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Advanced healthcare requires novel technologies capable of real-time sensing to long-term health monitoring. One example includes biomarker detection for disease diagnosis and deciding treatment options. But several limitations exist with current technologies; however, the COVID pandemic brought these limitations to a global presence as the authorities struggled to quickly authorize a facile test for the early detection of SARS-CoV-2. An important next step is to research alternate strategies, utilize the current infrastructure available, and build sensors that meet standards the current technologies fail to. One of the strategies involves identifying novel sensing parts. To this end, we turned our attention to bacteria as they provide a plethora of novel sensing parts. Bacteria respond to stimuli using a wide range of biomolecules that include enzymes and transcription factors. Our group reported an optical progesterone biosensor based on a novel progesterone responsive allosteric transcription factor (aTF). Firstly, the electrochemical transduction of the binding affinity between this aTF and its cognate DNA sequence is discussed. The binding and unbinding of aTF-DNA results in an impedance change and is directly proportional to progesterone concentration. The limit of detection is comparable to the optical progesterone sensor and relevant to the physiological ranges of progesterone present in bodily fluids. Secondly, to convert the sensor into a point of care system, the expression of the aTF-enzyme fusion protein that undergoes the binding-unbinding event is discussed. The enzyme in presence of its excess substrate acts as a signal amplifier to track the binding changes. The signal depends on the proximity of the fusion protein to the electrode surface and correlates to the progesterone concentration. As we recover from the deadly COVID pandemic, we realize that early diagnosis is a key pillar of disease containment, in addition to other approaches such as contact tracing, distancing, and personal protective equipment. A truly transformative technology in the fight against future viruses is a rapid and quantitative point-of-care (POC) test with a low limit of detection and a high specificity. To that end, an inverted glucometer technology for the detection of infectious diseases is presented. As a model system, SARS-CoV-2 antigens – nucleocapsid protein, antibodies against it, and an inflammatory biomarker are detected. Antigen of interest is sandwiched between capture and detection reagents with biotin and glucose oxidase tags respectively. Glucose oxidase, a widely used enzyme in glucometers, amplifies the output signal in presence of excess glucose. The following chapters encompass designs, different immobilization techniques, characterization, and optimization methods to develop biosensors that meet requirement standards. This research serves as a platform for development of state of the art technologies for diagnostics applications. / 2025-01-16T00:00:00Z

The universal traits of cellular expression and replication in modern life point to the existence of an ancient RNA world. Leading up to the origin of life, this stage of evolution utilized RNA as the genetic material, and as a catalyst in the form of ribozymes. Although it is expected that a polymerase ribozyme was required for the efficient replication of RNA, it is also likely that the earliest form of replication took place under non-enzymatic conditions. There are several problems with the current scenarios depicting non-enzymatic RNA replication, thus we aim to examine them in more detail using computational models. We first consider the relationship between the thermodynamics of RNA base pairing and non-enzymatic nucleotide addition in an attempt to model the rate of primer extension. Our predicted rates reveal the model parameters to be too simple to produce reliably accurate results. For now, we should simply use available experimental rate data, until we have access to more data and less unknown parameters. Nevertheless, the model indicates that the primer extension rate does depend on thermodynamics of base pairing, and a more accurate model can be of great use when creating realistic complex models of RNA world scenarios. In chapter 3, we investigate non-enzymatic RNA replication under temperature cycling using computer simulations. When starting with a diverse mixture of sequences, partially matching sequences can reanneal in configurations that allow continued strand growth. This is in contrast to the case of having multiple copies of matching sequences, where reannealing occurs quickly upon cooling. We find that, starting with short oligomers, strands can grow over multiple cycles to produce long sequences over 100 nucleotides in length. The small strand extension per cycle does not produce replicates of any one specific sequence. This relates to the work done in chapter 4, where we look for the presence of a virtual circular genome within our simulations. In a virtual circle, short overlapping RNA sequences will make up a mutually catalytic set. Within the diversity of our simulation, virtual circles are rare, and require a specific level of starting mixture diversity along with no input of new sequences. Continued replication of the diverse sequence mixture and emergence of long strands may eventually lead to the creation of rolling circles and ribozymes. / Thesis / Master of Science (MSc) / The origin of biological life can be traced back by looking at the common themes between modern cellular processes. The role of RNA polymers seems to be of great importance, making us believe that an RNA world existed leading up to life’s origin. During this time, RNA would act as both a genetic material and a catalyst. To examine this theory in more detail, we use computational modeling to recreate and explore the various potential chemistries and conditions on the early Earth. Specifically, we explore the problems that exist for the replication and production of RNA polymers. Our results can be used to guide future theoretical and experimental research of the RNA world.

The production of biofuels and biobased products from low-cost abundant renewable non-food lignocellulosic biomass will be vital to sustainable development because it will bring benefits to the environment, the economy, and the national security. The largest technical and economic challenge for emerging biorefineries is cost-effective release of fermentable sugars from recalcitrant structure of lignocellulosic biomass. Cellulose- and organic-solvent-based lignocelluloses fractionation (COSLIF) technology was employed to overcome biomass recalcitrance. Surface response methodology (SRM) showed that optimal COSLIF pretreatment conditions were 85% (w/v) H₃PO₄ and ~50 °C, regardless of moisture contents in biomass from 5-15% (w/w) for common reed. Under these conditions, the pretreated biomass was hydrolyzed fast with high glucan digestibilities at low enzyme loadings (i.e., one FPU of cellulase per gram of glucan). Crystallinity index (CrI) measurements by X-ray diffraction (XRD) and cross polarization/magic angle spinning (CP/MAS) ¹³C nuclear magnetic resonance (NMR), and cellulose accessibility to cellulase (CAC) determinations of COSLIF-pretreated biomass confirmed that highly ordered hydrogen-bonding networks in cellulose fibers of biomass were disrupted through cellulose dissolution in a cellulose solvent. This disruption of hydrogen bonding networks among cellulose chains resulted in a drastic increase in CAC values. Fourier transform infrared (FTIR) analyses on COSLIF-pretreated biomass revealed conformational changes in specific hydrogen bonding among cellulose chains due to COSLIF. While CrI is believed to be a key substrate characteristic that impacts enzymatic cellulose hydrolysis, studies in this thesis showed CrI values varied greatly depending on measurement techniques, calculation approaches, and sample preparation conditions. A correlation between CAC values and glucan digestibility of pretreated biomass showed that substrate accessibility is a key substrate characteristic impacting enzymatic cellulose hydrolysis. In summary, COSLIF can effectively overcome biomass recalcitrance. The resulting pretreated biomass has high CAC values, resulting in fast hydrolysis rates and high enzymatic glucan digestibilities of COSLIF-pretreated biomass at low enzyme usage. / Ph. D.

Rotavirus (RV) is a viral pathogen that causes severe, watery diarrhea and vomiting in the young of humans and other animals. RV infections result in over 200,000 pediatric deaths around the world each year, especially in developing nations. Within the infected host cell, RV forms inclusion bodies, called viroplasms, where many stages of viral replication occur. The RV polymerase, known as VP1, must localize to viroplasms during infection where it replicates the virus' RNA genome. The work described in this dissertation focused on identifying region(s) of VP1 essential for its viroplasmic localization and its function as a polymerase. We found that a single amino acid change in a region of the polymerase called the N-terminal domain negatively impacted its capacity to localize to viroplasms during infection as well as its enzymatic activity in a test tube. Follow up studies using VP1 proteins from divergent strains and a mutant containing only the N-terminal domain of VP1 provided more insight into polymerase localization determinants. In total, our work suggests that the VP1 N-terminal domain plays an important role in localizing the polymerase to viroplasms via interactions with other viral proteins and supporting its function as a polymerase. / Ph. D.

Maximizing enzymatic hydrolysis performance can be achieved through the combination of experimental work and modeling. The present work utilizes an enzymatic hydrolysis model based on reaction kinetics, Langmuir adsorption isotherms, and product inhibition of enzymes (β-glucosidase, cellobiohydrolase, and endoglucanase). The model was developed from a 10% w/w corn stover system. Glucose yield sensitivity to changes in parameter values was assessed and linked to biomass and enzyme characteristics. A commercial enzyme cocktail (CEC) was subsequently characterized by FPLC and gel electrophoresis to identify key enzymes/activities, and the CEC was used in the enzymatic hydrolysis of 20% w/w steam-exploded hardwood. The model was applied to experimental data from the enzymatic hydrolysis of the steam-exploded hardwood, which provided characteristic reaction rate and inhibition parameters consistent with cellulose and xylan hydrolysis. These model-based analyses enhanced understanding of hydrolysis at commercially relevant solids loadings, while identifying pathways to improve enzyme cocktails and enhance biomass conversion.

Foram realizados estudos do processo de extração de bromelina de caules de abacaxizeiro Ananás comosus (L.) Merr. cultivar Cayenne, com a finalidade de avaliar o efeito da temperatura de filtração do extrato, da adição de ativadores e de agentes precipitantes sobre a estabilidade da atividade proteolítica e no balanço material. A filtração de extratos a 20, 30, 40, 50 e 60°C, promoveu gradual elevação da atividade proteolítica para as temperaturas ate 50°C e redução a 60°C. A atividade proteolítica foi determinada em Unidades Kunitz. A adição de agentes reconhecidamente ativadores de enzimas proteolíticas como benzoato de sódio, cloridrato de cisteina e edetato dissódico aos extratos filtrados em diferentes temperaturas, promoveu elevação da atividade enzimática, demonstrando um efeito somatório com a temperatura. 0 extrato filtrado a 50°C, contendo ativadores, manteve-se estável durante 60 dias, quando armazenado a 5 e 20°C. 0 balanço material mostrou-se superavitário quando se precipitou bromelina por sulfato de amônio, de extrato filtrado a 40°C, contendo benzoato de sódio, cloridrato de cisteina e edetato dissódico. A acetona demonstrou menor eficiência que o sulfato de amônio, em relação ao balanço material. 0 estudo de estabilidade nas temperaturas de 5, 20, 30 e 40°C, durante 90 dias, das amostras de bromelina obtidas através do inter-relacionamento dos fatores temperatura, ativadores e precipitantes, revelou resultados mais favoráveis na amostra preparada a partir de extrato filtrado a 40°C, contendo benzoato de sódio, cloridrato de cisteina e edetato dissódico, e precipitado por sulfato de amônio. A performance conseguida na ativação e estabilização do extrato e no balanço material não foi ratificada em relação a estabilidade da bromelina em pó. A bromelina em pó, sequramente, necessita de estabilizadores para consolidar as vantagens comprovadas no balanço material, na ativação e estabilização do extrato. / Studies were made on the extraction process of bromelain obtained from the stem of pineapple plant Ananas comosus (L.) Merr. variety Cayenne, with the purpose to evaluate the effect of temperature of the filtration extract, addition of activators and of precipitants agents upon the stability of the proteolytic activity and upon the material balance. The filtration of the extracts at 20, 30, 40, 50 and 60°C results in a gradual increase in the proteolytic activity of the extract till 50°C and a reduction at 60°C. The proteolytic activity was determined in Kunitz units.The addition of commons agents, proteolytic activators like sodium benzoate, cysteine hydrochloride and sodium edetate to the filtrated extracts of stem pineapple at different temperatures promote one elevation of the activity showing an addictive effect with the temperature. The filtrated extract at 50°C and with activators was stable for 60 days when stored at 5 and 20°C.The material balance showed a superavit when the precipitation of bromelain was made by ammonium sulphate in relation to others filtrated at 40°C, obtained with sodium benzoate, cysteine hydrochloride and sodium edetate.The acetone showed less efficiency than the ammonium sulphate in relation to the material balance. The stability study of the bromelain at the temperature of 5, 20, 30 and 40°C during a period of 90 days and obtained through the interrelationship of factors like temperature, activators and precipitants showed best results to bromelain sample prepared from the filtrated extract at 40°C with sodium benzoate, cysteine hydrochloride and sodium edetate and precipitated with ammonium sulphate. The performance related to the activation, stabilization and in the material balance was not confirmed in relation to the stability of the bromelain powder. The bromelain powder, certainly needs stabilizers to consolidate the advantages showed in the material balance in the activation and stabilization of pineapple extract.

<p>Predmet doktorske disertacije je izučavanje različitih bioprocesa za modifikovanje biljnih globulina radi unapređenja njihovih funkcionalnih karakteristika. Istraživanja su zasnovana na karakterizaciji i enzimskoj modifikaciji glavnog rezervnog proteina (12S), kukurbitina, iz semena uljane tikve (<em>Cucurbita pepo</em>). Osnova istraživanja je enzimska konverzija globulina i dobijanje proteinskih modifikata delovanjem hidrolaza i transferaza. U okviru istraživanja, enzimski procesi modifikacije globulina izučavani su sa dva aspekta: enzimska hidroliza i enzimsko umrežavanje (cross-linking), primenom komercijalnih enzimskih preparata. Takođe istraživanja obuhvataju i razvoj i kontrolu samih bioprocesa definisanjem i optimizacijom procesnih parametara (temperature, pH, koncentracije enzima i supstrata, vreme reakcije). Ovako definisani procesi eksploatisani su u cilju kreiranja željenih funkcionalnih karakteristika proteina spram njihove potencijalne primene u formulacijama hrane. Odabir i optimizacija procesnih parametara i modelovanje bioprocesa izvedeno je implementiranjem nove kompjuterske i analitičke metodologije</p> / <p>The PhD thesis research is aimed at development of different bioprocesses for modification of plant globulins in order to improve their functional properties. Studies are based on characterization and enzymatic modification of major storage protein (12S), cucurbitin derived from pumpkin oil seed (<em>Cucurbita pepo</em>). The base of research is enzymatic conversion of cucurbitin by hydrolase and transferase. Two different enzymatic processes are used for protein modification: (i) enzymatic hydrolysis and (ii) enzymatic cross-linking. To monitor, control the bioprocesses, and definition of process parameters, such as temperature, pH, enzyme-substrate ratio, reaction time, Response Surface Methodology (RSM) was used. In addition, RSM was employed for production of protein modification with desired functional properties.</p>

Marine capture fisheries contribute over 50% of total world fish production and more than 70% of this production is utilized for processing. The Canadian commercial fishing industry is one of the world’s most valued industries but generates large quantities of solid waste and wastewater. The increasing growth of the fish processing industry, the need for reduction of pollutants and the need to increase returns on raw material has led fish processors to adopt new ways of utilizing the wastes. In particular, efforts have focused on converting the biological substance in solid fish processing waste to various valuable compounds including both nutritional and non-nutritional products. Sulfated glycosaminoglycans (sGAGs) are heteropolysaccharide molecules with potential therapeutic applications and anticoagulant properties. Anticoagulants are responsible for curing major death-causing diseases such as strokes and cardiovascular diseases. The aim of this study was to develop an economically feasible technique to extract sulfated glycosaminoglycans (sGAGs) from fish processing waste. Two different fish (mackerel and herring) were used to optimize the extraction of sGAG. The effects of hydrolysis time (3, 6, 12 and 24 hrs) and papain concentration (15 and 20u/ml) on the extraction of sGAGs from different fish parts (whole fish, flesh, head, gut, fins and tails, skin and bones) were evaluated. The highest concentration of sGAGs (206.7 mg/g) was obtained from the mackerel head sample at 6 hrs of hydrolysis time and 20 u/ml of enzyme concentration while the highest concentration of sGAGs (236.3 mg/g) was obtained from herring gut at 12 hrs of hydrolysis time and 20 u/ml of enzyme concentration. The concentration of sGAG obtained from other part of mackerel were flesh (23.96 mg/g), waste (163.23 mg/g), fins and tail (86.63 mg/g), gut (203.52 mg/g), skin (105.45 mg/g) and bones (97.2 mg/g). However, the concentration of sGAG obtained from other parts of herring were flesh (39.34 mg/g), waste (130.15 mg/g), head (162.76 mg/g), fins and tail (148.53 mg/g), skin (65.89 mg/g) and bones (75.57 mg/g). Comparing the overall concentration of sGAG in waste samples of the fish, the mackerel produced higher sGAG than the herring.

Foram realizados estudos do processo de extração de bromelina de caules de abacaxizeiro Ananás comosus (L.) Merr. cultivar Cayenne, com a finalidade de avaliar o efeito da temperatura de filtração do extrato, da adição de ativadores e de agentes precipitantes sobre a estabilidade da atividade proteolítica e no balanço material. A filtração de extratos a 20, 30, 40, 50 e 60°C, promoveu gradual elevação da atividade proteolítica para as temperaturas ate 50°C e redução a 60°C. A atividade proteolítica foi determinada em Unidades Kunitz. A adição de agentes reconhecidamente ativadores de enzimas proteolíticas como benzoato de sódio, cloridrato de cisteina e edetato dissódico aos extratos filtrados em diferentes temperaturas, promoveu elevação da atividade enzimática, demonstrando um efeito somatório com a temperatura. 0 extrato filtrado a 50°C, contendo ativadores, manteve-se estável durante 60 dias, quando armazenado a 5 e 20°C. 0 balanço material mostrou-se superavitário quando se precipitou bromelina por sulfato de amônio, de extrato filtrado a 40°C, contendo benzoato de sódio, cloridrato de cisteina e edetato dissódico. A acetona demonstrou menor eficiência que o sulfato de amônio, em relação ao balanço material. 0 estudo de estabilidade nas temperaturas de 5, 20, 30 e 40°C, durante 90 dias, das amostras de bromelina obtidas através do inter-relacionamento dos fatores temperatura, ativadores e precipitantes, revelou resultados mais favoráveis na amostra preparada a partir de extrato filtrado a 40°C, contendo benzoato de sódio, cloridrato de cisteina e edetato dissódico, e precipitado por sulfato de amônio. A performance conseguida na ativação e estabilização do extrato e no balanço material não foi ratificada em relação a estabilidade da bromelina em pó. A bromelina em pó, sequramente, necessita de estabilizadores para consolidar as vantagens comprovadas no balanço material, na ativação e estabilização do extrato. / Studies were made on the extraction process of bromelain obtained from the stem of pineapple plant Ananas comosus (L.) Merr. variety Cayenne, with the purpose to evaluate the effect of temperature of the filtration extract, addition of activators and of precipitants agents upon the stability of the proteolytic activity and upon the material balance. The filtration of the extracts at 20, 30, 40, 50 and 60°C results in a gradual increase in the proteolytic activity of the extract till 50°C and a reduction at 60°C. The proteolytic activity was determined in Kunitz units.The addition of commons agents, proteolytic activators like sodium benzoate, cysteine hydrochloride and sodium edetate to the filtrated extracts of stem pineapple at different temperatures promote one elevation of the activity showing an addictive effect with the temperature. The filtrated extract at 50°C and with activators was stable for 60 days when stored at 5 and 20°C.The material balance showed a superavit when the precipitation of bromelain was made by ammonium sulphate in relation to others filtrated at 40°C, obtained with sodium benzoate, cysteine hydrochloride and sodium edetate.The acetone showed less efficiency than the ammonium sulphate in relation to the material balance. The stability study of the bromelain at the temperature of 5, 20, 30 and 40°C during a period of 90 days and obtained through the interrelationship of factors like temperature, activators and precipitants showed best results to bromelain sample prepared from the filtrated extract at 40°C with sodium benzoate, cysteine hydrochloride and sodium edetate and precipitated with ammonium sulphate. The performance related to the activation, stabilization and in the material balance was not confirmed in relation to the stability of the bromelain powder. The bromelain powder, certainly needs stabilizers to consolidate the advantages showed in the material balance in the activation and stabilization of pineapple extract.