Tiger nuts (Cyperus esculentus L) are recognized as a high potential, alternative source of food nutrients. However, there is limited scientific literature on the technological possibilities for developing value-added foods, such as fermented products from tiger nut milk. Therefore, strategies for producing and improving the properties of fermented tiger nut milk were investigated for generating lactose-free, nutritious yogurt-like products with acceptable sensory properties and a prolonged shelf life quality. A wet-milling procedure was standardized for extracting tiger nut milk from tiger nuts, and the effects of the extraction process on nutrient distribution, colour properties and colloidal stability of the milk were analyzed. Next, tiger nut milk was enriched with proteins and/or hydrocolloids and the impact of the additives on the physical properties of the milk were determined. Enriched tiger nut milk was fermented by using classical yogurt cultures and the obtained products were analyzed for the microbiological, physico-chemical and sensory characteristics. Additionally, effects of enriching tiger nut milk with microbial transglutaminase cross-linked proteins on the microbiological and physico-chemical properties were evaluated. Higher wet-milling intensity improved the nutrient composition, colloidal stability and colour of the milk. Enrichment of tiger nut milk with milk proteins and xanthan gum enhanced the viscosity and stability, and after fermentation, led to homogenous gel-like products with superior microbiological, physico-chemical and different sensory properties compared to the fermented plain tiger nut milk. Microbial transglutaminase cross-linked proteins improved the physical characteristics of the fermented product, especially during storage. This product would be relevant in many developing countries with high prevalence of lactose intolerance, limited access to nutritious food but show a high distribution of tiger nut vegetation.:1. Introduction and aim 1 2. Literature review 4 2.1 Tiger nut, origin, nutritional value and food use 4 2.2 Tiger nut milk, preparation and nutrient composition 7 2.3 Colloidal characteristics of tiger nut milk 9 2.4 Factors accounting for the dispersion stability of tiger nut milk 10 2.5 Enhancing tiger nut milk stability 12 2.6 Properties of fermented tiger nut milk 17 2.7 Microbial transglutaminase and properties of fermented tiger nut milk 18 3. Methodology 21 3.1 Extraction and characterisation of tiger nut milk 21 3.1.1 Sample collection and preparation 21 3.1.2 Tiger nut milk extraction 21 3.1.3 Nutrient analysis of tiger nuts 22 3.1.4 Analysis of tiger nut products 23 3.1.5 Particle size distribution 24 3.1.6 Colloidal stability 25 3.1.7 Colour measurement 25 3.2 Stabilisation of tiger nut milk dispersion 26 3.2.1 Tiger nut milk preparation 26 3.2.2 Preparation of tiger nut milk enrichments 26 3.2.3 Gravitational stability of enriched tiger nut milk 27 3.2.4 Accelerated gravitational stability of enriched tiger nut milk 28 3.2.5 Viscosity of TNM mixtures 29 3.3 Extraction and characterisation of globular tiger nut proteins 29 3.3.1 Protein extraction and fractionation 29 3.3.2 Molecular mass of globular tiger nut proteins 31 3.3.3 Denaturation temperature of globular tiger nut proteins 32 3.3.4 Isoelectric point of globular tiger nut protein 33 3.4 Properties of fermented tiger nut milk enriched with proteins 34 3.4.1 Materials and Reagents 34 3.4.2 Preparation of plain and enriched tiger nut milk 34 3.4.3 Fermentation of plain and enriched tiger nut milk 35 3.4.4 Viable counts of starter cultures in fermented tiger nut milk systems 36 3.4.5 Chemical analysis of unfermented and fermented tiger nut milk 36 3.4.6 Physical analysis of fermented tiger nut milk products 37 3.4.7 Sensory analysis of fermented tiger nut milk products 38 3.5 Microbial transglutaminase and fermented tiger nut milk property 38 3.5.1 Preparation of plain and enriched tiger nut milk 38 3.5.2 Fermentation of plain and enriched tiger nut milk 39 3.5.3 Analysis of the enzymatically cross-linked proteins 39 3.5.4 Viable counts 40 3.5.5 pH and titratable acidity 40 3.5.6 Syneresis and viscosity 41 3.5.7 Colour of fermented tiger nut products 41 3.6 Statistical analysis 41 4. Results and discussion 43 4.1 Extraction and characteristics of tiger nut milk 43 4.1.1 Material recovery, mass transfer and yield of tiger nut solids 43 4.1.2 Nutrient composition of tiger nut products 45 4.1.3 Physical properties of tiger nut milk 48 4.1.3.1 Particle size distribution of extracted tiger nut milk 48 4.1.3.2 Colloidal stability of tiger nut milk 49 4.1.3.3 Colour stability of tiger nut milk 51 4.2 Stabilisation of tiger nut milk 53 4.2.1 Effects of enrichments on the stability of tiger nut milk 53 4.2.2 Effects of pH and temperature on the stability of enriched TNM 56 4.2.3 Effects of enrichments on the rheology of tiger nut milk 58 4.3 Tiger nut protein extraction and characterisation 60 4.3.1 Protein extraction and fractionation 60 4.3.2 Molecular mass of tiger nut protein 62 4.3.3 Thermal denaturation of tiger nut protein 63 4.3.4 Isoelectric point of tiger nut proteins 66 4.4 Properties of fermented tiger nut milk enriched with proteins 67 4.4.1 Acidification and gel formation during fermentation 67 4.4.2 Microbiological properties of fermented enriched tiger nut milk 70 4.4.3 Physico-chemical properties of fermented enriched tiger nut milk 71 4.4.4 Sensory properties of fermented tiger nut milk products 76 4.5 Microbial transglutaminase and fermented tiger nut milk property 77 4.5.1 Effects on tiger nut milk fermentation 77 4.5.2 Microbiological properties during storage of fermented product 81 4.5.3 Physico-chemical properties during storage of fermented product 83 4.5.4 Effects on colour of fermented tiger nut product 86 5. Conclusions and outlook 88 Bibliography 90 List of figures 111 List of tables 115 List of Publications 116 Poster and presentations 116 / Erdmandeln (Cyperus esculentus L) haben ein hohes Potential als alternative Quelle Lebensmittelinhaltsstoffen. Allerdings gibt es nur in begrenztem Ausmaß Literatur über technologische Möglichkeiten zur Entwicklung von Mehrwert-Lebensmitteln wie fermentierter Erdmandelmilch. Daher wurden Strategien zur Herstellung und Verbesserung der Eigenschaften von fermentierter Erdmandelmilch zur Erzeugung laktosefreier joghurtähnlicher Produkte mit akzeptablen sensorischen Eigenschaften untersucht. Für die Extraktion der Erdmandelmilch wurde ein Nassmahlverfahren standardisiert und der Einfluss des Verfahrens auf die Nährstoffverteilung, die Farbeigenschaften und die kolloidale Stabilität der Milch analysiert. Als nächstes wurde Erdmandelmilch mit Proteinen und/oder Hydrokolloiden angereichert, und der Einfluss der Additive auf die physikalischen Eigenschaften des Extrakts bestimmt. Angereicherte Erdmandelmilch wurde mit klassischen Joghurtkulturen fermentiert, und die mikrobiologischen, physikalisch-chemischen und sensorischen Eigenschaften der Produkte wurden untersucht. Zusätzlich wurden Effekte der Anreicherung von Erdmandelmilch mit enzymatisch vernetzten Proteinen auf die mikrobiologischen und physikalisch-chemischen Eigenschaften bewertet. Eine höhere Nassmahlintensität verbesserte die Nährstoffzusammensetzung, die kolloidale Stabilität und die Farbe der Milch. Die Anreicherung erhöhte die Viskosität und Stabilität und führte nach der Fermentation zu homogenen gelartigen Produkten mit verbesserten mikrobiologischen, physikalisch-chemischen und sensorischen Eigenschaften im Vergleich zur fermentierten Erdmandelmilch. Mikrobielle Transglutaminase-vernetzte Proteine verbesserten die physikalischen Eigenschaften des fermentierten Produkts, insbesondere während der Lagerung. Dieses Produkt wäre in vielen Entwicklungsländern mit hoher Prävalenz von Laktoseintoleranz und begrenztem Zugang zu nahrhaften Lebensmitteln als Alternative von Interesse.:1. Introduction and aim 1 2. Literature review 4 2.1 Tiger nut, origin, nutritional value and food use 4 2.2 Tiger nut milk, preparation and nutrient composition 7 2.3 Colloidal characteristics of tiger nut milk 9 2.4 Factors accounting for the dispersion stability of tiger nut milk 10 2.5 Enhancing tiger nut milk stability 12 2.6 Properties of fermented tiger nut milk 17 2.7 Microbial transglutaminase and properties of fermented tiger nut milk 18 3. Methodology 21 3.1 Extraction and characterisation of tiger nut milk 21 3.1.1 Sample collection and preparation 21 3.1.2 Tiger nut milk extraction 21 3.1.3 Nutrient analysis of tiger nuts 22 3.1.4 Analysis of tiger nut products 23 3.1.5 Particle size distribution 24 3.1.6 Colloidal stability 25 3.1.7 Colour measurement 25 3.2 Stabilisation of tiger nut milk dispersion 26 3.2.1 Tiger nut milk preparation 26 3.2.2 Preparation of tiger nut milk enrichments 26 3.2.3 Gravitational stability of enriched tiger nut milk 27 3.2.4 Accelerated gravitational stability of enriched tiger nut milk 28 3.2.5 Viscosity of TNM mixtures 29 3.3 Extraction and characterisation of globular tiger nut proteins 29 3.3.1 Protein extraction and fractionation 29 3.3.2 Molecular mass of globular tiger nut proteins 31 3.3.3 Denaturation temperature of globular tiger nut proteins 32 3.3.4 Isoelectric point of globular tiger nut protein 33 3.4 Properties of fermented tiger nut milk enriched with proteins 34 3.4.1 Materials and Reagents 34 3.4.2 Preparation of plain and enriched tiger nut milk 34 3.4.3 Fermentation of plain and enriched tiger nut milk 35 3.4.4 Viable counts of starter cultures in fermented tiger nut milk systems 36 3.4.5 Chemical analysis of unfermented and fermented tiger nut milk 36 3.4.6 Physical analysis of fermented tiger nut milk products 37 3.4.7 Sensory analysis of fermented tiger nut milk products 38 3.5 Microbial transglutaminase and fermented tiger nut milk property 38 3.5.1 Preparation of plain and enriched tiger nut milk 38 3.5.2 Fermentation of plain and enriched tiger nut milk 39 3.5.3 Analysis of the enzymatically cross-linked proteins 39 3.5.4 Viable counts 40 3.5.5 pH and titratable acidity 40 3.5.6 Syneresis and viscosity 41 3.5.7 Colour of fermented tiger nut products 41 3.6 Statistical analysis 41 4. Results and discussion 43 4.1 Extraction and characteristics of tiger nut milk 43 4.1.1 Material recovery, mass transfer and yield of tiger nut solids 43 4.1.2 Nutrient composition of tiger nut products 45 4.1.3 Physical properties of tiger nut milk 48 4.1.3.1 Particle size distribution of extracted tiger nut milk 48 4.1.3.2 Colloidal stability of tiger nut milk 49 4.1.3.3 Colour stability of tiger nut milk 51 4.2 Stabilisation of tiger nut milk 53 4.2.1 Effects of enrichments on the stability of tiger nut milk 53 4.2.2 Effects of pH and temperature on the stability of enriched TNM 56 4.2.3 Effects of enrichments on the rheology of tiger nut milk 58 4.3 Tiger nut protein extraction and characterisation 60 4.3.1 Protein extraction and fractionation 60 4.3.2 Molecular mass of tiger nut protein 62 4.3.3 Thermal denaturation of tiger nut protein 63 4.3.4 Isoelectric point of tiger nut proteins 66 4.4 Properties of fermented tiger nut milk enriched with proteins 67 4.4.1 Acidification and gel formation during fermentation 67 4.4.2 Microbiological properties of fermented enriched tiger nut milk 70 4.4.3 Physico-chemical properties of fermented enriched tiger nut milk 71 4.4.4 Sensory properties of fermented tiger nut milk products 76 4.5 Microbial transglutaminase and fermented tiger nut milk property 77 4.5.1 Effects on tiger nut milk fermentation 77 4.5.2 Microbiological properties during storage of fermented product 81 4.5.3 Physico-chemical properties during storage of fermented product 83 4.5.4 Effects on colour of fermented tiger nut product 86 5. Conclusions and outlook 88 Bibliography 90 List of figures 111 List of tables 115 List of Publications 116 Poster and presentations 116

Hemocytes (blood cells) play an important role in the immune response in invertebrates, and thus the regulation of hemocyte homeostasis (hematopoiesis) is essential for the host survival against pathogens. Astakine 1, a homologue to vertebrate prokineticins, was first identified in the freshwater crayfish Pacifastacus leniusculus as a cytokine, and was found to be necessary for new hemocyte synthesis and release in vivo, and also to induce spreading and proliferation of Hematopoietic tissue cells (Hpt cells, precursor of hemocytes) in vitro. The work of this thesis is aimed to further our understanding of the molecular mechanisms involved in astakine 1 induced hematopoiesis. Crayfish transglutaminase (Tgase) has been identified in the hemocytes, and is essential for the coagulation reaction. Interestingly this enzyme is exceedingly abundant in the Hpt cells, and the spreading of Hpt cells induced by astakine 1 was accompanied by sequential loss of TGase activity from the surface of these cells. This loss of TGase activity may be an important effect of astakine 1, resulting in recruiting new hemocytes into the circulatory system. Although astakine 1 contain a prokineticin domain, it lacks the conserved N-terminal AVIT motif present in its vertebrate homologues. This motif is important for vertebrate prokineticins to interact with their receptors, indicating a different receptor interaction for crayfish astakine 1. Astakine 1 was indeed found to interact with a completely different receptor, the β-subunit of ATP synthase, on a portion of Hpt cells, and subsequently block its extracellular ATP formation. Surface ATP synthase has been reported on numerous mammalian cells, but now for the first time in an invertebrate. The activity of ATP synthase on the Hpt cells may be important for the survival and proliferation of Hpt cells, but the underlying mechanisms remain further study. With the finding of a second type of astakine in crayfish, invertebrate astakines can be divided into two groups: astakine 1 and astakine 2. The properties of astakine 2 are different from those of astakine 1 both in structure and function. In primary cell culture of Hpt cells, only astakine 1 can promote proliferation as well as differentiation into semigranular cells, whereas astakine 2 may play a potential role in the maturation of granular cells. Moreover, a novel cysteine rich protein, Pacifastacus hematopoiesis factor (PHF), was found to be one target gene of astakine 1 in Hpt cells. Down regulation of PHF results in increased apoptosis in Hpt cells in vitro, and in vivo silencing PHF leads to a severe loss of hemocytes in the animal. Therefore astakine 1 acquires the anti-apoptosis ability by inducing its downstream gene PHF in the Hpt cells. With its ability to promote the survival, proliferation and differentiation of Hpt cells, astakine 1 is proven to be an important hematopoietic growth factor.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Human tissue transglutaminase (TG2) is a calcium-dependent multifunctional enzyme that natively catalyzes the post-translational modification of proteins, namely by the formation of isopeptide bonds between protein- or peptide-bound glutamine and lysine residues. This ubiquitously expressed enzyme plays important roles in cellular differentiation, extracellular matrix stabilization, and apoptosis, to name a few. However, its unregulated activity has been associated with many pathologies such as fibrosis, cancer, neurodegenerative disorders and celiac disease. Most of these disorders are associated with unregulated acyl-transferase activity. As such, the Keillor group has directed its efforts towards the development of TG2 inhibitors. Over the years, the Keillor group has synthesized large libraries of targeted covalent inhibitors against TG2. These compounds have undergone pharmacodynamic testing in order to examine their kinetic parameters of inhibition. Having gained knowledge of their enzyme kinetics, the logical next step was to consider their pharmacokinetic profiles. In the context of this thesis, we considered two important pharmacokinetic properties: membrane permeability and off-target reactivity. Firstly, we aimed to evaluate our inhibitors for their ability to permeate the cell membrane. In efforts to do so, we were able to adapt, optimize, and validate a parallel artificial membrane permeability assay (PAMPA) utilizing hexadecane as our artificial membrane. We were able to test a few of our own inhibitors and found that compounds NC9, VA4 and AA9 possess Log Pe values of -5.26 ± 0.01, -4.66 ± 0.04 and -6.5 ± 0.5 respectively. Secondly, we sought to investigate the susceptibility of our inhibitors to glutathione addition reactions under physiological conditions. We adapted and optimized a colorimetric assay using Ellman’s reagent (DTNB) and found that our inhibitors are minimally reactive with glutathione. The methods developed over the course of this work provide protocols that can be adopted for the characterization of future inhibitors in the Keillor group, along the process of developing TG2 inhibitors into drug candidates.

Os objetivos desse estudo foram: 1) Padronizar a pesquisa do anti-tTg, comparando-o com o anticorpo antiendomísio (AAE) e 2) Avaliar as interações da tTg com a fibronectina. 49 celíacos e 124 controles com AAE negativo foram avaliados. O AAE foi pesquisado por imunofluorescência indireta e a reatividade contra a tTg e a fibronectina por ELISA in house e com kits comerciais. O antitTg foi positivo em 46,9% e 100% dos celíacos com o ELISA in house e com kits comerciais, respectivamente. A adição de fibronectina não melhorou a sensibilidade do ELISA. Em conclusão: a detecção do antitTg por ELISA apresenta percentual elevado de falso-positivos, não podendo substituir a pesquisa do AAE / The aims of the current study were: to standardize the detection of anti-tTg antibodies, comparing them with antiendomysial antibodies (EMA) and to assess the interaction of tTg with fibronectin. 49 celiac patients and 124 controls were enrolled. EMA was detected by indirect immunofluorescence reaction and tTg and fibronectin reactivity by in house ELISA and with commercially available kits. Seropositivity to anti-tTG was found in 46.9% and 100% of patients by the in house technique and by commercial kits, respectively. Fibronectin addition did not improve the ELISA sensibility. In conclusion, ELISA for anti-tTG detection has a high rate of false positive results and does not replace EMA

La transglutaminase tissulaire est une enzyme dépendante du calcium qui catalyse la formation de liens isopeptidiques, entre les chaînes latérales de résidus glutamine et lysine, permettant, par le fait même, la réticulation des protéines dans les systèmes biologiques. Elle joue un rôle, entre autres, dans l’endocytose, la régulation du développement des cellules, et même dans l’apoptose. Néanmoins, une dérégulation de l’activité biologique de cette enzyme peut entrainer différentes pathologies, comme la formation de cataractes, de plaques amyloïdes dans la maladie d’Alzheimer, ou encore peut mener au développement de la maladie céliaque. C’est pourquoi une meilleure connaissance du mécanisme d’action de cette enzyme et la possibilité de réguler son action à l’aide de substrats ou d’inhibiteurs sont nécessaires. Dans cette optique, une méthode d’expression et de purification de la transglutaminase humaine a été développée, permettant de travailler directement avec la cible pharmacologique désirée. De plus, une étude du mode d’inhibition et de liaison d’une classe d’inhibiteurs réversibles précédemment découverte dans le groupe, soit la famille des trans-cinnamoyles, a permis d’identifier que la puissance de ces molécules est influencée par la présence du calcium et qu’une inhibition dépendante du temps est observée, en lien avec un potentiel équilibre conformationnel lent de la transglutaminase. D’un autre côté, la susceptibilité à une attaque nucléophile par des thiols de cette classe de molécule rend leur potentiel pharmacologique grandement diminué, et c’est pourquoi une nouvelle famille de molécules a été identifiée, basée sur un squelette ynone, avec une valeur d’IC50 très prometteuse de 2,6 μM, en faisant un des meilleurs inhibiteurs réversibles de la transglutaminase développés à ce jour. Finalement, une stratégie de photomarquage jumelée à une analyse de spectrométrie de masse en tandem a été développée pour la découverte du site de liaison du substrat dérivé de la lysine, dans le but de mieux comprendre le mécanisme complexe de cette enzyme. / Tissue transglutaminase is a calcium-dependent enzyme that catalyzes the formation of isopeptide bonds between the side chains of glutamine and lysine residues, thereby resulting in the crosslinking of proteins in biological systems. It plays a role, among others, in endocytosis, the regulation of cell growth, and even in apoptosis. However, a deregulation of the biological activity of this enzyme can result in various pathologies, such as cataract formation, amyloid plaque formation in Alzheimer’s disease, or the development of celiac disease. Therefore, a better understanding of the mechanism of action of this enzyme and the ability to regulate its action using inhibitors or substrates is necessary. In this context, a method of expression and purification of human transglutaminase has been developed, allowing one to work directly with the desired pharmacological target. In addition, a study of the mode of inhibition and binding mode of a reversible inhibitor class previously discovered in the group, the family of trans-cinnamoyl derivatives, revealed that the potency of these molecules is influenced by the presence of calcium and a time-dependent inhibition is observed, related to a putative slow conformational equilibrium of transglutaminase. On the other hand, the susceptibility of this class of molecules to nucleophilic attack by thiols greatly diminishes their pharmacological potential, and that is why a new family of molecules has been identified, based on a ynone skeleton, with a very promising IC50 value of 2.6 μM, making this molecule one of the best transglutaminase reversible inhibitors developed to date. Finally, a photolabelling strategy combined with a tandem mass spectrometry analysis has been developed for the discovery of the binding site of the lysine derivative substrate, in order to better understand the complex mechanism of this enzyme.

Transglutaminases are a family of enzymes expressed in various tissues of our body. Some are expressed ubiquitously while others are specific to a tissue. Their primary catalytic activity is to crosslink substrates via an isopeptidic bond. The work described in this thesis focuses on two of these transglutaminases; human tissue transglutaminase (hTG2) and human factor XIIIa (FXIIIa). Divided into two projects for each enzyme, the main objective of this thesis was directed towards the discovery of potent and selective covalent inhibitors for each isozyme, namely hTG2 and hFXIIIa. The first project was concentrated on the inhibition of hTG2 activity. Ubiquitously expressed in tissues, hTG2 is a multifunctional enzyme. Its primary activity is the formation of isopeptide bonds between glutamine and lysine residues found on the surface of proteins or substrates. In addition to its catalytic activity, hTG2 is also a G-protein, distinguishing it from other members of the transglutaminase family. Much evidence illustrates that hTG2’s multifunctional abilities are conformationally regulated between its “open” and “closed” forms. Overexpression and unregulated hTG2 activity has been associated with numerous human diseases; however, most evidence has been collected for its association with fibrosis and celiac sprue. More recently, elevated hTG2 expression has been linked to cancer stem cell survival and metastatic phenotype in certain cancer cells. These findings call for the development of suitable and potent inhibitors that selectivity inactivate human hTG2 as a potential therapeutic target. Starting with previously designed acrylamide based peptidomimetic irreversible inhibitors, a structure-activity relationship (SAR) study was conducted. In this work, >20 novel irreversible inhibitors were prepared and kinetically evaluated. Our lead inhibitors allosterically inhibited GTP binding by locking the enzyme in its open conformation, as demonstrated both in vitro and in cells. Furthermore, our most potent and efficient irreversible inhibitors revealed selectivity for hTG2 over other relevant members of the transglutaminase family (hTG1, hTG3, hTG6 and hFXIIIa), providing higher confidence towards our goal of developing an ideal drug candidate. The second project was concentrated on the inhibition of hFXIIIa activity. In the blood, coagulation factor XIII (FXIII) is a tetrameric protein consisting of two catalytic A subunits (FXIII-A2) and two carrier/inhibitory B (FXIII-B2) subunits. It is a zymogen, which is converted into active transglutaminase (FXIIIa) in the final phase of coagulation cascade by thrombin proteolytic activity and Ca2+ binding. hFXIII is essential for hemostasis and thus its deficiency results in severe bleeding conditions. Further, hFXIIIa mechanically stabilizes fibrin and protects it from fibrinolysis. Due to the enzyme’s involvement in the stability of blood clots, inhibition of hFXIIIa activity has been linked to thrombotic diseases. Furthermore, inhibitors of the enzyme have the therapeutic potential to be used as anticoagulant agents. The current number of selective and potent inhibitors of hFXIIIa are few, mainly due to the similarity between its catalytic pockets and hTG2. Inspired by a poorly reactive hTG2 inhibitor discovered in this work’s hTG2 SAR study, we synthesized a small library of covalent inhibitors for hFXIIIa. Our kinetic results from this pioneering SAR study will pave the way for future hFXIIIa inhibitor SAR studies.

Transglutaminases (EC 2.3.2.13) are enzymes that catalyze an acyl transfer reaction between a &gamma / -carboxyamide group of a peptide bound glutaminyl residue (acyl donor) and a variety of primary amines (acyl acceptors), including the amino group lysine. Transglutaminase has a potential in obtaining proteins with novel properties, improving nutritional quality of foods with the addition of essential amino acids, preparing heat stable gels, developing rheological properties and mechanical strength of foods and reducing the applications of food additives. The aim of this study is to develop intracellular and extracellular microbial protransglutaminase (pro-MTG) producing recombinant Pichia pastoris strains by using genetic engineering techniques. In this context first,protransglutaminase gene (pro-mtg) from Streptomyces mobaraensis was amplified by PCR both for intracellular and extracellular constructs using proper primers then they were cloned into the pPICZ&alpha / -A expression vectors, separately. Both intracellular (pPICZ&alpha / A::pro-mtgintra) and extracellular (pPICZ&alpha / A::pro-mtgextra) constructs were prepared with strong alcohol oxidase 1 promoter which is induced by methanol. Pichia pastoris X33 cells were transfected by linear pPICZ&alpha / A::pro-mtgintra and pPICZ&alpha / A::pro-mtgextra, separately and plasmids were integrated into the Pichia pastoris X33 genome at AOX1 locus. After constructing the recombinant P. pastoris strains, batch shaker bioreactor experiments were performed for each recombinant cell and the best producing strains were selected according to Dot blot and SDS-PAGE analyses. The selected recombinant P. pastoris strains, carrying pPICZ&alpha / A::promtgextra gene and pPICZ&alpha / A::pro-mtgintra gene in their genome were named as E8 and I1, respectively. Afterwards, a controlled pilot scale bioreactor experiment in a working volume of 1 L was performed with E8 clone and produced pro-MTG was activated by Dispase I. The variations in the recombinant MTG activity, cell concentration, total protease activity, AOX activity and organic acid concentrations throughout the bioprocess were analyzed and specific growth rates, specific consumption rates and yield coefficients were calculated regarding to measured data. Maximum MTG activity was obtained as 4448 U L- 1 and the maximum cell concentration was measured as 74.1 g L-1 at t=36 h of the bioprocess. In this study, an active transglutaminase enzyme was produced extracellularly by P. pastoris for the first time and the third highest extracellular MTG activity was achieved with E8 clone.

Sprouting endothelial cells (ECs) use soluble and insoluble cues to guide migration and expand the existing vascular network to meet changing trophic needs of the tissue during angiogenesis. A noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique was used to optically image endothelial sprouting morphogenesis in three dimensional (3D) collagen matrices with simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Sprout advancement and lumen expansion companying with ECM alteration were the synergistic results of membrane-associated matrix metalloproteinase and cell traction evidenced by proteinase inhibition and Rho-associated kinase (p160ROCK) inhibition experiments. These physical EC-ECM interactions suggest that ECM mechanical properties may influence angiogenic responses. In a 3D angiogenesis model, we measure angiogenic responses as a function of collagen matrix stiffness by inducing collagen cross-linking with microbial transglutaminase (mTG). Collagen matrices stiffen with both mTG treatment and incubation time as evidenced with biaxial mechanical test results and collagen TPF intensity increases with mTG treatment and that the ratio of TPF/SHG correlates with biaxial tested mechanical stiffness. SHG and optical coherence microscopy (OCM) are further used to show that other physical properties of the matrix do not change with mTG treatment, thus providing the same density but different stiffness with which to measure angiogenic responses. Stiffer matrices promote angiogenesis with more invading sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened 3D cultures, but there was evidence of greater matrix remodeling in stiffer gels using NLOM. Results of this study show angiogenic responses are enhanced with increasing stiffness and suggest that these properties may be used in tissue engineering and regenerative medicine applications to engineer angiogenesis.

Η παρουσία λευκωματουρίας αποτελεί δυσμενή προγνωστικό παράγοντα για τους ασθενείς με σπειραματονεφρίτιδα (ΣΝ) χωρίς εντούτοις αυτό να επιβεβαιώνεται πάντα. Συνεπώς, είναι επιβεβλημένη η εύρεση περισσότερο ειδικών προγνωστικών δεικτών που να αντανακλούν τη διαδικασία επούλωσης της βλάβης ή εξέλιξής της σε ίνωση. Σκοπός, της παρούσας μελέτης ήταν η αναγνώριση πρώιμων δεικτών εξέλιξης της νεφρικής βλάβης σε ασθενείς με σημαντικού βαθμού λευκωματουρία. Ως τέτοιοι προγνωστικοί δείκτες μελετήθηκαν η έκφραση της τρανσγελίνης (SM22) και της τρανσγλουταμινάσης–2 (TG2). Η SM22 είναι μια πρωτεΐνη που εκφράζεται στα λεία μυϊκά κύτταρα και αποτελεί δείκτη της επιθήλιο-μεσεγχυματικής μετάπτωσης. Η TG2 αποτελεί ένα ένζυμο που επάγει τη νεφρική ίνωση μεταβάλλοντας την ομοιόσταση της εξωκυτταρίου θεμέλιας ουσίας. Η έκφραση της SM22 μελετήθηκε με τη μέθοδο της ανοσοϊστοχημείας και του ανοσοφθορισμού, σε νεφρικές βιοψίες 67 ασθενών με διάφορους τύπους ΣΝ, και συσχετίστηκε με την κλινική τους πορεία μετά πάροδο πενταετούς παρακολούθησης. Επιπλέον, εξετάστηκε με τη μέθοδο του συνεντοπισμού, κατά πόσον η έκφραση της SM22 συμπίπτει με αυτήν της α–ακτίνης των λείων μυϊκών ινών (α–SMA). Η έκφραση της TG–2 μελετήθηκε με ανοσοφθορισμό σε νεφρικές βιοψίες από 32 ασθενείς με μεμβρανώδη νεφροπάθεια (ΜΝ). Όλοι οι ασθενείς με ΜΝ έλαβαν αγωγή με κορτικοειδή και κυκλοσπορίνη για 24 μήνες και σε 14 από αυτούς διενεργήθηκε επαναληπτική βιοψία και έγινε περαιτέρω μελέτη της TG2. Ως ομάδα ελέγχου για την έκφραση των παραπάνω μορίων χρησιμοποιήθηκαν τομές από υγιείς περιοχές νεφρών. Από τους 67 ασθενείς που μελετήθηκε η έκφραση της SM22, οι 46 διατήρησαν σταθερή νεφρική λειτουργία. Στις βιοψίες ελέγχου η έκφραση της SM22 περιορίστηκε στο τοίχωμα των αγγείων ενώ στους ασθενείς με ΣΝ επεκτάθηκε εντός των σπειραμάτων και του διάμεσου χώρου. Η SM22 βρέθηκε να σχετίζεται με το βαθμό σπειραματικής σκλήρυνσης και ίνωσης του διάμεσου χώρου, με το βαθμό μεσαγγειακής υπερπλασίας και την έκβαση της νεφρικής λειτουργίας. Η μελέτη συνεντοπισμού της SM22 και της α-SMA ανέδειξε περιοχές όπου οι δύο πρωτεΐνες εκφράζονταν ταυτόχρονα ενώ σε άλλες κάθε πρωτεΐνη εκφραζόταν χωριστά. Κατά το διάστημα παρακολούθησης οι 22 από τους 32 ασθενείς με ΜΝ παρουσίασαν σταθερή νεφρική λειτουργία. Η έκφραση της TG2 βρέθηκε αυξημένη στις βιοψίες των ασθενών με ΜΝ σε σχέση με την ομάδα ελέγχου. Η έκφραση της TG2 στις βιοψίες διάγνωσης ήταν περισσότερο αυξημένη στους ασθενείς με πιο έντονη διάμεση ίνωση και σπειραματική σκλήρυνση. Τέλος, η TG2 βρέθηκε αυξημένη στους περισσότερους ασθενείς στην επαναληπτική βιοψία, ενώ οι ασθενείς που παρουσίαζαν έντονη αύξηση της διάμεσης ίνωσης στην επαναληπτική βιοψία είχαν και εντονότερη έκφραση της TG2 κατά τη διάγνωση της νόσου. Συμπερασματικά, η έκφραση της SM22 βρέθηκε αυξημένη στο νεφρικό ιστό ασθενών με ΣΝ, ενώ η μελέτη συνεντοπισμού της SM22 και της α–SMA ανέδειξε την παρουσία πιθανώς διαφορετικών υποπληθυσμών μυοϊνοβλαστών. Όσον αφορά την TG2, η έντονη έκφραση της στο νεφρικό ιστό ασθενών με ΜΝ, η οποία καθίσταται εντονότερη μετά τη χορήγηση ανοσοκατασταλτικής αγωγής, υποδηλώνει τη συμμετοχή της TG2 στους μηχανισμούς βλάβης και εξέλιξης της ΜΝ. Επιπρόσθετα, η πρώιμη ανίχνευση της TG2 πιθανόν να έχει ιδιαίτερη σημασία στη ΜΝ καθώς η αυξημένη έκφρασή της φαίνεται να προηγείται της διάμεσης ίνωσης. / It is well established that albuminuria is an unfavorable prognostic factor for patients with various types of glomerulonephritis (GN), however, this is not observed in all patients. It is therefore important to identify more specific prognostic markers that reflect the healing process of injury or the progression to fibrosis. The aim of this study was to identify early markers of progression to renal injury in patients with significant degree of albuminuria. As such markers the expression of transgelin (SM22) and transglutaminase–2 (TG2) was studied. SM22 is expressed specifically in smooth muscle cells and is an early marker of epithelial to mesenchymal transition. TG–2 is an enzyme that contributes to renal scarring through altering extracellular matrix homeostasis. SM22 was studied in 67 patients with various kinds of GN and a 5 year follow up. SM22 was identified in kidney sections at the time of diagnosis using immunohistochemistry and immunofluorescence. SM22 expression was examined concerning its correlation with the clinical course of GNs. The expression of alpha smooth muscle actin (a–SMA) and co-localization with SM22 was also investigated. TG2 expression was studied by immunofluorescence in kidney sections from 32 patients with MN. These MN patients were subsequently treated by combination of cyclosporine and prednisolone for 24 months with a repeat biopsy taken in 14. Kidney sections from the normal part of kidneys were used as normal controls for the expression of the aforementioned molecules. Forty six out of 67 patients, that transgelin was studied, showed stable renal function. In control biopsies SM22 and a-SMA were restricted to vascular wall whereas in patients with GN expression was extended within glomeruli and the interstitium. SM22 expression correlated to the degree of glomerular sclerosis and interstitial fibrosis, to the degree of mesangial proliferation and renal function outcome. Double staining for co-localization of both SMM22 and a-SMA showed that in some areas of kidney tissue both proteins were identified whereas in other areas the expression of either SM22 or a-SMA was predominant. Twenty two out of 32 patients with MN showed stable renal function. TG2 immunostaining was increased in sections from patients with MN compared to healthy controls. TG2 at diagnosis was more intense in patients with more severe interstitial fibrosis and advanced glomerular sclerosis. TG2 significantly increased in most patients in the repeat biopsies whereas patients that showed a marked increase in interstitial fibrosis in the repeat biopsy had significantly more TG2 expression in the first biopsy. In conclusion, intense SM22 expression was observed in the renal tissue of patients with different types of GN. The co-localization study of SM22 and a–SMA suggests that different subpopulations of myofibroblasts might be involved in the development of kidney injury. As far it concerns TG2, its’ expression is increased in MN patients and continues to increase despite treatment which is suggestive of its’ involvement in the development and progression of renal scarring. Moreover, early detection of TG2 might be of value in MN since increased TG2 production seems to precede extensive interstitial fibrosis.