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An investigation into the aggregation of therapeutic peptides through adsorption to primary containers
Therapeutic peptides are highly functional drugs currently being investigated for a number of diseases, due to their naturally occurrence in the body, their targeted mechanism and efficacy and tolerability.
The four therapeutic peptides under investigation in this thesis; glucagon, liraglutide, g797 and exendin-4 are all used or on trial for mediating blood glucose levels as a safe and effective treatment for type 2 diabetes. One of the largest drawbacks of peptides is their ability to aggregate and adsorb to surfaces that they come into contact with during their lifecycle; vials and syringes, causing loss of function, drug and toxicity.
A large, underexplored area of drug related research is investigation into the interface; in part due to its complexity. A plethora of different methods were used in order to investigate the peptide solutions ability to aggregate and adsorb to surfaces. The initial step was to investigate the stability of the four therapeutic peptides in solution, providing information on the aggregation of the peptides in bulk, investigated by the Atomic Force Microscope and Zeta Potential. Images of secondary structures formed in solution by each peptide were produced, as well as providing information on the charges of the solution and the colloidal stability, showing the need for an added lipidated chain for peptide stability.
Surface induced aggregation was then investigated through studying a variety of surfaces using the Quartz Crystal Microbalance with dissipation, giving both the wet mass adsorbed, as well as indications of the rigidity of the layer formed. The two most common surfaces used in the pharmaceutical industry; borosilicate glass and polystyrene, were amongst the surfaces investigated and were shown to cause the highest adsorption of peptide, ranking as the least effective surfaces at preventing aggregation.
Another factor determining peptide adsorption to surfaces is roughness. Surfaces of different roughness were created through addition of gold nanoparticles, and using the QCM-D, adsorption was compared to that of flat gold surfaces. An increase of adsorption on flat surfaces compared to rougher surfaces was found, indicating a more intricate relationship than previously thought. E- beam lithography was then used to create different nanostructures on the gold surface.
Ultimately, the thesis aimed at tackling a large problem within the pharmaceutical industry, by providing alternative pathways to challenge the problem of aggregation through adsorption to primary containers
Mutated ATP10B increases Parkinson's disease risk by compromising lysosomal glucosylceramide export
Parkinson's disease (PD) is a progressive neurodegenerative brain disease presenting with a variety of motor and non-motor symptoms, loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the occurrence of alpha-synuclein-positive Lewy bodies in surviving neurons. Here, we performed whole exome sequencing in 52 early-onset PD patients and identified 3 carriers of compound heterozygous mutations in the ATP10B P4-type ATPase gene. Genetic screening of a Belgian PD and dementia with Lewy bodies (DLB) cohort identified 4 additional compound heterozygous mutation carriers (6/617 PD patients, 0.97%; 1/226 DLB patients, 0.44%). We established that ATP10B encodes a late endo-lysosomal lipid flippase that translocates the lipids glucosylceramide (GluCer) and phosphatidylcholine (PC) towards the cytosolic membrane leaflet. The PD associated ATP10B mutants are catalytically inactive and fail to provide cellular protection against the environmental PD risk factors rotenone and manganese. In isolated cortical neurons, loss of ATP10B leads to general lysosomal dysfunction and cell death. Impaired lysosomal functionality and integrity is well known to be implicated in PD pathology and linked to multiple causal PD genes and genetic risk factors. Our results indicate that recessive loss of function mutations in ATP10B increase risk for PD by disturbed lysosomal export of GluCer and PC. Both ATP10B and glucocerebrosidase 1, encoded by the PD risk gene GBA1, reduce lysosomal GluCer levels, emerging lysosomal GluCer accumulation as a potential PD driver
Mutated ATP10B increases Parkinson’s disease risk by compromising lysosomal glucosylceramide export
Parkinson’s disease (PD) is a progressive neurodegenerative brain disease presenting with a variety of motor and non-motor symptoms, loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the occurrence of α-synuclein-positive Lewy bodies in surviving neurons. Here, we performed whole exome sequencing in 52 early-onset PD patients and identified 3 carriers of compound heterozygous mutations in the ATP10B P4-type ATPase gene. Genetic screening of a Belgian PD and dementia with Lewy bodies (DLB) cohort identified 4 additional compound heterozygous mutation carriers (6/617 PD patients, 0.97%; 1/226 DLB patients, 0.44%). We established that ATP10B encodes a late endo-lysosomal lipid flippase that translocates the lipids glucosylceramide (GluCer) and phosphatidylcholine (PC) towards the cytosolic membrane leaflet. The PD associated ATP10B mutants are catalytically inactive and fail to provide cellular protection against the environmental PD risk factors rotenone and manganese. In isolated cortical neurons, loss of ATP10B leads to general lysosomal dysfunction and cell death. Impaired lysosomal functionality and integrity is well known to be implicated in PD pathology and linked to multiple causal PD genes and genetic risk factors. Our results indicate that recessive loss of function mutations in ATP10B increase risk for PD by disturbed lysosomal export of GluCer and PC. Both ATP10B and glucocerebrosidase 1, encoded by the PD risk gene GBA1, reduce lysosomal GluCer levels, emerging lysosomal GluCer accumulation as a potential PD driver.status: accepte
Mutated ATP10B increases Parkinson's disease risk by compromising lysosomal glucosylceramide export
Parkinson's disease (PD) is a progressive neurodegenerative brain disease presenting with a variety of motor and non-motor symptoms, loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the occurrence of alpha-synuclein-positive Lewy bodies in surviving neurons. Here, we performed whole exome sequencing in 52 early-onset PD patients and identified 3 carriers of compound heterozygous mutations in the ATP10B P4-type ATPase gene. Genetic screening of a Belgian PD and dementia with Lewy bodies (DLB) cohort identified 4 additional compound heterozygous mutation carriers (6/617 PD patients, 0.97%; 1/226 DLB patients, 0.44%). We established that ATP10B encodes a late endo-lysosomal lipid flippase that translocates the lipids glucosylceramide (GluCer) and phosphatidylcholine (PC) towards the cytosolic membrane leaflet. The PD associated ATP10B mutants are catalytically inactive and fail to provide cellular protection against the environmental PD risk factors rotenone and manganese. In isolated cortical neurons, loss of ATP10B leads to general lysosomal dysfunction and cell death. Impaired lysosomal functionality and integrity is well known to be implicated in PD pathology and linked to multiple causal PD genes and genetic risk factors. Our results indicate that recessive loss of function mutations in ATP10B increase risk for PD by disturbed lysosomal export of GluCer and PC. Both ATP10B and glucocerebrosidase 1, encoded by the PD risk gene GBA1, reduce lysosomal GluCer levels, emerging lysosomal GluCer accumulation as a potential PD driver