Influence of oyster defensins on the
membrane potential of S. Sven Burgdorf from the LIMES Institute, University of Bonn.
Evaginations of the outer membrane could be observed. Subsequently, outer membrane disruption
might depend on the cationic net charge of the peptides. For that purpose, murine splenocytes were
isolated and stained for T and B cell surface marker CD8, CD4 and B220, respectively. In brief, 5 ml
overnight culture solution was centrifuged and the plasmids isolated from the pellet by alkaline lysis
reaction. The arrows indicate the moment of peptide addition. In comparison to other carrier
molecules like antibodies or viruses, aptamers are described to be low or non-immunogenic78,119.
Blood 80, 2363-2373 (1992). 141 Patra, A. et al. 2'-Fluoro RNA shows increased Watson-Crick H-
bonding strength and stacking relative to RNA: evidence from NMR and thermodynamic data.
Science 337, 420-1. Overhage, J., Campisano, A., Bains, M., Torfs, E. C., Rehm, B. H., and
Hancock, R. E. (2008): Human host defense peptide LL-37 prevents bacterial biofilm formation.
Figure 19 Detection of cell wall lytic enzymes in the supernatant of RTD-treated cells. A: S. aureus
SG511-Berlin was exposed to 10x MIC of RTDs or Pep5 for 30 and 60 min. Can the aptamers be
conjugated to antigenic peptides without loss of binding ability. The samples were loaded on 16 %
Tricine-SDS gel (Table 7-14) and run for 1 hour 45 minutes at 175 V, 300 mA and 25 W in Tricine
SDS Anode and Cathode buffer. The cells were washed thrice with DPBS and blocked in 10 % milk
in DPBS for 1 hour. They need to bind specifically to DCs, internalize within adequate antigen
processing compartments and be non-immunogenic. It is common knowledge that OVA possess
MHC I and MHC II binding sites OVA257-264 (MHC I peptide) and OVA323-339 (MHC II
peptide), respectively128,129. Interestingly, no further ONPG hydrolysis could be measured after 60
min treatment with 5x MIC of hBD3, although the maximum reaction rate had not been reached
(Figure 24 B). For example, ligands internalized by the MR were entrapped in slowly maturing early
endosomes for cross-presentation on MHC I molecules19,38, whereas ligands taken up by DEC-205
are transported towards late endosomes or lysosomes for presentation on MHC II molecules37,39.
The biosynthesis consists of a series of cytoplasmic reactions yielding soluble UDP-linked precursors
of peptidoglycan, followed by membrane-bound steps, which starts with the anchoring of the last
soluble precursor, UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pp), to the lipid carrier
undecaprenyl phosphate (C55-P).
Org Biomol Chem 7, 2378-88. Trabi, M., Schirra, H. J., and Craik, D. J. (2001): Three-dimensional
structure of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes. After
reattachment the cells were washed twice with wash buffer. For instance, microbial agents like
lipopolysaccharides (LPS) are recognized by TLR4, which in turn triggers downstream signaling for
DC maturation17. Because of their nucleic acid composition, they can be easily modified to increase
their chemical diversity and biological properties. After transport across the membrane, the O antigen
is polymerised and ligated to lipid A-core (Raetz and Whitfield, 2002; Wang and Quinn, 2010).
Page 86. In addition, RTDs exhibit activity against Gram-negative bacteria in a similar concentration
range as against Gram-positives (Tang et al., 1999; Tran et al., 2008; Tran et al., 2002). Page 84.
Consequently, BM-DCs express moderate levels of co- stimulatory molecules like CD80, CD86 and
CD40 which function as secondary signal for adequate T cell priming71,170 (Section 3.2.1).
However, the situation in vivo is different. Briefly, the samples were diluted in DNA loading buffer,
loaded on 2-2.5 % agarose gels and run for 1 hour at 100 V. Although cell-specific aptamers are
proven to be suitable carriers (Section 3.5.2 and Figure 3.5.4), only few researchers addressed their
ability to bind or to deliver antigens to DCs for vaccination strategies. In the present work, no CD4 T
cell cytotoxicity was detectable in in vitro cytotoxicity assays (Figure 4.3.5). This was not
unexpected; in fact, there is no general Page 76. Under non-inflammatory steady-state conditions
immature DCs reside in most tissues and continuously sample a wide array of pathogens13. It is
plausible that the SELEX conditions chosen in this thesis influenced the results obtained. This is in
particular of great interest for in vivo applications in which the aptamers encounter all types of APCs
simultaneously. Although CTLDs exhibit different ligand specificity among the receptors, they share
conserved residues responsible for the typical formation of a hydrophobic fold35,137 (Figure
5.1.1AB). Previous studies demonstrated that antigens endocytosed by the MR are entrapped within
slowly maturating early endosomes for cross-presentation19,24,26. It is common knowledge that
OVA possess MHC I and MHC II binding sites OVA257-264 (MHC I peptide) and OVA323-339
(MHC II peptide), respectively128,129. Lipid II is subsequently translocated across the cytoplasmic
membrane to the outer leaflet and incorporated into the growing peptidoglycan network by the
activity of penicillin binding proteins (PBPs; Figure 27). Reife DZ generieren alle notigen Signale,
um T-Zellen optimal zu aktivieren. Long-term effector function of T cells requires prolonged
signaling of all three activation signals9. This procedure was repeated until the flow-through
containing the vesicles was almost colourless. Therefore, future work will focus on the ability of
aptamers to cause CD4 T cell polarization towards activating helper cells or suppressing regulatory T
cells. A second property is that they represent promising delivery vehicles. Initial defense
mechanisms are mediated by the innate immunity. It was demonstrated that the peptide permeabilise
the outer membrane by binding to LPS and that the composition of the LPS core region plays an
important role in this interaction. Biochim Biophys Acta 1758, 1408-25. Ernst, C. M., and Peschel,
A. (2011): Broad-spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and
flipping of phospholipids. Mean and standard deviation values were calculated with Microsoft
Office Excel 2007. Page 106. Apart from that, no or a low amount of 32P-DNA retained on aPC,
thrombin, Erk2 or Cyt1 Sec7 was observed. Peptides were added in molar ratios ranging from 0.5 to
2 with respect to lipid II. To this end, 4 g agarose was dissolved in 100 ml TBE buffer and boiled for
several minutes in the microwave. 40 ml was poured into the gel cast and stained with
ethidiumbromide at a 1:10000 dilution. It has been shown that HNP1-3 inhibit the function of lethal
toxin from Bacillus anthracis in vitro and in vivo as well as of diphtheria toxin from
Corynebacterium diphtheriae and P. PepI synthesis was induced by addition of 1% xylose.