No description available.

<p> A new technique for the systematic identification of metal-protein complexes combining gel chromatography with either neutron activation analysis or radioactive tracer methods has been proposed. The technique has been tested on the copper in serum situation to evaluate the results obtained on a well-known system.</p> <p> It was then applied to manganese in serum, manganese in erythrocytes and copper in erythrocytes. The results indicate that serum contains two manganese-binding proteins, one of low molecular weight and relatively labile in nature, the other of higher molecular weight and incorporating radioactive manganese in vivo at some definite time interval subsequent to the isotope's administration.</p> <p> Manganese in erythrocytes occurs as a porphyrin bound to apoglobin as a manganese analogue of hemoglobin.</p> <p> Copper in erythrocytes appears to exist in two forms - one firmly bound to erythrocuprein, the other more loosely bound to the same protein.</p> / Thesis / Doctor of Philosophy (PhD)

<p>Robust and efficient cleaning procedures for protein A chromatography resins used for production of monoclonal antibody based biopharmaceuticals are crucial for safe and cost efficient processes. In this master thesis the effect of different cleaning regimes with respect to ligand stability of two protein A derived media, MabSelectTM and MabSelect SuReTM, has been investigated. A 96-well format has been used for preliminary screening of different cleaning agents, contact times and temperatures. NaCl as a ligand stabilizer during cleaning-in-place (CIP) was also included as a parameter. For optimal throughput and efficiency of screening, Rectangular Experimental Design for Multi-Unit Platforms; RED-MUP, and TECAN robotic platform have been utilized. For verification of screening, selected conditions were run in column format using the parallel chromatography system ÄKTAxpressTM. In the efficiency study, where a manual preparation of CIP solutions was compared with an automated mode performed in TECAN, the total process time ended up at eight hours versus three days respectively. However, the time measured included the learning process for the TECAN platform and for further preparations the automated mode is the superior choice. The study confirmed the higher alkaline stability of MabSelect SuRe compared to MabSelect. After exposure to 0.55 M NaOH during 24h MabSelect SuRe still retained 90% of the initial capacity. In contrast MabSelect had 60% of the initial binding capacity. When CIP with 10 mM NaOH was performed at 40 °C MabSelect reduced its capacity by half while MabSelect SuRe still had a binding capacity of 80%. The 96-well screening showed that an addition of NaCl during CIP had a significant positive effect on the stability of MabSelect, but needs to be verified on column format. The correlation between results from screening in 96-well filter plate and column format was good.</p>

Robust and efficient cleaning procedures for protein A chromatography resins used for production of monoclonal antibody based biopharmaceuticals are crucial for safe and cost efficient processes. In this master thesis the effect of different cleaning regimes with respect to ligand stability of two protein A derived media, MabSelectTM and MabSelect SuReTM, has been investigated. A 96-well format has been used for preliminary screening of different cleaning agents, contact times and temperatures. NaCl as a ligand stabilizer during cleaning-in-place (CIP) was also included as a parameter. For optimal throughput and efficiency of screening, Rectangular Experimental Design for Multi-Unit Platforms; RED-MUP, and TECAN robotic platform have been utilized. For verification of screening, selected conditions were run in column format using the parallel chromatography system ÄKTAxpressTM. In the efficiency study, where a manual preparation of CIP solutions was compared with an automated mode performed in TECAN, the total process time ended up at eight hours versus three days respectively. However, the time measured included the learning process for the TECAN platform and for further preparations the automated mode is the superior choice. The study confirmed the higher alkaline stability of MabSelect SuRe compared to MabSelect. After exposure to 0.55 M NaOH during 24h MabSelect SuRe still retained 90% of the initial capacity. In contrast MabSelect had 60% of the initial binding capacity. When CIP with 10 mM NaOH was performed at 40 °C MabSelect reduced its capacity by half while MabSelect SuRe still had a binding capacity of 80%. The 96-well screening showed that an addition of NaCl during CIP had a significant positive effect on the stability of MabSelect, but needs to be verified on column format. The correlation between results from screening in 96-well filter plate and column format was good.

<p>On the surface heat shock protein 90 (Hsp90) is an unlikely drug target for the treatment of any disease, let alone cancer. Hsp90 is highly conserved and ubiquitously expressed in all cells. There are four major isoforms encoded by distinct genes and together they may constitute 1-3% of the cellular protein. Genetic deletion results in nonviable phenotypes in some organisms, and there are no recognized polymorphisms suggesting an association or causal relationship with any human disease. With respect to cancer, the proteins absence from some recent high profile articles underlines the perception that it is an unlikely bona fide target to treat this disease. Yet, to date, there are 17 distinct Hsp90 inhibitors in clinical trials for multiple indications in cancer. The protein has been championed for over 20 years by the National Cancer Institute as a cancer target since the discovery of the antitumor activity of geldanamycin. Rather than focus on the intracellular inhibition of Hsp90, we have shifted our aim to the differences of Hsp90 between cancer and normal tissue, namely its extracellular expression.</p><p>My graduate thesis work has focused on the characterization of a series of novel small molecule imaging agents (fluor-tethered Hsp90 inhibitors) that enable the specific detection of ectopically expressed Hsp90 on tumor cells. We believe that these molecules will have a large impact in the near future on the diagnosis and treatment of metastatic breast cancer as well as other cancers. This hypothesis is based on recent findings in the clinical literature that have linked upregulation of Hsp90 with poor outcomes in multiple subtypes of breast cancer. Additionally, several papers have also reported an association of the expression of extracellular Hsp90 and metastatic progression in several human cancers. Hsp90 is currently considered by some as a cutting edge cancer drug target. The Haystead lab synthesized a series of tethered Hsp90 inhibitors that were modified with fluorophores and other imaging moieties in such a way as to preserve the binding to Hsp90 and enable detection through non-invasive imaging techniques. In a series of cell-based, live animal and biochemical studies we demonstrated that these molecules are highly selective for Hsp90 and can be used to specifically recognize intact tumor cells expressing ectopic Hsp90. Furthermore, we also observed that once bound to ectopic Hsp90, our tethered-inhibitors are actively internalized and this process can be blocked with Hsp90 antibodies. These findings have two implications; first, Hsp90 is undergoing active cycling at the plasma membrane; second, the finding that once bound to surface Hsp90 our fluor-tethered inhibitors can be internalized despite their polar nature. These results suggest a new therapeutic strategy that will enable specific delivery of tumor killing agents (e.g. 131I or metabolic poisons) to metastatic cells. This is unique because the use of small molecule inhibitors and not antibody- or nanoparticle-based payload delivery strategies offers advantages in formulation, cost and reproducibility.</p><p> In addition to payload delivery possibilities, we also show the utility of the tethered-inhibitors diagnostically by demonstrating their use in the detection of tumors in mouse models of human breast cancer. As a result of our animal studies, we believe our molecules in their present form could be used to address a currently unmet need in the early diagnosis of aggressive breast cancer and discriminating this from more indolent forms. </p><p>Furthermore, the tethered Hsp90 inhibitors have been used to make ligand affinity chromatography resins that have facilitated the discovery of other unique Hsp90 expressions and functions associated with cancer. We have found a pool of Hsp90 that is misfolded as determined by affinity chromatography depletion and a leftward thermal stability shift in the population of Hsp90 that flows through the ligand affinity resins. Differential trypsin digest patterns detected by mass spectrometry reveal also that the native protein has sites that are more accessible to trypsinization. This could have further implications in treating and detecting differences between cancerous tissues and normal tissues by designing an antibody that recognizes the exposed portions of the misfolded Hsp90. Together this body of work illustrates that not only is Hsp90 different in total expression levels in cancers, but is ectopically expressed and misfolded so as to provide other opportunities for therapeutic intervention that improve the safety for more clinical applications.</p> / Dissertation

Many pathogenic bacteria use their iron acquisition mechanisms to live inside hosts. Streptococcus pyogenes is a pathogenic bacterium that uses streptococcal iron acquisition ABC transporter to obtain heme. SiaA (HtsA, spy1795), a lipoprotein located on the cell surface, serves as a heme binding protein. To understand the iron-uptake mechanism, histidine 229, one of the two proposed axial ligands in SiaA, was mutated to alanine. SiaA H229A was expressed in E. coli, lysed by French Press, and purified by fast protein liquid chromatography (FPLC). SDS-PAGE indicated that pure protein was isolated. Nickel affinity FPLC gave purer H229A when 0.5 M imidazole was added to the binding buffer. Overall, histidine 229 is likely to be an axial ligand in wild type SiaA, as shown by the fact the mutant readily lost heme as evidenced by UV-vis spectra.

Background: The focus of this study was to investigate how producing human bone marrow (hBM) derived mesenchymal stromal cell (MSC) extracellular vehicles (EVs) in a high purity isolation system would affect their established characterization criteria and address the validity of these methods of EV production. Additionally, we set out to functionally characterize the hBM-MSC-EVs for their identified immunomodulatory ability while also assessing the presence of novel MSC-EV marker NRP1 identified by our group to further affirm its validity as a functional MSC-EV identity marker. Methods: Each hBM-MSC-EV donor was cultured in a hollow-fiber bioreactor system in non-stimulating serum/xeno-free conditions for 25 days to produce EVs persistently under quiescent conditions to characterize the hBM-MSC-EVs in their native form. EVs were isolated by traditional PEG-based precipitation for preliminary characterization to monitor bioreactor production wherein they were characterized using multimodal tangential flow filtration coupled with fast protein liquid chromatograph (FPLC) size exclusion/high-affinity purifications to obtain the final highly purified EV sample. Additionally, functional analysis of their immunomodulatory ability, EVs and MSCs were incubated with activated peripheral blood mononuclear cells (PBMCs) as an in-vitro model to evaluate their potency. Results: The hBM-MSC-EVs produced from the bioreactor system showed consistent characterization in accordance with the MISEV2018 establish criteria. We were also able to demonstrate their functional ability by observing statistically significantly immunomodulatory ability of activated PBMCs equivalent to native MSC ability. We were also able to validate the present of NRP1 on all hBM-MSC-EV samples produced confirming its validity as a MSC-EV marker. Conclusion: The significance of the results obtained from this study confirms the production of MSC-EV using a bioreactor and high purity isolation for obtaining consistent MSC-EVs for downstream investigation. Additionally, we were able to demonstrate the significance of MSC-EVs on MSC signaling for immunomodulation by showing equivalent functional potency when tested in-vitro. These results contribute to further understanding the biological attributes of MSC-EVs and contribute to the validation of currently established characterization guidelines.

No description available.

The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.