Improving the catalytic activity of formate dehydrogenase from Candida boidinii by using magnetic nanoparticles

Formate dehydrogenase from Candida boidinii (FDH) was immobilized on three different magnetic supports: one composed by magnetite nanoparticles directly silanized with ARTS (aminopropyltriethoxysilane), i.e. MagNP-APTS: the second one containing a silica gel coated magnetite core which was further silanized with APTS (MagNP@SiO2-APTS), and the third one consisting of magnetite-APTS coated with Glyoxyl-Agarose (MagNP-Glyoxyl-Agarose). The catalytic activity of the three FDH systems was investigated as a function of pH and temperature. The silica gel coated nanoparticles provided the highest conversion rates; however, in terms of recycling, magnetite without the silica shell led to the most stable system. By using the enzyme tryptophan residues as internal fluorescence probes, the structure-activity behavior was investigated in the presence of the formate and NAD(+) substrates, revealing a rather contrasting behavior in the three cases. Because of its peculiar behavior, a direct interaction of the magnetic nanoparticles with the catalytic sites seems to be implicated in the case of MagNP-APTS. (C) 2012 Elsevier B.V. All rights reserved.

Design of a Dinuclear Nickel(II) Bioinspired Hydrolase to Bind Covalently to Silica Surfaces: Synthesis, Magnetism, and Reactivity Studies

Presented herein is the design of a dinuclear Ni-II synthetic hydrolase [Ni-2(HBPPAMFF)(mu-OAc)(2)(H2O)]-BPh4 (1) (H(2)BPPAMFF = 2-[(N-benzyl-N-2-pyridylmethylamine)]-4-methyl-6-[N-(2-pyridylmethyl)aminomethyl)])-4- methyl-6-formylphenol) to be covalently attached to silica surfaces, while maintaining its catalytic activity. An aldehyde-containing ligand (H(2)BPPAMFF) provides a reactive functional group that can serve as a cross-linking group to bind the complex to an organoalkoxysilane and later to the silica surfaces or directly to amino-modified surfaces. The dinuclear Ni-II complex covalently attached to the silica surfaces was fully characterized by different techniques. The catalytic turnover number (k(cat)) of the immobilized (NiNiII)-Ni-II catalyst in the hydrolysis of 2,4-bis(dinitrophenyl)phosphate is comparable to the homogeneous reaction; however, the catalyst interaction with the support enhanced the substrate to complex association constant, and consequently, the catalytic efficiency (E - k(cat)/K-M) and the supported catalyst can be reused for subsequent diester hydrolysis reactions.

Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

Abstract Background All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. Results The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. Conclusion Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.

“Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions”

Abstract Background Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Characterization and optimization of ArtinM lectin expression in Escherichia coli

Background ArtinM is a D-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM) in Escherichia coli system. Results The ArtinM coding region was inserted in pET29a(+) vector and expressed in E. coli BL21(DE3)-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C) and shaking speeds (130, 200 or 220 rpm) during induction, concentrations of the induction agent IPTG (0.01-4 mM) and periods of induction (1-19 h). BL21-CodonPlus(DE3)-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h) resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized D-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM). The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises β-sheet structure. Conclusions Overall, the optimized process to express rArtinM in E. coli provided high amounts of soluble, correctly folded and active recombinant protein, compatible with large scale production of the lectin.

Characterization of the mechanisms underlying the crosstalk between galectins and notch in gastric cancer

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related deaths worldwide. Galectins form a family of β-galactosides binding proteins that recognize a variety of glycan-containing proteins at the cell surface and are overexpressed in various tumors, including gastric cancer. Galectins overexpression as well as changes in their subcellular distribution has been associated with gastric cancer progression and poor prognosis. It is not well understood, however, how the interaction between galectins and glycosylated receptors modulates tumor development and growth. Since Notch receptors and ligands contain glycan structures known to bind galectins, we aim to demonstrate that galectins expression in the tumor microenvironment may interfere with Notch signaling activation during tumor development and progression. Materials and methods Immunoprecipitation procedures with gastric cancer cell line AGS (ATCC CRL-1739) and MKN45 (ACC 409) were used to test for association between galectin-1/-3 and Notch-1 receptor. Furthermore, we transfected AGS cell line with siRNA against galectin-1/-3 or scramble using standard protocols (IDT DNA technologies), stimulate them with immobilized human recombinant delta-4 or Jagged-1 and assessed Notch-1 receptor activation. Results Galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling pathway upon activation by Delta/Jagged ligands. Galectin-1 knockdown alters Notch-1 activation induced by Delta-4 whereas galectin-3 knockdown alters jagged-1-mediated Notch-1 activation. Furthermore, we found that exogenously added galectin-3 can enhance Notch-1 activation by Jagged-1. Conclusion Our results suggest that galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling activation induced by Jagged-1 and Delta-4.

Investigação eletroquímica e calorimétrica da interação de novos agentes antitumorais biscatiônicos com DNA

Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-α,ω-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Leucine and HMB differentially modulate proteasome system in skeletal muscle under different sarcopenic conditions

In the present study we have compared the effects of leucine supplementation and its metabolite β-hydroxy-β-methyl butyrate (HMB) on the ubiquitin-proteasome system and the PI3K/Akt pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass driven by immobilization. On the other hand, leucine supplementation was unable to provide protection against soleus mass loss in dexamethasone treated rats. Interestingly, HMB supplementation was unable to provide protection against mass loss in all treatments. While solely fiber type I cross sectional area (CSA) was protected in immobilized soleus of leucine-supplemented rats, none of the fiber types were protected by leucine supplementation in rats under dexamethasone treatment. In addition and in line with muscle mass results, HMB treatment did not attenuate CSA decrease in all fiber types against either immobilization or dexamethasone treatment. While leucine supplementation was able to minimize increased expression of both Mafbx/Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Mafbx/Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized increased expression of Mafbx/Atrogin. The amount of ubiquitinated proteins, as expected, was increased in immobilized and dexamethasone treated rats and only leucine was able to block this increase in immobilized rats but not in dexamethasone treated rats. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level. On the other hand, HMB treatment failed to maintain tetanic peak force regardless of treatment. The present data suggested that the anti-atrophic effects of leucine are not mediated by its metabolite HMB.

Interaction of leptospira elongation factor tu with plasminogen and complement factor h: a metabolic leptospiral protein with moonlighting activities

The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities

Processo biotecnologico a cellule immobilizzate per la produzione di acidi grassi volatili da acque di vegetazione

Le acque di vegetazione (AV) costituiscono un serio problema di carattere ambientale, sia a causa della loro elevata produzione sia per l’ elevato contenuto di COD che oscilla fra 50 e 150 g/l. Le AV sono considerate un refluo a tasso inquinante fra i più elevati nell’ambito dell’industria agroalimentare e la loro tossicità è determinata in massima parte dalla componente fenolica. Il presente lavoro si propone di studiare e ottimizzare un processo non solo di smaltimento di tale refluo ma anche di una sua valorizzazione, utlizzandolo come materia prima per la produzione di acidi grassi e quindi di PHA, polimeri biodegradabili utilizzabili in varie applicazioni. A tale scopo sono stati utilizzati due bioreattori anaerobici a biomassa adesa, di identica configurazione, con cui si sono condotti due esperimenti in continuo a diverse temperature e carichi organici al fine di studiare l’influenza di tali parametri sul processo. Il primo esperimento è stato condotto a 35°C e carico organico pari a 12,39 g/Ld, il secondo a 25°C e carico organico pari a 8,40 g/Ld. Si è scelto di allestire e mettere in opera un processo a cellule immobilizzate in quanto questa tecnologia si è rivelata vantaggiosa nel trattamento continuo di reflui ad alto contenuto di COD e carichi variabili. Inoltre si è scelto di lavorare in continuo poiché tale condizione, per debiti tempi di ritenzione idraulica, consente di minimizzare la metanogenesi, mediata da microrganismi con basse velocità specifiche di crescita. Per costituire il letto fisso dei due reattori si sono utilizzati due diversi tipi di supporto, in modo da poter studiare anche l’influenza di tale parametro, in particolare si è fatto uso di carbone attivo granulare (GAC) e filtri ceramici Vukopor S10 (VS). Confrontando i risultati si è visto che la massima quantità di VFA prodotta nell’ambito del presente studio si ha nel VS mantenuto a 25°C: in tale condizione si arriva infatti ad un valore di VFA prodotti pari a 524,668 mgCOD/L. Inoltre l’effluente in uscita risulta più concentrato in termini di VFA rispetto a quello in entrata: nell’alimentazione la percentuale di materiale organico presente sottoforma di acidi grassi volatili era del 54 % e tale percentuale, in uscita dai reattori, ha raggiunto il 59 %. Il VS25 rappresenta anche la condizione in cui il COD degradato si è trasformato in percentuale minore a metano (2,35 %) e questo a prova del fatto che l’acidogenesi ha prevalso sulla metanogenesi. Anche nella condizione più favorevole alla produzione di VFA però, si è riusciti ad ottenere una loro concentrazione in uscita (3,43 g/L) inferiore rispetto a quella di tentativo (8,5 g/L di VFA) per il processo di produzione di PHA, sviluppato da un gruppo di ricerca dell’università “La Sapienza” di Roma, relativa ad un medium sintetico. Si può constatare che la modesta produzione di VFA non è dovuta all’eccessiva degradazione del COD, essendo questa nel VS25 appena pari al 6,23%, ma piuttosto è dovuta a una scarsa concentrazione di VFA in uscita. Questo è di buon auspicio nell’ottica di ottimizzare il processo migliorandone le prestazioni, poiché è possibile aumentare tale concentrazione aumentando la conversione di COD in VFA che nel VS25 è pari a solo 5,87%. Per aumentare tale valore si può agire su vari parametri, quali la temperatura e il carico organico. Si è visto che il processo di acidogenesi è favorito, per il VS, per basse temperature e alti carichi organici. Per quanto riguarda il reattore impaccato con carbone attivo la produzione di VFA è molto ridotta per tutti i valori di temperatura e carichi organici utilizzati. Si può quindi pensare a un’applicazione diversa di tale tipo di reattore, ad esempio per la produzione di metano e quindi di energia.

Sviluppo di biosensori: modifiche di superfici elettrodiche e sistemi di immobilizzazione enzimatica

An amperometric glucose biosensor was developed using an anionic clay matrix (LDH) as enzyme support. The enzyme glucose oxidase (GOx) was immobilized on a layered double hydroxide Ni/Al-NO3 LDH during the electrosynthesis, which was followed by crosslinking with glutaraldehyde (GA) vapours or with GA and bovine serum albumin (GABSA) to avoid the enzyme release. The electrochemical reaction was carried out potentiostatically, at -0.9V vs. SCE, using a rotating disc Pt electrode to assure homogeneity of the electrodeposition suspension, containing GOx, Ni(NO3)2 and Al(NO3)3 in 0.3 M KNO3. The mechanism responsible of the LDH electrodeposition involves the precipitation of the LDH due to the increase of pH at the surface of the electrode, following the cathodic reduction of nitrates. The Pt surface modified with the Ni/Al-NO3 LDH shows a much reduced noise, giving rise to a better signal to noise ratio for the currents relative to H2O2 oxidation, and a linear range for H2O2 determination wider than the one observed for bare Pt electrodes. We pointed out the performances of the biosensor in terms of sensitivity to glucose, calculated from the slope of the linear part of the calibration curve for enzimatically produced H2O2; the sensitivity was dependent on parameters related to the electrodeposition in addition to working conditions. In order to optimise the glucose biosensor performances, with a reduced number of experimental runs, we applied an experimental design. A first screening was performed considering the following variables: deposition time (30 - 120 s), enzyme concentration (0.5 - 3.0 mg/mL), Ni/Al molar ratio (3:1 or 2:1) of the electrodeposition solution at a total metals concentration of 0.03 M and pH of the working buffer solution (5.5-7.0). On the basis of the results from this screening, a full factorial design was carried out, taking into account only enzyme concentration and Ni/Al molar ratio of the electrosynthesis solution. A full factorial design was performed to study linear interactions between factors and their quadratic effects and the optimal setup was evaluated by the isoresponse curves. The significant factors were: enzyme concentration (linear and quadratic terms) and the interaction between enzyme concentration and Ni/Al molar ratio. Since the major obstacle for application of amperometric glucose biosensors is the interference signal resulting from other electro-oxidizable species present in the real matrices, such as ascorbate (AA), the use of different permselective membranes on Pt-LDHGOx modified electrode was discussed with the aim of improving biosensor selectivity and stability. Conventional membranes obtained using Nafion, glutaraldehyde (GA) vapours, GA-BSA were tested together with more innovative materials like palladium hexacyanoferrate (PdHCF) and titania hydrogels. Particular attention has been devoted to hydrogels, because they possess some attractive features, which are generally considered to favour biosensor materials biocompatibility and, consequently, the functional enzyme stability. The Pt-LDH-GOx-PdHCF hydrogel biosensor presented an anti-interferant ability so that to be applied for an accurate glucose analysis in blood. To further improve the biosensor selectivity, protective membranes containing horseradish peroxidase (HRP) were also investigated with the aim of oxidising the interferants before they reach the electrode surface. In such a case glucose determination was also accomplished in real matrices with high AA content. Furthermore, the application of a LDH containing nickel in the oxidised state was performed not only as a support for the enzyme, but also as anti-interferant sistem. The result is very promising and it could be the starting point for further applications in the field of amperometric biosensors; the study could be extended to other oxidase enzymes.

Rehabilitation Engineering in Parkinson's disease

Impairment of postural control is a common consequence of Parkinson's disease (PD) that becomes more and more critical with the progression of the disease, in spite of the available medications. Postural instability is one of the most disabling features of PD and induces difficulties with postural transitions, initiation of movements, gait disorders, inability to live independently at home, and is the major cause of falls. Falls are frequent (with over 38% falling each year) and may induce adverse consequences like soft tissue injuries, hip fractures, and immobility due to fear of falling. As the disease progresses, both postural instability and fear of falling worsen, which leads patients with PD to become increasingly immobilized. The main aims of this dissertation are to: 1) detect and assess, in a quantitative way, impairments of postural control in PD subjects, investigate the central mechanisms that control such motor performance, and how these mechanism are affected by levodopa; 2) develop and validate a protocol, using wearable inertial sensors, to measure postural sway and postural transitions prior to step initiation; 3) find quantitative measures sensitive to impairments of postural control in early stages of PD and quantitative biomarkers of disease progression; and 4) test the feasibility and effects of a recently-developed audio-biofeedback system in maintaining balance in subjects with PD. In the first set of studies, we showed how PD reduces functional limits of stability as well as the magnitude and velocity of postural preparation during voluntary, forward and backward leaning while standing. Levodopa improves the limits of stability but not the postural strategies used to achieve the leaning. Further, we found a strong relationship between backward voluntary limits of stability and size of automatic postural response to backward perturbations in control subjects and in PD subjects ON medication. Such relation might suggest that the central nervous system presets postural response parameters based on perceived maximum limits and this presetting is absent in PD patients OFF medication but restored with levodopa replacement. Furthermore, we investigated how the size of preparatory postural adjustments (APAs) prior to step initiation depend on initial stance width. We found that patients with PD did not scale up the size of their APA with stance width as much as control subjects so they had much more difficulty initiating a step from a wide stance than from a narrow stance. This results supports the hypothesis that subjects with PD maintain a narrow stance as a compensation for their inability to sufficiently increase the size of their lateral APA to allow speedy step initiation in wide stance. In the second set of studies, we demonstrated that it is possible to use wearable accelerometers to quantify postural performance during quiet stance and step initiation balance tasks in healthy subjects. We used a model to predict center of pressure displacements associated with accelerations at the upper and lower back and thigh. This approach allows the measurement of balance control without the use of a force platform outside the laboratory environment. We used wearable accelerometers on a population of early, untreated PD patients, and found that postural control in stance and postural preparation prior to a step are impaired early in the disease when the typical balance and gait intiation symptoms are not yet clearly manifested. These novel results suggest that technological measures of postural control can be more sensitive than clinical measures. Furthermore, we assessed spontaneous sway and step initiation longitudinally across 1 year in patients with early, untreated PD. We found that changes in trunk sway, and especially movement smoothness, measured as Jerk, could be used as an objective measure of PD and its progression. In the third set of studies, we studied the feasibility of adapting an existing audio-biofeedback device to improve balance control in patients with PD. Preliminary results showed that PD subjects found the system easy-to-use and helpful, and they were able to correctly follow the audio information when available. Audiobiofeedback improved the properties of trunk sway during quiet stance. Our results have many implications for i) the understanding the central mechanisms that control postural motor performance, and how these mechanisms are affected by levodopa; ii) the design of innovative protocols for measuring and remote monitoring of motor performance in the elderly or subjects with PD; and iii) the development of technologies for improving balance, mobility, and consequently quality of life in patients with balance disorders, such as PD patients with augmented biofeedback paradigms.

Chemische und enzymatische Synthese von orthogonal stabil geschützten Kohlenhydrat-Scaffolds für kombinatorische Anwendungen

Auf der Suche nach potenten pharmakologischen Wirkstoffen hat die Kombinatorische Chemie in der letzten Dekade eine große Bedeutung erlangt, um innerhalb kurzer Zeit ein breites Spektrum von Verbindungen für biologische Tests zu erzeugen. Kohlenhydrate stellen als Scaffolds interessante Kandidaten für die kombinatorische Synthese dar, da sie mehrere Derivatisierungspositionen in stereochemisch definierter Art und Weise zur Verfügung stellen. So ist die räumlich eindeutige Präsentation angebundener pharmakophorer Gruppen möglich, wie es für den Einsatz als Peptidmimetika wünschenswert ist. Zur gezielten Derivatisierung einzelner Hydroxylfunktionen ist der Einsatz eines orthogonalen Schutz-gruppenmusters erforderlich, das gegenüber den im Lauf der kombinatorischen Synthese herrschenden Reaktionsbedingungen hinreichend stabil ist. Weiterhin ist ein geeignetes Ankersystem zu finden, um eine Festphasensynthese und damit eine Automatisierung zu ermöglichen.Zur Minimierung der im Fall von Hexosen wie Galactose benötigten fünf zueinander orthogonal stabilen Schutzgruppen wurde bei der vorliegenden Arbeit von Galactal ausgegangen, bei dem nur noch drei Hydroxylfunktionen zu differenzieren sind. Das Galactose-Gerüst kann anschließend wiederhergestellt werden. Die Differenzierung wurde über den Einsatz von Hydrolasen durch regioselektive Acylierungs- und Deacylierungs-reaktionen erreicht, wobei auch immobilisierte Enzyme Verwendung fanden. Dabei konnte ein orthogonales Schutzgruppenmuster sequentiell aufgebaut werden, das auch die nötigen Stabilitäten gegenüber sonstigen, teilweise geeignet modifizierten Reaktionsbedingungen aufweist. Für die Anbindung an eine Festphase wurde ein metathetisch spaltbarer Anker entwickelt, der über die anomere Position unter Wiederherstellung des Galactose-Gerüsts angebunden wurde. Auch ein oxidativ spaltbares und ein photolabiles Ankersystem wurden erprobt.

Zur Kooperativität des Vogelspinnen-Hämocyanins

Zusammenfassung Kooperativität bei Atmungsproteinen bedeutet eine Änderung der O2-Affinität während der Beladung mit O2 und läßt sich durch die Wechselbeziehung zwischen O2-Beladung und Konformation beschreiben. In dieser Arbeit wurde das 24-mere Hämocyanin der Vogelspinne Eurypelma californicum bzgl. Beladung und Konformationen sowohl auf der Ensemble- wie auch auf der Einzel-Molekül-Ebene charakterisiert. EnsembleDie Bindung von O2 an Hämocyanine ist mit einer drastischen Abnahme der Fluoreszenz-Quantenausbeute verbunden. Durch Vergleich von theoretisch und experimentell bestimmten Quantenausbeuten konnte gezeigt werden, daß die Löschung auf Förster-Transfer zurückzuführen ist, und daß kein Einfluß der Oligomerisierung, Protein-Konformation, Beweglichkeit oder Tierart besteht. Die Konformation von Hämocyaninen konnte mit Crosslinkern im oxy- und deoxy-Zustand fixiert werden. Die Charakterisierung der Produkte führte zu einer neuen Vorstellung, wie unterschiedliche Affinitäten realisiert sein können. Hierbei kommt der Dynamik der Protein-Matrix eine entscheidende Rolle zu. Einzel-MoleküleMittels Zwei-Photonen-Anregung konnten erstmalig einzelne Proteine über ihre intrinsische Tryptophan-Fluoreszenz nachgewiesen werden. Zum einen gelang es, Modellsysteme mit nur 340 Trp abzubilden, andererseits konnte die Diffusion einzelner Hämocyanine (148 Trp) mit Hilfe der Fluoreszenz-Korrelationsspektroskopie nachgewiesen werden.Einzelne Hämocyanine konnten durch Adsorption und mildes Eintrocknen an verschiedenen Oberflächen immobilisiert werden. Mittels Atomarer Kraft-Mikroskopie (AFM) ließen sich individuelle Hämocyanine abgebilden, wobei Details der Quarärstruktur aufgelöst werden konnten.

Development of muliplexed analytical methods based on bioluminescent whole-cell biosensors

The subject of this Ph.D. research thesis is the development and application of multiplexed analytical methods based on bioluminescent whole-cell biosensors. One of the main goals of analytical chemistry is multianalyte testing in which two or more analytes are measured simultaneously in a single assay. The advantages of multianalyte testing are work simplification, high throughput, and reduction in the overall cost per test. The availability of multiplexed portable analytical systems is of particular interest for on-field analysis of clinical, environmental or food samples as well as for the drug discovery process. To allow highly sensitive and selective analysis, these devices should combine biospecific molecular recognition with ultrasensitive detection systems. To address the current need for rapid, highly sensitive and inexpensive devices for obtaining more data from each sample,genetically engineered whole-cell biosensors as biospecific recognition element were combined with ultrasensitive bioluminescence detection techniques. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial, yeast or mammalian cells a vector expressing a reporter protein whose expression is controlled by regulatory proteins and promoter sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein that can be readily measured and directly related to the analyte bioavailable concentration in the sample. Bioluminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in small sample volumes allowing an accurate signal localization and quantification. In the first chapter of this dissertation is discussed the obtainment of improved bioluminescent proteins emitting at different wavelenghts, in term of increased thermostability, enhanced emission decay kinetic and spectral resolution. The second chapter is mainly focused on the use of these proteins in the development of whole-cell based assay with improved analytical performance. In particular since the main drawback of whole-cell biosensors is the high variability of their analyte specific response mainly caused by variations in cell viability due to aspecific effects of the sample’s matrix, an additional bioluminescent reporter has been introduced to correct the analytical response thus increasing the robustness of the bioassays. The feasibility of using a combination of two or more bioluminescent proteins for obtaining biosensors with internal signal correction or for the simultaneous detection of multiple analytes has been demonstrated by developing a dual reporter yeast based biosensor for androgenic activity measurement and a triple reporter mammalian cell-based biosensor for the simultaneous monitoring of two CYP450 enzymes activation, involved in cholesterol degradation, with the use of two spectrally resolved intracellular luciferases and a secreted luciferase as a control for cells viability. In the third chapter is presented the development of a portable multianalyte detection system. In order to develop a portable system that can be used also outside the laboratory environment even by non skilled personnel, cells have been immobilized into a new biocompatible and transparent polymeric matrix within a modified clear bottom black 384 -well microtiter plate to obtain a bioluminescent cell array. The cell array was placed in contact with a portable charge-coupled device (CCD) light sensor able to localize and quantify the luminescent signal produced by different bioluminescent whole-cell biosensors. This multiplexed biosensing platform containing whole-cell biosensors was successfully used to measure the overall toxicity of a given sample as well as to obtain dose response curves for heavy metals and to detect hormonal activity in clinical samples (PCT/IB2010/050625: “Portable device based on immobilized cells for the detection of analytes.” Michelini E, Roda A, Dolci LS, Mezzanotte L, Cevenini L , 2010). At the end of the dissertation some future development steps are also discussed in order to develop a point of care (POCT) device that combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. In this POCT perspective, field-flow fractionation (FFF) techniques, in particular gravitational variant (GrFFF) that exploit the earth gravitational field to structure the separation, have been investigated for cells fractionation, characterization and isolation. Thanks to the simplicity of its equipment, amenable to miniaturization, the GrFFF techniques appears to be particularly suited for its implementation in POCT devices and may be used as pre-analytical integrated module to be applied directly to drive target analytes of raw samples to the modules where biospecifc recognition reactions based on ultrasensitive bioluminescence detection occurs, providing an increase in overall analytical output.