Development and applications of hollow fiber flow field-flow fractionation in the bioanalytical field. Studies of aggregation phenomena in complex protein samples

Recent advances in the fast growing area of therapeutic/diagnostic proteins and antibodies - novel and highly specific drugs - as well as the progress in the field of functional proteomics regarding the correlation between the aggregation of damaged proteins and (immuno) senescence or aging-related pathologies, underline the need for adequate analytical methods for the detection, separation, characterization and quantification of protein aggregates, regardless of the their origin or formation mechanism. Hollow fiber flow field-flow fractionation (HF5), the miniaturized version of FlowFFF and integral part of the Eclipse DUALTEC FFF separation system, was the focus of this research; this flow-based separation technique proved to be uniquely suited for the hydrodynamic size-based separation of proteins and protein aggregates in a very broad size and molecular weight (MW) range, often present at trace levels. HF5 has shown to be (a) highly selective in terms of protein diffusion coefficients, (b) versatile in terms of bio-compatible carrier solution choice, (c) able to preserve the biophysical properties/molecular conformation of the proteins/protein aggregates and (d) able to discriminate between different types of protein aggregates. Thanks to the miniaturization advantages and the online coupling with highly sensitive detection techniques (UV/Vis, intrinsic fluorescence and multi-angle light scattering), HF5 had very low detection/quantification limits for protein aggregates. Compared to size-exclusion chromatography (SEC), HF5 demonstrated superior selectivity and potential as orthogonal analytical method in the extended characterization assays, often required by therapeutic protein formulations. In addition, the developed HF5 methods have proven to be rapid, highly selective, sensitive and repeatable. HF5 was ideally suitable as first dimension of separation of aging-related protein aggregates from whole cell lysates (proteome pre-fractionation method) and, by HF5-(UV)-MALS online coupling, important biophysical information on the fractionated proteins and protein aggregates was gathered: size (rms radius and hydrodynamic radius), absolute MW and conformation.

A formal analysis of blockchain consensus

In this thesis, we analyse these protocols using PRISM+, our extension of the probabilistic model checker PRISM with blockchain types and operations upon them. This allows us to model the behaviour of key participants in the protocols and describe the protocols as a parallel composition of PRISM+ processes. Through our analysis of the Bitcoin model, we are able to understand how forks (where different nodes have different versions of the blockchain) occur and how they depend on specific parameters of the protocol, such as the difficulty of the cryptopuzzle and network communication delays. Our results corroborate the statement that considering confirmed the transactions in blocks at depth larger than 5 is reasonable because the majority of miners have consistent blockchains up-to that depth with probability of almost 1. We also study the behaviour of the Bitcoin network with churn miners (nodes that leave and rejoin the network) and with different topologies (linear topology, ring topology, tree topology and fully connected topology). PRISM+ is therefore used to analyse the resilience of Hybrid Casper when changing various basic parameters of the protocol, such as block creation rates and penalty determination strategies. We also study the robustness of Hybrid Casper against two known attacks: the Eclipse attack (where an attacker controls a significant portion of the network's nodes and can prevent other nodes from receiving new transactions) and the majority attack (where an attacker controls a majority of the network's nodes and can manipulate the blockchain to their advantage).