Thèse diffusée initialement dans le cadre d'un projet pilote des Presses de l'Université de Montréal/Centre d'édition numérique UdeM (1997-2008) avec l'autorisation de l'auteur.

Ph.D., Faculty of Science, University of the Wiwatersrand, 2011 / Biological production of hydrogen gas has received increasing interest from the international community during the last decade. Most studies on biological fermentative hydrogen production from carbohydrates using mixed cultures have been conducted in conventional continuous stirred tank reactors (CSTR) under mesophilic conditions. Investigations on hydrogen production in reactor systems with attached or self-immobilized microbial growth have also appeared recently in the literature. These investigations on attached or self-immobilised bacteria involve hydrogen production in the mesophilic and thermophilic temperature range. The present study investigated the design and operational features of anaerobic fluidized granular bed bioreactor (AFGB) system which would facilitate the simultaneous achievement of high productivities (HPs) and high hydrogen yields (HYs).Where high HPs is greater than 120 mmol H2 /(L.h) and HYs greater than 4 mol H2/mol glucose. Theoretical maximum yield for an exponentially growing non-granulated bacterial monoculture will always be less than the thermodynamic maximum of 4 mol H2 /mol glucose: C6H12O6 +4H2O → 2CH3COO- + 4H2 + 4H+ + 2HCO3. The design features included reducing the total non-working or dead volume of bioreactor system. The operational improvements included application of thermophilic temperatures and high rates of de-gassed effluent recycling through the fluidized granular bed. An example of an optimal ratio of effluent recycle rate (R) to bioreactor working volume (V) was (3.0 L/min)/(3.2 L/min) = 0.94 minutes. Under conditions where temperatures were maximised and V/R were minimized the HPs increased to 21.58 L H2 /h. Also under these conditions the HYs increased above 3.0 mol H2/mol glucose. Specific hydrogen productivity for the fluidized granular bed increased from 0.25 L H2 / (g BM.h) or 8.83 mmol H2 / (g BM.h) at 45 oC to 0.525 L H2 / (g BM.h) or 18.03 mmol H2 / ( g BM.h) at 70 oC. A 3.64 fold increase in hydrogen yield occurred with an increase in temperature from 45 oC to 70 oC. XX When expressed in terms of glucose, this represents an increase from 1.34 mol H2 /mol glucose to 4.65 mol H2 /mol glucose. Finally, an evaluation of the net energy production by the AFGB system revealed a positive energy balance, making thermophilic biohydrogen production energetically viable from a commercial perspective.

"When cells are exposed to environmental stress, they respond by compartmentalizing mRNA and translation proteins in stress granulates to protect mRNA. However, the mechanism through which external stress is communicated into the cell to form stress granules is unknown. Phospholipase Cβ (PLCβ) is activated by Gq on the plasma membrane in response to sensory stimuli to initiate calcium signals resulting in a variety of cellular responses. Here, we show that PLCβ binds to major proteins that organize stress granules as well as the main component of the RNA-induced silencing machinery, Argonaute-2 (Ago2). Under stress, PLCβ moves from the plasma membrane to the cytosol to escort Ago2 into stress granules and potentially inhibit mRNA degradation by regulating microRNAs (miRs) expression. Using a model muscle cell line functionally adapted to handle stress, we find that upon osmotic stress, the movement of PLCβ into the cytosol to move Ago2 into stress granules changes the population and distribution of miRs, and in particular, members of the let family. The impact of changes in let is to acutely affect glucose metabolism allowing cells to adapt to stress conditions. Our studies present a model in which PLCβ relays information about external stress to promote stress granule formation and protect mRNAs."

The study presented here was firstly to investigate the physiochemical properties of native sago starch (obtained from Metroxylon sp. and designated as sago INA), in comparison with those of Metroxylon sago starch obtained from a different source, sago starch derived from Arenga sp. palms, wheat, corn, and tapioca starches. The properties analysed were chemical composition, total starch content, apparent amylose content, pasting properties, endothermic thermal behaviour, starch paste clarity, freeze-thaw stability, hardness of gel, and microscopic structure of the granules. The results obtained indicated that sago INA starch sample contained less fat and protein, compared to cereal starches. The sago starch sample had larger sized granules and had a more transparent paste. The gels of the starch were harder, and showed a relatively better stability to freeze-thaw treatment. The other part of the study was extrusion of sago INA starch both in the absence and presence of enzyme by utilising a response surface design. In the absence of the enzyme, the experiment was conducted to establish the extrusion process conditions including moisture contents, melt temperature, and screw speed. The extruded products were then analysed for degree of molecular degradation, light microscopic structure, reducing sugars of the water soluble materials, water absorption index, water solubility index, enzyme susceptibility, and gelatinisation endothermic energy. Increased mechanical and thermal energy input received by the products in the extruder resulted in a significant degradation of the molecular weight of the macromolecules. Light photomicrographs also suggested that the granule structures of the extrudates have been reshaped. All extrudate samples had a very low gelatinisation endothermic energy compared to its native starch. The specific mechanical energy received by the products in the extruder was calculated and related to the process variables. The possibility of using the products in food application was also discussed. / Doctor of Philosophy (PhD)

Neutrophil (PMN) inflammatory functions, including cell adhesion, diapedesis, and phagocyto-sis, are dependent on the mobilization and release of various intracellular granules/vesicles. In this study, I found that treating PMN with damnacanthal, a Ras family GTPase inhibitor, resulted in a specific release of secondary granules, but not primary or tertiary granules, and caused dy-sregulation of PMN chemotactic transmigration and cell surface protein interactions. Analysis of the activities of Ras members identified Ral GTPase as a key regulator during PMN activation and degranulation. In particular, Ral was active in freshly isolated PMN, while chemoattractant stimulation induced a quick deactivation of Ral that correlated with PMN degranulation. Over-expression of a constitutively active Ral (Ral23V) in PMN inhibited chemoattractant-induced secondary granule release. By subcellular fractionation, I found that Ral, which was associatedwith the plasma membrane under the resting condition, was redistributed to secondary granules after chemoattractant stimulation. Blockage of cell endocytosis appeared to inhibit Ral transloca-tion intracellularly. In conclusion, these results demonstrate that Ral is a critical regulator in PMN that specifically controls secondary granule release during PMN response to chemoattrac-tant stimulation.

The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.

Resistance to flavivirus-induced disease is conferred by a single gene that encodes oligoadenylate synthetase (Oas) 1b (Oas1b). Oas1b is not a functional synthetase suggesting its anti-flavivirus mechanism is RNase L-independent and that it may be mediated by interactions with other host cell protein(s). A yeast two-hybrid screen was used to identify host cell binding partners of Oas1b. Candidate partners were confirmed by yeast co-transformation and co-immunoprecipitation analyses. Oxysterol binding protein-related 1L (ORP1L) and ATP binding cassette subfamily F 3 (ABCF3) were found to interact with Oas1b. RNAi knockdown studies suggested that ORP1L and ABCF3 form a tripartite complex with Oas1b that is critical for the flavivirus-induced disease resistance mechanism. Stresses including oxidation, nutrient starvation, and viral infections often induce the formation of stress granules (SGs) in eukaryotic cells. In response to stress, eIF2α kinases phosphorylate eIF2α leading to stalled 48S pre-initiation complexes and SG formation. West Nile virus (WNV) Eg101 infections were previously shown not to induce the formation of SGs. Infections with viruses of other natural WNV strains, as well as a WNV lineage 1/2-based infectious clone (W956IC) were analyzed and only W956IC infections were found to induce SGs. eIF2α kinase knockout MEFs were used to show that the W956IC-induced SGs were PKR-dependent. WNV chimeras were made by inserting Eg101 genes into the W956IC backbone. Chimeras replacing NS5 or NS1 and NS5 or NS1 and NS3 and NS4a reduced SG formation as well as early viral RNA synthesis similar to Eg101 infections. W956IC infections but not Eg101 infections were shown to produce exposed viral dsRNA at early times after infection. The data suggest that natural WNV infections evade the cell SG response by suppressing the amplification of viral RNA until cytoplasmic membranes have been remodeled to protect replication complexes from detection. It was previously reported that WNV Eg101 infections inhibited the formation of arsenite-induced SGs. The ability of other natural WNV strain infections to inhibit SG formation by arsenite (HRI), DTT (PERK), W956IC co-infection (PKR), and heat shock treatments was assessed. WNV infections only inhibited arsenite-induced SG formation suggesting that WNV infections specifically suppress the response to oxidative intermediates.

A gas-fluidized bed bioreactor was developed in this research as a new method for treating polluted air. The fluidization characteristics of selected packing materials were investigated. Then, bioremediation was tested using two types of packing in a fluidized bioreactor, as well as in a comparable packed bed. Microorganisms on the particles biodegrade contaminants in the polluted air, which flows up through the bed. At high flowrates, the polluted air fluidizes the particles, while at low velocities the operation is in packed bed mode.<p>Initially, sawdust was selected for use as a packing material. Due to the poor fluidization properties of sawdust, glass spheres were added. A mixture of sawdust and glass spheres remained well mixed during fluidization. In the mixture, interparticle forces increased with increasing moisture in the sawdust, eventually causing defluidization of the bed. In the absence of bioremediation, mass transfer was studied between ethanol-contaminated air and sawdust/glass sphere packing, and found to be higher in the fluidized versus packed mode. In bioremediation experiments, ethanol removal efficiencies were as high as 95% in both operating modes. The maximum elimination capacities (EC) of ethanol were 75 and 225 g m^-3 sawdust h^-1 in the fluidized and packed beds respectively.<p>The packing of the fluidized bed bioreactor was optimized in order to boost bioremediation rates. Experiments showed that peat granules fluidized well in a bubbling regime, likely due to their relatively high density and sphericity. In peat bioremediation trials, the fluidized mode outperformed the packed bed; the maximum ECs were 1520 and 530 g m^-3 peat h^-1, respectively. Removal efficiency in the fluidized mode decreased with velocity, because the size and amount of large bubbles increased.<p>A steady-state model of the fluidized bioreactor was developed. By taking account of bubble properties during fluidization, the model helps to explain how bubble size, microbial properties and bioreactor residence time affect removal efficiency and elimination capacity of the bioreactor.<p>A peat gas-fluidized bioreactor shows promise as an efficient, low-cost technology for air treatment. Particle mixing in the fluidized bed may prevent operating problems associated with the packed bed bioreactor. Fluidized bioreactors are ideal for the treatment of high volume, low concentration air emissions.

A gas-fluidized bed bioreactor was developed in this research as a new method for treating polluted air. The fluidization characteristics of selected packing materials were investigated. Then, bioremediation was tested using two types of packing in a fluidized bioreactor, as well as in a comparable packed bed. Microorganisms on the particles biodegrade contaminants in the polluted air, which flows up through the bed. At high flowrates, the polluted air fluidizes the particles, while at low velocities the operation is in packed bed mode.<p>Initially, sawdust was selected for use as a packing material. Due to the poor fluidization properties of sawdust, glass spheres were added. A mixture of sawdust and glass spheres remained well mixed during fluidization. In the mixture, interparticle forces increased with increasing moisture in the sawdust, eventually causing defluidization of the bed. In the absence of bioremediation, mass transfer was studied between ethanol-contaminated air and sawdust/glass sphere packing, and found to be higher in the fluidized versus packed mode. In bioremediation experiments, ethanol removal efficiencies were as high as 95% in both operating modes. The maximum elimination capacities (EC) of ethanol were 75 and 225 g m^-3 sawdust h^-1 in the fluidized and packed beds respectively.<p>The packing of the fluidized bed bioreactor was optimized in order to boost bioremediation rates. Experiments showed that peat granules fluidized well in a bubbling regime, likely due to their relatively high density and sphericity. In peat bioremediation trials, the fluidized mode outperformed the packed bed; the maximum ECs were 1520 and 530 g m^-3 peat h^-1, respectively. Removal efficiency in the fluidized mode decreased with velocity, because the size and amount of large bubbles increased.<p>A steady-state model of the fluidized bioreactor was developed. By taking account of bubble properties during fluidization, the model helps to explain how bubble size, microbial properties and bioreactor residence time affect removal efficiency and elimination capacity of the bioreactor.<p>A peat gas-fluidized bioreactor shows promise as an efficient, low-cost technology for air treatment. Particle mixing in the fluidized bed may prevent operating problems associated with the packed bed bioreactor. Fluidized bioreactors are ideal for the treatment of high volume, low concentration air emissions.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 138-170).