Avian influenza viruses can cause devastating outbreaks in domesticated poultry, with rapid transmission of virus between birds and high mortality. Current measures for control of influenza involve surveillance, closure of live poultry markets and mass culling. However, despite implementing these measures, outbreaks continue. Considering the uncertainty over whether certain strains of avian influenza viruses will adapt to human transmission and limitations over how their spread may be controlled, this study considers the use of anti-viral peptides as protective agents against low pathogenicity avian influenza (LPAI) virus. This study investigated the activity and mode of action of two cell-penetrating anti-viral peptide families against influenza A virus infection: ‘FluPep’ (a family of short, hydrophobic peptides related to suppressor of cytokine signaling- 1 proteins) and ‘Entry Blocker’ (derived from the signal sequence of fibroblast growth factor-4). Plaque reduction assays demonstrated dose-dependent anti-viral activity of both peptide families against a panel of influenza viruses with diverse haemagglutinin subtypes. Determination of IC50 values showed strain-specific differences in sensitivity to FluPep but not Entry Blocker. The IC50 of FluPep 4 for A/PR/8/34 was reduced by reassorting in the HA and NA from a relatively sensitive avian strain using a reverse genetics approach, suggesting inhibitory effects on the viral glycoproteins. Accordingly, viral entry assays focusing on binding, internalisation, fusion and import were designed and optimised to dissect the mechanism(s) of action of the peptides. Results indicated that the peptides acted upstream of nuclear import of viral ribonucleoprotein complexes but did not reduce overall virus binding to cells. However, the peptides caused aggregation of the virus particles on the surface of the host cells and reduced their internalisation. Further work evaluated how the peptides may be delivered to the site(s) of viral replication in poultry. A screen of the current literature was completed to allow for the design of an expression cassette for poultry-derived Lactobacillus to express FluPep and Entry Blocker. Though the cassette has been reported to be suitable for expression of heterologous proteins in Lactobacilli, rescue of recombinants for expression of anti-viral peptides or a reporter protein proved challenging, possibly owing to toxicity. A stable construct for the expression of FluPep 4 in Lactobacillus was obtained but culture supernatant did not inhibit virus replication.

No description available.

Plasma natriuretic peptide concentrations rise in response to either atrial or ventricular wall stretch and have been found to be useful in the diagnosis and assessment of patients with congestive cardiac failure. Although previous studies have suggested that plasma natriuretic peptides may offer some prognostic information in patients with valvular heart disease, it is unclear whether concentrations reflect disease severity and how plasma concentrations vary across different valve lesions. The aim of this research was to identify the factors that affect natriuretic peptide releases in valvular heart disease (VHD) and to investigate whether natriuretic peptides can be used in clinical practice to identify those patients who may benefit from early intervention. Plasma brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) concentrations were measured in patients with normal left ventricular (LV) systolic function and isolated VHD (mitral regurgitation, MR, n=33; aortic regurgitation, AR, n=39; aortic stenosis, AS, n=34; mitral stenosis, MS, n=30), and age and sex matched controls (n=39) immediately prior to exercise stress echocardiography. Peptide levels were compared against age and sex matched controls and against markers of severity for each valve lesions and across different valve lesions. Compared to controls, patients with all types of VHD had elevated plasma BNP concentrations [(MR median 35(inter quartile range 23-52), AR 34(22-45), AS 31(22-60), MS 58(34-90); controls 24(16-33) pg/mL; p < 0.01 for all]. LV end diastolic volume index varied by valve lesion; [MR (mean ± standard deviation 77±14), AR (91±28), AS (50±17), MS (43±11), controls (52±13) mL/m2; p < 0.0001]. There were no associations between LV volume and BNP. Left atrial (LA) area index varied [MR (18±4cm2/m2), AR (12±2), AS (11±3), MS (19±6), controls (11±2); p < 0.0001], but correlated with plasma BNP concentrations: MR (r=0.42,p=0.02), MS (r=0.86,p < 0.0001), AR (r=0.53,p=0.001), AS (r=0.52, p=0.002). Higher plasma BNP concentrations were associated with increased pulmonary artery pressure and reduced exercise capacity. Despite adverse cardiac remodelling, 81(60%) patients had a BNP concentration within the normal range. In patients with MS BNP was strongly associated with left atrial area index (r=0.86; p < 0.0001) and a BNP level of greater than 2 times the upper limit of normal identified patients who fulfilled guideline criteria for intervention (Area under the curve (AUC) 0.87 [0.74,0.99], p =0.006) and lower exercise capacity (AUC 0.82 [0.67,0.97]; p=0.004). In AR patients significant remodelling could occur whilst BNP remained within the normal range and in general BNP appeared less useful in assessing disease severity. However raised levels of BNP was associated with more severe AR as assessed by left ventricular outflow tract:AR Jet area ratio (r=0.43 p=0.0007). AR patients with an abnormal BNP had signs of early LV dysfunction on exercise with a lower LV longitudinal strain rate post exercise compared to AR patients with a normal BNP (0.68±0.31 vs. 1.06±0.45 1/sec; p=0.02). In MR patients, higher plasma BNP concentrations were associated with larger left atrial area index (r=0.42, p=0.02), higher pulmonary artery pressure (r=0.53, p=0.002) and a lower exercise time (r=-0.60, p=0.0002). BNP was not associated with any marker of left ventricular size or function in MR. These findings suggest that despite significant LV remodelling, plasma BNP concentrations are often normal in patients with VHD. Consequently, plasma BNP concentrations should be interpreted with caution when assessing patients with VHD. However natriuretic peptide levels offer complementary information to the standard assessment of patients with VHD and an unexplained finding of an elevated BNP in an otherwise asymptomatic patient should prompt further investigation.

No description available.

<p>The combination of collision-activated dissociation, (CAD) and electron capture dissociation, (ECD) yielded a 125% increase in protein identification. The S-score was developed for measuring the information content in MS/MS spectra. This measure made it possible to single out good quality spectra that were not identified by a search engine. Poor quality MS/MS data was filtered out, streamlining the identification process.</p><p>A proteomics grade de novo sequencing approach was developed enabling to almost completely sequence 19% of all MS/MS data with 95% reliability in a typical proteomics experiment.</p><p>A new tool, Modificomb, for identifying all types of modifications in a fast, reliable way was developed. New types of modifications have been discovered and the extent of modifications in gel based proteomics turned out to be greater than expected.</p><p>PhosTShunter was developed for sensitive identification of all phosphorylated peptides in an MS/MS dataset.</p><p>Application of these programs to human milk samples led to identification of a previously unreported and potentially biologically important phosphorylation site.</p><p>Peptide fragmentation has been studied. It was shown emphatically on a dataset of 15.000 MS/MS spectra that CAD and ECD have different cleavage preferences with respect to the amino acid context.</p><p>Hydrogen rearrangement involving z• species has been investigated. Clear trends have been unveiled. This information elucidated the mechanism of hydrogen transfer.</p><p>Partial side-chain losses in ECD have been studied. The potential of these ions for reliably distinguishing Leu/Iso residues was shown. Partial sidechain losses occurring far away from the cleavage site have been detected. </p><p>A strong correlation was found between the propensities of amino acids towards peptide bond cleavage employing CAD and the propensity of amino acids to accept in solution backbone-backbone H-bonds and form stable motifs. This indicated that the same parameter governs formation of secondary structures in solution and directs fragmentation in peptide ions by CAD.</p>

Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models.

Antimicrobial peptides (AMPs) are relatively-short chain molecules that living organisms use to defend themselves against a wide range of invading microorganisms such as bacteria and viruses. They selectively bind to and kill microbes over host cells by permeabilizing cell membranes or by inhibiting the biological functions of intra-cellular components. Despite its significance in determining their cell selectivity, however, the cell-concentration dependence of AMP's membrane-perturbing activity has not been criticality examined. In this thesis, we present a physical model for cell selectivity of AMPs, especially its cell-concentration dependence. To this end, we use a coarse-grained model that captures essential molecular details such as lipid composition (e.g., fraction of anionic lipids) and peptide amphiphilicity and charge. In particular, we calculate the surface coverage of peptides in the membrane-perturbing mode as a function of peptide and cell densities: those that bind to the interface between lipid headgroups and tails. This allows us to extract the minimum inhibitory concentration (MIC) and the minimum hemolytic concentration (MHC) of the peptides. Our results show that both MIC and MHC increase as the cell density increases so that the peptide selectivity (given by MHC/MIC) decreases with increasing cell density. Our results will help resolve conflicting interpretations of peptide-selectivity experiments.

Ferrocene-peptide conjugates represent a hybrid area between organometallic chemistry and biochemistry. In these bioconjugates, the ferrocene (Fc) moiety can serve as molecular scaffold, chromophore, sensitive probe, biological marker, redox active site, etc. Disubstituted Fc systems, in which both cyclopentadienyl rings are substituted, provide influence over the supramolecular structure of the assemblies, and serve as starting materials for the design of electronic biomaterials. Recently, 1'-amino-ferrocene-1-carboxylic acid (Fca) and 1,1'-diaminoferrocene Fc[NH2]2 were recognized as useful tools in bioorganometallic chemistry. This work sketches some novel preparative and structural aspects of Fc-peptide conjugates and explores their applications as biosensors and as polymeric materials. <p>First, I demonstrated that Fca invariably induces a turn-like structure, which is stable in solution and the solid state. The obtained results showed different behaviour for Fca peptides depending on the chirality and position of the attached amino acid. The axial chirality of the Fca is completely dependent on the chirality of the first amino acid attached to the amino terminal of the Fca group.<p>Second, I was able to develop a surface based sensor for the electrochemical detection of papain based on Fc-peptide conjugates. The idea was to place a surface-bound redox probe in close proximity to the electrode surface. In addition, the redox-active Fca label will be part of the recognition site but will not interfere with the recognition process. My sensor provides an attractive alternative for the electrochemical detection of non-labelled non-redox active proteins, which under current detection schemes remains a significant challenge.<p>This work represents a truly important proof of concept for establishing this novel bioorganometallic approach for the electrochemical detection of important biological targets.<p>Last, I was successful in developing a very convenient method to synthesize 1,1'-bis(tert-butoxycarbonylamino)ferrocene as a stable derivative of Fc[NH2]2. This new synthetic approach has circumvented the problems encountered with the explosive diazide usually used as a precursor in the conventional synthons of Fc[NH2]2. Building on this achievement, a series of novel peptide-like oligomeric and polymeric ferrocenyl-amides were synthesized and fully characterized. The electrochemical investigations on these polymers suggested unresolved or no electronic interaction between the ferrocene groups in all systems. These results may reveal the influence of the amide groups on suppressing the electronic interaction between the iron centers in my polymers. <p>Thus, my systematic work on Fc-peptide conjugates lays solid foundations in the fields of structural control, biosensors, and material science.

The combination of collision-activated dissociation, (CAD) and electron capture dissociation, (ECD) yielded a 125% increase in protein identification. The S-score was developed for measuring the information content in MS/MS spectra. This measure made it possible to single out good quality spectra that were not identified by a search engine. Poor quality MS/MS data was filtered out, streamlining the identification process. A proteomics grade de novo sequencing approach was developed enabling to almost completely sequence 19% of all MS/MS data with 95% reliability in a typical proteomics experiment. A new tool, Modificomb, for identifying all types of modifications in a fast, reliable way was developed. New types of modifications have been discovered and the extent of modifications in gel based proteomics turned out to be greater than expected. PhosTShunter was developed for sensitive identification of all phosphorylated peptides in an MS/MS dataset. Application of these programs to human milk samples led to identification of a previously unreported and potentially biologically important phosphorylation site. Peptide fragmentation has been studied. It was shown emphatically on a dataset of 15.000 MS/MS spectra that CAD and ECD have different cleavage preferences with respect to the amino acid context. Hydrogen rearrangement involving z• species has been investigated. Clear trends have been unveiled. This information elucidated the mechanism of hydrogen transfer. Partial side-chain losses in ECD have been studied. The potential of these ions for reliably distinguishing Leu/Iso residues was shown. Partial sidechain losses occurring far away from the cleavage site have been detected. A strong correlation was found between the propensities of amino acids towards peptide bond cleavage employing CAD and the propensity of amino acids to accept in solution backbone-backbone H-bonds and form stable motifs. This indicated that the same parameter governs formation of secondary structures in solution and directs fragmentation in peptide ions by CAD.

Ferrocene-peptide conjugates represent a hybrid area between organometallic chemistry and biochemistry. In these bioconjugates, the ferrocene (Fc) moiety can serve as molecular scaffold, chromophore, sensitive probe, biological marker, redox active site, etc. Disubstituted Fc systems, in which both cyclopentadienyl rings are substituted, provide influence over the supramolecular structure of the assemblies, and serve as starting materials for the design of electronic biomaterials. Recently, 1'-amino-ferrocene-1-carboxylic acid (Fca) and 1,1'-diaminoferrocene Fc[NH2]2 were recognized as useful tools in bioorganometallic chemistry. This work sketches some novel preparative and structural aspects of Fc-peptide conjugates and explores their applications as biosensors and as polymeric materials. <p>First, I demonstrated that Fca invariably induces a turn-like structure, which is stable in solution and the solid state. The obtained results showed different behaviour for Fca peptides depending on the chirality and position of the attached amino acid. The axial chirality of the Fca is completely dependent on the chirality of the first amino acid attached to the amino terminal of the Fca group.<p>Second, I was able to develop a surface based sensor for the electrochemical detection of papain based on Fc-peptide conjugates. The idea was to place a surface-bound redox probe in close proximity to the electrode surface. In addition, the redox-active Fca label will be part of the recognition site but will not interfere with the recognition process. My sensor provides an attractive alternative for the electrochemical detection of non-labelled non-redox active proteins, which under current detection schemes remains a significant challenge.<p>This work represents a truly important proof of concept for establishing this novel bioorganometallic approach for the electrochemical detection of important biological targets.<p>Last, I was successful in developing a very convenient method to synthesize 1,1'-bis(tert-butoxycarbonylamino)ferrocene as a stable derivative of Fc[NH2]2. This new synthetic approach has circumvented the problems encountered with the explosive diazide usually used as a precursor in the conventional synthons of Fc[NH2]2. Building on this achievement, a series of novel peptide-like oligomeric and polymeric ferrocenyl-amides were synthesized and fully characterized. The electrochemical investigations on these polymers suggested unresolved or no electronic interaction between the ferrocene groups in all systems. These results may reveal the influence of the amide groups on suppressing the electronic interaction between the iron centers in my polymers. <p>Thus, my systematic work on Fc-peptide conjugates lays solid foundations in the fields of structural control, biosensors, and material science.