Thèses sur le sujet « Proteinaceou »

Le nanoparticelle metalliche, grazie alle loro affascinanti proprietà ottiche ed elettrochimiche, attirano l'attenzione di diverse discipline scientifiche e di ricerca ingegneristica. Tra queste proprietà, l'effetto fototermico indotto dalle risonanze plasmoniche, è una caratteristica notevole ed esclusiva delle nanoparticelle di metalli nobili che, ad oggi, vengono già sfruttate per vari scopi, sia per ricerca che, soprattutto, per applicazioni biomediche. Il fenomeno della risonanza plasmonica superficiale, caratterizzato da bande ben definite, fornisce a queste nanoparticelle una notevole la flessibilità nella risposta ottica che si può vantaggiosamente trasferire a matrici polimeriche in cui queste vengano disperse. In particolare, la possibilità di indurre un aumento di temperatura altamente localizzato che può essere attivato tramite un dispositivo di stimolazione esterno come una sorgente di luce, renderebbe tali matrici strumenti preziosi nel campo dei trattamenti cellulari e, in generale, dell'ingegneria dei tessuti In questa tesi, la tecnica di scrittura (photo-cross-link) laser diretta (DLW), attivata da assorbimento a due fotoni, è stata impiegata per fabbricare micro-architetture con il diverso modulo elastico (da 80kPa a 800kPa) a partire da un inchiostro proteico composto da albumina di siero bovino (BSA), un foto-iniziatore (Rose Bengale o blu di metilene) e nanoparticelle d'oro a simmetria non sferica (GNP). Mostriamo qui che la presenza di queste ultime, se opportunamente schermate dall’interazione con il foto-iniziatore, fornisce alle microstrutture foto-polimerizzate la capacità di generare un aumento della temperatura locale mediante stimolazione nella regione spettrale del vicino infrarosso. L'efficienza fototermica misurata sotto l’effetto di radiazione laser focalizzata a 800 nm (in continua) su microstrutture caricate con una bassa concentrazione di atomi d'oro (1% w/w) ha raggiunto 12.2 pm 0.4 C/W, che costituisce un record di effetto fototermico indotto su una microstruttura a base proteica proteinica stampata tramite DLW. La funzionalità foto-termica derivante dalle GNP incorporate nelle microstrutture proteiche fabbricate riveste una notevole potenzialità nello studio delle risposte di sistemi viventi, come cellule e colture di batteri, al rilascio di calore altamente localizzato e controllato sia per quanto riguarda il tempo di irraggiamento che la dose rilasciata.
Metallic nanoparticles, due to their fascinating optical and electrochemical properties, attract the attention of different science and engineering research disciplines. Among these properties, the plasmonic photothermal effect is a notable and exclusive feature of noble-metal nanoparticles that, by today, are exploited through lots of research activities for various purposes, especially for biomedical applications. This optically-tunable phenomenon uniquely increases the flexibility of the optical response of host matrices, by allowing to induce highly localized temperature increases that can be triggered via simple external stimulation device like a light source. Such matrices can be valuable tools in the field of cell treatments and, in general, tissue engineering. In the present study, the two-photon-assisted direct laser writing (DLW) technique was employed to fabricate microarchitectures with the different elastic modulus (80kPa to 800kPa) from a proteinaceous ink composed of bovine serum albumin (BSA), rose Bengal (RB), or methylene blue (MB), and non-spherical symmetric gold nanoparticles (GNPs), with the ability to generate local temperature increase by stimulation in the near-infrared spectral region. The recorded photothermal efficiency measured using focused continuous wave (CW) laser irradiation at 800nm on microstructures loaded with GNPs at low gold atom concentration (1%w/w) reached 12.2 pm 0.4 C/W, that is a record photothermal effect induced from a printed proteinaceous feature. This photo-thermal functionality arising from the GNPs embedded within the fabricated proteinaceous microstructures can then be applied for studying responses of living systems like cells and bacteria cultures under an externally triggered highly localized heat release.

La present tesi està formada per sis treballs de recerca independents que estan situats en el camp dels inhibidors de metal·locarboxipeptidases: s'estudia el seu plegament, estabilitat, estructura i funció.
El primer treball inclou l'aïllament i el clonatge de l'ADNc d'un nou inhibidor de carboxipeptidases de la paparra, anomenat TCI. Els estudis realitzats sobre la forma recombinant del TCI indiquen que aquesta proteïna està fortament limitada pels seus ponts disulfur, sent molt estable enfront un ampli rang de pH i altament resistent a condicions desnaturalitzants. El TCI recombinant és un inhibidor que s'uneix fortament a TAFIa, estimulant així la fibrinòlisi in vitro, fet que li confereix potencial en aplicacions de prevenció o tractament de desordres trombòtics.
El segon treball presenta l'estructura cristal·lina del TCI unit a la carboxipeptidasa A bovina i a la carboxipeptidasa B humana. El TCI està format per dos dominis que són estructuralment molt semblants a pesar de tenir un baix grau d'homologia seqüencial. Cada domini inclou una hèlix a curta seguida per una petita fulla b antiparal·lela girada i presenta una alta homologia estructural enfront proteïnes de la família de les b-defensines. El TCI s'ancora a la superfície de les carboxipeptidases de mamífer en un mode d'unió doble que no s'havia observat abans per cap altre inhibidor de carboxipeptidases.
En el tercer treball s'examina la contribució de cadascun dels aminoàcids del lloc d'unió secundari de l'inhibidor de carboxipeptidases de patata (PCI) de cara a les propietats generals d'aquesta proteïna. Els estudis estructurals i enzimàtics demostren que el posicionament correcte del lloc d'unió primari per una unió i inhibició eficient de la carboxipeptidasa A depèn significativament del lloc d'unió secundari. A més, l'estudi comparatiu del plegament oxidatiu d'una sèrie de mutants del PCI utilitzant una aproximació de captura dels ponts disulfur indiquen que les forces no covalents dirigeixen el replegament d'aquesta petita proteïna rica en ponts disulfur a l'etapa de "reshuffling", que és el pas limitant del procés de plegament del PCI.
En el quart treball s'ha determinat els camins de plegament oxidatiu i desplegament reductor de l'inhibidor de carboxipeptidases de sangonera (LCI). L'LCI reduït i desnaturalitzat és replega ràpidament a través d'un flux seqüencial d'espècies amb un, dos, tres i quatre ponts disulfur fins assolir la forma nativa. Dins dels intermediaris de plegament de l'LCI hi trobem dues espècies predominants de tres ponts disulfur (anomenades III-A i III-B) i una població heterogènia d'isòmers "scrambled" (quatre ponts disulfur no natius) que s'acumulen consecutivament al llarg de la reacció de plegament. Aquest estudi revela que les formes III-A i III-B contenen exclusivament ponts disulfur natius i corresponen a espècies estables i parcialment estructurades que s'interconverteixen entre elles assolint l'equilibri abans que la formació dels isòmers "scrambled" tingui lloc.
En el cinquè treball s'ha purificat l'intermediari III-A directament de la reacció de plegament i se l'ha caracteritzat estructuralment per RMN. Els resultats mostren que aquesta espècie presenta una estructura força nativa, tot i que li manquen alguns elements d'estructura secundària per la qual cosa és més flexible que l'LCI natiu. III-A representa el primer exemple d'un intermediari "disulfide insecure" ("ponts disulfur insegurs") determinat estructuralment. L'oxidació directa d'aquesta espècie cap a la proteïna totalment nativa sembla estar restringida per l'enterrament de les seves dues cisteïnes dins d'una estructura semblant a la nativa. També s'han utilitzat aproximacions teòriques basades en constriccions topològiques que prediuen força bé la presència d'aquest intermediari de plegament.
L'últim treball d'aquesta tesi tracta sobre l'altre intermediari majoritari de plegament de l'LCI. En aquest treball s'ha construït i analitzat extensivament un anàleg d'aquest intermediari. Els resultats obtinguts mostren que aquesta proteïna presenta una estructura global i una activitat semblants a les de la forma salvatge de l'LCI. A més, presenta un procés de plegament més ràpid i eficient que l'LCI salvatge. Tanmateix, aquest anàleg està fortament desestabilitzat, fet que indica que el quart pont disulfur dóna alta estabilitat i especificitat estructural a l'LCI.
The present thesis consists of six independent research works that are situated in the field of metallocarboxypeptidase inhibitors: their folding, stability, structure and function are studied.
The first work comprises the isolation and cDNA cloning of a new carboxypeptidase inhibitor from ticks, named TCI. The studies performed on the recombinant form of TCI indicate that this protein is strongly constrained by its disulfide bonds, being unusually stable over a wide pH range and highly resistant to denaturing conditions. As a tight binding inhibitor of TAFIa, TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.
The second work presents the crystal structure of TCI in complex with either bovine carboxypeptidase A or human carboxypeptidase B. The structure of TCI consists of two domains that are structurally similar despite the low degree of sequence homology. The domains, each consisting of a short a-helix followed by a small twisted antiparallel b-sheet, show high structural homology to proteins of the b-defensin-fold family. Also, TCI anchors to the surface of mammalian carboxypeptidases in a double-headed manner not previously seen for carboxypeptidase inhibitors.
In the third part, the role of each residue of the secondary binding site of potato carboxypeptidase inhibitor (PCI) in the folding, structure and function of this protein is determined. Structural and enzymatic studies demonstrate that the proper positioning of the primary site for efficiently binding and inhibition of carboxypeptidase A is significantly dependent on such a secondary contact region. In addition, a comparative study of the oxidative folding of several PCI mutants indicates that noncovalent forces drive the refolding of this small disulfide-rich protein at the reshuffling stage, the rate-limiting step of the process.
The fourth work elucidates the oxidative folding and reductive unfolding pathways of leech carboxypeptidase inhibitor (LCI). Reduced and denatured LCI refolds rapidly through a sequential flow of 1-, 2-, 3-, and 4-disulfide (scrambled) species to reach the native form. Folding intermediates of LCI comprise two predominant 3-disulfide species (III-A and III-B) and a heterogeneous population of scrambled isomers that consecutively accumulate along the folding reaction. Our study reveals that forms III-A and III-B exclusively contain native disulfide bonds and correspond to stable and partially structured species that inter-convert, reaching an equilibrium prior to the formation of the scrambled isomers.
In the fifth work, the III-A intermediate is directly purified from the folding reaction and structurally characterized by NMR spectroscopy. The data shows that this species displays a highly native-like structure although it lacks some secondary structure elements being more flexible than native LCI. III-A represents the first structurally determined example of a disulfide insecure intermediate; direct oxidation of this species to the fully native protein seems to be restricted by the burial of its two free cysteine residues inside a native-like structure. We show also that theoretical approaches based on topological constrains predict with good accuracy the presence of this folding intermediate.
The last work of this thesis deals with the other major folding intermediate of LCI: III-B intermediate. In this work, an analog of this intermediate is constructed and extensively analyzed. The derived data shows that this protein displays the same overall structure, a similar activity, a faster and more efficient folding process than wild-type LCI, but a lower stabilization, which indicates that the fourth disulfide bond provides LCI with both high stability and structural specificity.

La maturation des ARNt en 5' est réalisée par RNase P. C'est un ribozyme chez les bactéries, les fungi et les nuclei des mammifères et un enzyme protéique dans les plantes ou des organelles des mammifères qui s’appelle PRORP. Il y a trois PRORP dans A. thaliana. PRORP contiennent deux domaines : un domaine PPR qui reconnaît spécifiquement des séquences d'ARN et un domaine nucléase qui assure la coupure endonucléolytique 5' des précurseurs d’ARNt. Pendant ma thèse j'ai pu montré par des méthodes biophysiques et structurales comme SRCD et SAXS que PRORP1 et 2 sont composées en majorité des hélices alpha Elles ont un rayon de giration de 33 Å et contiennent deux domaines distincts avec et une dimension maximale de 110 Å. Pour le complex entre un substrat d'ARNt et PRORP une constante de dissociation de 1 uM a pu être confirmé par la microcalorimétrie, la thermophorèse et l'ultracentrifugation analytique. Ces analyses nous ont permis de construire un modèle PRORP et un substrat d'ARNt
RNase P cleaves 5’ leaders of precursor tRNAs. RNase P is a ribozyme in bacteria, fungi and animal nuclei and a protein in animal organelles, plants and many other organism. There are three PRORPs in A. thaliana. MALS, SRCD and SAXS provided first structural information: 1) PRORPs are monomers in solution. 2) PRORP 1-2 have a high alpha-helical content. 3) PRORPs are composed of two distinct domains with a radius of gyration of 33 A. These results together with homology modelling enabled us to build a first model of PRORPs in complex with tRNA. Using three different methods, isothermal titration calorimetry, microscale thermophoresis and analytical ultracentrifugation, a binding constant of about 1 µM could be determined for the system PRORP2mDD and L5T0 tRNA. This helped us conducting a SAXS experiment taking into account the low resolution affinity and designed to provide the direct structural data of a complex of proteinaceous RNase P with a substrate tRNA

Soil carbon (C) and nitrogen (N) are predominantly in organic form. Proteins/ peptides, as an important organic form of N, constitute a substantial part of soil organic matter. On one hand, proteins/peptides are an important N source for plants and microorganisms, particularly in soils where inorganic N is limited. On the other hand, their stabilization in soils by forming organo-mineral associates or macromolecule complex reduces the C loss as CO2 into the atmosphere. Therefore, studies on the turnover, abundance, composition, and stability of proteins/peptides are of crucial importance to agricultural productivity and environmental sustainability. In the first part of this study, the bioavailability and distribution of amino acids, (building block of proteins/peptides), were investigated, in soils across the North-South and West-East transects of continental United States. The second part of this study aimed to understand the variations of organic C speciation in soils of continental United States. Previous investigations of the interactions between soil minerals and proteins/peptides were mostly limited to batch sorption experiments in labs, seldom of which gave the details at the molecular scales. Therefore, in the third part of this study, the molecular orientation of self-assembled oligopeptides on mineral surfaces was investigated by employing synchrotron based polarization-dependent Near Edge X-ray Adsorption Fine Structure Spectroscopy (NEXAFS) techniques. Specific aims of this study were: 1) to assess potentially bioavailable pool of proteinaceous compounds and the immediately bioavailable pool of free amino acids in surface and subsurface soils of various ecosystems; 2) to evaluate the relationship between environmental factors and levels/composition of the two pools; 3) to investigate the organic C speciation in soils of various land use; and 4) to understand molecular level surface organization of small peptides on mineral surfaces. The levels of free amino acids and hydrolysable amino acids which represent the potentially bioavailable pool of proteinaceous compounds in A-horizon soils were significantly high than in C-horizon soils due to the accumulation of organic matter in surface. On average, free amino acids accounted for less than 4 % of hydrolysable amino acids which represent the total proteinaceous compounds in soils. The composition of free amino acids was significantly different between surface soil and subsurface soil and was significantly influenced by mean annual temperature and precipitation. A relatively uniform composition of hydrolysable amino acids was observed irrespective of a wide range of land use. Significant variations were observed for the levels of free and hydrolysable amino acids along mean annual temperature and precipitation gradients, as well as among vegetation types of continental USA, suggesting levels of free and hydrolysable amino acids were associated with the above-ground biomass and root distribution. Organic C speciation investigation revealed the presence of carboxylic-C (38%), aliphatic-C (~ 22%), aromatic-C (~ 18%), O/N-alkyl-C (~ 16%), and phenolic-C (< 6%). Factors such as temperature and vegetation cover were revealed in this study to account for the fluctuations of the proportions of aromatic-C and phenolic-C, in particular. Phenolic-C may serve as a good indicator for the effect of temperature or vegetation on the composition of SOC. The average composition of soil organic C, over the continental scale, was relatively uniform over various soil ecosystems and between two soil horizons irrespective of surface organic C content. Polarization dependent NEXAFS analysis showed the oligopeptides tend to orient on mineral surface with an average tilt angle of 40 ° between the molecular chain and the mineral surface.
Ph. D.

The emerging evidence of preferential accumulation and long residence time of proteinaceous compounds in soil are counter to the traditional view that their structure is readily broken down through microbial activity. The shift in thinking of their residence time is, however, heavily influenced by physical and chemical protections in soil, representing an important change for understanding global biogeochemical carbon and nitrogen cycling. We investigated the accumulation patterns of proteinogenic amino acids for a long term (thousands of years) related to their sources and sinks. We found clear patterns of change in the amino acids in a 4000 year-chronosequence adjacent to Lake Michigan, USA (Michigan chronosequence) and they were tightly related to the shifts in their biological sources, namely aboveground vegetative community (r2=0.66, p<0.0001) and belowground microbial community (r2=0.71, p<0.0001). Results also showed great variations of approximately 49% between seasons (summer and winter). Moreover, seasonal dynamic patterns (22% variations) of the amino acids in soil mineral associated fraction were rather counter to the conceptual view that it represents a slow soil organic pool with long residence times. The amino acids enriched in the mineral associated fraction, (e.g., positively charged, aromatic, and sulfur containing amino acids), tended to preferentially accumulate in whole soil pool during the 4000 years of ecosystem development. Their interaction with soil minerals, therefore, may play a critical role in the long-term sink and selective accumulation of proteinaceous compounds with some degree of the displacement. This was further confirmed by another chronosequence system near Haast River, New Zealand, which is geologically separated and climatically- and ecologically- different from the Michigan chronosequence. Common trends between two chronosequences suggested that either polar interactions or redox reactions may be relatively more important in the mineral interaction of amino acids than non-polar interactions. The consistency of results at two disparate locations in the southern and northern hemispheres is strong evidence that the processes of pedogenesis and ecosystem development are parsimonious and predictable. Our research demonstrated fundamental understanding of behavior of proteinaceous compounds at the molecular species level, and further provided their partitioning mechanisms associated with soil components.
Ph. D.

Organic nitrogen containing compounds represent the vast majority of natural N inputs to soils and of these, proteins, peptides and amino acids are the most significant. In order to facilitate the elucidation of both the nature and rates of the processes by which these vital nutrients are transformed in soils, an understanding of their fate at a mole; level is vital. This thesis aimed to utilise soil incubation experiments with dual (¹⁵N- and ¹³C-) labelled substrates, i.e. a suite of amino acids, individual amino acids and more complex substrates, i.e. protein and cow dung, as isotopically labelled trace enabling their N and C to be followed over time. A molecular level approach was adopted which included the application of chemically and isotopically defined substrates to soil and the use of compound-specific stable isotope (N and C) analysis to trace the fate of label into the soil amino acids (and in selected cases phospholipids, C only).

Thesis (MTech (Chemical Engineering))--Cape Peninsula University of Technology, 2016.
Poultry slaughterhouse wastewater (PSW) contains organic matter that can be degraded by microorganisms. Such matter can further be used by the microbial community as a nutrient source for growth. Moreover, this type of wastewater also contains a high quantity of particulate matter, lipids and proteins, including antimicrobial compounds such as triclosan (TCS) and trichlorocarbanilide (TCC) used during cleaning and sanitising of processing facilities. Lipids and particulate matter lead to clogging of pipes and fouling of diffusers in the wastewater treatment plants (WWTPs). To overcome this problem, a pre-treatment system such as a dissolved air flotation system (DAFs) in which synthetic flocculants are used, is commonly used prior to the biological treatment of the wastewater. Synthetic flocculants add to the environmental burden associated with the use of synthetic compounds, particularly when these compounds are used in WWTPs. This study focused on the reduction of suspended solids, lipids and proteinaceous matter using a bioflocculant- supported DAF for the treatment of PSW.

Thesis (Ph. D.)--Ohio State University, 2004.
Document formatted into pages; contains xiv, 143 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 June 21.

Dissertação Apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Ciências da Conservação, especialização em Pintura
A novel use of ionic liquids as alternative solvents for enzymes in cleaning treatments for the removal of proteinaceous materials from painted or gilded surfaces is presented. The ionic liquids are potentially green solvents to be applied in restoration treatments being also called designer solvents, because of their peculiar properties which can be adjusted by selecting different cationanion combinations. Two ionic liquids were selected: IL1)1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4])and IL2) 1-ethyl-3-methylimidazolium ethylsulfate ([EMIM][EtSO4]). Formulations were prepared with these ionic liquids and two different proteases: one acid (pepsin) and one alkaline (from Aspergillus sojae). Additionally aqueous gel formulations were prepared with these enzymes for reference purpose. A third enzyme provided by the Bromatology Department at the Faculty of Pharmacy from the Porto University was tested only in gel formulation in order to assess its potential use in cleaning treatments. To understand the enzyme activity of these formulations and predict their ability as cleaning agents, analyses were performed with ultraviolet–visible (UV-Vis) spectroscopy and highperformance liquid chromatography (HPLC) prior cleaning; and with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) after cleaning. These formulations were tested on mock-up samples prepared in accordance with documented and historical sources of artistic techniques of egg tempera and oil painting, and gilding. A non-invasive non-destructive multi-scale analytical protocol was carried out for cleaning effectiveness evaluation and surface characterization before and after treatment. Different surface analytical techniques were adopted to this purpose: stereomicroscopy (SM), optical microscopy (OM) with visible and fluorescence light, atomic force microscopy (AFM), scanning electron microscopy (SEM) and electron dispersive spectroscopy (EDS) and colorimetry (CIE L*a*b* system). The surface analytical protocol proved to be adequate, not only, for monitoring the cleaning process but also for complete characterization of the surface, before and after treatment, including information on the presence of residues and possible surface deterioration. It was also proved that the formulations of enzymes combined with ILs can be used successfully for the removal of proteinaceous material as alternatives to gel formulations. More studies should be conducted to determine the most suitable IL or group of ILs, the main concern should focus on improving aspects such as compatibility with other surface materials, and possible long-term effects of residues after cleaning.

Seme tikve (Cucurbita pepo) obiluje kako uljem tako i proteinima. Nakon izdvajanja ulja, proteini se koncentrišu u uljanoj pogači, sekundarnom proizvodu procesa proizvodnje ulja, gde njihov sadrţaj dostiţe do 65%. Proteini semena tikve su primamljiv sastojak za prehrambenu, farmaceutsku i kozmetičku industriju zbog svoje farmakološke aktivnosti i visoke biološke vrednosti. Pored toga, budući da su mnogi proizvodi ovih industrija po svojoj koloidnoj prirodi emulzije, proteini semena tikve bi se u njima mogli naći i kao prirodne površinski aktivne materije. Međutim, koloidna funkcionalnost proteina semena tikve se još uvek potcenjuje zbog globularne strukture za koju se vezuju slabija funkcionalna svojstva u odnosu na proteine sa fleksibilnijom strukturom. Prema tome, cilj ove disertacije je ispitivanje funkcionalnih osobina proteinskog izolata semena tikve, pre svega njegovih adsorpcionih i emulgujućih svojstava, kao i ispitivanje uticaja promene proteinske strukture putem enzimske hidrolize na ispitivana svojstva.Pripremljeni su izolat proteina semena tikve (IPST) i dva enzimska hidrolizata, H1 i H2. IPST, H1 i H2 su okarakterisani određivanjem sadržaja vlage, proteina i pepela, zatim, određivanjem prinosa, molekulske mase i zeta potencijala. Ispitan je uticaj koncentracije proteina/peptida (0,0001–1 g/100 cm³), pH (3–8) i jonske jačine (0–1 mol/dm³ NaCl) na rastvorljivost i adsorpciona svojstva: dinamički međupovršinski pritisak (ulje/voda), statički površinski (vazduh/voda) i međupovršinski (ulje/voda) pritisak, kinetiku adsorpcije i dilatacionu reologiju proteinskih adsorpcionih filmova. Nakon toga, ispitan je i uticaj pomenutih parametara na emulziona svojstva IPST, H1 i H2. Emulgujuća svojstva IPST, H1 i H2 su okarakterisana na osnovu prosečnog prečnika kapljica emulzija, raspodele veličina kapljica i stabilnosti emulzija.Utvrđeno je da je prinos IPST veći od prinosa oba hidrolizata za oko 65 %. IPST ima najniţu rastvorljivost na pH=5, što ujedno predstavlja i njegovu izoelektričnu tačku. Enzimskom hidrolizom IPST značajno se povećava rastvorljivost, posebno na pI=5. Povećanje jonske jačine je izazvalo salting–in ili salting–out efekat rastvorljivosti kod svih uzoraka u zavisnosti od pH. IPST, H1 i H2 poseduju površinsku aktivnost pri čemu je površinski/međupovršinski pritisak H1 i H2 manje zavistan od promene pH i jonske jačine u poređenju sa površinskim/međupovršinskim pritiskom IPST. Adsorpcijom na granicu faza IPST i oba hidrolizata obrazuju adsorpcione filmove sa dominantnom elastičnom komponentom. Emulgujuća svojstva IPST, H1 i H2 zavise od koncentracije uzorka, pH vrednosti i jonske jačine kontinualne faze. Pri koncentraciji od 1 g/100 cm³ i Ic=0 mol/dm³ pripremljene emulzije su stabilne na svim pH osim emulzije IPST na pH 5. Sve emulzije podležu gravitacionoj nestabilnosti.
Pumpkin (Cucurbita pepo) seed is rich source of both, oil and proteins. Once the oil has been extracted, proteins concentrate in oil cake, a by–product of the oilextraction process, where their content can reach up to 65%. Pumpkin seed proteins are desirable ingredient in food, pharmaceutical and cosmetic industry due to their pharmacological activities and high biological value. Moreover, since many of products of these industries are, in colloidal terms, emulsions, pumpkin seed proteins could serve as surface active materies. However, colloidal functionality of pumpkin seed proteins is still underestimated for their globular structure which entails inferior functional properties to functional properties of proteins with more flexible structure. Based on that, the aim of this dissertation is to investigate functional properties of pumpkin seed protein isolate, adsorption and emulsifying properties, in the first place, and then to investigate the influence of modification of the protein structure, by means of enzymatic hydrolysis, on the aforementioned properties.Pumpkin seed protein isolate, IPST, and two enzymatic hydrolysates, H1 and H2, were prepared. IPST, H1 and H2 were characterized by determination of moisture, ash and protein content, then, by determination of protein recovery, molecular mass and zeta potential. Influence of the protein/peptide concentration (0.0001–1 g/100 cm³), pH (3–8) i ionic strength (0–1 mol/dm³ NaCl) on the solubility and adsorption properties: dynamic interfacial (oil/water) pressure, static surface (air/water) and interfacial (oil/water) pressure, adsorption kinetics and interfacial dilatational properties, wasinvestigated next. In the end, influence of the aforementioned pharameters on the emulsifying properties of IPST, H1 and H2 was investigated. Emulsifying properties of IPST, H1 and H2 were discussed in terms of mean droplet diameter, droplet size distribution and emulsion stability.Protein recovery of IPST was determined to be 65 % higher than recovery of H1 and H2. Solubility of IPST was the lowest at pH 5, what presents the isoelectric point. The enzymatic hydrolysis of IPST significantly increased solubility, especialy at the isoelectric point. Increase in the ionic strenght led to salting–in or salting–out effect depending on pH of the sample. Three investigated samples, IPST, H1 and H2 exhibited surface activity, however, sufrace/interfacial pressure of H1 and H2 were found to be less influenced by change in pH or ionic strenght of the solution in comparison to the IPST. Once adsorbed to the interface IPST and both hydrolysates form interfacial film with dominant elastic component. Emulsifying properties of IPST, H1 and H2 depend on the concentration, pH and ionic strength of the continuous phase. Stabile emulsions were formed at concentration of 1 g/100 cm3 and Ic=0 mol/dm³ regardless of pH, with the exception of the IPST at pH 5. All emulsions were susceptibile to gravitational separation.

Les invertases de plantes (Invs) hydrolysent de manière irréversible le saccharose en fructose et glucose. En fonction de leur pH optimum et de leur localisation subcellulaire, les Invs sont classées en trois groupes : alcaline et neutre (A/N-Inv), vacuolaire (VI) et de paroi (CWI). Le but de notre étude a été de mieux comprendre les mécanismes impliqués dans les régulations post-traductionnelles d'une VI de Solanum lycopersicum (VINV). L'ADNc codant pour VINV a été cloné et exprimé dans le système hétérologue Pichia pastoris. Après purification, la caractérisation biochimique a été réalisée et a montré des résultats comparables à ceux obtenus précédemment pour d'autres Invs. La structure tridimensionnelle de VINV a été résolue par cristallographie aux rayons X à 2,75 Å et il s'agit de la première structure d'une VI décrite jusqu'ici. Des expériences de mutagénèse dirigée ont permis d'identifier certains acides aminés impliqués dans la catalyse : le nucléophile, le catalyseur acide/base, le stabilisateur d'état de transition et un résidu qui module le pKa du catalyseur acide/base. Par ailleurs, la régulation de l'activité de VINV a été étudiée. La N-glycosylation de VINV recombinante semble être importante pour la stabilité de la structure. De plus, l'activité VINV peut aussi être modulée par un inhibiteur protéique spécifique. Une approche de génomique fonctionnelle a été utilisée, et un inhibiteur d'invertase vacuolaire putatif (SolyVIF) de S. lycopersicum a été identifié dans la banque de données des Solanacées. L'ADNc codant pour SolyVIF a été cloné et exprimé dans le système hétérologue Escherichia coli Rosetta gami (DE3)
Plant invertases (Invs) hydrolyze irreversibly sucrose into fructose and glucose. Based on their pH optima and subcellular localization, Invs are categorized into three groups: alkaline and neutral invertase (A/N-Inv), vacuolar invertase (VI), and cell wall invertase (CWI). The goal of our study was to better understand mechanisms involved in the molecular regulation of a VI from Solanum lycopersicum (VINV) at post-translational levels. The VINV cDNA was cloned and heterologously expressed in Pichia pastoris. After purification, the biochemical characterization was performed and showed comparable results with those obtained previously for other characterized Invs. The three-dimensional structure of VINV was solved by X-ray crystallography to 2.75 Å resolution and it was the first structure of a plant VI described so far. Mutations experiments allowed to identify important amino acids: the nucleophile, the acid/base catalyst, the transition-state stabilizer and a residue that modulate pKa of the acid/base catalyst. Moreover, the regulation of VINV at different post-translational levels was studied. N-glycosylation of recombinant VINV seems to be important for structure stability. VINV activity can also be modulated by specific proteinaceous inhibitor. A functional genomics approach was used, and a putative vacuolar invertase inhibitor (SolyVIF) of S. lycopersicum was identified in the Solanaceae data bank. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli Rosetta gami (DE3). Recombinant protein was purified and characterized

Easel paintings are important Cultural Heritage assets with significant historic and cultural value. They usually possess a multi-tiered structure, composed of different layers, some of which may present protein binders. Proteins have been commonly used as paintings medium, adhesives and coating layers in easel paintings. Hence, their recognition is a crucial step for easel painting’s conservation and restoration processes. The present work presents a novel fluorescent labelling methodology, using a coumarin derivative chromophore, C392STP (sodium (E/Z)-4-(4-(2-(6,7- dimethoxycoumarin-3-yl)vinyl)benzoyl)-2,3,5,6-tetrafluorobenzenesulfonate) as a fluorophore probe to bond proteinaceous binders used in paintings. The method was developed and optimized using commercial proteins and proteins extracted from hen’s egg yolk and white, bovine milk, and rabbit skin. In order to mimic the real conditions, paint models of easel paintings have been prepared by mixing proteins such as ovalbumin, casein and rabbit glue with different pigments (lead white, chrome yellow and black bone) and the fluorescent labelling method was miniaturized and tested. The results revealed that proteins in concentration as low as 6.0 μg/ml could be detected. Finally, for validation methodology, real micro samples of easel paintings were analyzed. The extracted proteins were submitted to the fluorescent labelling method developed and clearly identified in electrophoretic profiles. The results evidence the applicability of this methodology as an effective and useful analytical tool for the identification of protein binders obtained from easel paintings and, possibly in other art work. Additionally, theoretical quantum chemical calculations based on the Density Functional Theory (DFT) and Time Dependent Density Functional Theory (TD-DFT) have been performed in the C392STP coumarin and in a related coumarin derivative ((E/Z)-4-(2-(6,7- dimethoxycoumarin-3- yl)vinyl)-N-propylbenzamide), that mimics the coumarin bonded to lysine. The calculations confirm the experimental trends in absorption wavelengths and are in good agreement with the experimental absorption spectra, providing a comprehensive characterization of the main spectral features of the studied compounds; RESUMO: Identificação de ligantes proteicos em arte As pinturas de cavalete são um componente importante do Património Cultural, com um significativo valor histórico e cultural. Geralmente possuem uma estrutura composta por diferentes camadas, algumas das quais podem apresentar ligantes proteicos. As proteínas surgem geralmente em pinturas de cavalete como meio de suporte da pintura, adesivos e camadas de revestimento. A sua identificação é, portanto, um passo crucial para os processos de conservação e restauração da pintura de cavalete. O presente trabalho apresenta uma nova metodologia de marcação fluorescente, utilizando um cromóforo derivado da cumarina, C392STP ((E/Z)-4-(2-(6,7- dimetoxicoumarin-3-yl)vinil)-N-propilbenzamida) como sonda fluorescente para marcar os ligantes proteicos usados em pinturas. O método foi desenvolvido e otimizado utilizando proteínas comerciais e proteínas extraídas da gema e clara de ovo de galinha, de leite de bovino e de pele de coelho. Para simular as condições reais, foram preparados modelos de pintura de pinturas de cavalete, misturando-se proteínas como ovalbumina, caseína e cola de coelho, com diferentes pigmentos (branco de chumbo, amarelo de crómio e negro de osso) e o método de marcação fluorescente foi miniaturizado e testado. Com base nos resultados obtidos, o método revelou-se capaz de detetar proteínas a concentração tão baixa quanto 6,0 μg / ml. Finalmente, para validação do método, foram analisadas micro amostras reais de pinturas de cavalete. As proteínas extraídas foram submetidas ao método de marcação fluorescente desenvolvido, tendo sido claramente identificadas em perfis eletroforéticos. Os resultados evidenciam a aplicabilidade desta metodologia como uma ferramenta analítica eficaz e útil para a identificação de ligantes proteicos extraídos de pinturas de cavalete e, possivelmente, de outras obras de arte. Adicionalmente, foram realizados cálculos quânticos baseados na Teoria Funcional da Densidade (DFT) e na Teoria do Funcional da Densidade Dependente do Tempo (TD-DFT) da cumarina C392STP de um derivado desta cumarina, ((E/Z)-N-propyl-4-(2-(6,7-dimethoxy-2-oxo-2H-chromen-3-yl)vinyl)benzamide)), que modela a cumarina ligada a lisina. Os cálculos confirmam as tendências experimentais observadas nos comprimentos de onda de absorção e estão de acordo com os espectros de absorção experimentais, fornecendo uma caracterização abrangente das principais características espectrais dos compostos estudados.

Tese de doutoramento em Engenharia Têxtil
The formation of intramolecular disulphide bonds is critical in the process of protein folding and on the stabilization of protein tertiary structure. Their formation involves the oxidation of two thiol groups that should be correctly paired. The incorrect paring inhibit the protein folding into its native conformation. The rearrangement of incorrectly disulfide bonds on proteins is catalysed in vivo by the protein disulphide isomerase (PDI). This versatile enzyme is able to catalyse the oxidation, reduction and isomerisation of disulfide bonds in a broad range of protein substrates. This dissertation successfully presents the use of PDI for functionalization of cysteine-containing (CC) proteinaceous substrates such as keratin fibres and RNase A microspheres. These approaches take advantage of the presence of thiol moieties and disulphide bonds in these substrates. It was shown that the type of reaction catalysed by PDI can be predicted, by controlling the redox environment. When the active site of PDI is in its oxidized state due to is characteristic potential redox (Eº = -180 mV) an oxidation reaction is catalyzed. When the active site of PDI is transformed to its reduced state (ΔE = - 260 mV), the isomerisation of disulfide bonds is promoted. PDI was able to incorporate CC functional molecules on wool and hair trough disulfide bonds, as suggested by matrix-assisted laser desorption and ionization time-offlight results (MALDI-TOF) analysis. Similarly, PDI increased the affinity of a synthesised keratin-based peptide (KP) towards hair and facilitated the penetration into its cortex. Targeting a biomedical approach, Ribonuclease A (RNase A) was oxidatively attached to the wool surface through disulphide bonds, and PDI was shown to induce its release. Aiming a cosmetic application, KP and other synthesized surfactant-based peptide (SPB) were applied in over-bleached, damaged hair. Both peptides induced an improvement on its mechanical properties and thermal stability. Thus, recovering of the fibre integrity loss during the hair bleaching process was achieved. In the presence of PDI, peptides were linked by disulphide bonds and the thermal stability increased at higher levels. Due to the great properties developed on over-bleached hair after KP application, in the absence of PDI, other type of hair (relaxed hair) was treated with this peptide and the same properties were measured. The relaxing treatment, commonly applied on excessively curly hair, often result on the weakening of the fibre. KP was then applied to this weakened hair and its ability to recover its mechanical and thermal properties was proved. Two different peptide formulations were evaluated. In one formulation KP was dissolved in aqueous solution (WF) while in the other KP was dissolved in organic solvent solution (OF). The last imparted better mechanical and thermal properties to the hair, however, the safety assessment showed that OF is potentially cytotoxic, inhibit cell growth, and genotoxic. The KP itself did not inhibit the cell growth and was found to be non-cytotoxic and non-genotoxic, hence suitable for the application on cosmetic formulations at concentrations up to 0.5 g/L. Showing its ability to act on a broad range of cysteine-containing compounds, PDI was also able to oxidize the thiol groups found in sphere-like particles, recovering their biological function, at the conditions that lead PDI‟s active site on its oxidized state. Using a reducing environment, PDI promoted the released of native RNase A from protein-based microspheres. The research presented in this thesis shows the versatility of PDI to promote diverse functionalization on proteinaceous substrates, resulting in a wide applicability in different areas such as cosmetic, textile and biotechnology. The work was carried out partially in collaboration with a cosmetic company; hence the research included promising biotoolsbased strategies for hair-care product development, especially for the application on several types of damaged hair.
As pontes dissulfídicas são uma característica muito importante para a estabilização da estrutura terciária de proteínas. A formação das mesmas envolve a oxidação de dois grupos tiol, os quais devem estar correctamente ligados. Caso contrário, a proteína perde a sua actividade biológica característica, devido à formação de pontes dissulfídicas nãonativas, e desnatura. In vivo, aquando da formação das proteínas na célula, uma enzima tem uma função muito importante: promover o rearranjo das pontes dissulfídicas nãonativas e prevenir a agregação das proteínas desnaturadas. Esta enzima é a Protein Disulfide Isomerase (PDI), uma isomerase de pontes dissulfídicas que catalisa a oxidação, redução ou isomerisação de uma vasta gama de substratos contendo cisteína na sua constituição. Esta dissertação apresenta com sucesso a aplicação desta enzima na funcionalização substratos proteicos, tais como as fibras queratinosas ou microesferas de Ribonuclease A (RNase A). As pontes dissulfídicas dos substratos, acima mencionados, são as ligações-alvo nesta abordagem. Foi demonstrado que o potencial de redução do centro activo da PDI é uma ferramenta importante na pré-determinação das reacções que esta enzima pode catalisar. Quando o centro activo da PDI está no seu estado oxidado, devido ao seu característico potencial redox (Eº = -180 mV), uma reacção de oxidação é catalisada. Quando o centro activo da PDI é transformado para o seu estado reduzido (ΔE = - 260 mV), a isomerisação das pontes dissulfídicas é promovida. A PDI catalisou a incorporação de moléculas contendo cisteína (CC) em lã e cabelo através de pontes dissulfídicas, sugerido pela análise de espectroscopia de massa (MALDITOF). A PDI facilitou também, a penetração de um péptido de queratina (KP) no córtex da fibra de cabelo e induziu a libertação de uma proteína modelo (Ribonuclease A) da superfície da lã. Com o objectivo de uma aplicação cosmética, o KP e outro péptido, derivado de um surfactante humano (SPB) foram aplicados em cabelo danificado, previamente sujeito a vários ciclos de branqueamento oxidativo. Ambos os péptidos melhoraram as propriedades mecânicas e a estabilidade térmica do cabelo danificado, provando a sua capacidade para recuperar a integridade da fibra. Quando a PDI foi aplicada, os péptidos ligaram-se ao cabelo através de pontes dissulfídicas e a estabilidade térmica aumentou para valores ainda mais elevados. Devido ao efeito renovador que o KP teve sobre o cabelo branqueado, outro tipo de cabelo (cabelo relaxado) foi tratado com este péptido e as mesmas propriedades foram medidas. Os tratamentos de relaxamento em cabelo extremamente encaracolado resultam em enfraquecimento do mesmo. O KP foi por isso aplicado neste tipo de cabelo e a sua capacidade para o melhorar foi provado. Dois tipos de formulações peptídicas foram também testados. Numa formulação o KP foi diluído numa solução aquosa (WF), enquanto na outra o KP foi diluído numa solução contendo solventes orgânicos (OF). Esta última promoveu melhores resultados, contudo, revelou-se potencialmente cytotoxica, genotoxica e inibidora do crescimento celular. Demonstrou-se, todavia, que o péptido em solução aquosa pode ser aplicado em formulações cosméticas até à concentração de 0.5 g/L não manifestando citotóxicidade, genotóxicidade ou inibição do crescimento celular. Demonstrando a sua aptidão para actuar em compostos contendo cisteína, a PDI foi também capaz de oxidar os grupos tiol presentes nas microesferas, recuperando a sua função biológica nas condições que promovem o estado oxidado do seu centro activo. Usando um ambiente mais redutor, promoveu a libertação da proteína nativa das microesferas proteicas. Os resultados apresentados nesta tese demonstram a versatilidade da PDI para promover a funcionalização de substratos proteicos, resultando numa ampla aplicabilidade em áreas distintas como cosmética, têxtil e biotecnologia. O trabalho foi desenvolvido em parceria com uma empresa de cosmética, o que fomentou a procura de estratégias biológicas para o desenvolvimento de novos produtos para cabelo, especialmente para a aplicação em vários tipos de cabelo danificado.
Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/38363/2007

Pyrenophora teres f. teres (Ptt) causes net form net blotch disease (NFNB), an important disease of barley in Australia and worldwide. This fungus uses proteinaceous toxins to cause necrosis and different isolates of Ptt differ in their ability to cause symptoms on different cultivars of barley. However, little is known about the roles of pathogen growth and individual toxins in symptom development. This project therefore aimed to determine whether there is a relationship between toxin production, fungal growth and virulence in NFNB. Conidial germination, extent of fungal growth and culture filtrate toxicity were compared for six South Australian Ptt isolates with different virulence on the barley cultivar ‘Sloop’. In addition, Ptt toxin production was optimised before identification and selection of virulence-related candidate proteins (VRCPs) for further characterisation. The biological activity of recombinant VRCPs on susceptible and resistant cultivars and VRCPs gene expression during the interaction of Sloop with each isolate were also compared. In general, the more virulent isolates had higher rates of conidial germination (both in vitro and in planta) and fungal development in planta, represented by longer hyphae and more appressoria, compared with less virulent isolates. Similarly, PttGAPDH and its transcript were more abundant during the interaction of barley with more virulent isolates. A proteomics approach was used to identify proteins unique to the more virulent isolate, proteins from bioactive fractions on either susceptible (Sloop) or resistant cultivars (CI9214 and Beecher) and proteins from the intercellular washing fluids (ICWFs) of infected barley. These analyses revealed that Ptt produced proteins between 37 and 150 kDa that have biological activity. Liquid Chromatography-Electrospray Ionisation Ion-Trap Mass Spectrometry (LC-eSI-IT MS), of individual biologically active proteins was used to identify peptides which matched to 17 proteins that belong to three groups of fungal proteins including virulence-related proteins; fungal growth and development proteins; and those with unknown function (hypothetical proteins). However, Ptt toxins were not detected in the ICWF protein profiles suggesting that Ptt toxins were either in trace amounts or might be internalised into the cell. The four VRCPs selected, were identified as hypothetical proteins with unknown function in the Ptt database. Further bioinformatic analysis characterised these VRCPs as an isochorismatase (PttCHFP1), an endo-1, 4-β- xylanase A (PttXyn11A), a glycophosphatidylinositol (GPI)-anchored common in fungal extracellular membrane (CFEM) domain-containing protein (PttGPICFEM) and an unknown proteinaceous secreted (but conserved) hypothetical protein (PttSP1). These VRCPs were heterologously expressed and characterised using gene expression studies. PttXyn11A had strong homology with the well characterised endoxylanases, TrXyn11A from Trichoderma reesei and BcXyn11A from Botrytis cinerea, known to contribute to virulence. A necrosis-inducing region on the surface of the enzyme was also identified in PttXyn11A, suggesting a potential role in necrosis induction. The culture filtrates for more virulent isolates had significantly greater xylanase activity than those from less virulent isolates. Even though heterologously expressed PttXyn11A was toxic to Escherichia coli, xylanase activity was detectable at very low levels and was not enough to cause symptoms in the bioassay. In addition, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and RT-quantitative PCR (RT-qPCR) analysis demonstrated that PttXyn11A was expressed more abundantly by the more virulent isolates compared with the other isolates in culture and during the plant-pathogen interaction. Together, these results suggest that PttXyn11A plays a role in virulence, either through its ability to degrade the plant cell wall to assist fungal growth or through its necrosisinducing ability. PttCHFP1 showed homology to an isochorismatase, an enzyme that has been proposed to have a role in plant defence via inhibition of salicylic acid production. PttSP1 showed homology to a membrane lipoprotein proposed to have a role in fungal development. Bioassay of recombinant PttCHFP1 and PttSP1 induced chlorosis symptoms in the susceptible barley cultivar (Sloop). The cysteine-rich CFEM domain identified in PttGPI-CFEM has been suggested to have an important role in hyphal attachment and fungal networking. However, E. coli was not able to express this gene probably due to its attachment to the plasma membrane and/or cell wall. Analysis of the gene expression profiles for PttCHFP1, PttGPI-CFEM and PttSP1 showed no significant differences between isolates in vitro and in planta suggesting that all isolates regulated the expression of these genes to the essential level possibly required for pathogenesis. This is the first study to identify the relationship between fungal growth and proteinaceous toxin production, characterise individual proteinaceous toxins in the mixture of Ptt culture filtrate and investigate the expression profiles of genes encoding VRCPs during the Ptt-barley interaction. This study therefore provides a better understanding of the Ptt-barley interaction by identifying the potential toxins which might lead to identify the toxin targets and ultimately support the breeding of resistant cultivars of barley.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2013

碩士
明新科技大學
化學工程與材料科技系碩士班
101
Newcastle disease virus (NDV) is an enveloped RNA virus of Paramyxoviridae family. Two envelope glycoproteins, Hemagglutinin-Neuroaminidase (HN) and Fusion (F), play major roles in the fusion of envelope membrane and cellular membrane to deliver viron particle into host cells. The interactions between HN and host cell glycoprotein(s) act the first step of virus infection. HN is a type II membrane protein with a transmembrane domain at N-terminal end, a middle stalk structure and C-terminal Head domain which is composed of six blade subdomains. It is believed that the Head domain contains the binding sites of host cell glycoprotein receptor and these sites could be used as epitopes to elicit antisera with virus neutralizing ability. A patented PTD-J-X/Hsp72 antigen presenting system had been developed and applied herein. Polypeptide X fused with the J domain can be presented to immune system more effectively via the Hsp72 which can associate J domain specifically and stimulate innate immune response by Tole-like receptor 2 (TLR2). A 5L fermentation method was established to prepare recombinant Hsp72 protein which was used as a protein type adjuvant. Four cDNA fragments encoding NDV HN Head subdomains, i.e., blade 1-2 (161-290), blade 3-4 (292-466), blade 5-6 (468-571) and blade 4-5 (401-454/468-530), were subcloned into pET22b-PTD1J1(Tb) plasmid to construct the pET22b-PTD1J1-HN161-290, pET22b-PTD1J1-HN292-466, pET22b-PTD1J1-HN468-571 and pET22b-PTD1J1-HN401-454/468-530 expression vectors in order to express the PTD-J-HN292-466, PTD-J-HN468-571, PTD-J-HN161-290, and PTD-J-HN401-454/468-530 recombinant proteins, respectively. Antisera elicited with the four PTD-J-HN/Hsp72 protein complexes could recognize full length HN overexpressed in HeLa cells demonstrated by western blotting and immunofluorescence methods. The highest neutralizing valency of the four antisera was 32, however, the mixture of them was 256 indicating that the Head domain contains multi-receptor binding sites.

Pyrenophora tritici-repentis (Ptr) is the necrotrophic fungus responsible for tan spot of wheat (Triticum aestivum). Ptr causes disease on susceptible wheat cultivars through the production and secretion of host-selective toxins (HSTs). HSTs are compounds that are only known to be produced by fungi and considered to be primary determinants of pathogenicity. Infiltration of these toxins into sensitive wheat elicits the same symptoms as the pathogen, which simplifies investigations of host- pathogen interactions due to exclusion of the pathogen. These characteristics make HSTs ideal molecules to dissect molecular plant-microbe interactions. Known HSTs of Ptr include Ptr ToxA (ToxA), Ptr ToxB (ToxB) and Ptr ToxC (ToxC). ToxA is the most characterized toxin of Ptr, as well as the first proteinaceous HST identified. The proposed mode-of-action for ToxA includes internalization into sensitive wheat mesophyll cells, localization to the chloroplast, photosystem perturbations and elicitation of high amounts of reactive oxygen species (ROS), all of which lead to necrosis. However, it is still unknown how ToxA is transported to the chloroplast. To identify additional interacting components involved in ToxA symptom development, genes were silenced in tobacco plants (Nicotiana benthamiana) using the tobacco rattle virus (TRV) virus-induced gene-silencing (VIGS) system. Four genes were identified that potentially could play a role in ToxA-induced cell death: a 40S ribosomal subunit, peroxisomal glycolate oxidase (GOX), a thiamine biosynthetic enzyme (Thi1), and the R-gene mediator, Sgt1. Ptr exhibits a complex race structure determined by the HST(s) produced and the symptom(s) elicited on sensitive wheat cultivars. Currently, there are eight characterized races and other HSTs and races have been proposed. Isolate SO3 was discovered in southern Oregon and elicits ToxA-like symptoms on a wheat differential set, yet lacks the ToxA gene. The transcriptome of SO3 was sequenced, assembled, and aligned to a ToxA-producing isolate, Pt-1C-BFP, which will aid in the identification of the protein(s) that may be responsible for these ToxA-like symptoms. SO3 contains a set of 497 sequences that were not found in the ToxA-producing isolate Pt-1C-BFP (BFP). These sequences should be further investigated to identify those that encode small secreted proteins (SSPs) and could potentially serve as HSTs and pathogenicity factors of SO3.
Graduation date: 2013

Meiosis is a specialized type of cell division essential for the production of four normal haploid gametes. In early prophase I of meiosis, the intimate synapsis between homologous chromosomes, and the formation of chiasmata, is facilitated by a proteinaceous structure known as the synaptonemal complex (SC). Ultrastructural analysis of germ cells of a number of organisms has disclosed that SC is a specialized tripartite structure composed of two lateral elements, one on each homolog, and a central element, which, in turn, are linked by transverse elements. Genetic studies have revealed that defects in meiotic chromosome alignment and/or segregation result in aneuploidy, which is the leading cause of spontaneous miscarriages in humans, hereditary birth defects such as Down syndrome, and are also, associated with the development and progression of certain forms of cancer. The mechanism(s) underlying the alignment/pairing of chromosomes at meiosis I differ among organisms. These can be divided into at least two broad pathways: one is independent of DNA double-strand breaks (DSB) and other is mediated by DSBs. In the DSB-dependent pathway, SC plays crucial roles in promoting homolog pairing and disjunction. On the other hand, the DSB-independent pathway involves the participation of telomeres, centromeres and non-coding RNAs in the pre-synaptic alignment, pre-meiotic pairing as well as pairing of homologous chromosomes. Although a large body of literature highlights the central role of SC in meiotic recombination, the possible role of SC components in homolog recognition and alignment is poorly understood. Genetic screens for Saccharomyces cerevisiae mutants defective in meiosis and sporulation lead to the isolation of genes required for interhomolog recombination, including those that encode SC components. In S. cerevisiae, ten meiosis-specific proteins viz., Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8 have been recognized as bona fide constituents of SC or associated with SC function. Mutations in any of these genes result in defective SC formation, thus leading to reduction in the rate of recombination. HOP1 (Homolog Pairing) encodes a ̴ 70 kDa structural protein, which localizes to the lateral elements of SC. It was found to be essential for the progression of meiotic recombination. In hop1Δ mutants, meiosis specific DSBs are reduced to 10% of that of wild type level and it fails to produce viable spores. It also displays relatively high frequency of inter-sister recombination over inter-homolog recombination. Bioinformatics analysis suggests that Hop1 comprises of an N-terminal HORMA domain (Hop1, Rev7 and Mad2), which is conserved among Hop1 homologs from diverse organisms. This domain is also known to be present in proteins involved in processes like chromosome synapsis, repair and sex chromosome inactivation. Additionally, Hop1 harbors a 36-amino acid long zinc finger 348374 motif (CX2CX19CX2C) which is critical for DNA binding and meiotic progression, and a putative nuclear localization signal corresponding to amino acid residues from 588-594. Previous studies suggested that purified Hop1 protein exists in multiple oligomeric states in solution and displays structure specific DNA binding activity. Importantly, Hop1 exhibited higher binding affinity for the Holliday junction (HJ), over other early recombination intermediates. Binding of Hop1 to the HJ at the core resulted in branch migration of the junction, albeit weakly. Intriguingly, Hop1 showed a high binding affinity for G4 DNA, a non-B DNA structure, implicated in homolog synapsis and promotes robust synapsis between double-stranded DNA molecules. Hop1 protein used in the foregoing biochemical studies was purified from mitotically dividing S. cerevisiae cells containing the recombinant plasmid over-expressing the protein where the yields were often found to be in the low-microgram quantities. Therefore, one of the major limitations to the application of high resolution biophysical techniques, such as X-crystallography and spectroscopic analyses for structure-function studies of S. cerevisiae Hop1 has been the non-availability of sufficient quantities of functionally active pure protein. In this study, we have performed expression screening in Escherichia coli host strains, capable of high level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed +2 for expression and purification of S. cerevisiae Hop1 protein, using Ni-NTA and double-stranded DNA-cellulose chromatography. Recombinant S. cerevisiae Hop1 protein thus obtained was >98% pure and exhibited DNA binding activity with high-affinity for Holliday junction. The availability of the bacterial HOP1 expression vector and functionally active Hop1 protein has enabled us to glean and understand several vital biological insights into the structure-function relationships of Hop1 as well as the generation of appropriate truncated mutant proteins. Mutational analyses in S. cerevisiae has shown that sister chromatid cohesion is required for proper chromosome condensation, including the formation of axial elements, SC assembly and recombination. Consistent with these findings, homolog alignment is impaired in red1hop1 strains and associations between homologs are less stable. red1 mutants fail to make any discernible axial elements or SC structures but exhibit normal chromosome condensation, while hop1 mutants form long fragments of axial elements but without any SCs, are defective in chromosome condensation, and produce in-viable spores. Using single molecule and ensemble assays, we found that S. cerevisiae Hop1 organizes DNA into at least four major distinct DNA conformations: (i) a rigid protein filament along DNA that blocks access to nucleases; (ii) bridging of non-contiguous segments of DNA to form stem-loop structures; (iii) intra-and intermolecular long range synapsis between double-stranded DNA molecules; and (iv) folding of DNA into higher order nucleoprotein structures. Consistent with B. McClintock’s proposal that “there is a tendency for chromosomes to associate 2-by-2 in the prophase of meiosis involving long distance recognition of homologs”, these results to our knowledge provide the first evidence that Hop1 mediates the formation of tight DNA-protein-DNA nucleofilaments independent of homology which might help in the synapsis of homologous chromosomes during meiosis. Although the DNA binding properties of Hop1 are relatively well established, comparable knowledge about the protein is lacking. The purification of Hop1 from E. coli, which was functionally indistinguishable from the protein obtained from mitotically dividing S. cerevisiae cells has enabled us to investigate the structure-function relationships of Hop1, which has provided important insights into its role in meiotic recombination. We present several lines of evidence suggesting that Hop1 is a modular protein, consisting of an intrinsically unstructured N-terminal domain and a core C-terminal domain (Hop1CTD), the latter being functionally equivalent to the full-length Hop1 in terms of its in vitro activities. Importantly, however, Hop1CTD was unable to rescue the meiotic recombination defects of hop1null strain, indicating that synergy between the N-terminal and C-terminal domains of Hop1 protein is essential for meiosis and spore formation. Taken together, our findings provide novel insights into the molecular functions of Hop1, which has profound implications for the assembly of mature SC, homolog synapsis and recombination. Several lines of investigations suggest that HORMA domain containing proteins are involved in chromatin binding and, consequently, have been shown to play key roles in processes such as meiotic cell cycle checkpoint, DNA replication, double-strand break repair and chromosome synapsis. S. cerevisiae encodes three HORMA domain containing proteins: Hop1, Rev7 and Mad2 (HORMA) which interact with chromatin during diverse chromosomal processes. The data presented above suggest that Hop1 is a modular protein containing a distinct N-terminal and C-terminal (Hop1CTD) domains. The N-terminal domain of Hop1, which corresponds to the evolutionarily conserved HORMA domain, although, discovered first in Hop1, its precise biochemical functions remain unknown. In this section, we show that Hop1-HORMA domain expressed in and purified from E. coli exhibits preferential binding to the HJ and G4 DNA, over other early recombination intermediates. Detailed functional analyses of Hop1-HORMA domain, using mobility shift assays, DNase I footprinting and FRET, have revealed that HORMA binds at the core of Holliday junction and induces marked changes in its global conformation. Further experimental evidence also suggested that it causes DNA stiffening and condensation. However, like Hop1CTD, HORMA domain alone failed to rescue the meiotic recombination defects of hop1 null strain, indicating that synergy between the N-and C-terminal domains of Hop1 is essential for meiosis as well as for the formation of haploid gametes. Moreover, these results strongly implicate that Hop1 protein harbours a second DNA binding motif, which resides in the HORMA domain at its N-terminal region. To our knowledge, these findings also provide the first insights into the biochemical mechanism underlying HORMA domain activity. In summary, it appears that the C-terminal (CTD) and N-terminal (HORMA) domains of Hop1 may perform biochemical functions similar (albeit less efficiently) to that of the full-length Hop1. However, further research is required to uncover the functional differences between these domains, their respective interacting partners and modulation of the activity of these domains.