<p>Seme tikve (Cucurbita pepo) obiluje kako uljem tako i proteinima. Nakon izdvajanja ulja, proteini se koncentri&scaron;u 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 farmakolo&scaron;ke aktivnosti i visoke biolo&scaron;ke 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 povr&scaron;inski aktivne materije. Međutim, koloidna funkcionalnost proteina semena tikve se jo&scaron; 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.<br />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&ndash;1 g/100 cm³), pH (3&ndash;8) i jonske jačine (0&ndash;1 mol/dm³ NaCl) na rastvorljivost i adsorpciona svojstva: dinamički međupovr&scaron;inski pritisak (ulje/voda), statički povr&scaron;inski (vazduh/voda) i međupovr&scaron;inski (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.<br />Utvrđeno je da je prinos IPST veći od prinosa oba hidrolizata za oko 65 %. IPST ima najniţu rastvorljivost na pH=5, &scaron;to ujedno predstavlja i njegovu izoelektričnu tačku. Enzimskom hidrolizom IPST značajno se povećava rastvorljivost, posebno na pI=5. Povećanje jonske jačine je izazvalo salting&ndash;in ili salting&ndash;out efekat rastvorljivosti kod svih uzoraka u zavisnosti od pH. IPST, H1 i H2 poseduju povr&scaron;insku aktivnost pri čemu je povr&scaron;inski/međupovr&scaron;inski pritisak H1 i H2 manje zavistan od promene pH i jonske jačine u poređenju sa povr&scaron;inskim/međupovr&scaron;inskim 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.</p> / <p>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&ndash;product of the oil<br />extraction 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.<br />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&ndash;1 g/100 cm³), pH (3&ndash;8) i ionic strength (0&ndash;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, was<br />investigated 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.<br />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&ndash;in or salting&ndash;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.</p>

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.

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.