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Phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (Pepc) are two important CO₂-fixation enzymes which share a similar reaction mechanism. Both operate through a lid-gated active site and have a hypothesized enol-pyruvate intermediate in their catalytic pathway. While PEPCK is an important metabolic enzyme in animals and plays a broad role in cataplerosis, gluconeogenesis and glyceroneogenesis, Pepc reaction in plants catalyzes the first committed step in CO₂ fixation in CAM and C₄ plants via Rubisco. We are studying the structure-function aspects of both enzymes, with a goal of discovering new elements in these enzymes which can modulate catalysis. We have undertaken an interdisciplinary approach for this work and have shown that a combination of experimental and computational techniques can be complementary and can provide novel information. We have determined that in human PEPCK, Tyr235 forms an anion-quadrupole interaction with the carboxylate of PEP and thus positions the latter with respect to the enzyme-bound Mn²+ for optimal phosphoryl transfer and catalysis. We have also identified Pro82 as a catalytically influential residue in this enzyme. Using molecular dynamics simulations we have noted that absence of ligands induces active-site lid opening in GTP-PEPCKS and we have made the first observation of the intermediary structures of the lid opening event, the dynamics of which is an important element that controls GTP-PEPCK catalysis. We have determined the first three-dimensional crystal structure of an archaeal-type Pepc, i.e. C. perfringens PepcA. Our experimental data also provide information about the oligomerization of PepcAs and reveal that aspartate inhibits the C. perfringens enzyme competitively compared to the allosteric inhibition in Pepcs. Structure-based modeling has led to the identification of putative aspartate- and bicarbonate-binding residues in C. perfringens PepcA, of which Arg82, His11, Ser201, Arg390, Lys340, Arg342 and Arg344 probably play an important role. / Ph. D.

Emergent infectious diseases are a major driver of the accelerated rates of biodiversity loss that are being documented around the world. Global losses of amphibians provide evidence of this, especially those associated with chytridiomycosis, a lethal skin disease caused by the fungus Batrachochytrium dendrobatidis (Bd). Amphibian skin can harbor diverse bacterial communities that, in some cases, can inhibit the growth of Bd. Thus, there is interest in using skin bacteria as probiotics to mitigate Bd infections in amphibians. However, experiments testing this conservation approach have yielded mixed results, suggesting a lack of understanding about the ecology of these microbial communities. My dissertation research aimed to assess basic ecological questions in microbial ecology and to contribute to the development of probiotics using amphibian skin bacteria. First, to assess whether environmental conditions influence the function of amphibian skin bacterial communities, I conducted a field survey across low and high elevation populations of an amphibian host to assess their skin bacterial communities and metabolite profiles. I found that similar bacterial communities produced different metabolites at different locations, implying a potential functional plasticity. Second, since culturing is critical for characterizing bacteria, I aimed to identify the culture media (low vs high nutrient concentration) that recovers the most representative fraction of the amphibian skin bacterial community. I found that media with low nutrient concentrations cultured a higher diversity and recovered a more representative fraction of the diversity occurring on amphibian skin. I also determined that sampling more individuals is critical to maximize culture collections. Third, I assessed the diversity of the amphibian skin fungal community in relation to Bd infection across eight amphibian species. I determined that amphibian species was the most important predictor of fungal diversity and community structure, and that Bd infection did not have a strong impact. My dissertation highlights the importance of environmental conditions in the function of amphibian skin bacteria, expands our knowledge of the understudied fungal component of the amphibian skin microbiome, and complements current efforts in amphibian conservation. / Ph. D. / In light of the global losses of amphibian diversity due to, in part, the skin disease chytridiomycosis (caused by the fungus Batrachochytrium dendrobatidis [Bd]); the discovery that some amphibian-skin bacteria can inhibit Bd growth provides hope for amphibian conservation via their use as probiotics to control Bd infections. However, experiments testing these bacteria have yielded inconsistent results, suggesting a limited understanding about the factors influencing the diversity of amphibian-skin microbes and their ability to inhibit Bd. Also, efforts to identify effective candidates for probiotic therapy are still premature. Thus, my dissertation had an ecological emphasis and focused on complementing conservation efforts focused on probiotics. First, I assessed whether environmental conditions influence bacteriallyproduced products, which can have antifungal properties. Specifically, I surveyed low and highelevation populations of an amphibian species to assess the skin-bacteria and their products. I determined that, while skin bacterial communities were similar across an environmental gradient, their products differed, suggesting potential different antifungal properties. Second, I assessed the ability of different culture media types (low vs high nutrient concentrations) to grow a high portion and most representative fraction of the amphibian-skin bacteria. I found that culture media with low nutrient concentrations allowed the growth of a higher diversity of the bacteria occurring on the amphibian-skin, including the abundant members, and also determined that including a large number of amphibians is the best way to improve culture collections. Third, I assessed the fungal diversity occurring in the skin of different amphibian species and how it might response to Bd infections, and examined whether skin-fungi interact with co-occurring bacteria. I found that the amphibian species was the most important driver of the fungal diversity, and that Bd infection did not influence the diversity of these communities. Moreover, I identified the most diverse fungal phyla occurring in the amphibian-skin and determined that these fungi might interact with co-occurring bacteria. My dissertation contributes to our understanding about the influence of the environmental conditions in the amphibian-skin bacteria, expands our limited knowledge on the amphibian-skin fungi, and complement current amphibian conservation efforts.

Kinases are involved in a multitude of signaling pathways, such as cellular growth, proliferation, and apoptosis, and have been discovered to be important in numerous diseases including cancer, Alzheimer's disease, cardiovascular health, rheumatoid arthritis, and fibrosis. Due to the involvement in a wide variety of disease types, kinases have been studied for exploitation and use as targets for therapeutics. There are many limitations with developing kinase target therapeutics due to the high similarity of kinase active site composition, making the utilization of new techniques to determine kinase exploitability for therapeutic design with high specificity essential for the advancement of novel drug strategies. In silico approaches have become increasingly prevalent for providing useful insight into protein structure-function relationships, offering new information to researchers about drug discovery strategies. This work utilizes streamlined computational techniques on an atomistic level to aid in the identification of orthologue and isoform exploitability, identifying new features to be utilized for future inhibitor design. By exploring two separate kinases and kinase targeting domains, we found that orthologues and isoforms contain distinct features, likely responsible for their biological roles, which can be utilized and exploited for selective drug development. In this work, we identified new exploitable features between kinase orthologues for treatment in Human African Trypanosomiasis and structural morphology differences between two kinase isoforms that can potentially be exploited for cancer therapeutic design. / Master of Science in Life Sciences / Numerous diseases such as cancer, Alzheimer's disease, cardiovascular disease, rheumatoid arthritis, and fibrosis have been attributed to different cell growth and survival pathways. Many of these pathways are controlled by a class of enzymes called kinases. Kinases are involved in almost every metabolic pathway in human cells and can act as molecular switches to turn on and off disease progression. Due to the involvement of these kinases' in a wide variety of disease types, kinases have been continually studied for the development of new drugs. Developing effective drugs for kinases requires an extensive understanding of the structural characteristics due to the high structural similarity across all kinases. In silico, or computational, techniques are useful strategies for drug development practices, offering new information into protein structure-function relationships, which in turn can be utilized in drug discovery advancements. Utilizing computational methods to explore structural features can help identify specific protein structural features, thus providing new strategies for protein specific inhibitor design. In this work, we identified new exploitable features between kinase orthologues for treatment in Human African Trypanosomiasis and structural morphology differences between two kinase isoforms that can potentially be exploited for cancer therapeutic design.

Yes / Purpose: Inter-individual variance in optic nerve head (ONH) position, axial length and location of the temporal raphe suggest that customizing mapping between visual field locations and optic nerve head sectors for individuals may be clinically useful. Here we quantify the proportion of the population predicted to have structure-function mappings that markedly deviate from “average”, and thus would benefit from customized mapping. Design: Database study and case report Participants: Population database of 2836 eyes from the Beijing Eye Study; single case report of an individual with primary open angle glaucoma Methods: Using the morphometric fundus data of the Beijing Eye Study on 2836 eyes and applying a recently developed model based on axial length and ONH position relative to the fovea, we determined for each measurement location in the 24-2 Humphrey visual field the proportion of eyes for which, in the customized approach as compared to the generalized approach, the mapped ONH sector was shifted into a different sector. We determined the proportion of eyes for which the mapped ONH location was shifted by 15°, 30° or 60°. Main outcome measures: Mapping correspondence between locations in visual field space to localized sectors on the optic nerve head Results: The largest inter-individual differences in mapping are in the nasal step region where the same visual field location can map to either the superior or inferior ONH depending on other anatomical features. For these visual field locations, approximately 12% of eyes showed a mapping opposite to conventional expectations. Conclusions: Anatomically customised mapping shifts the map markedly in approximately 12% of the general population in the nasal step region where visual field locations can map to the opposite pole of the ONH than conventionally considered. Early glaucomatous damage commonly affects this region, hence individually matching structure to function may prove clinically useful for the diagnosis and monitoring of progression within individuals. / Australian Research Council Linkage Project 130100055 (industry partner, Heidelberg Engineering, GmBH, Germany).

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plant defensin peptides have become promising and attractive candidates to be used as antifungal agents in agricultural biotechnology. These peptides have a broad spectrum antifungal activity and play a vital role in the innate immune system of plants. Plant diseases caused by fungi are a major contributor to the decrease in the quality and safety of agricultural products. Due to the dangerous effects and negative environmental impact of pesticides, an effective, safe, natural and durable method to control crop pathogens has therefore become one of the major concerns in modern agriculture. Although these peptides are promising and attractive candidates, their precise mechanism of action is to date still unknown. Several common observations have been made. These include the antagonistic effect of cations on the activity of plant defensins. It is of vital importance to understand the underlying mechanism of the cation-antagonistic effect on the antifungal potency of defensin peptides in order to evaluate the possible contribution to defence reactions against microorganisms in planta. To this end we set out to characterize the effect of cations in the form of biological salts, NaCl, KCl, MgCl2 and CaCl2 on the structural stability and activity in terms of growth inhibition, morphological effects and permeabilization. In order to perform these characterization experiments, a production method resulting in a greater yield and involving simple and rapid purification methods was required. Heliophila coronopifolia peptides have previously been produced in a bacterial system, however the purification methods were tedious resulting in poor yields. Pichia pastoris was selected as production system as several other plant defensins have been successfully produced in this eukaryotic system. Hc-AFP1 and Hc-AFP3 was successfully produced using the Pichia production system and rendered active peptides. Hc-AFP2 and Hc- AFP4 was, however, not produced correctly, due to a post-translational modification event leading to the cyclization of the N-terminal glutamine to generate pyroglutamic acid. This modification negatively influenced the activity of these peptides. An active Hc-AFP2 could be produced by replacing the production buffer with a reduced ionic buffer. The effect of divalent and monovalent cations on the secondary structure of Hc-AFP1 was evaluated by circular dichroism spectroscopy. These cations induced a conformational change in the secondary structure of Hc-AFP1, with NaCl and MgCl2 inducing a more defined secondary structure and KCl and CaCl2 inducing a less defined secondary structure. Monovalent cations caused a slight reduction in the growth inhibition activity of Hc-AFP1 on Botrytis cinerea, however, characteristic hyperbranching and other morphogentic effects were still visible. Divalent cations had a greater antagonistic effect on the activity of Hc-AFP1, completely abolishing the growth inhibitory activity of the peptide, but the induced morphological effects on hyphae remained present. The activity of Hc-AFP1 to permeabilize B. cinerea hyphae was not influenced by the addition of cations, however it was in fact increased to up to 10-fold. However, since the growth inhibition activity of Hc-AFP1 was reduced in the presence of the biological salts indicates that permeabilization is not the sole activity responsible for growth inhibition caused by Hc-AFP1. This peptide probably has an alternative/primary target and more complex MOA. This is the first known report of the investigation of the influence of cations on the structure of plant defensin peptides. It is clear that cations induce a secondary structural conformational change in Hc-AFP1. This may be linked to the antagonism on the activity of this peptide. This study provides significant progress towards the structure-function analysis of plant defensins. / AFRIKAANSE OPSOMMING: Plantdefensinpeptiede word beskou as belowende en aantreklike kandidate vir gebruik as swammiddles in agribiotegnologie. Hierdie peptiede beskik oor breë spektrum antifungiese aktiwiteit en speel ‘n essensiële rol in die ingebore immuunsisteem van plante. Plant siektes wat deur swamme veroorsaak word dra betekenisvol by tot die afname in die kwaliteit en veiligheid van landbouprodukte. As gevolg van die skadelike effekte en negatiewe omgewingsimpak van plaagdoders, het effektiewe, veilige, natuurlike en duursame metodes om gewaspatogene te beheer, van die belangrikste vraagstukke van moderne landbou geword. Alhoewel hierdie peptiede belowende en aantreklike kandidate is vir die toepassing, is hulle presiese meganisme van aksie tot vandag toe steeds onbekend. Verskeie algemene waarnemings is egter al gemaak. Dit sluit die antagonistiese effek van katione op die aktiwiteit van plantdefensinpeptiede in. Dit is kernbelangrik om die onderliggende meganisme van die katioon-antagonistiese effek op die antifungiese effektiwiteit te verstaan om die moontlike bydrae van die peptiede tot die verdedigingsreaksies teen mikro-organismes in planta te evalueer. Met die doel voor oë het ons gemik om die effek van katione, spesifiek in die vorm van die biologiese soute NaCl, KCl, MgCl2 en CaCl2, op die strukturele stabiliteit en aktiwiteit in terme van groei inhibisie, morfologiese effekte en permeabilisasie te karakteriseer. Om uiteindelik hierdie karakterisasie eksperimente uit te voer was dit nodig om ‘n metode met ‘n groter opbrengs en wat vinnige suiwering van die peptied ondersteun, te optimiseer. Heliophila coronopifolia peptiede was voorheen in ‘n bakteriese sisteem geproduseer, maar die suiweringsmetodes was tydsaam en het gelei tot ‘n swak opbrengs. Pichia pastoris is dus geselekteer as die produksie sisteem aangesien verskeie ander plantdefensinpeptiede al suksesvol geproduseer is in hierdie eukariotiese sisteem. Hc-AFP1 and Hc-AFP3 is suksesvol vervaardig in die Pichia sisteem en het aktiewiteit getoon. Hc-AFP2 and Hc-AFP4 kon egter nie korrek vervaardig word nie as gevolg van ‘n na-vertalingsverandering wat gelei het tot die siklisering van die N-terminale glutamien, om piroglutamiensuur te lewer. Hierdie verandering het die aktiwiteit van die peptied negatief beinvloed. ‘n Aktiewe Hc-AFP2 kon wel vervaardig word deur die produksiebuffer te vervang met ‘n lae-ionise buffer. Die effek van divalente en monovalente katione op die sekondêre struktuur van Hc-AFP1 is ge-evalueer deur van sirkulêre dikroisme spektroskopie gebruik te maak. Hierdie katione het ‘n vouingsverandering in die sekondêre struktuur van Hc-AFP1 geïnduseer, NaCl and MgCl2 het ‘n meer gedefinieërde sekondêre struktuur induseer, terwyl KCl and CaCl2 ‘n minder gedefinieërde sekondêre struktuur geinduseer het. Monovalente katione het ‘n effense vermindering in die groei-inhibisie aktiwiteit van Hc-AFP1 op Botrytis cinerea veroorsaak, alhoewel kenmerkende hife-oorvertakking en ander morfologiese effekte nogsteeds sigbaar was. Divalente katione het ‘n sterker antagonistiese effek gehad op die aktiwiteit van Hc-AFP1, waar dit totaal en al die groei-inhibisie aktiwiteit van die peptied vernietig het, alhoewel die geïnduseerde morfologiese effekte op die hiffes steeds sigbaar was . Die aktiwiteit van Hc-AFP1 om B. cinerea hyphae te permeabiliseer is nie negatief beinvloed deur die byvoeging van katione nie, tewens dit het die aktiwiteit tot 10-voudig verhoog. Aangesien die groei-inhibisie aktiwiteit van Hc-AFP1 nie verminder is in die teenwoordigheid van die biologiese soute nie, dui dit aan dat permeabilisasie nie die enigste aktiwiteit is wat die groei inhibisie veroorsaak het nie. Die peptied het dus moontlik ‘n alternatiewe of primêre teiken en ‘n meer komplekse meganisme van aksie. Dit is die eerste verslag wat die invloed van katione op die struktuur van plantdefensinpeptiede ondersoek het. Dit is duidelik dat katione ‘n sekondêre strukturele vouingsverandering in Hc-AFP1 induseer. Hierdie verandering mag dalk bydra tot die antagonistiese uitwerking op die aktiwiteit van hierdie peptied. Hierdie studie het betekensisvolle vordering gemaak met die analise van die struktuur-funksie interaksie van plantdefensinpeptiede. / The National Research Foundation (NRF), Institute of Wine Biotechnology (IWBT), THRIP and Winetech for financial assistance.

Les chaînes de glycosaminoglycanes (GAGs) des protéoglycanes (PGs) jouent un rôle majeur dans la régulation de multiples événements cellulaires et le maintien de l'architecture des tissus. Des perturbations de la synthèse des GAGs sont impliquées dans des pathologies d'origine dégénérative, tumorale et génétique, tel que le syndrome progéroïde d'Ehlers-Danlos (ED). Ce déficit résulte de mutations de la [bêta]1,4-galactosyltransférase 7 ([bêta]4GalT7) humaine associées à des atteintes sévères du système musculo-squelettique. En effet, cette enzyme catalyse une étape essentielle de l?initiation de la synthèse des GAGs à partir de la protéine "core" des PGs et de xylosides exogènes. Notre travail a porté sur l'étude structure-fonction de la [bêta]4GalT7 recombinante humaine. Nous avons associé des approches in vitro et ex vivo afin d?explorer le rôle des acides aminés des motifs 163DVD165, 221FWGWGREDDD230 et 257HLH259, strictement conservés au sein des [bêta]4GalTs. L'étude des conséquences de mutations systématiques sur les propriétés cinétiques et fonctionnelles de la [bêta]4GalT7 recombinante a permis d'identifier des acides aminés essentiels du site actif. Nous avons montré que les résidus D165 et H257 forment des liaisons de coordination avec le cation Mn2+ et proposé le rôle du résidu D228 dans la catalyse. Nous avons mis en évidence un rôle central du résidu W224 dans les interactions avec les substrats donneur et accepteur. Nous avons également établi les bases moléculaires des mutations de la [bêta]4GalT7 associées au syndrome ED. Enfin, l'étude de mécanismes de régulation épigénétique des voies de biosynthèse des GAGs dans les cellules H-EMC-SS de chondrosarcome humain a mis en évidence une hyperméthylation spécifique des gènes de la famille des 3-O-sulfotransférases, associée à un phénotype invasif. L'ensemble de ce travail ouvre des perspectives vers de nouvelles stratégies thérapeutiques dans le traitement des arthropathies / Proteoglycans (PGs) and their glycosaminoglycan chains (GAGs), play a major role in the architecture of extracellular matrices and are implicated in numerous cell events. The impairment of GAG synthesis and sulfation is involved in degenerative, tumor and genetic diseases, such as the progeroid form of Ehlers-Danlos (ED) syndrome. This inherited disorder is due to mutations of human [bêta]4GalT7 ([bêta]4GalT7) causing a defect in GAG synthesis, associated with severe musculo-skeletal alterations. Indeed, this enzyme catalyzes a key step in GAG synthesis linked to the core protein of PGs and from exogenous xylosides. Our work has been focused on the structural and functional characterization of human recombinant [bêta]4GalT7 enzyme. We combined in vitro and ex vivo approaches to explore the role of amino acids located in 163DVD165, 221FWGWGREDDD230 and 257HLH259 motifs, which are highly conserved within [bêta]4GalTs. The study of the consequences of site-directed mutations on kinetic and functional properties of the [bêta]4GalT7 enzyme allowed us to identify key active site amino acids. Our results indicate that D165 and H257 residues form coordination bonds with Mn2+ divalent cations. Furthermore, we suggested a catalytic role for D228 residue and highlighted a central role of W224 residue via interactions with the donor and acceptor substrates. We also determined the molecular basis of [bêta]4GalT7 mutations associated with ED syndrome. Finally, the study of epigenetic regulation mechanisms by DNA methylation of GAG biosynthesis in human chondrosarcoma cells (H-EMC-SS) revealed the specific hypermethylation of the 3-O-sulfotransferase gene family, associated with the invasive phenotype of these cells. Together, this work paves the way towards innovative strategies in the treatment of arthropathies

Les glycosyltransférases (GTs) sont une famille importante d’enzymes responsable de la biosynthèse des chaînes de glycosaminoglycane (GAG) des protéoglycanes, composants clés de la matrice extracellulaire et de la membrane plasmique cellulaire impliqués dans la communication, l'adhésion, la migration et la prolifération cellulaires. Les GTs jouent donc un rôle central dans de nombreux processus physiologiques et physiopathologiques tels que les cancers ou encore les maladies dégénératives et génétiques. Parmi ces GTs, la ß1,4-galactosyltransférase 7 (ß4GalT7) est une cible thérapeutique potentielle puisqu'elle : i) catalyse une étape précoce et majeure de la biosynthèse des chaînes de GAG, ii) est impliquée dans une forme rare de maladie génétique des tissus conjonctifs, le syndrome d’Ehlers-Danlos, iii) prend en charge des xylosides exogènes modulant son activité in vitro et in vivo. Ce travail de thèse s'organise sur une étude structure/fonction de cette enzyme afin de cerner les résidus d'acides aminés clés dans l'interaction de l'enzyme avec un substrat modèle, le 4-methylombelliferyl-ß-D-xylose (4-MOX). Les résidus Y194, Y196 et Y199 ont ainsi été identifiés comme clés dans l'architecture du site de fixation du substrat accepteur et dans l'interaction de l'enzyme avec le xyloside. Au contraire, les résidus H195, R226 et le résidu R270, muté dans la forme progéroïde du syndrome d'Ehlers-Danlos, apparaissent comme des résidus « modulant » l'activité de l'enzyme, notamment du fait d'interactions moléculaires impliquant leur squelette peptidique pour H195 et R226 et une boucle flexible pour R270. Ces travaux ont permis de guider la synthèse d’analogues xylosidiques visant à inhiber l'activité de la ß4GalT7 humaine. Parmi les propositions, un dérivé fluoré du 4-MOX apparaît comme un inhibiteur efficace de la ß4GalT7 in vitro et in cellulo. Faiblement cytotoxique, ce dérivé réduit la prolifération des cellules de lignées cancéreuses SW1353 et MB MDA 231. Ces résultats ouvrent la perspective de nouvelles stratégies thérapeutiques utilisant les xylosides comme agents potentiels dans le traitement de cancers ou encore des maladies génétiques des tissus conjonctifs / Glycosyltransferases (GTs) are an important family of enzymes involved in the biosynthesis of glycosaminoglycan (GAG) chains of proteoglycans which are key components of cell plasma membranes and of the extracellular matrix, and are thus implicated in cell communication, adhesion, migration and proliferation. GTs are thus key players in numerous pathophysiological processes such as cancers, degenerative and genetic diseases. Among these GTs, ß1,4-galactosyltransférase 7 (ß4GalT7) is a potential therapeutic target since : i) it catalyzes a rate-limiting step in the early phase of the GAG chains biosynthesis, ii) it is implicated in a rare genetic connective tissue disorder (Ehlers-Danlos Syndrome), iii) its in vitro and in vivo activity can be modulated by exogenous xyloside molecules. This PhD work is focused on a structure/function study of the enzyme aiming to identify key amino acid residues that interact with 4-methylumbelliferyl-ß-D-xylose (4-MUX), taken as reference substrate. Y194, Y196 and Y199 have been identified as key residues for the architecture of the acceptor substrate binding site and establish interactions with 4-MUX. By contrast, H195, R226 and R270, a residue mutated in the progeroid form of Ehlers-Danlos Syndrome, should rather be considered as “modulating” residues towards the ß4GalT7 activity, H195 and R226 interacting with 4-MUX with their polypeptide backbone, and R270 via a flexible loop. This work guided the design of xylosidic compounds that would potentially inhibit the ß4GalT7 activity. Thus, a fluorinated derivative of 4-MUX appeared as an efficient in vitro and in cellulo inhibitor of the enzyme. Poorly cytotoxic, this compound also reduced the proliferation rate of cancer cells SW1353 and MB MDA 231. Altogether, these results offer new therapeutic strategies using xylosides as potential therapeutic agents in the treatment of cancer or rare genetic disorders

The objective is to contribute to several issues regarding the traditional view of the local structure of passive scalar fields: (1) probability density function (PDF) of the scalar concentration and scalar gradient, (2) the scalar power spectrum, (3) the structure functions, and (4) correlation functions and multi-point correlators. In addition, the research provides a geometric description of two-dimensional transects of the passive scalar iso-surfaces using the tools of fractal geometry. The local structure is analyzed as a function of large-scale anisotropy, intermittency factor, Reynolds number, and initial condition of the scalar injection. Experiments were performed in the bed boundary layer produced by a uniform depth open channel flow of water in a tilting flume for Re_lamda = 63, 94, and 120. A small nozzle iso-kinetically delivers a passive scalar of high Schmidt number ( Sc = 1000) at mid-depth to generate the turbulent scalar field. Three nozzle diameters are used to study the effects of the injection length scale. High-resolution planar laser induced fluorescence (PLIF) technique is used to measure the scalar field. The local structure far from isotropic and is influenced even at the smallest scales by large-scale anisotropy, initial injection length scale and the Reynolds number of the flow. The PDF of the scalar fluctuations is non-Gaussian and dependent on large-scale anisotropy. The PDF of scalar gradients show the influence of large-scale anisotropy on the structure at the smallest scales. The spectrum of the scalar field deviates from the in the inertial convection regime and is dependent on large-scale anisotropy, external intermittency, and low Reynolds number. There is no evidence of Batchelors k^-1 scaling law. The scaling exponents of the even-ordered structure functions appear to be inversely correlated with the kurtosis of the scalar fluctuations. The fractal geometry of the two dimensional transects of passive scalar iso-surfaces is scale dependent. The fractal dimension is 1.0 at the smallest length scale and increases in a universal manner in the viscous-convective regime. The coverage length underestimate reflects this universal behavior with practical significance. The lacunarity function shows that the instantaneous scalar field is most in-homogenous around the Kolmogorov scale.