Engineering poly(ethylene glycol) nanogel coatings: Developments in achieving ultralow protein adsorption and applications as substrates for stem cell culture

The biocompatibility of biomaterials is primarily dictated by the interactions that occur at the material\u27s interface with its biological environment. Proteins irreversibly adsorb to these interfaces within seconds to minutes of exposure, altering their structural conformation, inducing cell adhesion, and activating various cellular responses. To this end, surface modification strategies have been designed in attempts to develop stealth biomaterials or even biomaterials that modulate this response by inducing specific biological reactions. We sought to advance the design of biomaterial surfaces by quantitatively studying protein adsorption to ultralow protein adsorbing surfaces formed from poly(ethylene glycol) nanogel coatings of variable structural and chemical properties. We found that resistance to protein adsorption can be improved by increasing the nanogel coating\u27s surface packing density, which is achievable using orthogonal cross-linking chemistries, such as click chemistry, under phase separation conditions. We also confirmed that PEG and albumin act synergistically within nanogel coatings to resist protein adsorption. As a demonstration of the utility for such protein resistant surfaces, we fabricated improved cell culture substrates with nanogel coatings, by spatially patterning cell adhesion and functionalizing surfaces with specific ligands. These surfaces showed superior potential for driving the direct reprogramming of fibroblasts to cardiomyocytes over the standard stem cell substratum of Matrigel and revealed insight into optimal cellular organizations for cardiomyocyte differentiation. However, we also unexpectedly found that adsorption of laminin to mercaptosilanated glass promotes a relatively high efficiency of cardiomyocyte differentiation. The findings outlined in this dissertation demonstrate that consideration of often overlooked material design parameters, in addition to the choice of material, provides further opportunity for improving biocompatibility. We further demonstrated that the precision control of cell adhesion and substratum signaling provided by these materials has broad potential in biological applications, including stem cell culture

The Conformational Stability and Biophysical Properties of the Eukaryotic Thioredoxins of Pisum Sativum Are Not Family-Conserved

Thioredoxins (TRXs) are ubiquitous proteins involved in redox processes. About forty genes encode TRX or TRX-related proteins in plants, grouped in different families according to their subcellular localization. For instance, the h-type TRXs are located in cytoplasm or mitochondria, whereas f-type TRXs have a plastidial origin, although both types of proteins have an eukaryotic origin as opposed to other TRXs. Herein, we study the conformational and the biophysical features of TRXh1, TRXh2 and TRXf from Pisum sativum. The modelled structures of the three proteins show the well-known TRX fold. While sharing similar pH-denaturations features, the chemical and thermal stabilities are different, being PsTRXh1 (Pisum sativum thioredoxin h1) the most stable isoform; moreover, the three proteins follow a three-state denaturation model, during the chemical-denaturations. These differences in the thermal- and chemical-denaturations result from changes, in a broad sense, of the several ASAs (accessible surface areas) of the proteins. Thus, although a strong relationship can be found between the primary amino acid sequence and the structure among TRXs, that between the residue sequence and the conformational stability and biophysical properties is not. We discuss how these differences in the biophysical properties of TRXs determine their unique functions in pea, and we show how residues involved in the biophysical features described (pH-titrations, dimerizations and chemical-denaturations) belong to regions involved in interaction with other proteins. Our results suggest that the sequence demands of protein-protein function are relatively rigid, with different protein-binding pockets (some in common) for each of the three proteins, but the demands of structure and conformational stability per se (as long as there is a maintained core), are less so

Towards the Development of the TPR Scaffold into Novel Biomaterials & Bioswitches.

PhD.TetratricoPeptide Repeats or TPRs are a class of repeat proteins made up of - helices. Each repeat contains 34 amino acids that form a helix-turn-helix motif and is stabilised by short range interactions creating a non-globular fold. Tandem arrays of these repeats form stable superhelical structures. The modular nature of the TPR fold has allowed a series of consensus TPRs (CTPRs) to be designed where the number of repeat units has been varied. We have exploited the modular nature of CTPR proteins in order to create fibres via a bottom-up approach. Using Native Chemical Ligation (NCL) we have been able to trigger specific assembly of monomeric CTPR units to form extended fibrous structures up to microns in length (as viewed by TEM). This reaction proceeds at room temperature and neutral pH, with filaments observed within 12 hours. The equilibrium unfolding of CTPRs is prone to the population of partially folded states. Through studying the stability of a series of deletion mutants and using a Heteropolymer Ising model to analyse the unfolding data we have been able to design a CTPR with a conformational ‘switch’. This new CTPR was designed to populate a stable intermediate, with an exposed dimerisation interface, under certain conditions. When this new construct was analysed using 2D NMR and CD spectroscopy, it was found to selectively unfold its C-terminal -helix at a specific concentration of GuHCl. Our aim is to develop a system in which a ‘switching’ CTPR is used as a sensor that, when triggered by environmental conditions, partially unfolds and oligomerises

Bacterial detection using an anharmonic acoustic aptasensor

Infectious diseases are currently, one of the greatest global challenges in medicine. Rapid and precise diagnosis and identification of pathogen is important for timely initiation of appropriate antimicrobial therapy. However, many patients with infectious diseases receive empirical treatment rather than appropriate pathogen-directed therapy. As a result antimicrobials have been overused and/or misused, which has ultimately led to antimicrobial resistance (AMR). AMR is broadly considered as the most significant public health threat facing the world today. Policy makers from all over the world have recognised the urgent need for rapid point-of-care (POC) diagnostics that would not only identify pathogens but also provide antimicrobial susceptibility profiles in meaningful timeframe to initiate appropriate antimicrobial therapy and thereby, prevent AMR. Traditional culture-dependent diagnostic methods are still considered as gold standard methods. But they are very slow and generally require 18 to 48 hours with further 8 to 48 hours to perform antibiotic susceptibility test. Among culture-independent methods, PCR and ELISA are label-based, costly, laborious and require specialised equipment and trained personnel to operate them. Lateral flow assays (LFAs) that are low-cost, simple, rapid and paper-based portable detection platforms are very popular, as they can be applied at the POC. [Continues.

Raman spectroscopy for point of care urinary tract infection diagnosis

Urinary tract infections (UTIs) are one of the most common bacterial infections experience by humans, with 150 million people suffering one or more UTIs each year. The massive scale at which UTIs occurs translates to a tremendous health burden comprising of patient morbidity and mortality, massive societal costs and a recognised contribution to expanding antimicrobial resistance. The considerable disease burden caused by UTIs is severely exacerbated by an outdated diagnostic paradigm characterised by inaccuracy and delay. Poor accuracy of screening tests, such as urinalysis, lead to misdiagnosis which in turn result in delayed recognition or overtreatment. Additionally, these screening tests fail to identify the causative pathogen, causing an overreliance on broad-spectrum antimicrobials which exacerbate burgeoning antimicrobial resistance. While diagnosis may be accurately confirmed though culture and sensitivity testing, the prolonged delay incurred negates the value of the information provided doing so. A novel diagnostic paradigm is required that that targets rapid and accurate diagnosis of UTIs, while providing real-time identification of the causative pathogen. Achieving this precision management is contingent on the development of novel diagnostic technologies that bring accurate diagnosis and pathogen classification to the point of care. The purpose of this thesis is to develop a technology that may form the core of a point-of-care diagnostic capable of delivering rapid and accurate pathogen identification direct from urine sample. Raman spectroscopy is identified as a technology with the potential to fulfil this role, primarily mediated though its ability to provide rapid biochemical phenotyping without requiring prior biomass expansion. Raman spectroscopy has demonstrated an ability to achieve pathogen classification through the analysis of inelastically scattered light arising from pathogens. The central challenge to developing a Raman-based diagnostic for UTIs is enhancing the weak bacterial Raman signal while limiting the substantial background noise. Developing a technology using Raman spectroscopy able to provide UTI diagnosis with uropathogen classification is contingent on developing a robust experimental methodology that harnesses the multitude of experimental and analytical parameters. The refined methodology is applied in a series of experimental works that demonstrate the unique Raman spectra of pathogens has the potential for accurate classification. Achieving this at a clinically relevant pathogen load and in a clinically relevant timeframe is, however, dependent on overcoming weak bacterial signal to improve signal-to-noise ratio. Surface-enhanced Raman spectroscopy (SERS) provides massive Raman signal enhancement of pathogens held in close apposition to noble metal nanostructures. Additionally, vacuum filtration is identified as a means of rapidly capturing pathogens directly from urine. SERS-active filters are developed by applying a gold nanolayer to commercially available membrane filters through physical vapour deposition. These SERS-active membrane filter perform multiple roles of capturing pathogens, separating them from urine, while providing Raman signal enhancement through SERS. The diagnostic and classification performance of SERS-active filters for UTIs is demonstrated to achieve rapid and accurate diagnosis of infected samples, with real-time uropathogen classification, using phantom urine samples, before piloting the technology using clinical urine samples. The Raman technology developed in this thesis will be further developed toward a clinically implementable technology capable of ameliorating the substantial burden of disease caused by UTIs.Open Acces

Modulation of LRRK2 mediated signaling in immune cells

Mutations in LRRK2 are considered the most common cause of familial and sporadic Parkinson’s disease (PD), a neurodegenerative movement disorder. PD is characterised pathologically by the death of dopaminergic neurons and symptomatically by tremor, bradykinesia and impaired coordination. LRRK2 variants is also linked to an increased risk of Crohn's inflammatory bowel disease and increased susceptibility to leprosy. All those diseases share a common immune dysfunction theme. The exact physiological function of LRRK2 remains largely elusive. However, multiple reports suggest that LRRK2 is involved in Wnt and NFAT signalling. Those pathways are known to mediate a variety of physiological functions, including immune system regulation and synaptic functions. In this study, I investigate the role of LRRK2 in mediating the inflammatory responses in macrophages and T cells and associated changes in Wnt signalling components. Challenging the LRRK2 WT and LRRK2 KO RAW 264 macrophages with Lipopolysaccharides (LPS) resulted in changes in the release of Nitric oxide (NO) and expression of several cytokines, including tumour necrosis factor-alpha (TNFα), cyclooxygenase-2 (COX-2) and others. Furthermore, the phagocytic activity of the LRRK2 KO cells appears to be substantially lower compared to LRRK2 WT cells. I observed changes in Wnt signalling activity in response to LPS across the different genotypes. Interestingly, the expression of the LRRK2 kinase substrate RAB10 and its phosphorylated form, decreased significantly in response to LPS in LRRK2 KO cells compared to WT, providing a possible mechanistic explanation for the decrease in cytokines and NO release. Wnt-related changes were confirmed ex-vivo in bone-marrow-derived macrophage (BMDM) and T cells across WT, KO and G2019S genotypes. In conclusion, this study substantiates a role for LRRK2, in mediating inflammatory responses in immune cells and highlights the significance of Wnt signalling and Rab10 in this LRRK2 immune function

Functionally Relevant Macromolecular Interactions of Disordered Proteins

Disordered proteins are relatively recent newcomers in protein science. They were first described in detail by Wright and Dyson, in their J. Mol. Biol. paper in 1999. First, it was generally thought for more than a decade that disordered proteins or disordered parts of proteins have different amino acid compositions than folded proteins, and various prediction methods were developed based on this principle. These methods were suitable for distinguishing between the disordered (unstructured) and structured proteins known at that time. In addition, they could predict the site where a folded protein binds to the disordered part of a protein, shaping the latter into a well-defined 3D structure. Recently, however, evidence has emerged for a new type of disordered protein family whose members can undergo coupled folding and binding without the involvement of any folded proteins. Instead, they interact with each other, stabilizing their structure via “mutual synergistic folding” and, surprisingly, they exhibit the same residue composition as the folded protein. Increasingly more examples have been found where disordered proteins interact with non-protein macromolecules, adding to the already large variety of protein–protein interactions. There is also a very new phenomenon when proteins are involved in phase separation, which can represent a weak but functionally important macromolecular interaction. These phenomena are presented and discussed in the chapters of this book

A Novel Methodology for Isolating Broadly Neutralizing HIV-1 Human Monoclonal Antibodies

Abstract also published in AIDS Research and Human Retroviruses. November 2013, 29(11): A-53. doi:10.1089/aid.2013.1500Poster presentationpublished_or_final_versio

Biophysical and Biochemical Screening Approaches for Antimicrobial Drug Discovery Targeting S. aureus ClpP

The discovery of antibacterial drugs has been among most significant achievements of mankind in saving millions of lives across the planet from infectious diseases. With rise in resistance to almost all existing chemotypes, the design of next generation novel antibiotics has become much more challenging and difficult. The early 21st century witnessed the advancement of multiple novel chemotypes during golden age of antibiotics however the pace of antibiotic drug discovery has slowed down tremendously, contributing to life threatening antimicrobial discovery void since 1980’s. Therefore the need to develop novel antibiotics with unique mechanism of action to leverage against multi drug resistance pathogens, is paramount. In this direction the Caseinolytic Protease P (ClpP) is an emerging drug discovery target with significant potential for treatment of recalcitrant biofilm forming infections from pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA) This dissertation highlights the ongoing efforts to facilitate the discovery of novel non peptidic ClpP activator compounds and improvement of pharmacological profile of existing ClpP targeting Acyldepsipeptides (ADEPs) series antibiotics. The chapter one discusses the history and synopsis of conventional antibiotics drug discovery screening approaches, and transitions to modern era structure or fragment based screening approaches. The merits and challenges of such approaches of targeting a well conserved bacterial protease (ClpP) are discussed along with dissertation aims toward development of biophysical and biochemical screening approaches. Chapter two discusses optimization of thermal shift assay as primary screening assay for ClpP and its utility toward screening of fragment collections and buffer conditions. Chapter three discussed the development of a site specific Fluorescence Polarization based FP probe based on ADEP scaffold and its utility as a robust high throughput capable primary screening assay for screening of diverse collections ranging from bioactives to fragments. Chapter four discusses development of a label free Surface Plasmon Resonance (SPR) based assay geared toward screening of fragment as well as in house small and large (ADEP analogs) series compounds in addition to determining full kinetics for lead prioritization. Chapter five discusses the results of multiple screening campaigns utilizing combination of above assays to generate multiple hits with superior ligand efficiency and chemical tractability. Chapter six concludes with analysis of the best of compounds among individual series or from screening campaigns and highlights effectiveness of above screening assays toward hit exploration along with outlook on anticipated challenges and future directions

Estudo do stress proteotóxico no peixe zebra

Doutoramento em BiologiaA fidelidade da síntese proteica é fundamental para a estabilidade do proteoma e para a homeostasia celular. Em condições fisiológicas normais as células têm uma taxa de erro basal associada e esta muitas vezes aumenta com o envelhecimento e doença. Problemas na síntese das proteínas estão associados a várias doenças humanas e aos processos de envelhecimento. De facto, a incorporação de erros nas proteínas devido a tRNAs carregados pelas aminoacil-tRNA sintetases com o amino ácido errado causa doenças neurodegenerativas em humanos e ratos. Ainda não é claro como é que estas doenças se desenvolvem e se são uma consequência directa da disrupção do proteoma ou se são o resultado da toxicidade produzida pela acúmulação de proteínas mal traduzidas ao nível do ribossoma. Para elucidar como é que as células eucarióticas lidam com proteínas aberrantes e agregados proteicos (stress proteotóxico) desenvolvemos uma estratégia para destabilizar o proteoma. Para isso estabelecemos um sistema de erros de tradução em embriões de peixe zebra que assenta em tRNAs mutantes capazes de incorporar erradamente serina nas proteínas. As proteínas produzidas neste sistema despoletam as vias de resposta ao stress, nomeadamente a via da ubiquitina-proteassoma (UPP – “ubiquitin protesome pathway”) e a via do retículo endoplasmático (UPR – “unfolded protein response”). O stress proteotóxico gerado pelos erros de tradução altera a expressão génica e perfis de expressão de miRNAs, o desenvolvimento embrionário e viabilidade, aumenta a produção de espécies reactivas de oxigénio (ROS), leva ainda à acumulação de agregados proteicos e à disfunção mitocondrial. As malformações embrionárias e fenótipos de viabilidade que observámos foram revertidos por antioxidantes, o que sugere que os ROS desempenham papéis importantes nos fenótipos degenerativos celulares induzidos pela produção de proteínas aberrantes e agregação proteica. Estabelecemos ainda uma linha de peixe zebra transgénica para o estudo do stress proteotóxico. Este trabalho mostra que a destabilização do proteoma em embriões de peixe zebra com tRNAs mutantes é uma boa metodologia para estudar a biologia do stress proteotóxico visto que permite a agregação controlada do proteoma, mimetizando os processos de agregação de proteínas que ocorrem naturalmente durante o envelhecimento e em doenças conformacionais humanas.Protein synthesis fidelity is pivotal for proteome stability and cellular homeostasis. Even though normal physiologic conditions have an associated low protein synthesis error rate, this is frequently increased with aging and disease and the resulting protein misfolding is associated with various human diseases and aging processes. Importantly, gene mistranslations produced by tRNA misreading in the ribosome or tRNA mischarging by aminoacyl-tRNA synthetases cause neurodegenerative diseases in humans and mice. It is not yet clear how such diseases develop and whether they are a direct consequence of proteome disruption or a result of the toxicity produced by accumulation of mistranslated proteins. To elucidate how eukaryotic cells cope with protein misfolding and aggregation (proteotoxic stress) we have developed a strategy to destabilize the proteome in a regulated manner. We engineered transfer RNA (tRNAs) to misincorporate serine into proteins in zebrafish embryos. The mutant proteins misfold, trigger the unfolded protein response (UPR), up-regulate the ubiquitin proteasome pathway (UPP) and down-regulate protein synthesis. Proteotoxic stress generated by these gene mistranslations has strong effects on gene expression and miRNA profiles, embryo development and viability, increases the production of reactive oxygen species (ROS), accumulation of protein aggregates and mitochondrial dysfunction. Interestingly, embryo malformations and viability phenotypes could be reversed by ROS scavengers, suggesting that ROS play major roles in the cell degeneration phenotypes induced by protein misfolding and aggregation. We have also established a transgenic zebrafish line for proteotoxic stress studies. Hence, proteome mutagenesis by misreading tRNAs in zebrafish embryos is a powerful methodology to destabilize the proteome to study the biology of proteotoxic stress, it allows for controlled proteome aggregation and mimicking protein aggregation processes that occur naturally during aging and in human conformational diseases