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Doctor of Philosophy / Department of Grain Science and Industry / Jon M. Faubion / Starch granule surface components were studied as a function of puroindoline haplotype, starch isolation method, and processing fraction. Commonly grown cultivars and near-isogenic wheat lines that varied in their wheat endosperm hardness were collected. Wheat whole-meal, flour and starch were evaluated for their polar lipid composition. Water-washed starch was isolated using a modified batter method and a dough method. Direct infusion tandem mass spectrometry was used to identify the lipid species in the extracts. A total of 155 polar lipid species in wheat meal, flour and starch were quantitatively characterized. The predominant polar lipid classes were digalactosyldiglycerides, monogalactosyldiglycerides, phosphatidylcholine, and lysophosphatidylcholine. Wheat whole-meal, flour and surface-starch contained greater concentrations of total galactolipids while internal-starch lipids contained greater concentrations of monoacyl phospholipids. Wide ranges in starch surface polar lipid concentrations were observed between the two starch isolation methods. Starch isolation methods provided a greater source of variation than did wheat kernel hardness. When dough is optimally mixed the lipids originally on the surface of wheat starch become incorporated into the gluten phase of the dough, whereas in a batter system the starch-surface lipids stay associated with the starch granule surface. The greatest quantities of polar lipids on the starch surface occurred when both puroindoline proteins were present on starch in their wild-type form. Starch surface polar lipid content decreased dramatically when one of the puroindoline proteins was null, or if the puroindoline-b (pin-b) was in the mutated form (Tryptophan-44 to Arginine). Within the hard textured samples, more polar lipids were present on the starch surface when pin-b was in its wild-type form and puroindoline-a (pin-a) was null than when pin-a was in its wild-type form and pin-b was null. The lowest amount of polar lipids were present when pin-b was mutated (Tryptophan-44 to Arginine) and pin-a was in its wild-type form. This indicates the relative importance of pin-b’s presence and structure as it relates to lipid association with the starch granule surface.

Master of Science / Department of Grain Science and Industry / Jon M. Faubion / This work investigated the relationship between flour age (days post-milling), storage condition (temperature), and the bread baking quality of whole wheat flour. A laboratory scale milling method was designed to mimic the particle size distribution of commercially milled whole wheat flours and the 100 g ‘pup’ loaf baking method was adapted for use with whole wheat doughs. Laboratory milled whole wheat flour (Karl 92) was subjected to a 21 day storage study at two storage temperatures (72 & -15 F) with quality (baking) and chemical (lipids) analyses conducted every three days. Parameters for quality analysis included: loaf weight, volume & specific volume, as well as slice area, cell number, wall thickness, cell diameter, elongation, and non-uniformity. Three lipid classes (glycolipids, phospholipids, and neutral lipids) were extracted and analyzed by TLC with quantification by computerized analysis of spot size and density. Results were analyzed by ANOVA. Analysis of the loaf quality data revealed no trends in volume or specific volume as a function of storage time or temperature, although values for some specific days were significantly different. Likewise, analysis of crumb characteristics revealed no consistent trends for either time or storage temperature. Again, values for some, but not all, parameters (area, brightness, wall thickness, cell diameter, and non-uniformity) were significantly different for specific days of the study. Analysis of lipids revealed no consistent trends for either time or storage temperature. However, values for some lipid classes (total glycolipids, free phospholipids, and total phospholipids) were significantly different for storage temperature, and values for total neutral lipids were significantly different for specific days of the study. Suggested future research opportunities include: using new crop wheat, increasing storage duration, performing WW flour lipid exchange studies, and using lipid profiling to identify and more closely track changes in individual lipid species.

Intracellular transport of membrane-bound vesicles and organelles is a process fundamental for many cellular functions including cell morphogenesis and signaling. The transport is mediated by ensembles of motor proteins, such as kinesins, walking on microtubule tracks. When transporting membrane-bound cargo inside a cell, the motors are linked to diffusive lipid bilayers either directly or via adaptor molecules. The fluidity of the lipid bilayers induces loose inter-motor coupling which is likely to impact the collective motor dynamics and may induce cooperativity. Here, we investigate the influence of loose coupling of kinesin motors on its transport characteristics. In the first part of this thesis, we used truncated kinesin-1 motors with a streptavidin-binding-peptide (SBP) tag and performed gliding motility assays on streptavidin-loaded biotinylated supported lipid bilayers (SLBs), so called ‘membrane-anchored’ gliding motility assays. We show that the membrane-anchored motors act cooperatively; the microtubule gliding velocity increases with increasing motor density. This is in contrast to the transport behavior of multiple motors rigidly bound to a substrate. There, the motility is either insensitive to the motor density or shows negative interference at higher motor density, depending on the structure of the motors. The cooperativity in transport driven by membrane-anchored motors can be explained as following: while stepping on a microtubule, membrane-anchored motors slip backwards in the viscous membrane, thus propelling the microtubule in the solution at a velocity, given by the difference of the motor stepping velocity and the slipping velocity. The motor stepping on the microtubule occurs at maximal stepping velocity because the load on the membrane-anchored motors is minute. Thus, the slipping velocity of membrane-anchored motors determines the microtubule gliding velocity. At steady state, the drag force on the microtubule in the solution is equal to the collective drag force on the membrane-anchored motors slipping in the viscous membrane. As a consequence, at low motor density, membrane-anchored motors slip back faster to balance the drag force of the microtubule in the solution. This results in a microtubule gliding velocity significantly lower than the maximal stepping velocity of the individual motors. In contrast, at high motor density, the microtubules are propelled faster with velocities equal to the maximal stepping velocity of individual motors. Because, in this case, the collective drag force on the motors even at very low slipping velocity, is large enough to balance the microtubule drag in the solution. The theoretical model developed based on this explanation is in good agreement with the experimental data of gliding velocities at different motor densities. The model gives information about the distance that the diffusing motors can isotropically reach to bind to a microtubule, which for membrane-anchored kinesin-1 is ~0.3 µm, an order of magnitude higher as compared to rigidly bound motors, owing to the lateral mobility of motors on the membrane. In addition, the model can be used to predict the number of motors involved in transport of a microtubule based on its gliding velocity. In the second part of the thesis, we investigated the effect of loose inter-motor coupling on the transport behavior of KIF16B, a recently discovered kinesin motor with an inherent lipid-binding domain. Recent studies based on cell biological and cell extract experiments, have postulated that cargo binding of KIF16B is required to activate and dimerize the motor, making it a superprocessive motor. Here, we demonstrate that recombinant full-length KIF16B is a dimer even in the absence of cargo or additional proteins. The KIF16B dimers are active and processive, which demonstrates that the motors are not auto-inhibited in our experiments. Thus, in cells and cell extracts Kif16B may be inhibited by additional factors, which are removed upon cargo binding. Single molecule analysis of KIF16B-GFP reveals that the motors are not superprocessive but exhibit a processivity similar to kinesin-1 indicating that additional factors are most likely necessary to achieve superprocessivity. Transport on membrane-anchored KIF16B motors exhibited a similar cooperative behavior as membrane-anchored kinesin-1 where the microtubule gliding velocity increased with increasing motor density. Taken together, our results demonstrate that the loose coupling of motors via lipid bilayers provides flexibility to cytoskeletal transport systems and induces cooperativity in multi-motor transport. Moreover, our ‘membrane-anchored’ gliding motility assays can be used to study the effects of lipid diffusivity (e.g. the presence of lipid micro-domains and rafts), lipid composition, and adaptor proteins on the collective dynamics of different motors.

The work described in this thesis investigates the lipid metabolism of human hepatocytes in the context of Hepatitis C Virus (HCV) infection. This includes lipoprotein signalling and cholesterol metabolism targeted analysis of gene expression as well as the influence of polyunsaturated ER targeting liposomes (PERLs) on infection. These analyses indicate that HCV suppresses the expression of key regulators throughout the cholesterol biosynthesis pathway. This effect was quantified and the influence of liposome treatment evaluated. The latter resulted in the formulation of the hypothesis that PERL treatment interfers with virus-induced abberations of the cholesterol biosynthesis pathway and normalises the expression of four genes directly involved in cholesterol regulation. In addition, the lipidome of isolated lipid droplet was analysed by mass spectrometry. These data, combined with microscopy data suggest that PERLs interfere with S-palmitoylation of the HCV core protein resulting in dissociation of core from lipid droplets. This is likely to interrupt the viral assembly process, leading to inhibition of the production of infectious viral particles. Further described here are two different yet unsuccessful approaches to fluorescently label HCV RNA for live cell microscopy studies, namely an MS2 coat protein mediated approach, and Alexa®UTP labelling.

This thesis presents applications of high resolution secondary ion mass spectrometry (NanoSIMS) analysis for stable isotope imaging in biological samples. These projects were designed to explore the potential applications of NanoSIMS analysis, and to develop protocols and novel methodologies to visualize and quantify biological processes. Working with collaborators in the UK and USA, I have applied NanoSIMS analysis to study 3 research areas, including molecule interactions, single cell metabolisms and lipid imaging in tissues. Antimicrobial peptides (AMPs) play important role in the immune system, and understanding how AMPs interact with cell membranes can provide useful information to design new therapies to control infection. The pore structures and dynamics of the interaction of AMPs with membranes has been visualized for the first time and confirmed with combined AFM and NanoSIMS analysis. A correlative backscattered electron (BSE) imaging and NanoSIMS analysis methodology has been developed to study glutamine metabolism in single cancer cells. This method enables us to measure the chemical information in specific organelles in these cells and can be widely applied to study metabolisms and to trace the uptake of labelled molecules in biological matrices. Quantitative analysis on the effects of hypoxic conditions and the PYGL gene were studied. Applying correlative BSE and NanoSIMS analysis, I also studied lipid uptake mechanisms in various mouse tissues, including brown adipose tissue, heart, intestines, liver and skeletal muscle, mainly focused on a recently discovered protein, GPIHBP1, and its function in the lipid uptake process. TRL margination was proved to depend on the GPIBP1-LPL complex, and 3 stages of lipid transport from capillary lumen to lipid droplets was also visualized by combined BSE and NanoSIMS analysis.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Selection of raw materials for a consistent and high quality end product has been a challenge for brewers globally. Various different factors may influence quality and although a great number of methods for malt analysis exist today for the prediction of end product quality, some still do not accurately represent malt performance in beer. This research focussed on determining parameters in malts to predict two of the major beer quality determining factors namely, foam- and flavour stability. Specific biochemical markers in barley malt such as lipid transfer protein 1 (LTP1) lipoxygenase-1 (LOX-1), anti-radical/oxidant potential (AROP), free amino nitrogen and intact protein were determined and used in beer quality prediction from malt character. These biochemical quality predictions were then correlated with the end product beer quality as assessed in sensory analysis trials on micro-brewed beers. Being such a multi-faceted factor in beer, LTP1 have already become an attractive field of study. LTP1 is primarily associated with stable beer foam, as a foam protein in its own right, and acting as a lipid scavenger. This protein is also theorised to play a role in the stability of beer flavour by possibly acting as anti-oxidant. Lastly LTP1 is known to have anti-yeast activity, which could negatively impact fermentation. In this study LTP1 and its lipid bound isoform LTP1b were successfully purified in an economical and easy five step protocol. Both isoforms showed temperature stability at temperatures >90°C and prefer more neutral and basic pH environments. Although the reported antioxidant activity was not observed, both purified LTP1 and LTP1b inhibited lipoxygenase-1 (LOX-1) activity, which is responsible for the enzymatic breakdown of linoleic acid to form 2(E)-nonenal. This is a novel finding that links LTP1 also to flavour stability. LTP1 exhibited anti-yeast activity whereas LTP1b lost most if not all the activity. However, since most of the LTP1 is converted to LTP1b and glycosylated isoforms during the brewing process fermentation will not be greatly influenced, while foam and flavour stability could still be promoted by the presence of LTP1b. Flavour deterioration of the final packaged product is partially due to the enzymatic production of 2(E)-nonenal by LOX-1 and the presence of free oxygen radical species, limited anti-radical/oxidant potential (AROP) and LTP1. The development of two 96-well micro-assays based on the ferrous oxidation-xylenol orange (FOX) assay for the determination of LOX-1 and AROP was successfully accomplished and compared well with established assays. The LOXFOX and AROP-FOX assays were specifically developed for the on-site, high throughput comparative determination of LOX-1 and AROP in malt and other brewery samples. The AROP-FOX and LOX-FOX micro-assays and a number of established assays were used to categorise malts in different predicted quality groups, various biochemical markers were measured which included LOX activity, LTP1 content, FAN values, intact protein concentration and AROP. An excellent trend (R2=0.93) was found between FAN/LOX and LTP1/LOX which also correlated with the novel observation that LOX-1 activity is inhibited by LTP1 at various concentrations. These trends could assist brewers in optimal blending for not only high quality end products but also fermentation predictions. To determine whether these biochemical markers selected for screening in barley malt are predictive of shelf life potential of the end product, sensory trials were performed. Three barley malt cultivars were selected for LOX, AROP, LTP1, protein and FAN content and used in micro-brewery trials at 0 and 3 months and evaluated using sensory analysis. Good correlation was found between the biochemical predictors and sensory trial for the best quality malt and beer. These parameters were therefore highly relevant for predicting shelf life potential, although additional research is required to elucidate the effect of LTP1 and LOX-1 on each other during the brewing process, since it seems that high LOX-1 concentrations could be leading to LTP1 decreases. With this study it is proposed that if more detailed protein or FAN characterisation is used together with the screening of LOX-1, LTP1 and AROP, an more accurate shelf life prediction, based on malt analysis, is possible and with the help of these parameters brewers can simply blend malts accordingly. / AFRIKAANSE OPSOMMING: Die keuse van roumateriaal om 'n konstante eindproduk van goeie kwaliteit te lewer, was nog altyd 'n uitdaging vir brouers wêreldwyd aangesien verskeie faktore 'n invloed het op die kwaliteit van die produk. Alhoewel daar tans verskeie metodes vir moutanalise bestaan wat die eindproduk–kwaliteit voorspel, is daar min wat werklik die eindproduk kwaliteit soos voorspel deur moutanalise verteenwoordig. Hierdie navorsing fokus op die bepaling van mout-eienskappe om twee van die belangrikste bierkwaliteitvereistes, naamlik skuim- en geurstabiliteit te voorspel. Spesifieke biochemiese eienskappe in garsmout soos lipiedtransportproteien-1 (LTP1), lipoksigenase-1 (LOX-1), antioksidant-antiradikaal potensiaal (AROP), vry aminostikstof (FAN) is geïdentifiseer en gebruik in voorspelling van bierkwaliteit vanaf moutkarakter. Hierdie biochemiese kwaliteit voorspellings is dan gekorreleer met die eindproduk soos ge-evalueer d.m.v sensoriese analise op mikro-gebroude bier. Omdat LTP1 soveel fasette in bier beïnvloed, het dit reeds 'n aanloklike studiefokus geword. LTP1 word hoofsaaklik geassosieer met stabiele skuimkwaliteit in bier en tree op as 'n lipiedmop (“lipid scavenger”). Die proteien speel teoreties ook 'n rol in die stabiliteit van bier geur deur moontlik as 'n anti-oksidant op te tree. Laastens is LTP1 bekend vir sy antigis aktiwiteit wat moontlik 'n negatiewe uitwerking op fermentasies het. Gedurende hierdie navorsing is LTP1 en sy lipiedbinding isoform LTP1b suksesvol gesuiwer met 'n ekonomies en eenvoudige 5-stap protokol. Beide isoforme het stabiliteit by temperature >90°C en meer neutrale en basiese pH omgewings getoon. Alhoewel die voorheen gerapporteerde anti-oksidant aktiwiteit vir LTP1 nie bevestig kon word nie, is daar wel gevind dat beide LTP1 en LTP1b, LOX-1, wat verantwoordelik is vir die ensimatiese afbraak van linoleensuur na 2(E)-nonenal, se aktiwiteit inhibeer. Dit is 'n unieke bevinding wat LTP1 ook koppel aan geurstabiliteit. LTP1 het antigis aktiwiteit getoon, maar LTP1b het die meeste, indien nie alle antigis-aktiwiteit verloor. Omdat die meeste van die LTP1's omgeskakel word na LTP1b's en geglikosileerde isoforme tydens die brouproses, sal fermentasie nie beduidend beinvloed word nie, maar die skuim- en geurstabiliteit sal steeds bevorder word deur die blote teenwoordigheid van die LTP1b. Geurverval van die finale verpakte produk is gedeeltelik a.g.v die ensimatiese produksie van 2(E)-nonenal deur LOX-1 en die teenwoordigheid van vry suurstofradikaal spesies, beperkte AROP en LTP1. Die ontwikkeling van twee 96-putjie mikroessaïs, gebasseer op die yster oksidasie-xilenol oranje (FOX) essai vir die bepaling van LOX-1 en AROP, was suksesvol en het goed vergelyk met reeds gevestigde essaïs. Die LOX-FOX en AROP-FOX mikroessaïs is spesifiek ontwikkel vir die residente, hoë deurvloei vergelykende bepaling van LOX-1 en AROP in mout en ander brouery-monsters. Die AROP-FOX en LOX-FOX mikroessaïs en 'n paar gevestigde essaïs is gebruik om moute te kategoriseer in die verskillende voorspelde kwaliteitsgroepe. Die biochemiese merkers wat gemeet is het die volgende ingesluit: LOX aktiwiteit, LTP1 inhoud, FAN waardes, proteïen konsentrasie en AROP. 'n Merkwaardige korrelasie (R2=0.93) is gevind tussen FAN/LOX en LTP1/LOX wat ook ooreenstem met die waarneming dat LOX-1 aktiwiteit onderdruk word deur LTP1 by verskeie konsentrasies. Hierdie korrelasies kan brouers help met optimale versnitting van moute vir, nie net die hoogste kwaliteit eindproduk nie, maar ook vir fermentasie voorspellings. Om te bepaal of hierdie geselekteerde biochemiese merkers in mout die potensieële raklewe van die eindproduk verteenwoordig, is sensoriese evaluerings uitgevoer. Drie gars-mout kultivars is geselekteer o.g.v LOX-, AROP-, LTP1-, proteïen- en FAN-inhoud en gebruik in mikro-brouery proewe en op 0 en 3 maande en is ge-evalueer deur sensoriese analise. Goeie korrelasie is gevind tussen die biochemiese voorspellers en sensoriese evaluering vir die beste kwaliteit mout en bier. Hierdie maatstawwe is daarom uiters relevant vir voorspelling van die potensiele rakleeftyd, alhoewel addisionele navorsing nodig is om die effek van LTP1 en LOX-1 op mekaar gedurende die brouproses te bepaal. Dit blyk dat 'n hoë LOX-1 konsentrasies kan lei tot 'n afname in LTP1. Met hierdie studie word dit voorstel dat, as meer gedetaileerde proteien of FAN karakterisering saam met LOX-1, LTP1, en AROP analise uitgevoer word, 'n meer akkurate raklewe voorspelling moontlik is en met behulp van hierdie parameters kan brouers moute dienooreenkomstig versnit.

Zooplankton are the major primary consumers in pelagic ecosystems, providing the principal pathway for energy transfer from primary production to higher trophic levels. The marine copepod Calanus finmarchicus is an important component of the pelagic food web in the North Atlantic and peripheral ecosystems, and forms an essential dietary component of a number of commercially important fish. As part of its life cycle, many C. finmarchicus overwinter in a diapause phase (a dormant overwintering phase where development is suppressed in adaptation to the seasonal food supply) at depths of 500 to 2000 m, but little is known about the triggers that initiate and terminate diapause, or the internal processes associated with these triggers. Understanding these processes is important, given that subtle changes in the environmental conditions which may affect diapause could have consequences for the entire Calanus-based ecosystem. In this study I took advantage of relatively easy access to a deep (> 100 m), isolated population of C. finmarchicus in Loch Etive (a sea loch on the west coast of Scotland) to sample Calanus finmarchicus monthly between April 2006 and June 2007 and measure lipid dynamics and gene expression associated with diapause. Chapter 1 of this thesis provides a general introduction to diapause and Calanus finmarchicus, Chapter 2 reports on the population of C. finmarchicus in Loch Etive, Chapter 3 reports changes in the lipid class and fatty acid composition of individual copepods, Chapter 4 reports on differential gene expression between diapausing and active C. finmarchicus and Chapter 5 provides a general discussion and puts this research into context. This study provides some initial insight into possible gene expression patterns, but further work is needed to attribute specific gene expression patterns with initiation and termination of diapause.

The growing obesity epidemic in America carries with it numerous health risks, including diabetes, increased serum lipid levels, and excess fat accumulation in the liver. If these conditions persist or become exacerbated, they may lead to the development of cardiovascular disease, the current leading cause of death among Americans, or to nonalcoholic fatty liver disease (NAFLD) which can progress to hepatocellular carcinoma (HCC), one of the deadliest forms of cancer. Better understanding of the genes involved in these diseases can lead to improved identification of at-risk individuals and treatment strategies. Our lab previously identified zinc fingers and homeoboxes 2 (Zhx2) as a regulator of hepatic gene expression. The BALB/cJ mouse strain has a hypomorphic mutation in the Zhx2 gene, causing a 95% reduction in Zhx2 protein expression. The near ablation of Zhx2 in BALB/cJ mice confers protection from cardiovascular disease when fed a high fat diet, yet these mice show increased hepatic lipid accumulation and liver damage. Microarray data indicates Zhx2 may be involved in the regulation of numerous genes involved in lipid metabolism. Recent GWAS studies indicate ZHX2 may contribute to the risk of cardiovascular disease and liver damage in humans as well. In this dissertation, I characterize the role of Zhx2 expression in the liver and how it affects the risk of both cardiovascular disease and liver damage. I generated liver-specific Zhx2 knockout mice and confirmed Zhx2 regulates several novel targets that could contribute to the fatty liver phenotype seen in BALB/cJ mice. Further studies revealed that hepatic Zhx2 expression is necessary for proper sex-specific expression of several Cyptochrome P450 (CYP) genes and could contribute to gender differences in disease susceptibility. Lastly, I performed studies into the functional role of the Zhx2 target gene Elovl3. A mouse model of HCC revealed that Elovl3 is completely repressed in HCC tumors. Cell viability and cell cycle assays indicate that Elovl3 expression slows cell proliferation and may be important for proper cell cycle checkpoints. Together, these data indicate that Zhx2 and/or its targets could be clinically relevant in the detection, prevention, or treatment of cardiovascular disease, fatty liver, and HCC.