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The dissolving pulps used in this thesis are high-grade cellulose pulps, with low amounts of hemicellulose, degraded cellulose and lignin, produced by acid bi-sulphite pulping of a fast growing South African hardwood Eucalypt clone. Microcrystalline cellulose (MCC) grade, viscose grade and cellulose acetate grade dissolving pulp were produced using a 4 stage bleaching process. MCC, viscose and cellulose acetate are the cellulose derivatives of 91% α-cellulose, 92% α-cellulose and 96% α-cellulose respectively. The key properties of the dissolving pulp considered for cellulose derivatisation are: (1) Structure (2) Accessibility and (3) ‘Reactivity’. The ‘reactivity’ depends to a large extent on the supra-molecular structure of cellulose I. Supra-molecular structure deals with the arrangement of cellulose I molecules into cellulose fibrils which then make up the cellulose fibril aggregate. The accessibility of cellulose I depends on the surface area, as determined by the size of the cellulose fibril aggregates, that are accessible; the structure of the cellulose molecules, which will determine which hydroxyl groups are accessible; and the size and type of reagent used during derivatisation. Supra-molecular changes in cellulose fibril aggregation of cellulose I, in hardwood acid bi-sulphite pulp, during bleaching and drying were studied using Atomic Force Microscopy (AFM) and Cross-polarization/Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance Spectroscopy (CP/MAS 13C-NMR – Solid state NMR) in combination with spectral fitting. There was a marked increase in cellulose fibril aggregation (i.e. supra-molecular structure) during bleaching of hardwood acid bi-sulphite pulp using 96% α-cellulose conditions. In contrast there was no increase in cellulose fibril aggregation pulp bleached using 91% α-cellulose and 92% α-cellulose bleaching conditions. An increase in hemicellulose and degraded cellulose / short chain glucan was shown to correlate with a decrease in cellulose fibril aggregation recorded using solid state NMR. Further changes in supra-molecular structure were noticed when each of the dissolving pulp samples were dried. First time drying of hardwood acid bi-sulphite pulp samples induces a significantly different degree of irreversible cellulose fibril aggregation in the 92% α-cellulose and the 96% α-cellulose pulp samples. The irreversible increase in cellulose fibril aggregation correlates with the estimated amount of hemicellulose and degraded cellulose / short chain glucan present in the pulp. The percentage increase in cellulose fibril aggregation upon drying is as follows: 96% α-cellulose > 92% α-cellulose > 91% α-cellulose. Hemicellulose and degraded cellulose / short chain glucan are among the wet chemical properties that influence cellulose fibril aggregation and the presence in dissolving pulp samples could provide steric hindrance preventing the aggregation of fibrils. Reactivity studies were carried out on the 91% α-cellulose, 92% α-cellulose and 96% α-cellulose grades of dissolving pulp. During 91% α-cellulose reactivity studies, there was no relationship between cellulose fibril aggregation, acid hydrolysis or MCC preparation. Other possible techniques for 91% α-cellulose reactivity evaluation such as the degree of polymerization (DP) determination using AFM have been discussed. Size exclusion chromatography with multi-angle laser light scattering detection was shown as a more suitable method of estimating the reactivity of 92% α-cellulose pulp samples. 96% α-cellulose reactivity studies were carried with the aid of a model system consisting of the acetylation of high purity pulp samples viz. cotton linters cellulose and 96% α-cellulose. Results indicate that the initial reaction rate constant is proportional to the specific surface area for the two cellulose pulp samples showing that specific surface area is directly related to initial reactivity of the performed acetylation. This work has shown that it is possible to control the cellulose fibril aggregation and hence specific surface area in laboratory produced 91% α-cellulose, 92% α-cellulose and 96% α-cellulose by the method in which the pulp is dried. Thus controlling cellulose fibril aggregation can probably be one viable route for controlling the initial reactivity of dissolving pulp towards acetylation. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2009.

Physcomitrella patens has become a model system to study plant biology. 8 cellulose synthase (CesA) genes were identified by searching against Physcomitrella EST database. Two of these genes, PpCesA6 and PpCesA7 are the first full-length CesAs to be identified. These two genes are highly similar to each other, both on the cDNA and genomic DNA levels. They both have 13 introns and 12 exons. The first introns are more than 1kb. The proteins they encode both have 1096 amino acids. There are only three amino acid differences in the proteins they encode. PpCesA6 and PpCesA7 share 74% amino acid identity with Monterey pine (Pinus radiate) PrCesA10, 72% amino acid identity with quaking aspen (Populus tremuloide) PtrCesA6, 71% amino acid identity with maize (Zea mays) ZmCesA7 and three rice (Oryza sativa) CesAs, 65%-68% amino acid identity with Arabidopsis CesAs. The deduced proteins of PpCesA6 and PpCesA7 contain the D, D, D, QXXRW motif in the form of DDG, DCD, TED, QVLRW, which is the catalytic region of cellulose synthases. Two other pairs of CesA genes, PpCesA3 and PpCesA8, PpCesA4 and PpCesA10, also show high similarity. PpCesA2 and PpCesA9 are pseudogenes. By taking advantage of the high efficiency homologous recombination in Physcomitrella nuclear DNA, a C-terminus GFP fusion construct was produced for PpCesA6. Expression analysis showed that PpCesA6 is expressed in both protonemata and young gametophore. In protonemata, PpCesA6 is expressed in both chloronema and caulonema cells, but not in every cell. In young gametophore, PpCesA6 is expressed in axillary hairs and rhizoids. Confocal miscrocopy study shows that PpCesA protein is localized on the plasma membrane and it is randomly dispersed. The gene targeted knockout constructs of PpCesA6 and PpCesA7 were produced. The null mutants of PpCesA6 and PpCesA7 single knockout as well as double knockout were generated by the PEG (polyethylene glycol)-mediated protoplast transformation. Both single knockout mutants did not show obvious phenotypic differences from the wild type. The double knockout mutants had reduced stem length. The stem lengths of the wild type, PpCesA6 knockout mutant, PpCesA7 knockout mutant and double knockout mutant growing on BCD and BCDAT media were 3.93±0.45mm and 3.51±0.08mm, 3.82±0.46mm and 3.5±0.3mm, 3.65±0.68mm and 3.73±0.49mm, 2.75±0.22mm and 2.65±0.43mm, respectively. A cellulose synthase-like C gene (CslC4) was identified by searching against the Physcomitrella EST and genomic DNA databases. The protein it encodes is 694 amino acids. The D, D, D, QXXRW motif is in the form of DDS, DAD, VED, QQHRW. PpCslC4 genomic DNA has 4 small introns in the coding region. There is also one small intron at the 5'-UTR. The deduced PpCslC4 protein shows 72% similarity with PpCslC2 and PpCslC3, 65% similarity with PpCslC1. When compared with other organisms, PpCslC4 protein shows more than 60% similarity with Arabidopsis and Oryza sativa CslC proteins. A gene targeted knockout construct was produced for PpCslC4. The null mutants were generated by the PEG-mediated protoplast transformation. PpCslC4 mutant did not show any obvious phenotypic differences from the wild type.

Glucans are polysaccharides integral to many materials and biological functions. Under the umbrella of Biomime, the Swedish Center for Biomimetic Fiber Engineering, this work has aimed to improve basic understanding of the biosynthesis of such glucans. This has been achieved through direct investigation of cellulose structure, and by developing the tools to analyze glucan biosynthesis. Notably we have identified a novel chemical effector of glucan synthesis processes and developed a proteomic toolkit useful for analyzing membrane-bound glycosyltransferases, the enzyme group responsible for glucan biosynthesis. During this work, glucan synthesis has been studied using both Gluconacetobacter and Populus cell suspension cultures. Publication I. Gluconacetobacter cellulose (BC) was used as a base to create a novel and well characterized nano-material with improved mechanical properties. This novel composite of BC and hydroxyethylcellulose (HEC) had improved tensile strength compared to pure BC. Through thorough study utilizing dispersion measurements, electron microscopy, nuclear magnetic resonance and X-ray diffraction it was shown that the improved properties derived from a layer of HEC coating each fibril. Publication II. Bacterial cellulose was labeled in specific positions with 13C (C4 and C6). These samples were analyzed by CP/MAS NMR along with cellulose samples from cotton and Halocynthia sp. For each sample spectral fitting was performed and general properties of crystal allomorph composition and fibril widths were determined. Calculations were also made for water accessible surfaces of the fibrils. The results showed that water accessible C4 surface signals are reflective of the allomorph composition of the sample, along with a distorted signal that derives due to fibril imperfections. Water accessible surface signals from the C6 region are instead derived from rotamer conformations of the C6 hydroxymethyl groupsfrom glucose residues. In Publication III, a high-throughput screen was used to identify an inhibitor of Golgi-derived glycosyltransferase activity, termed chemical A. The structural basis for inhibition was determined and in vitro assays of callose synthesis were performed. The in vitro assays revealed chemical A to also be an activator of callose synthesis. To understand this activation kinetic studies were performed, showing that chemical A is a mixed type of activator, which can bind either the free enzyme or the enzyme-substrate complex. Chemical A has uses in chemical genetics for dissecting processes involving callose synthesis, such as stress response and cell-plate formation. In publication IV, we present an in-house developed platform for proteomics with a distributed processing model. This in-house system has been central to many proteomics tasks, including for those presented in publication V, and is being distributed as the Automated Proteomics Pipeline (APP). In publication V, conditions for enrichment of Detergent-Resistant Microdomains (DRM) have been optimized for Populus trichocarpa cell cultures. The proteins enriched in DRM were identified using mass spectrometry based proteomics, and a functional model for DRM was proposed. This model involves proteins specialized in stress response, including callose synthase, and cell signaling. This further strengthens the arguments for DRMs as sites of specific cellular functions and confirms they play a role in glucan synthesis. / <p>QC 20140710</p>

The synthesis of fluorinated cellulose derivatives was pursued for the purpose of investigating the effects of fluorine on the interfacial properties of cellulose derivatives, in multiphase materials. Synthetic approaches included: 1) the replacement of hydroxyl groups on cellulose with fluorine using fluoride displacement chemistry, or direct, one step fluorination 2) the replacement of hydroxyl groups on hydroxypropyl cellulose, HPC, with direct one step fluorinating agents, or 3) the conventional derivatization of cellulose with prefluorinated agents. Fluoride displacement of cellulose sulfonate esters was plagued low yields, and was therefore ineffective. Direct cellulose fluorination with aminofluorosulfur, and aminofluorocarbon reagents was effective for the surface fluorination of cellulose. However, the bulk, homogeneous fluorination of cellulose was complicated by side reactions that often precluded fluorination. Cellulose dissolved in DMAC/LiCI underwent simultaneous chlorination, and branching reactions when treated with dialkylaminosulfur trifluoride, DAST. Branching resulted from an anhydrous HF catalyzed trans-glycosidation reaction, which produced mainly (3-1,6 branching. Serendipitously, this discovery allows for the first known synthesis of long chain branched cellulose derivatives, with increased molar mass, and increased polydispersity. Treatment of HPC with DAST and FAR gave good levels of fluorination; however, the HF catalyzed gelation was always a complicating factor. The lower reactivity of FAR allowed for partial control of gelation with the use of nonnucleophilic bases. The easiest way to introduce fluorine was to perform conventional derivatization using prefluorinated reagents such as for the synthesis of fluorobenzyl cellulose. Mixed benzyl ethers of controlled fluorine content were made by altering the ratio of pfluorobenzyl chloride to benzyl chloride during reaction. 2-Dimensional NMR techniques were used to identify most proton and carbon resonances of cellulose and amylose fluorobenzyl ethers. Thermal analysis, solution analysis, and l3C spin-lattice relaxation experiments were used to compare the rod-like and coil-like behavior of the fluorobenzyl cellulose and amylose. Polycaprolactone, PCL, was blended with tri-O-benzylated cellulose with different degrees of fluorination. PCL was found to be immiscible with all derivatives, of fluorine contents from 0 % to as high as 11.7%. PCL did display some degree of mechanical compatibility with all derivatives. The greatest compatibility was found with benzylated cellulose having fluorine contents slightly below the maximum. It was found that no specific interactions were occurring between peL and fluorobenzyl cellulose. The enhanced compatibility of the mixed fluorobenzyl/benzyl cellulose ethers was postulated to arise from intramolecular interactions, which served to enhance the mixing of the copolymeric mixed benzyl ethers with PCL. / Ph. D.

The basis of this study are mutant plants with ARP2/3 complex lacking in one of its subunits (arpc5 and arp2). These plants also express CSC subunit CESA6 of primary cell wall tagged by YFP. Thanks to modern imaging technologies, it is possible to observe the movement of tagged cellulose synthase complexes in vivo at plasmatic membrane. Kymograph analyses was used to measure the velocity of CESA complexes. In addition to observing CESA complexes directly on the plasma membrane, experiments were made to regenerate cell walls of protoplasts of Arabidopsis thaliana plants arpc5 and WT. It was found, that observed mutants arpc5 and arp2 have reduced velocity of CESA complexes in comparison to WT and arpc5 protoplasts regenerate cellulose mesh of cell wall slower. Keywords: Cellulose synthesis, ARP2/3 complex, CESA, CSC velocity, arpc5, arp2, Arabidopsis thaliana.

Water and alkali extracted steam exploded yellow poplar fiber (log R₀ 4.25) was purified through a multi-stage process. The initial stage consisted of peralkanoic acid treatment, either formic or acetic acid, at atmospheric pressure. Subsequent bleaching was achieved through treatment with alkaline peroxide in several stages. Performic acid experiments were performed by researchers at the Finnish Pulp and Paper Research Institute: K. Poppius, I. Tuominen and J. Sundquist. Peracetic experiments were conducted at Virginia Tech. The alkanoic acid stage process parameters examined included time (3 and 6 hours primarily), temperature (40 and 60° for the peracetic trials, 80° for the performic trials), acid concentration (25, 50, and 80% for the peracetic trials), and initial peroxide charge (10 and 20 % for the peracetic trials; 2,5, and 10% for the performic trials). The bleached fiber was analyzed on the basis of brightness (diffuse reflectance of hand sheets at 457nm), Kappa number, molecular weight (carbanilation followed by GPC), and carbohydrate composition (complete hydrolysis followed by HPLC). Peroxide concentration was monitored through iodometric titration. The results indicated that the purified fiber may be useful as a microcrystalline cellulose. The purification process occurred in three phases: lignin activation, followed by dissolution, followed by alkaline peroxide bleaching. Brightness correlated well with Kappa number. Molecular weight loss (as weight average) averaged 43%. Glucose purity of 93% was realized for the peracetic trials with residual lignin of approximately 1%. Residual hemicellulose content was approximately 1.5%. Final brightness levels of nearly 90% MgO were attained by the performic acid samples. In terms of percent gain in brightness per percent peroxide consumed, higher temperature resulted in greater efficiency. An alkaline extraction (no peroxide) following the initial acid stage improved efficiency. Initial peracetic peroxide charge of 20% was inefficient compared to an initial charge of 10%. Molecular weight loss was affected by water concentration of the acid stage, but unaffected by an increase in the acid stage temperature from 40° to 60°. Molecular weight loss appeared to increase with acid stage peroxide charge up to 10%, but 20% charge did not result in greater losses. Further research using higher temperatures and a sulfuric acid catalyst are suggested. / Master of Science