Dissertations / Theses on the topic 'Bio-conjugation'

Transglutaminases (protein-glutamine:amine y-glutamyl- transferase, EC 2.3.2.13) are a family of calcium-dependent enzymes which catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When lysine acts as the acyl-acceptor substrate, α-glutamyl lysine isopeptide bond is formed. Isopeptide catalyzation results in protein cross-linkage which is prevalent throughout biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond. Furthermore, mTGs promiscuity in donor substrate preference highlights its biocatalytic potential. To realize the potential of the enzyme, a high-reactivity tag was necessary for protein labelling. To address this, an enzyme-coupled assay was developed to characterize peptides in the hopes of developing orthogonal substrates to facilitate mTG-mediated labelling and biocatalysis. The discovery of high-reactivity peptide tags allowed the realization of in vitro protein labelling- facilitated by mTG. The 7M48 peptide was fused to a test protein, where it was subsequently propargylated with propargyl amine to fluorescently label or immobilize a test protein. Although there are endless possibilities for in vitro bio-conjugation through mTG, proteolytic activation limits any in-cell labelling strategies with this enzyme. To circumvent this issue, development of an alternative bacterial enzyme, Bacillus subtilis transglutaminase (bTG), was chosen to replace mTG. bTG maintains the advantages associated with mTG but is expressed in its active form. Unlike mTG, there is limited preliminary research associated with the enzyme or its substrate scope. To better understanding substrate reactivity, a FRET-based assay was developed allows for the discovery of new high-reactivity peptides for bTG. These peptides were then used in labelling strategies to demonstrate the potential bTG-mediated bioconjugation. This strategy includes the added advantage of potential for in-cellulo labelling.

Toll-like receptors (TLRs) are among the first receptors activated during host-pathogen interactions. They act as key mediators in the pathogen-associated molecular patterns (PAMPs) detection and are responsible for the innate and adaptive immune responses in mammal. Belonging to this family, Toll-like receptor 4 (TLR4) is the mammalian sensor of bacterial endotoxin, lipopolysaccharide (LPS). Its activation results in the rapid triggering of pro-inflammatory processes essential for optimal host immune responses. TLR4 activation is a complex event which involves several proteins (LBP, CD14, and MD-2) and ends with the dimerization as an activated (TLR4/MD-2/LPS)2 complex. Dysregulated TLR4 activation is related to an impressively broad spectrum of disorders still lacking specific pharmacological treatment, including autoimmune disorders, chronic inflammations, allergies, asthma, infectious and central nervous system diseases, cancer, and sepsis. TLR4 modulation by small molecules of synthetic and natural origin provides access to new TLR-based therapeutics. Indeed, compounds able to block TLR4 activation (antagonists) are promising drug candidates against thee pathologies while TLR4 activating compounds (agonists) may be use as vaccines adjuvants and antitumoral agents. The work of this PhD thesis is described in four papers. The aim of the thesis is to study the processes and requirements leading to TLR4 activation/inhibition. The first study, Chap. IX, focuses on the contribution of carbohydrates to bacterial endotoxin (lipopolysaccharide, LPS, lipooligosaccharide, LOS, and lipid A) activity and in the switch from TLR4 agonism to antagonism. In particular, the structure-activity relationship and contribution of core saccharides 3-deoxy-D-manno-octulosonic acid (Kdo) and heptosyl-2-keto-3-deoxy-octulosonate (Hep) to TLR4/MD-2 binding and activation by LPS and LOS have been investigated in detail. This study allows the rational design of new structures of potential TLR4 modulators. These compounds were projected by computer-assisted design and their binding to MD-2 was evaluated through docking studies. A set of the most promising compounds was defined. They are composed of a glucosamine-bis-succinyl core (two carboxylate groups as phosphates bioisosteres) linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to MD-2 was studied by four independent methods using functional recombinant human MD-2 protein purified. It was demonstrated that all compounds bound to MD-2 with similar affinities and inhibited, in a concentration-dependent manner, the LPS-stimulated TLR4 signaling and cytokine production in human and murine cells.
Toll-like receptors (TLRs) are among the first receptors activated during host-pathogen interactions. They act as key mediators in the pathogen-associated molecular patterns (PAMPs) detection and are responsible for the innate and adaptive immune responses in mammal. Belonging to this family, Toll-like receptor 4 (TLR4) is the mammalian sensor of bacterial endotoxin, lipopolysaccharide (LPS). Its activation results in the rapid triggering of pro-inflammatory processes essential for optimal host immune responses. TLR4 activation is a complex event which involves several proteins (LBP, CD14, and MD-2) and ends with the dimerization as an activated (TLR4/MD-2/LPS)2 complex. Dysregulated TLR4 activation is related to an impressively broad spectrum of disorders still lacking specific pharmacological treatment, including autoimmune disorders, chronic inflammations, allergies, asthma, infectious and central nervous system diseases, cancer, and sepsis. TLR4 modulation by small molecules of synthetic and natural origin provides access to new TLR-based therapeutics. Indeed, compounds able to block TLR4 activation (antagonists) are promising drug candidates against thee pathologies while TLR4 activating compounds (agonists) may be use as vaccines adjuvants and antitumoral agents. The work of this PhD thesis is described in four papers. The aim of the thesis is to study the processes and requirements leading to TLR4 activation/inhibition. The first study, Chap. IX, focuses on the contribution of carbohydrates to bacterial endotoxin (lipopolysaccharide, LPS, lipooligosaccharide, LOS, and lipid A) activity and in the switch from TLR4 agonism to antagonism. In particular, the structure-activity relationship and contribution of core saccharides 3-deoxy-D-manno-octulosonic acid (Kdo) and heptosyl-2-keto-3-deoxy-octulosonate (Hep) to TLR4/MD-2 binding and activation by LPS and LOS have been investigated in detail. This study allows the rational design of new structures of potential TLR4 modulators. These compounds were projected by computer-assisted design and their binding to MD-2 was evaluated through docking studies. A set of the most promising compounds was defined. They are composed of a glucosamine-bis-succinyl core (two carboxylate groups as phosphates bioisosteres) linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to MD-2 was studied by four independent methods using functional recombinant human MD-2 protein purified. It was demonstrated that all compounds bound to MD-2 with similar affinities and inhibited, in a concentration-dependent manner, the LPS-stimulated TLR4 signaling and cytokine production in human and murine cells.

Currently, nanoparticles (NPs) play an increasing role in biomedical research and clinical applications, thanks to their peculiar optical, physical and chemical properties. A great challenge in nanodiagnostics is the development of new nano-sized devices aimed to optimize the detection of primary cancer cells and metastases. The design of ideal nanoconjugates, containing bioactive ligands specific for targeting cancer cell, requires optimization of fundamental parameters involved in conjugation reactions: both functional conformation and proper orientation must be preserved. This characteristic determines bioactivity, avidity and targeting efficiency of the functionalized NPs. In the context of this thesis, different conjugation strategies were analyzed, focusing on the improvement of the biological activity of the immobilized protein. First of all, trastuzumab-functionalized pegylated iron oxide nanoparticles were synthetized and protein conformation analyzed using FTIR spectroscopy. This technique provides direct evidence of the extent of native structure preservation of the immobilized protein, in dependence of the conjugation strategy. Moreover, the possibility to control the ligand/peptide orientation on the nanoparticle surface is a fundamental step to optimize receptor recognition. An elegant strategy involves the use of fusion proteins containing a small enzyme (defined “capture protein”) capable of irreversibly cross-coupling with a suicide inhibitor anchored to the solid surface. Three different approaches have been analyzed: SNAP (O6-alkylguanine-DNA-transferase), HALO (haloalkane dehalogenase) and cutinase enzymes fused with specific proteins or small peptides for the selective targeting of breast cancer cells. Although targeted therapy with monoclonal antibody, or small portion of these proteins, is a major treatment currently employed in many cancers, the use of short peptides as targeting moieties of tumor receptors have several advantages. The possibility to exploit gold nanoparticles (AuNPs) properties, to form a self-assembled monolayer on AuNPs surfaces, allows to increase ligand-receptor target affinity/recognition. The capability of all these bioconjugation methods to specifically and selectively target breast cancer cells, was confirmed by flow cytometry (FACS), confocal laser scanning microscopy and transmission electron microscopy (TEM).

Nanotechnology applied to the Medicine is providing new tools to the current therapeutic and diagnostic approaches to fight cancer and other diseases. However, many of the proposed nanodevices show some deficits related to both their inherent properties and performance, and the synthetic strategies proposed for their production. In the present work, a new promising approach based on e-beam radiation-induced radical crosslinking of a water soluble, biocompatible synthetic polymer has been developed. In particular, the possibility of generating Poly-N-(Vinyl- Pyrrolidone)(PVP)-based nanocarriers, i.e. nanogels with a base PVP structure, tailored physico-chemical properties (particles size distribution, surface charge density) and multifunctionality has been explored. A thorough product analysis on the generated nanoparticles through different characterization techniques, such as dynamic and static light scattering, photo-correlation spectroscopy, FT-IR, Raman, solid state NMR and XPS spectroscopies, SEM and AFM, has been carried. PVPbased nanogels have been then used as building blocks for the assembly of tumortarget “composite” nanodevices. “Model” ligands with various biological functions and drugs have been conjugated to the nanogels. Moreover, the biocompatibility and localization pattern of the nanocarriers in cell cultures have been evaluated. It has been demonstrated that all the NGs produced are biocompatible and able to be internalized by cells. Furthermore, the many functional groups grafted on the NGs are available for coupling reactions with bioactive molecules, such as targeting moieties, drugs and metal-ions chelating agents. This collective evidence validates the generated nanostructures for the intent they have been designed for, i.e. as nanocarriers in the biomedical field. E-beam irradiation using industrial type accelerators has demonstrated to be a viable manufacturing process since it grants high yields in terms of recovered product and high throughputs. Moreover, through a proper selection of the experimental parameters, this approach has allowed to obtain NGs with the desired properties, in terms of size, surface charge density, degree of crosslinking and functionality. All the evidences collected in this study, in terms of favorable properties-byprocess of the nanostructures generated and inherent advantages in the manufacturing process developed, can represent the fundaments for a further development and evaluation of this versatile “nanomaterial platform” for the treatment and diagnosis of various diseases, and cancer in particular.

Studi recenti sull’utilizzo di sonde organometalliche per bioconiugazione sfruttano la possibilità di interazione di queste con una proteina, l’avidina, per la sua capacità di coordinarsi selettivamente ad una particolare molecola organica, la biotina. In questa tesi viene descritta la sintesi di due leganti bi-funzionali contenenti sia un’unità capace di fungere da legante per un opportuno metallo, che un’unità di biotina in grado di legarsi all’avidina. La differenza fra i due leganti risiede nello spaziatore che collega le due unità funzionali della molecola. Una volta ottenuti i leganti progettati, sono stati sintetizzati i corrispondenti complessi di Ir (III) e Re (I). Le proprietà fotofisiche sono state misurate sia in solvente organico che in soluzione acquosa e quindi sono state effettuate titolazioni dell’avidina con soluzioni acquose a titolo noto dei complessi sintetizzati, con il fine di comprendere come le proprietà luminescenti dei complessi vengano influenzate dalla bio-coniugazione tra biotina ed avidina.

The work presented in this thesis describes methodologies developed for integration and accurate interpretation of barcoded DNA, to empower large-scale-omics analysis. The objectives mainly aim at enabling multiplexed proteomic measurements in high-throughput format through DNA barcoding and massive parallel sequencing. The thesis is based on four scientific papers that focus on three main criteria; (i) to prepare reagents for large-scale affinity-proteomics, (ii) to present technical advances in barcoding systems for parallel protein detection, and (iii) address challenges in complex sequencing data analysis. In the first part, bio-conjugation of antibodies is assessed at significantly downscaled reagent quantities. This allows for selection of affinity binders without restrictions to accessibility in large amounts and purity from amine-containing buffers or stabilizer materials (Paper I). This is followed by DNA barcoding of antibodies using minimal reagent quantities. The procedure additionally enables efficient purification of barcoded antibodies from free remaining DNA residues to improve sensitivity and accuracy of the subsequent measurements (Paper II). By utilizing a solid-phase approach on magnetic beads, a high-throughput set-up is ready to be facilitated by automation. Subsequently, the applicability of prepared bio-conjugates for parallel protein detection is demonstrated in different types of standard immunoassays (Papers I and II). As the second part, the method immuno-sequencing (I-Seq) is presented for DNAmediated protein detection using barcoded antibodies. I-Seq achieved the detection of clinically relevant proteins in human blood plasma by parallel DNA readout (Paper II). The methodology is further developed to track antibody-antigen interaction events on suspension bead arrays, while being encapsulated in barcoded emulsion droplets (Paper III). The method, denoted compartmentalized immuno-sequencing (cI-Seq), is potent to perform specific detections with paired antibodies and can provide information on details of joint recognition events. Recent progress in technical developments of DNA sequencing has increased the interest in large-scale studies to analyze higher number of samples in parallel. The third part of this thesis focuses on addressing challenges of large-scale sequencing analysis. Decoding of a huge DNA-barcoded data is presented, aiming at phase-defined sequence investigation of canine MHC loci in over 3000 samples (Paper IV). The analysis revealed new single nucleotide variations and a notable number of novel haplotypes for the 2nd exon of DLA DRB1. Taken together, this thesis demonstrates emerging applications of barcoded sequencing in protein and DNA detection. Improvements through the barcoding systems for assay parallelization, de-convolution of antigen-antibody interactions, sequence variant analysis, as well as large-scale data interpretation would aid biomedical studies to achieve a deeper understanding of biological processes. The future perspectives of the developed methodologies may therefore stem for advancing large-scale omics investigations, particularly in the promising field of DNA-mediated proteomics, for highly multiplex studies of numerous samples at a notably improved molecular resolution.

QC 20150203

The next generation of vaccine adjuvant are represented by a wide ranging set of molecules called Toll like agonists (TLR’s). Although many of these molecules are complex structures extracted from microorganisms, small molecule TLR agonists have also been identified. However, delivery systems have not been optimized to allow their effective delivery in conjunction with antigens. Here we describe a novel approach in which a small molecule TLR agonist has been conjugated directly to antigens to ensure effective co delivery. We describe the conjugation of a relevant protein, a recombinant protective antigen from S.pneumoniae (RrgB), which is linked to a TLR7 agonist. Following thorough characterization to ensure there was no aggregation, the conjugate was evaluated in a murine infection model. Results showed that the conjugate extended animals’ survival after lethal challenge with S.pneumoniae. Comparable results were obtained with a 10 fold lower dose than that of the native unconjugated antigen. Notably, the animals immunized with the same dose of unconjugated TLR7 agonist and antigen showed no adjuvant effect. The increased immunogenicity was likely a consequence of the co-localization of TLR7 agonist and antigen by chemical binding and is was more effective than simple co-administration. Likely, this approach can be adopted to reduce the dose of antigen required to induce protective immunity, and potentially increase the safety of a broad variety of vaccine candidates

The next generation of vaccine adjuvant are represented by a wide ranging set of molecules called Toll like agonists (TLR’s). Although many of these molecules are complex structures extracted from microorganisms, small molecule TLR agonists have also been identified. However, delivery systems have not been optimized to allow their effective delivery in conjunction with antigens. Here we describe a novel approach in which a small molecule TLR agonist has been conjugated directly to antigens to ensure effective co delivery. We describe the conjugation of a relevant protein, a recombinant protective antigen from S.pneumoniae (RrgB), which is linked to a TLR7 agonist. Following thorough characterization to ensure there was no aggregation, the conjugate was evaluated in a murine infection model. Results showed that the conjugate extended animals’ survival after lethal challenge with S.pneumoniae. Comparable results were obtained with a 10 fold lower dose than that of the native unconjugated antigen. Notably, the animals immunized with the same dose of unconjugated TLR7 agonist and antigen showed no adjuvant effect. The increased immunogenicity was likely a consequence of the co-localization of TLR7 agonist and antigen by chemical binding and is was more effective than simple co-administration. Likely, this approach can be adopted to reduce the dose of antigen required to induce protective immunity, and potentially increase the safety of a broad variety of vaccine candidates

La synthèse contrôlée par la cible sous contrôle cinétique (Kinetic Target-Guided Synthesis, KTGS) est une approche relativement peu explorée, alternative à la chimie combinatoire traditionnelle,dans laquelle la protéine cible participe à la synthèse du ou de ses propres ligands. Ainsi, les travaux présentés dans la première partie de cette thèse ont pour principal objectif d'élargir l'éventail des réactions actuellement disponibles en KTGS grâce à la réaction d'aldolisation voire d'amidation, et ce en utilisant la β-sécrétase (BACE-1) comme cible biologique, qui est une enzyme étroitement impliquée dans la maladie d'Alzheimer. La seconde partie de cette thèse a été consacrée à la synthèse de marqueurs de masse fluorescents bioconjugables basés sur l'association d'un noyau coumarinique et d'une fonction phosphonium. Les deux générations présentées dans ce manuscrit ont entre autre permis de synthétiser une sonde FRET permettant de détecter l'activité enzymatique de la BACE-1, qui pourrait par ailleurs être un outil intéressant pour l'analyse des bruts réactionnels des réactions de click in situ,et diminuer les quantités d'enzyme engagées dans ces expériences. Enfin dans la dernière partie de cette thèse nous décrivons la mise au point de nouvelles réactions de conjugaison bio-orthogonale pour le marquage de molécules comportant une fonction aldéhyde. Nous avons ainsi développé d'une part une réaction trois composants via une séquence de condensation/Mannich/lactamisation et d'autre part une réaction d'oléfination de Wittig
The kinetic target-guided synthesis (KTGS), is an underexplored alternative approach to combinatorial chemistry, in which the biological target is able to assemble its own inhibitors from a pool of fragments. Thus, the first part of this thesis aimed at extending the scope of the reactions available for the KTGS, by investigating the aldolisation and amidation reaction, using the β-secretase (BACE-1) as biological target, which is an enzyme narrowly involved in the Alzheimer's disease. The second part of this thesis was dedicated to the synthesis of bioconjagatable fluorophores containing a phosphonium group as mass tag associated to a coumarin core. Both generations presented in this manuscript allowed us, among other things, to synthesize a FRET probe that proved suitable for the determination of BACE-1 enzymatic activity. The utility of such a fluorogenic tool could be leveraged to facilitate the analysis of crude mixtures obtained during KTGS experiments, and lessen the amount of enzyme required in these experiments. Finally, in the last part of this thesis, we describe the development of two new bioorthogonal reactions allowing the selective labeling of molecules containing an aldehyde moiety : 1) a three component reaction involving a condensation/Mannich/lactamisation procedure, between an amine, an aldehyde and an enol partner; 2) a Wittig ligation between an aldehyde and a phosphonium bearing an active methylene

Vaccines are complex multi-component products, in which an interdependent relationship among constituents exists: the concomitant inclusion of antigens, adjuvants, and excipients is essential to guarantee the efficacy of the final product. The selection of adjuvants and formulation criteria is a fundamental aspect, having implications towards efficacy, safety and stability of vaccines. In this field, the work was aimed at the design, formulation and characterization of novel vaccines suitable for intradermal delivery and able to induce earlier onset of immune response as well as more potent and longer lasting immune responses in comparison to conventional vaccines. With this purpose two different model antigens were used to evaluate the intradermal route as immunological target for vaccination: MenC-CRM_197 glycoconjugate and CRM_197 protein (detoxified diphtheria toxin). Moreover different adjuvants were tested to potentiate and modulate the immune responses towards a desired quality profile. The impact of adjuvant-antigen conjugation on immune response against the antigen was investigated in comparison to the adjuvant/antigen co-delivery. For the first time, the intradermal delivery was evaluated as route for the administration of a MenC-CRM_197 glycoconjugate vaccine. LT mutant (LTK63), MPL (TLR4a), Toll like receptor 7 agonist (TLR7a), αGalCer analog, Dectin-1 receptor agonist (β-(1-3)glucan hexasaccharide) and an oil in water (o/w) emulsion have been chosen to target the skin-resident antigen-presenting cells and to enhance the immune response against the antigen. The in vivo evaluation of such novel formulations provided encouraging results with respect to quantity, quality and functionality of the induced immune responses. Notably, the intradermal delivery of MenC-CRM_197 glycoconjugate showed superiority in term of immunogenicity and serum bactericidal titers compared to the intramuscular administration, highlighting the power of the intradermal route for glycoconjugate vaccine delivery. Moreover, the addition of LTK63, TLR4a, TLR7a, αGalCer analog adjuvants allowed the reduction of the number of doses administered and especially LTK63, TLR4a, TLR7a adjuvants were able to modulate the quality of the immune response towards a more beneficial, for the model antigen used, Th1 response. No adjuvant effect was observed when formulations were combined with (β-(1-3) glucan hexasaccharide. When MenC-CRM_197+TLR4a formulation was intradermally delivered in combination with the o/w emulsion adjuvant no additional improvements in immunogenicity were observed in comparison to the single adjuvant effect. The immune system activation was also investigated using a protein model antigen 〖(CRM〗_197) intradermally; however no improvements in term of immunogenity were observed in comparison to the intramuscular administration. This finding has specified the interest in selecting a glycoconjugate antigen to exploit the advantages of the intradermal route in terms of immunogenicity. To further investigate the role of adjuvant and to ensure codelivery of adjuvant/antigen, the conjugations of TLR7a to the glycoconjugate model antigen have been investigated. Two different approaches were developed: the conjugation of TLR7a to MenC polysaccharide and to CRM_197 carrier protein. When the conjugation of TLR7a to CRM_197 carrier protein was tested in vivo, results clearly showed that conjugation of the TLR7a enhances the anti-carbohydrate response. This effect was less pronounced than after co-administration of a commercial glycoconjugate with a standard dose of TLR7a adsorbed on AlumOH. Conjugation of the small immunopotentiator molecule was particularly suited for vaccination via intradermal delivery, where insoluble salts of aluminum cannot be used because of their reactogenicity. These findings have opened the path to the rational design of improved adjuvanted glycoconjugate vaccines for intradermal routes. Regarding the conjugation of TLR7a to MenC polysaccharide tested in vivo, the obtained results have shown a negative impact of the conjugation to MenC polysaccharide receptor recognition and consequently a reduction of the anti-MenC carbohydrate response. In order to investigate the potential use of a short β-glucan chain as adjuvant for intradermal vaccination toward the CRM_197 protein model antigen, in comparison to the previously presented glycoconjugate model antigen, the results obtained by the in vitro and in vivo evaluation of the β-glucan conjugation to the CRM_197 protein were reported. The results have demonstrated that receptor activation was significantly impacted by the presentation of the glucan conjugated to the protein antigen. Considering that glycoconjugate vaccines are some of the safest and most effective vaccines for reducing, even eradicating, infectious diseases, the conjugation of well-defined synthetic glucans could represent a useful strategy for developing new glycoconjugate vaccines for intradermal delivery, with inherent adjuvant properties. The combination of antigens and adjuvants needs to be rationally defined to develop stable formulations and to give the safest and most efficient response with respect to the considered pathogen and route of administration. In the present work, formulation science has been proposed as a key element of novel vaccine development suitable for intradermal delivery.
I vaccini sono prodotti complessi costituiti da più componenti, nei quali esiste un rapporto di interdipendenza tra i costituenti: la concomitante presenza di antigeni, adiuvanti, ed eccipienti è essenziale per garantire l'efficacia del prodotto finale. La selezione degli adiuvanti e i criteri di formulazione sono un aspetto fondamentale, avendo implicazioni su efficacia, sicurezza e stabilità dei vaccini. In questo ambito, il lavoro è stato finalizzato alla progettazione, formulazione e caratterizzazione di nuovi vaccini atti alla consegna per via intradermica e capaci di indurre uno sviluppo precoce, più intenso e più duraturo della risposta immunitaria rispetto ai vaccini convenzionali. Con questo scopo due diversi antigeni sono stati utilizzati come modello per valutare la via di somministrazione intradermica per la vaccinazione: il glicoconiugato MenC-CRM_197 e la proteina CRM_197 (tossina difterica detossificata). Inoltre diversi adiuvanti sono stati usati per potenziare e modulare le risposte immunitarie verso un profilo di qualità desiderata. In aggiunta è stato valutato l'impatto che la coniugazione dell’adiuvante all’antigene ha sulla risposta immunitaria contro l'antigene in confronto alla semplice miscelazione. La via di immunizzazione intradermica è stata valutata, per la prima volta, per la somministrazione del glicoconiugato MenC-CRM_197. Per dirigere l’antigene verso le cellule APC presenti nella pelle e migliorare la risposta immunitaria verso l’antigene, sono stati testati diversi adiuvanti, quali la tossina enterica detossificata di Escherichia coli (LTK63), un agonista del recettore Toll Like 4 (TLR4a) il monofosfolipide A (MPL), un agonista del recettore Toll like 7 (TLR7a), un analogo dell’αGalCer, un agonista del recettore Dectin-1 (β- (1-3) hexasaccharide glucano) e l’emulsione olio/acqua (o/w). La valutazione in vivo di tali nuove formulazioni ha fornito alcuni risultati incoraggianti riguardanti la quantità e la qualità delle risposte immunitarie indotte. In particolare, i risultati immunologici ottenuti hanno dimostrato una netta superiorità della via di immunizzazione intradermica rispetto a quella intramuscolare quando è utilizzato il glicoconiugato MenC-CRM_197. Inoltre, l'aggiunta degli adiuvanti LTK63, TLR4a, TLR7a, l’analogo dell’αGalCer ha consentito la riduzione del numero di dosi somministrate e soprattutto le formulazioni adiuvantate con LTK63, TLR4a e TLR7a sono state in grado di modulare la qualità della risposta immunitaria verso una più vantaggiosa risposta Th1. Nessun effetto adiuvante è stato osservato utilizzando il glucano esasaccarico come adiuvante miscelato all’antigene. Il glicoconigato MenC-CRM_197 adiuvantato con il TLR4a è stato consegnato per via intradermica anche in combinazione con l’emulsione o/w, ma non è stato osservato alcun effetto adiuvante aggiuntivo della miscela in confronto all’effetto dei singoli immunopotenziatori. In aggiunta è stata valutata l’immunogenicità della proteina CRM_197 a seguito della consegna intradermica ma non è stato osservato alcun miglioramento in termini di immunogenicità in confronto alla somministrazione intramuscolare. Questo risultato ha messo in evidenza l'importanza dell’adeguata scelta dell’antigene per sfruttare i vantaggi della via di immunizzazione intradermica: un antigene glicoconiugato è preferibile ad un antigene proteico. Per meglio definire l’effetto adiuvante e garantire la concomitante somministrazione di antigene e adiuvante, sono stati svilippati e studiati due approcci di coniugazione del TLR7a all'antigene modello MenC-CRM_197: la coniugazione del TLR7a prima al polisaccaride MenC e poi alla proteina CRM_197. I risultati ottenuti dai tests in vivo del coniugato TLR7a alla proteina CRM_197 hanno dimostrato che la coniugazione del TLR7a migliora la risposta anti-MenC. L’effetto adiuvante del TLR7a coniugato si è rivelato meno pronunciato rispetto alla somministrazione di una dose standard di TLR7a adsorbito su AlumOH. Bisogna però sottolineare che il sale di alluminio non può essere consegnato per via intradermica, dato la sua reattogenicità, perciò ne risulta che la coniugazione del TLR7a alla proteina del glicoconiugato MenC-CRM_197 può essere molto utile per la vaccinazione intradermica. I risultati ottenuti costituiscono la base per una razionale progettazione di vaccini glicoconiugati adiuvantati da somministrare per via intradermica. Per quanto riguarda l’approccio di coniugazione del TLR7a al MenC, i risultati ottenuti in vivo hanno portato alla luce un impatto negativo della coniugazione sul riconoscimento recettoriale del polisaccaride e di conseguenza una riduzione della risposta anti-MenC. Il glucano esasaccaridico è stato utilizzato per studiare il suo effetto adiuvante quando coniugato ad un antigene proteico a seguito di una somministrazione intradermica. I risultati ottenuti dalla sperimentazione in vitro ed in vivo dell’esasaccaride coniugato alla proteina CRM_197 hanno dimostrato che l'attivazione del recettore Dectin-1 è significativamente influenzata dalla presenza del glucano. Considerando che i vaccini glicoconiugati sono alcuni dei vaccini più sicuri e più efficaci per ridurre e sradicare le malattie infettive, la coniugazione di glucani sintetici potrebbe rappresentare una strategia utile per lo sviluppo di nuovi vaccini glicoconiugati adatti alla somministrazione intradermica, con intrinseche proprietà adiuvanti. La combinazione appropriata di antigeni e adiuvanti deve guidare la formulazione di vaccini stabili che forniscano una risposta più sicura ed efficace contro il patogeno desiderato mediante la via di somministrazione scelta. Nel presente lavoro, la scienza di formulazione è stata proposta come un elemento chiave dello sviluppo di nuovi vaccini adatti alla consegna intradermica.

Antigene e anticorpo sono i due reagenti chiave di un saggio immunodiagnostico. L’indagine di nuove tecniche e il miglioramento di processi quali la purificazione e la marcatura sito-specifica di antigeni e anticorpi possono promuovere lo sviluppo di nuovi reagenti più efficienti capaci di migliorare la performance dei saggi immunodiagnostici. La prima parte di questa tesi è stata focalizzata sull’esplorazione di tecniche biotecnologiche innovative nella produzione di antigeni al fine di migliorare i saggi per la rilevazione degli anticorpi IgM e IgG specifici per il virus Epstein-Barr. Il virus EBV è causa della mononucleosi infettiva ed è associato ad un crescente numero di tumori; per questa motivo è importante sviluppare saggi diagnostici per la rilevazione di EBV ad alta specificità e sensibilità. La proteina VCA p18 è uno degli antigeni più importanti per la diagnosi di EBV. I saggi attuali Diasorin LIAISON EBV VCA IgM and IgG si basano su un singolo antigene corrispondente alla regione C-terminale immunodominante della proteina p18, immobilizzata su fase solida. I vari metodi esplorati in questa tesi hanno permesso di ottenere diverse varianti dell’antigene p18 con lo scopo di migliorare le prestazioni dei saggi EBV VCA IgM e IgG a diversi livelli: 1_produzione dell’antigene p18; 2_immobilizzazione dell’antigene p18 su fase solida; 3_formato di saggio. 1_La lunghezza della regione C-terminale della proteina p18 (57aa), risulta essere considerevole per il processo sintetico ma, allo stesso tempo, troppo piccola per essere prodotta in modo efficiente per via ricombinante. Per superare questo problema, abbiamo esplorato il sistema Elastin Like Polypeptides (ELP)-Inteina basato sull’uso di una proteina capace di effettuare auto-cleavage (inteina) e un tag responsivo alla temperatura (ELP). Questa tecnica si è rivelata un eccellente sistema per la produzione del peptide p18. 2_Lo sviluppo di diverse varianti dell’antigene p18 ha permesso di esplorare varie tecniche per l’immobilizzazione dello stesso antigene su fase solida: coating covalente diretto, attraverso il complesso streptavidina-biotina e attraverso l’uso dei peptidi leucine zipper (o velcro). L’immobilizzazione del peptide p18 su fase solida attraverso questi vari metodi è avvenuta con successo e l’attività immunochimica dell’antigene, immobilizzato con queste tecniche innovative, è comparabile o migliore rispetto a quella del peptide sintetico utilizzato nei saggi attuali. 3_Nonostante il saggio Diasorin LIAISON EBV VCA IgM abbia una buona performance analitica, al fine di ottenere un aumento di specificità, è stato esplorato un nuovo tipo di formato di saggio. Sfortunatamente i risultati indicano che questo diverso tipo di formato raggiunge un livello di specificità minore rispetto a quello del saggio attuale. La seconda parte di questa tesi è stata focalizzata sull’esplorazione di un metodo innovativo per la marcatura sito specifica degli anticorpi. Uno degli approcci più promettenti è basato sulla generazione di gruppi tiolo liberi attraverso la riduzione parziale e selettiva dei ponti disolfuro inter-catena presenti a livello della “hinge region” e la loro reazione con molecole marcanti caratterizzate dal possedere gruppi funzionali reattivi verso i gruppi sulfidrilici. Questa tecnologia è stata utilizzata per la biotinilazione di due diversi anticorpi usati attualmente per il rilevamento della proteina virale p24 di HIV e per l’antigene FGF23. I risultati suggeriscono che la biotinilazione sito-specifica rispetto a quella classica random promuove un miglioramento dell'attività immunochimica degli anticorpi con una conseguente ottimizzazione della performance dei saggi immunodiagnostici.
Antigen and antibody are the two key reagents for an immunodiagnostic assay. Investigation of new techniques and improvement of processes such as purification and site-specific labeling of antigen and antibody molecules can promote the development of new more powerful bioreagents able of improving the performance of immunodiagnostic assays. The first part of this thesis aimed to explore innovative biotechnology techniques in antigen production for the improvement of immunoassays that allow the detection of antibodies directed against the Epstein-Barr virus. EBV virus is the causative agent of infectious mononucleosis and it is considered to be associated with a still increasing number of tumors; for this reason it is important to develop diagnostic assays for EBV detection with high specificity and sensitivity. The viral capsid protein VCA p18 is one of the most important antigens for the diagnosis of EBV. The current Diasorin LIAISON EBV VCA IgM and IgG assays rely on a single antigen, consisting in a synthetic peptide corresponding to the immunodominant C-terminal portion of the p18 protein, which is immobilized on solid phase (indirect format). The several methods explored in this thesis have allowed to obtain different variants of the p18 antigen with the aim to improve the performance of DiaSorin LIAISON EBV VCA IgM and IgG assays at different levels: 1_production of p18 antigen; 2_immobilization of p18 antigen on solid phase; 3_immunoassay format. 1_ The length of the immunodominant C-terminal portion of the p18 protein (57aa) appears to be considerable for the synthetic route but, at the same time, too small to be effectively produced in a recombinant fashion. To overcome this problem, we explored the Elastin Like Polypeptides (ELP)-Intein system, a method based on the use of a self-cleavable protein (the intein) and a temperature responsive tag (ELP). This technique has proved to be an excellent system for the preparation of the p18 peptide. 2_The development of different variants of p18 antigen has enabled to explore different techniques for the immobilization of the same antigen on solid phase: direct covalent coating, through streptavidin-biotin complex and through an innovative procedure based on the use of leucine zipper (or “velcro”) peptides. The immobilization of the p18 peptide on solid phase through these different methods occurred successfully and the immunochemical activity of the antigen, immobilized with these innovative techniques, is comparable or better than that of the synthetic peptide used in the current immunoassays. 3_Despite the DiaSorin LIAISON EBV VCA IgM immonoassay has a good analytical performance, in order to obtain an increase of specificity, a new assay format was explored. Unfortunately, the results indicate that this different type of format reaches a lower level of specificity than that of current assay. The second part of this thesis aimed to explore an innovative method for the site-specific labeling of antibodies. One the most promising approach is based on the generation of free thiol groups by selective partial reduction of the interchain disulfide bridges present at the level of the “hinge region” and their reaction to labels carrying sulfhydryl-reactive chemical groups. This technology was used for the biotinylation of two different antibodies currently used in immunoassays for the HIV p24 viral protein and for FGF23 antigen detection. The results suggest that the site-specific biotinylation compared to the random traditional biotinylation promotes a great improvement of antibodies immunochemical activity with a consequent optimization of immunodiagnostic assays performance.

碩士
中原大學
化學工程研究所
93
CdSe/ZnS quantum dots (Qdots) were made water soluble by surface modification with the help of mercaptopropionic acid (MPA) or a polymer called Poly(maleic anhydride alt-1-tetradecene). Both methods produced soluble Qdot close to 85% recovery, and the quantum yields were both approaching 50%. The stability of soluble Qdots was also under investigation. It was found that MPA modified dots easily aggregated in 0.1 M NaCl, but the polymer coated dots were fairly stable. The covalently bound bovine serium albumin (BSA) increased the stability of Qdots. We also tried to detect the single mismatched ssDNA by Qdot linked ssDNA. It was found that the hybridization efficiency of single mismatched ssDNA was far lower than the completely complementary one. It indicated that Qdots linked ssDNA could accurately detect mismatched ssDNA. By mean of fluorescence resonance energy transfer (FRET) the number of red fluorescence protein (RFP) absorbed on Qdots could be accurately estimated. There are only one RFP molecule adsorbed on each dot. By adopting Forster’s theory the distance between Qdot and fluorescent center can be calculated by FRET efficiency. Therefore, the orientation of RFP on Qdot could be elucidated.

碩士
崑山科技大學
電腦與通訊研究所
104
Dielectrophoresis (DEP) has been widely used to manipulate, separate, and concentrate microscale particles. Unfortunately, DEP force is difficult to be used in regard to the manipulation of nanoscale molecules/particles. For manipulation of 50 to 100-nm particles, the electrical field strength must be higher than 3 × 106 V/m, and with a low applied voltage of 10 Vp-p, the electrode gap needs to be reduced to submicrons. Our research consists of a novel and simple approach, using a several tens micrometers scale electrode (low cost and easy to fabricate) to generate a dielectrophoretic microparticle assembly to form nanogaps with a locally amplified alternating current (AC) electric field gradient, which is used to rapidly trap nanocolloids. The results show that the amplified DEP force could effectively trap 20-nm colloids in the nanogaps between the 5-μm particle aggregates. The concentration factor at the local detection region was shown to be approximately 5 orders of magnitude higher than the bulk solution. This approach was also successfully used in bead-based surface-enhanced Raman spectroscopy (SERS) for the rapid identification of bacteria from diluted blood.

Il microbiota intestinale umano svolge un ruolo importante nell'omeostasi funzionale e nella salute del corpo umano. La dieta e gli antibiotici sono tra gli altri, i fattori più importanti, noti per influenzare il microbiota intestinale umano. L’uso indiscriminato degli antibiotici in acquacoltura e agricoltura, come degli ormoni della crescita, negli esseri umani e negli animali per profilassi e terapia ha contribuito, attraverso la catena alimentare, alla diffusione e all’espansione della resistenza agli antibiotici.Lo scopo principale di questo progetto di Dottorato di Ricerca è stato quello di studiare gli effetti di diverse diete, dette onnivora, vegetariana e vegana sull’incidenza della resistenza batterica agli antibiotici nel GIT umano. Inizialmente è stata determinata la diversità microbica e l’abbondanza dei microrganismi in diversi cibi consumati dai soggetti nelle tre diverse diete. Secondariamente, è stata investigata la prevalenza dei batteri acido lattico resistenti agli antibiotici (LAB) nel GIT attraverso l’isolamento e la caratterizzazione dei LAB resistenti agli antibiotici nelle feci di volontari consumatori in questi tre tipi di dieta. In terzo luogo, è stata stimata la trasferibilità dei geni AR. I risultati rivelano una forma di E. faecium che mostra geni resistenti a eritromicina, streptomicina e tretracicline, isolate da un soggetto onnivoro capace di trasferire i suoi geni resistenti agli antibiotici, attraverso coniugazione, a E. faecium [64/3], E. faecalis (JH2-2), L. ivanovii (7842RF) e L. welshimeri (11857RF), che sono tutte forme di riferimento libere da plasmidi rispettivamente di 5.7 x 10-4 , 1.7 x 10-6 , 4.3 x 10-7 e 8.5 x 10-8 . L’estrazione di plasmidi rivela due plasmidi di almeno 23Kb nel donatore e nel coniugante. Il sequenziamento di Sanger del plasmide rivela molte forme aperte segnalate come enzimi batterici coinvolti nella resistenza a molteplici antibiotici. Le scoperte di questo studio, in accordo con la letteratura, confermano che il microbioma umano effettivamente ospita un bacino mobile di geni antibiotici, ma non sono conclusive sugli effetti delle diverse diete sulla prevalenza dei batteri resistenti agli antibiotici nel microbiota intestinale. Ulteriori esperimenti necessitano di essere svolti per verificare la trasferibilità di tali geni in vivo.
The human intestinal microbiota plays a huge role in the functional homeostasis and health of the human body. Diet and antibiotics are the most important factors, among others, known to influence the human intestinal microbiota. Overuse of antibiotics in aquaculture and agriculture as growth hormones; and in humans and animals for prophylaxis and therapy has, through the food chain, contributed to the dissemination and spread of antibiotic resistance (AR). The main aim of this PhD project was to study the effect of different diets, namely; omnivorous, vegetarian and vegan on the incidence of antibiotic-resistant bacteria in the human gastrointestinal tract (GIT). Initially, the microbial diversity and abundance of the microorganisms in the different foods consumed by subjects taking the three different diets was determined. Secondly, the prevalence of antibiotic-resistant lactic acid bacteria (LAB) in the GIT was investigated through isolation and characterisation of antibiotic-resistant LAB in the faeces of volunteers taking the three types of diets. Thirdly, transferability of AR genes was assessed. The results revealed an E. faecium strain carrying erythromycin, streptomycin and tetracycline resistant genes, isolated from an omnivore subject, that transferred its antibiotic-resistant genes, through conjugation, to E. faecium [64/3], E. faecalis (JH2-2), L. ivanovii (7842RF) and L. welshimeri (11857RF), all plasmid-free reference strains at frequencies of 5.7 x 10-4, 1.7 x 10-6, 4.3 x 10-7 and 8.5 x 10-8, respectively. Plasmid extraction revealed two plasmids of more than 24Kb in the donor and transconjugants. Sanger sequencing of the plasmids revealed several ORFs annotated to bacterial enzymes involved in multiple ARs. Findings from this study, affirm that the GIT harbours a mobilizable reservoir of antibiotic genes but the effects of different diets on the prevalence of AR bacteria in the GIT are not conclusive. Further experiments need to be duplicated in vivo.

Unconventional nanofabrication techniques; both those which have been newly developed and those under development, had brought inexpensive, facile, yet high quality means to fabricate nanostructures that have feature sizes of less than 100 nm in industry and academia. This dissertation focuses on developing unconventional fabrication techniques, building studying platforms, and studying the mechanisms behind them. The studies are divided into two main facets and four chapters. The first facet, in Chapter II and Chapter III, deals with the research and development of different nanofabrication techniques and nanostructures. These techniques include litho-synthesis, colloidal lithography, and photolithography. The nanostructures that were fabricated by these techniques include the metal nanoparticle arrays, and the self-assembled CdSe nanoring arrays. At the same time, the dissertation provides mechanisms and models to describe the physical and chemical nature of these techniques. The second area of this study, in Chapter III to Chapter V, presents the applications of these nanostructures in fundamental studies, i.e. the mechanisms of plasmon enhanced fluorescence and photo-oxidation kinetics of CdSe quantum dots, and applications such as molecular sensing and material fabrication. More specifically, these applications include tuning the optical properties of CdSe quantum dots, biomodification of CdSe quantum dots, and copper ion detection using plasmon and photo enhanced CdSe quantum dots. We have successfully accomplished our research goals in this dissertation. Firstly, we were able to tune the emission wavelength of quantum dots, blue-shifted for up to 45 nm, and their surface functionalization with photo-oxidation. A kinetic model to calculate the photo-oxidation rates was established. Secondly, we established a simple mathematical model to explain the mechanism of plasmon enhanced fluoresce of quantum dots. Our calculation and experimental data support the fluorescence resonance energy transfer (FRET) mechanism between quantum dots and the metal nanoparticles. Thirdly, we successfully pattered the CdSe quantum dots (diameter ~4 nm) into nanorings with tunable diameters and annular sizes on different substrates. We also established a physical model to quantitatively explain the mechanism with the forces that involved in the formation of the nanorings.