DEVELOPMENT OF PEPTIDE-MODIFIED GEMINI SURFACTANTS AS NON-VIRAL GENE DELIVERY SYSTEM.

Gene therapy is a promising therapeutic approach for the treatment of inherited and acquired genetic disorders. Gemini surfactants are an emerging class of cationic lipids that has shown promising results in delivering genetic materials, particularly to the skin. The unique structure of the gemini surfactants imparts a design flexibility that permit the modulation of their physicochemical properties toward the enhancement of transfection efficiency. The behavior of gemini surfactants in complex biological systems may ultimately determine their efficiency and cytotoxicity. Correlating the biodistribution and biological fate of the nanoparticles to their chemical structure and physicochemical properties will inform the rational design process, resulting in the production of compounds with higher efficiency and reduced toxicity. In this research, a series of 22 novel peptide-modified gemini surfactants was evaluated. The aim of my research is to elucidate their structure-activity relationship and to determine their skin penetration behaviour. Transfection efficiency and cytotoxicity of the compounds were evaluated in murine keratinocytes and African green monkey kidney fibroblast cell lines. Physicochemical and structural properties of the nanoparticles were examined. Results revealed that the highest transfection efficiency and lowest cytotoxicity were associated with 16-7N(G-K)-16 gemini surfactant, showing an 8-fold increase in gene expression and a 20% increase in cell viability compared to the first-generation unsubstituted gemini surfactants. Furthermore, assessment of the contribution of the alkyl tail, in terms of length and saturation, indicated that compounds with hexadecyl tails were 5-10 fold more efficient than compounds with dodecyl and oleyl tails. To track the level of localization of the gemini surfactants in the skin, mass spectrometric analytical strategies were developed. The tandem mass spectrometric (MS/MS) dissociation behavior of the compounds was evaluated. Diagnostic product ions were selected for accurate identification of the gemini surfactants in complex biological matrices. Such knowledge was utilized to develop rapid and simple flow injection analysis (FIA)‐MS/MS method for the quantification of three peptide-modified gemini surfactants ex vivo in skin and phosphate buffered saline. Results showed a more than 11% deposition in the skin with minimum penetration into the saline compartment, suggesting the suitability of the delivery system to be used for topical application

Design of an one-step platform purification of STEAP1 using Hydrophobic Interaction Chromatography

Prostate cancer is one of the most lethal and prevalent carcinoma among elder men worldwide. Currently, prostate cancer diagnosis based on prostate-specific antigen levels is unspecific and with limited efficient, mainly in advanced stages of cancer. Thus, there is a need to identify and characterize specific and reliable protein biomarkers for prostate cancer. Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a transmembrane protein whose high expression levels were correlated with PCa. STEAP1 may take part in intracellular and intercellular communication in cancer cells by modulating cell proliferation and tumor invasiveness through its potential activity as ion channel or transporter. So, the characterization of STEAP1 structure might allow the design of specific inhibitors that decrease and modulate its oncogenic function. The structural and functional studies require high purified amounts of protein, which can be obtained through a recombinant production of human STEAP1 protein integrated with a properly chromatographic strategy. In this work, the performance of Octyl- and Butyl-Sepharose were evaluated according to binding and elution conditions required for STEAP1 isolation from cell lysates, obtained in mini-bioreactor Pichia pastoris X33 methanol-induced cultures. The concentration of sodium phosphate buffer and monosodium phosphate plus sodium chloride in the equilibration buffer was optimized in order to promote a complete STEAP1 adsorption on the hydrophobics supports. Succinctly, a higher retention of STEAP1 was observed with concentrations above 500 mM of sodium phosphate buffer and monosodium phosphate plus sodium chloride, pH 8.0. If the adsorption is achieved at high concentrations of sodium phosphate buffer, the elution must be performed with increasing concentrations of Triton X-100 in 50 mM phosphate buffer. The obtained results indicate that the exposition of membrane binding domains of STEAP1 to Octyl- and Butyl- Sepharose requires high salt concentrations due the strong interactions established between them. However, after its complete adsorption, STEAP1 elution requires chaotropic agents such as detergents. Although application of HIC in the purification of integral membrane proteins are uncommon, the obtained results in the development of this dissertation indicate that the use of traditional hydrophobic matrices may open a promising alternative for the isolation of STEAP1 from cell lysates.O cancro de próstata é um dos carcinomas mais letais e prevalentes entre homens idosos em todo o mundo. Atualmente, o diagnóstico do cancro da próstata baseado nos níveis de Prostate Specific Antigen (PSA) é inespecífico e com eficiência limitada, principalmente em estágios avançados de cancro. Assim, existe a necessidade de identificar e caracterizar biomarcadores proteicos específicos e confiáveis para o cancro da próstata. A Six-transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) é uma proteína transmembranar cujos altos níveis de expressão foram correlacionados com o cancro da próstata. A STEAP1 pode participar na comunicação intracelular e intercelular em células cancerígenas através da modulação da proliferação celular e invasão tumoral através da sua potencial atividade como canal iónico ou transportador. Assim, a caracterização da estrutura da STEAP1 pode permitir a conceção de inibidores específicos que diminuem e modulam a sua função oncogénica. Os estudos estruturais e funcionais requerem quantidades elevadas de proteína purificada, que podem ser obtidas através de uma produção recombinante da proteína STEAP1 humana integrada com uma estratégia cromatográfica adequada. Neste trabalho, foi avaliado o desempenho da Octil- e Butyl-Sepharose de acordo com as condições de ligação e eluição necessárias para o isolamento da STEAP1 a partir de lisados celulares, obtidos em culturas induzidas com metanol num mini biorreator de Pichia pastoris X33. A concentração do tampão fosfato de sódio e fosfato monossódico com cloreto de sódio no tampão de equilíbrio foi otimizada para promover uma adsorção completa da STEAP1 nos suportes hidrofóbicos. Sucintamente, observou-se uma retenção mais elevada da STEAP1 com concentrações acima de 500 mM de tampão fosfato de sódio e fosfato monossódico com cloreto de sódio, pH 8,0. Se a adsorção for alcançada com altas concentrações de tampão fosfato de sódio ou fosfato monossódico com cloreto de sódio, a eluição deve ser realizada com concentrações crescentes de Triton X-100 em 50 mM de tampão fosfato. Os resultados obtidos indicam que a exposição dos domínios de ligação de membrana da STEAP1 à Octyl- e Butyl-Sepharose requerem à priori altas concentrações de sal devido às fortes interações estabelecidas entre eles. No entanto, após a sua adsorção completa, a eluição da STEAP1 requer agentes caotrópicos, como detergentes. Embora a aplicação da Cromatografia de Interação Hidrofóbica (HIC) na purificação de proteínas integrais de membrana seja incomum, os resultados obtidos no desenvolvimento da dissertação indicam que a utilização de matrizes hidrofóbicas tradicionais pode abrir uma alternativa promissora para o isolamento da STEAP1 a partir de lisados celulares

Development of a database and its use in the Investigation of Interferences in SRM assay design

Selected Reaction Monitoring (SRM), is a form of mass spectrometry that guarantees high throughput and also a high level of selectivity and specificity. Performing SRM experiments requires the development of assays to aid in peptide identification. This is a time consuming and expensive process thus biological researchers have come up with bioinformatics solutions for the design of SRM assay. The accuracy of these bioinformatics methods is quite high and the next step is to optimise the process by tackling the interference issue. As various analytes may have the same signals within an SRM experiment and thus interfere with each other’s signals, different solutions are being derived to tackle the issue. This thesis describes the development of a SRM transition database to store peptide and transition data, software to populate the database and also software to retrieve the data from the database. Finally the database is tested with the MRMaid transitions for the human proteome which were mined from the PRIDE database and the results analysed to investigate the transition interference issue. The database currently contains data for 20220 proteins and approximately 870,000 tryptic peptides from the human proteome

Quantitative analysis of mass spectrometry proteomics data : Software for improved life science

The rapid advances in life science, including the sequencing of the human genome and numerous other techiques, has given an extraordinary ability to aquire data on biological systems and human disease. Even so, drug development costs are higher than ever, while the rate of new approved treatments is historically low. A potential explanation to this discrepancy might be the difficulty of understanding the biology underlying the acquired data; the difficulty to refine the data to useful knowledge through interpretation. In this thesis the refinement of the complex data from mass spectrometry proteomics is studied. A number of new algorithms and programs are presented and demonstrated to provide increased analytical ability over previously suggested alternatives. With the higher goal of increasing the mass spectrometry laboratory scientific output, pragmatic studies were also performed, to create new set on compression algorithms for reduced storage requirement of mass spectrometry data, and also to characterize instrument stability. The final components of this thesis are the discussion of the technical and instrumental weaknesses associated with the currently employed mass spectrometry proteomics methodology, and the discussion of current lacking academical software quality and the reasons thereof. As a whole, the primary algorithms, the enabling technology, and the weakness discussions all aim to improve the current capability to perform mass spectrometry proteomics. As this technology is crucial to understand the main functional components of biology, proteins, this quest should allow better and higher quality life science data, and ultimately increase the chances of developing new treatments or diagnostics

Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells.

Candida albicans is the most common cause of hematogenously disseminated and oropharyngeal candidiasis. Both of these diseases are characterized by fungal invasion of host cells. Previously, we have found that C. albicans hyphae invade endothelial cells and oral epithelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the fungal surface protein and host cell receptors that mediate this process. We found that the C. albicans Als3 is required for the organism to be endocytosed by human umbilical vein endothelial cells and two different human oral epithelial lines. Affinity purification experiments with wild-type and an als3delta/als3delta mutant strain of C. albicans demonstrated that Als3 was required for C. albicans to bind to multiple host cell surface proteins, including N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. Furthermore, latex beads coated with the recombinant N-terminal portion of Als3 were endocytosed by Chinese hamster ovary cells expressing human N-cadherin or E-cadherin, whereas control beads coated with bovine serum albumin were not. Molecular modeling of the interactions of the N-terminal region of Als3 with the ectodomains of N-cadherin and E-cadherin indicated that the binding parameters of Als3 to either cadherin are similar to those of cadherin-cadherin binding. Therefore, Als3 is a fungal invasin that mimics host cell cadherins and induces endocytosis by binding to N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. These results uncover the first known fungal invasin and provide evidence that C. albicans Als3 is a molecular mimic of human cadherins

Enquiry into the topology of plasma membrane-localized PIN auxin transport components

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five alpha-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants

Decoding sequence-level information to predict membrane protein expression

The expression and purification of integral membrane proteins remains a major bottleneck in the characterization of these important proteins. Expression levels are currently unpredictable, which renders the pursuit of these targets challenging and highly inefficient. Evidence demonstrates that small changes in the nucleotide or amino-acid sequence can dramatically affect membrane protein biogenesis; yet these observations have not resulted in generalizable approaches to improve expression. In this study, we develop a data-driven statistical model that predicts membrane protein expression in E. coli directly from sequence. The model, trained on experimental data, combines a set of sequence-derived variables resulting in a score that predicts the likelihood of expression. We test the model against various independent datasets from the literature that contain a variety of scales and experimental outcomes demonstrating that the model significantly enriches expressed proteins. The model is then used to score expression for membrane proteomes and protein families highlighting areas where the model excels. Surprisingly, analysis of the underlying features reveals an importance in nucleotide sequence-derived parameters for expression. This computational model, as illustrated here, can immediately be used to identify favorable targets for characterization