Prodepth: Predict Residue Depth by Support Vector Regression Approach from Protein Sequences Only

Residue depth (RD) is a solvent exposure measure that complements the information provided by conventional accessible surface area (ASA) and describes to what extent a residue is buried in the protein structure space. Previous studies have established that RD is correlated with several protein properties, such as protein stability, residue conservation and amino acid types. Accurate prediction of RD has many potentially important applications in the field of structural bioinformatics, for example, facilitating the identification of functionally important residues, or residues in the folding nucleus, or enzyme active sites from sequence information. In this work, we introduce an efficient approach that uses support vector regression to quantify the relationship between RD and protein sequence. We systematically investigated eight different sequence encoding schemes including both local and global sequence characteristics and examined their respective prediction performances. For the objective evaluation of our approach, we used 5-fold cross-validation to assess the prediction accuracies and showed that the overall best performance could be achieved with a correlation coefficient (CC) of 0.71 between the observed and predicted RD values and a root mean square error (RMSE) of 1.74, after incorporating the relevant multiple sequence features. The results suggest that residue depth could be reliably predicted solely from protein primary sequences: local sequence environments are the major determinants, while global sequence features could influence the prediction performance marginally. We highlight two examples as a comparison in order to illustrate the applicability of this approach. We also discuss the potential implications of this new structural parameter in the field of protein structure prediction and homology modeling. This method might prove to be a powerful tool for sequence analysis

In vitro uptake studies of cell targeting agents and nanoparticles

Recent progress in synthetic chemistry has enabled the preparation of new highly-defined polymers that exhibit changes in their structure in response to environmental changes. These responsive nanomaterials may be desirable as carriers of drugs to deliver at the cellular and sub-cellular level. However, the endocytic pathways used by these nanoparticles to access cells must be defined. Carboxylated polystyrene beads (C-PB) of 50 and 100 nm size were chosen as ‘model’ nanomedicines and their route of uptake into cells characterised and compared to thermoresponsive PLGA-b-(PEGMA-co-PPGMA) and PLA-b-(DEGMA-co-OEGMA) block copolymers of 50-150 nm (‘candidate’ drug delivery systems) uptake. A number of protocols were optimised for endocytosis inhibition studies. Results reported that the inhibition of clathrin mediated endocytosis (CME) with chlorpromazine (CPZ) was cell- and time-dependent. After the maximal effect of the inhibitor, the endocytosis of human transferrin (Htf), a marker of CME, recovered up to uninhibited levels in 3T3 and HCT116 cells. Furthermore, high passage number and ageing of cells showed a resistance towards the inhibition of the uptake of Htf with CPZ. Both PLGA-b-(PEGMA-co-PPGMA) and PLA-co-(DEGMA-co-OEGMA) thermoresponsive block copolymers presented colloidal instability and aggregation that impeded further endocytic pathway internalization experiments. However, the results reported in this thesis question some of the interpretation in the literature of the susceptibility of cells to CPZ in the internalization of nanomaterials. New experimental settings for CPZ inhibition studies should be considered and protocols optimised in order to avoid incorrect and potentially misleading outcomes

In vitro uptake studies of cell targeting agents and nanoparticles

Recent progress in synthetic chemistry has enabled the preparation of new highly-defined polymers that exhibit changes in their structure in response to environmental changes. These responsive nanomaterials may be desirable as carriers of drugs to deliver at the cellular and sub-cellular level. However, the endocytic pathways used by these nanoparticles to access cells must be defined. Carboxylated polystyrene beads (C-PB) of 50 and 100 nm size were chosen as ‘model’ nanomedicines and their route of uptake into cells characterised and compared to thermoresponsive PLGA-b-(PEGMA-co-PPGMA) and PLA-b-(DEGMA-co-OEGMA) block copolymers of 50-150 nm (‘candidate’ drug delivery systems) uptake. A number of protocols were optimised for endocytosis inhibition studies. Results reported that the inhibition of clathrin mediated endocytosis (CME) with chlorpromazine (CPZ) was cell- and time-dependent. After the maximal effect of the inhibitor, the endocytosis of human transferrin (Htf), a marker of CME, recovered up to uninhibited levels in 3T3 and HCT116 cells. Furthermore, high passage number and ageing of cells showed a resistance towards the inhibition of the uptake of Htf with CPZ. Both PLGA-b-(PEGMA-co-PPGMA) and PLA-co-(DEGMA-co-OEGMA) thermoresponsive block copolymers presented colloidal instability and aggregation that impeded further endocytic pathway internalization experiments. However, the results reported in this thesis question some of the interpretation in the literature of the susceptibility of cells to CPZ in the internalization of nanomaterials. New experimental settings for CPZ inhibition studies should be considered and protocols optimised in order to avoid incorrect and potentially misleading outcomes