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Carbon Dioxide (CO2) is a major fermentation product generated during the production of beer, the subsequent release of this gas within the fermentor results in agitation that is necessary for sustained industrial fermentation. CO2 is sometimes monitored allowing brewers to stoichiometrically relate CO2 released to other products. In this manner the rate of gas release from the fermentor may be used to assess, control and predict other aspects of fermentation. The dynamics of CO2 generation, transport and release are explored throughout this thesis over several studies. The tools used to examine CO2 production were scrutinized including a miniature assay using various modeling techniques. A miniature scale fermentation assay included in the methods of the American Society of Brewing Chemists was compared to industrial scale fermentations. It was found that discrepancies were possibly due (at least in part) to fermentor geometry. Following this study, a literature review of CO2 solubility in aqueous sugar, and ethanol solutions was conducted. This study exposed previously undescribed inaccuracies in literature, i.e., it was found that several gas solubility tables were empirical derived and are therefore unlikely to accurately reflect all styles of beer. The next study scrutinized the consumption of sugars during barley fermentation and found that these fermentations often exhibit asymmetric sigmoidal attenuation. A five parameter logistic model was introduced to model this sugar consumption more accurately than previously described techniques. Using methods refined during the aforementioned studies, a fermentation was conducted where a mass balance was used to track all major fermentation parameters (the consumption of individual sugars, and the production of ethanol, carbon dioxide, yeast biomass and glycerol). This allowed an assessment of Balling’s theorem as compared to modern theory. It was shown that while accurate in predicting original extract, Balling’s theorem incorrectly quantified other fermentation parameters. This has large ramifications for both industry and research as the estimation of fermentation parameters (such as ethanol and fermentation time) is now better understood. From these studies, the production of beer becomes less of a “black box” operation, and CO2 saturation, transport and release can be better explained. Of the many fermentation aspects monitored during these studies, most were predicted by theory, however, there were notable exceptions. For instance, it was found that both the inhibition of maltose consumption and yeast sugar consumption dynamics (which remained relatively constant throughout the fermentation at ~ 50 pg·h-1 for cells with an average mass of ~ 40 pg). were found to deviate from previously described reports. These, and other findings improve our understanding of brewing fermentations allowing for additional applications of theory and recommendations in industrial operations.

Late expression factor 3 is one of the six AcMNPV genes essential for DNA replication identified through transient replication assays. LEF-3 is a single stranded DNA binding protein responsible for the transportation of the viral helicase (P143) into the nucleus of the infected cell. In this study, a protein complementation-based assay was adapted to identify the region(s) of LEF-3 that is (are) involved in LEF-3-LEF-3 protein interactions. The full-length LEF-3, or various truncated LEF-3 regions were fused with Venus1 (N- terminus portions of full length Venus, a modified yellow fluorescence protein) or Venus2 (C- terminus). Venus1 and Venus2 fragments generated a functional fluorescent Venus protein when the two fragments were brought together by protein-protein interaction of the fused LEF-3 constructs. Fluorescence generated by coexpression of full-length LEF-3 fusion proteins confirmed that LEF-3 exists as homo-oligomer. Interaction between the full-length and the N- terminal (aa 1-189) or C- terminal regions (aa 190-385), and between the various truncated LEF-3 regions suggested the complexity of LEF-3 oligomeric structure. LEF-3 constructs deleted for NLS function revealed cytoplasmic fluorescence, suggesting that LEF-3-LEF-3 interactions occur in the absence of DNA or nuclear proteins. Because LEF-3 is essential for nuclear transporting the viral helicase (P143), the ability of LEF-3 to interact with another viral protein was investigated. P47, a sub-unit of the viral RNA polymerase was chosen because it is cytoplasmic when expressed on its own. The interaction between LEF-3 and P47 produced complete nuclear localized fluorescent signals. Overall, the results suggest that there are multiple regions of LEF-3 that are capable of closely interacting, and that multiple domains are likely involved in the oligomerization of full-length LEF-3. The interaction of LEF-3 with P47 suggests that P47 may be another LEF-3 cargo protein. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-05-27 15:02:53.983

Salinity problems seriously affect agricultural production by reducing crop yield and arable land. The evaluation of potato genotypes (Solanum spp.) for their salinity (NaCl) tolerance in conventional field trials is time consuming and labour intensive. The results are often confounded by many field and environmental variations. In vitro bioassays can overcome some of these difficulties by providing faster, more convenient and dependable methods for screening and selection of salt tolerant potato genotypes. The objective of this research was to develop in vitro bioassay methods for screening and selection of salt tolerant potato. Under in vitro NaCl stress conditions, seed germination, early seedling growth, and single-node cutting bioassays were used to evaluate salinity tolerance. The selected genotypes were further tested with three in vitro bioassays (single-node cuttings, root tip segments, and microtuberization). The rankings of potato cultivar salinity tolerance were similar in these bioassays. The single-node cutting bioassay was recommended because it was simpler to perform than the root tip segment and microtuberization bioassays and did not exclude certain genotypes as did the microtuberization bioassay. The in vitro bioassay rankings were compared with yield ranking in field lysimeters. In both the in vitro and in vivo saline stress experiments, cvs. Kennebec and Russet Burbank were more salt tolerant than Norland. The tubers and microtubers harvested from previous experiments were tested in the greenhouse to investigate salinity carry-over effect for seed tuber production. There was no apparent residual carry-over effect found. Microtuber yield increase in the presence of low NaCl concentration was induced primarily by specific ion (Na+), and not osmotic effects. This research clearly indicated that in vitro bioassays are relatively simple, rapid, convenient, repeatable, and agree with the field lysimeter results. They can be used to substitute for f

Endocrine disrupting compounds (EDCs) in the environment have recently emerged as a major issue in Canada and around the globe. The primary objective of this thesis was to investigate the fate of EDCs in two wastewater treatment processes, membrane bioreactors (MBRs) and ultraviolet (UV) disinfection. Two submerged MBR systems using hollow fiber membranes from two membrane manufacturers were tested. The results from a bench-scale and a pilot scale MBR for the treatment of swine wastewater with high concentration of EDCs showed that over 94% of the estrogenic activity (EA) in the influent was reduced through the MBR process. Biological degradation was the dominant removal mechanism for the removal of EDCs in MBRs. Over 85% of the influent EA was reduced by biodegradation through the MBR process. The other MBR system was built to study the removal mechanisms of two estrogens in a hybrid MBR with the addition of powdered activated carbon (PAC). The effects of PAC dosing on MBR overall performance was studied as well. It was found that PAC dosing could increase the removal rates of 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) by 3.4% and 15.8%, respectively and result in a slower rate of trans-membrane pressure (TMP) increase during MBR operation, which could significantly reduce the operating cost for membrane cleaning and/or replacement. The operating cost for PAC dosing could be offset by the benefit achieved from reducing the cost for membrane maintenance. The slower rate of TMP increase in the PAC-MBR was associated with the lower concentrations of soluble extracellular polymeric substances and colloidal organic compounds in the PAC-MBR sludge. The degradation kinetics of three estrogens, estrone (E1), E2, and EE2 in de-ionized water by UV irradiation was studied. The experimental results showed both the apparent concentrations and overall EA of all three investigated estrogens in water decreased with direct UV irradiation. To further study the impact of UV on the overall EA of wastewater, the EA of pre-UV and post-UV samples from five wastewater treatment plants were measured in both liquid and solid phase by Yeast Estrogen Screen assay. It was found that the EA of wastewater decreased after UV disinfection in three of the investigated plants whereas it increased in the other two plants. This observation needs to be further studied because it might have significant impacts on the application of UV systems for wastewater disinfection.

Combination chemotherapies that introduce multi-agent treatments to target cancer cells are emerging as new paradigms to overcome chemotherapy resistance and side effects involved with conventional monotherapies. In environmental toxicology, characterizing effects of mixtures of toxins rather than simply analyzing the effect of single toxins are of significant interest. In order to determine such combination effects, it is necessary to systematically investigate interactions between different concentration-dependent components of a mixture. Conventional microtiter plate format based assays are efficient and cost-effective, however are not practical as the number of combinations increases drastically. Although robotic pipetting systems can overcome the labor-intensive and time-consuming limitations, they are too costly for general users. Microfluidic live cell screening platforms can allow precise control of cell culture microenvironments by applying accurate doses of biomolecular mixtures with specific mixing ratios generated through integrated on-chip microfluidic gradient generators. This thesis first presents a live cell array platform with integrated microfluidic network-based gradient generator which enables generation and dosing of 64 unique combinations of two cancer drugs at different concentrations to an 8 by 8 cell culture chamber array. We have developed the system into a fully automated microfluidic live cell screening platform with uniform cell seeding capability and pair-wise gradient profile generation. This platform was utilized to investigate the gene expression regulation of colorectal cancer cells in response to combination cancer drug treatment. The resulting cell responses indicate that the two cancer drugs show additive effect when sequential drug treatment scheme is applied, demonstrating the utility of the microfluidic live cell assay platform. However, large reagent consumption and difficulties of repeatedly generating the exact same concentrations and mixture profiles from batch to batch and device to device due to the fact that the generated gradient profiles or mixing ratios of chemicals have to rely on stable flow at optimized flow rate throughout the entire multi-day experiment limit the widespread use of this method. Moreover, producing three or more reagent mixtures require complicated microchannel structures and operating procedures when using traditional microfluidic network-based gradient generators. Therefore, an on-demand geometric metering-based mixture generator which facilitates robust, scalable, and accurate multi-reagent mixing in a high-throughput fashion has been developed and incorporated with a live cell array as a microfluidic screening platform for conducting combination drug or toxin assays. Integrated single cell trapping array allowed single cell resolution analysis of drugs and toxin effects. Reagent mixture generation and precise application of the mixtures to arrays of cell culture chambers repeatedly over time were successfully demonstrated, showing significantly improved repeatability and accuracy than those from conventional microfluidic network-based gradient generators. The influence of this improved repeatability and accuracy in generating concentration specified mixtures on obtaining more reliable and repeatable biological data sets were studied.

As the move towards the miniaturization of many diagnostic and detection systems continues, the need for increasingly versatile yet sensitive labels for use in these systems also grows. Luminescent reporters provide us with a solution to many of the issues at hand through their unique and favorable characteristics. Bioluminescent proteins offer detection at extremely low concentrations and no interference from physiological fluids leading to excellent detection limits, while the vast number of fluorescent proteins and molecules available allows the opportunity to select a tailored reporter for a specific task. Both provide relatively simply instrumentation requirements and have exhibited great promise with many of the miniaturized systems such as lab-on-a-chip and lab-on-a-CD designs. Herein, we describe the novel employment of luminescent reporters for four distinct purposes. First off, by combining both time and wavelength resolution we have expanded the multiplexing capabilities of the photoprotein aequorin beyond duel-analytes, demonstrating the ability to simultaneously detect three separate analytes. Three semi-synthetic aequorin proteins were genetically conjugated to three pro-inflammatory cytokines (interleukins 1, 6, and 8) resulting in aequorin labeled cytokines with differing emission maxima and half lives to allow for the simultaneous detection of all three in a single solution through the elevated physiological concentration range. Secondly a semi-synthetic aequorin variant has been genetically enhanced to serve as an immunolabel and exhibited the ability to sensitively detect the acute myeloid leukemia marker, CD33, down to the attomole level in addition to improving aequorin imaging capabilities. In the third example, the aequorin complex was rationally, genetically split into two parts and attached to the termini of the cAMP selective cAMP receptor protein (CRP) creating a genetically fused molecular switch. The conformational change experienced by CRP upon the binding of cAMP translates into a loss of bioluminescent signal from aequorin and has shown the ability to respond linearly to cAMP over several orders of magnitude. Lastly, through custom design, a reagentless, portable, fluorescent fiber optic detection system has been developed, capable of being integrated into the body through a heart catheter. The system was able to respond to changes in potassium concentration selectively, reproducibly and reversibly with a fast response time of one minute.

A biological assay is designed to set up a rapid and robust drug-screening system on a small scale. An assay is considered as a single unit of a platform to screen various compounds for aiding in drug discovery. Each assay is carried out in a 96-well plate, each of whose wells consists of the biological component called the Spheroids. The value of each assay lies in it facilitating for versatile screening applications. The spheroid is considered as a micro-structural product. And the addition of various compounds for testing is performed in each well (consisting of the spheroids). The focus has been to put forth the production principles and validation strategies to run the biological assay and test its efficacy to be used for screening in high volumes. The assay development illustrates processing and validation techniques. The goal is to develop optimized standards to process the assay, addressing various quality control issues, from the raw material to the end-product stage. Such an approach also brings interesting analogies of biological process in a manufacturing scenario. The developed system incorporates a value stream approach, by pulling the product from the customer end. The process involves simply encapsulating HUVECs (Human Umbelical Vein Endothelial cells) from the raw material stage, culturing to form the spheroid and transferring the component to assemblage in a 96-well format undergoing stages of heat treatments. The small scale screening system allows the use of small amounts of drug, which is especially essential for new drug synthesis or in rapid decision making to find out any unknown potent compounds. The design of optimal processes in product development of the spheroid assay is illustrated. Thus in light of the value of this assay, developing the production system has been pivotal so as to produce quality spheroids in the 96-well plate formats. The quantification of the stimulatory and inhibitory effects of the different agents is required to help understand the complex biological behavior involved. The goal is to validate the data using image analysis software. The image analysis helps determine the quantification to be accurate, objective, and consistent. The quality of the product is tested by the reproducibility and robustness of the assay.

Endocrine disrupting compounds (EDCs) in the environment have recently emerged as a major issue in Canada and around the globe. The primary objective of this thesis was to investigate the fate of EDCs in two wastewater treatment processes, membrane bioreactors (MBRs) and ultraviolet (UV) disinfection. Two submerged MBR systems using hollow fiber membranes from two membrane manufacturers were tested. The results from a bench-scale and a pilot scale MBR for the treatment of swine wastewater with high concentration of EDCs showed that over 94% of the estrogenic activity (EA) in the influent was reduced through the MBR process. Biological degradation was the dominant removal mechanism for the removal of EDCs in MBRs. Over 85% of the influent EA was reduced by biodegradation through the MBR process. The other MBR system was built to study the removal mechanisms of two estrogens in a hybrid MBR with the addition of powdered activated carbon (PAC). The effects of PAC dosing on MBR overall performance was studied as well. It was found that PAC dosing could increase the removal rates of 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) by 3.4% and 15.8%, respectively and result in a slower rate of trans-membrane pressure (TMP) increase during MBR operation, which could significantly reduce the operating cost for membrane cleaning and/or replacement. The operating cost for PAC dosing could be offset by the benefit achieved from reducing the cost for membrane maintenance. The slower rate of TMP increase in the PAC-MBR was associated with the lower concentrations of soluble extracellular polymeric substances and colloidal organic compounds in the PAC-MBR sludge. The degradation kinetics of three estrogens, estrone (E1), E2, and EE2 in de-ionized water by UV irradiation was studied. The experimental results showed both the apparent concentrations and overall EA of all three investigated estrogens in water decreased with direct UV irradiation. To further study the impact of UV on the overall EA of wastewater, the EA of pre-UV and post-UV samples from five wastewater treatment plants were measured in both liquid and solid phase by Yeast Estrogen Screen assay. It was found that the EA of wastewater decreased after UV disinfection in three of the investigated plants whereas it increased in the other two plants. This observation needs to be further studied because it might have significant impacts on the application of UV systems for wastewater disinfection.

The colony formation assay (CFA) is the gold standard for measuring cytotoxic effects on cells. To increase efficiency, the CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation (IR) on the FaDu and A549 cancer cell lines. Its ability to evaluate combination therapies was investigated using cisplatin and IR. The 96-well CFA was transferred to a robotic platform for evaluation as a high-throughput screen (HTS) readout for the discovery of novel anti-cancer compounds, and radiosensitizers. Screening yielded eight putative anti-cancer hits, and five putative radiosensitizing hits. Secondary screening confirmed 6/8 anti-cancer compounds, and 0/5 radiosensitizing compounds. Thus, the 96-well CFA can be adopted as an alternative assay to the 6-well CFA in the evaluation of cytotoxicity in vitro, providing a possible readout to be utilized in HTS for discovering anti-cancer compounds, but with limited applicability in discovering radiosensitizers.