Molecular biology

Access to human blood for forensic research and training in bloodstain pattern analysis (BPA) can be difficult due to many ethical, safety and cost concerns. Mammalian blood alternatives can be sourced, especially from local and willing abattoirs, but some concerns remain, and the added difficulties of high variation and species-specific differences in cellular components pose other issues. Therefore, synthetic alternatives to human blood provide practical options for the BPA community. This thesis explores the use of alginate hydrogels as a base material for forensic blood substitute (FBS) development. Hydrogels are first explored as a suitable environment for DNA stability and functionality and compared to other polymer systems. The ability of DNA to remain intact while undergoing electrospray ionization (ESI) is also investigated. The FBS design focuses on mimicking the fluid properties and genetic capabilities of whole human blood – a material not developed in FBSs previously. ESI was used to develop microparticles (MPs) that serve as cellular components of human blood (the red blood cells – RBCs, and white blood cells – WBCs). The microparticles were ionically crosslinked using calcium to provide small MPs (RBCs) or covalently crosslinked with functional DNA to provide larger WBC-like functional particles. The integration of these novel MPs into alginate-based materials is optimized and their use in BPA scenarios is explored. The FBS is tested in BPA scenarios of dripping experiments, impact patterns, and the ability to extract and amplify the contained DNA. In addition, the stability (or shelf-life) of the FBS was also assessed. The FBS exhibited similar spreading ratios to blood and demonstrated feasibility in use for impact angle (a) determination and impact pattern creation. Importantly, the DNA contained within the FBS could be processed with analogous protocols used in DNA evidence processing, enhancing its applicability to BPA research and training.

Author Keywords: Alginate hydrogels, Bloodstain pattern analysis, Electrospray ionization, Forensic blood substitutes, Forensic materials, Synthetic DNA design

Glyphosate burndown and tillage, followed by the cultivation of cash crops, are frequently used techniques in LUC from perennial cropping systems (PS) to annual cropping systems (AS). Agricultural LUC can result in the loss of soil nitrogen (N) via emission of nitrous oxide (N2O), a potent greenhouse gas (GHG). The purpose of this thesis is to investigate the short-term impacts of agricultural LUC from PS to AS on soil health parameters and the nitrogen (N)-cycling bacterial communities responsible for nitrification and denitrification processes that result in the emission of N2O. The study field site was in Stone Mills, Ontario and comprised of four fields: two annual cropping systems were regularly cultivated for cash crops (AS), and two perennial cropping systems had not been cultivated for cash crops for over 50 years (PS). One PS was left intact while the other PS was subjected to LUC (converted system [CS]) from PS to AS within the study period. The results of this study indicate that PS promotes soil health, as illustrated through higher soil organic matter % (2.3 ± 0.2 %), beta-glucosidase activity (0.41 ± 0.04 mmol g-1 dry soil h-1), and N-acetylglucosaminidase activity (0.18 ± 0.03 mmol g-1 dry soil h-1). The PS soils exhibited higher nitrifier (6.0  0.3 log10 copies per g dry soil) and denitrifier (nirS, nirK and nosZI: 7.8  0.05, 8.1  0.1 and 5.0  0.1 log10 copies per g dry soil, respectively) gene abundances compared to AS (amoA, nirS, nirK and nosZI: 5.7  0.1, 7.7  0.04, 7.9  0.1 and 4.8  0.1 log10 copies per g dry soil, respectively). Moreover, LUC from PS to AS deteriorated soil health parameters and significantly decreased the nosZI/16S rRNA gene ratio, leading to potential N loss through N2O emissions. A laboratory incubation study revealed that the use of N-containing fertilizer in conjunction with easily metabolized C cumulatively resulted in 64.2% increase in N2O and 42.1% increase in CO2 fluxes in AS soils compared to PS soils. The AS soils also produced 69.8% more N2O and 13.4% more CO2 when compared to CS soils. The results suggest that the availability of C and N promote R-strategists, leading to increased production of CO2 and N2O. Additionally, results also suggest that LUC mediates fluxes depending on resource availability. The findings of this research demonstrate the significance of LUC in shaping N-cycling microbial communities and GHG emissions, emphasizing the importance of transitioning towards less intensive management practices to ensure the long-term sustainability of the agri-food system.

Author Keywords: annual, denitrification, greenhouse gas, laboratory incubation, nitrification, perennial

The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of neurodegenerative diseases that affect all ages, primarily children. Batten disease is caused by mutations in 1 of the 13 ceroid lipofuscinosis neuronal (CLN) genes (CLN1-CLN8, CLN10-CLN14), each of which causes an NCL subtype when mutated. One of the NCL subtypes, CLN5 disease, is caused by mutations in the CLN5 gene. CLN5 is a soluble lysosomal protein that localizes to the endoplasmic reticulum (ER), the Golgi complex, the cytoplasm, and extracellularly. CLN5 has four putative molecular functions, including as a ceramide synthase, glycoside hydrolase, depalmitoylase, and bis(monoacylglycerol)phosphate synthase. CLN5 plays various roles within the cell, such as lipid metabolism, autophagy, and proteasome degradation. However, the function and the exact pathway in which CLN5 is involved are unclear. In addition, CLN5 is a secreted protein that, as shown via bioinformatics analysis, contains a signal peptide sequence. Furthermore, there are currently 70 CLN5 disease-causing mutations reported in the NCL mutation database. 12 CLN5 disease-causing mutations have been studied thus far in terms of their cellular impact, as well as the release of CLN5 to a certain extent. However, there is a lack of research into the functionality of the signal peptide in CLN5 and an in-depth analysis of the molecular impact of mutations in CLN5 disease. Consequently, this Ph.D. thesis focused on using comparative transcriptomics to reveal biological pathways affected by cln5-deficiency, revealing mechanisms that regulate the secretion of Cln5 and CtsD, and using Dictyostelium to gain insights into the molecular effects of mutations in CLN5 disease. Comparative transcriptomics reveal many differentially expressed genes that are linked to phenotypes observed in cln5-deficient cells and identified pathways affected in other CLN5 disease models, such as autophagy. Furthermore, novel findings, like affected expression of lysosomal enzymes and pathways, including secretion, are identified within the comparative transcriptomics analysis. Subsequently, this research also shows the secretory role of the signal peptide in Cln5 and CtsD. Finally, this Ph.D. thesis revealed that mutations in CLN5 disease affect the lysosomal biology and secretion of Cln5 and other lysosomal enzymes.

Author Keywords: Batten disease, CLN5, Dictyostelium discoideum, Enzymes, Lysosome, Secretion

Acid mine drainage (AMD) and metal-contaminated tailings represent some of the most inhospitable aquatic environments on Earth, characterized by low pH, elevated metal concentrations, and chronic carbon limitation. Yet these systems support microbial consortia with remarkable resilience. Among the most conspicuous inhabitants is Euglena mutabilis, an acidophilic protist traditionally regarded as an indicator species of AMD but seldom thoroughly investigated. This thesis reframes E. mutabilis at the center of a fungal-algal-bacterial (FAB) consortium, demonstrating that its cadmium tolerance and persistence are emergent properties of the consortium.

Culture-based experiments revealed that E. mutabilis survival under cadmium stress declined when fungal and bacterial partners were disrupted, underscoring their indispensability. Glucose supplementation revealed the consortium's capacity for structural and metabolic reorganization: fungal hyphae bound algal cells into flocs, bacterial associates proliferated, and hormone production shifted. Hormone profiling suggested a distributed signaling system in which fungi contributed cytokinins (CKs) and gibberellins while algae produced methyl-thiolated CKs, jasmonic acid, and salicylic acid. Transmission electron microscopy revealed bacterial-like inclusions within algal vacuoles, suggesting facultative endosymbiosis or phagotrophic retention. Transcriptomic analyses revealed that cadmium stress suppresses light-harvesting complexes and growth-promoting hormone biosynthesis while activating metal transporters and chloroplast sequestration mechanisms.

Beyond stress physiology, the FAB consortium unlocked chemical diversity inaccessible to axenic cultures. Molecular networking revealed that environmental consortia consistently produced unique metabolite families, often linked to silent biosynthetic pathways. Metagenomic sequencing linked these products to bacterial gene clusters further supporting the view that metabolic innovation is an emergent property of the collective.

Together, these findings suggest that the FAB consortium should be understood not as a loose association but as a microbial superorganism. This framing extends beyond the holobiont concept by dissolving the hierarchy between host and symbiont: E. mutabilis, fungi, and bacteria are all indispensable, and the identity of the host itself becomes blurred.

By reframing E. mutabilis as the nucleus of a microbial superorganism, this work highlights both theoretical and applied significance. It advances ecological understanding of how life persists under geochemical extremes, while pointing to new opportunities for sustainable bioremediation and natural product discovery through the deliberate cultivation of naturally evolved microbial consortia.

Author Keywords: Algal symbiosis, Bioremediation, Co-culture, Hormones, Microscopy, Transcrioptomics

Giardia intestinalis is a parasitic protozoan that possesses a flavohemoglobin (gFlHb), an enzyme that plays a role in the detoxification of reactive nitrogen species (RNS) and reactive oxygen species (ROS) via its nitric oxide dioxygenase (NOD) activity as well as its NADH-oxidase activity. This enzyme is a potential target for imidazole-based antigiardial drugs that act as ligands of the iron within its heme cofactor. In this work, two rapid and relatively inexpensive assays, the colorimetric Griess assay and a fluorescence assay, were adapted, optimized, and implemented to screen for flavohemoglobin inhibitors in parallel studies that compared the response of gFlHb to that of Hmp (Escherichia coli flavohemoglobin) when a group of six different imidazole-based compounds was tested. These assays displayed isotype selectivity, showing how the different drugs elicited different responses from the two enzymes. Comparative results for gFlHb and Hmp revealed that bulkier compounds elicited higher inhibition of Hmp, while smaller compounds resulted in better inhibition of gFlHb, which might be explained by the presence of different amino acid residues in the active sites of the enzymes, with two large amino acid sequence inserts being a unique feature of gFlHb, thus blocking the active site from being reached and blocked by larger compounds.

Author Keywords: 2.3-diaminonaphthalene, Flavohemoglobin, Giardia intestinalis, Griess Assay, imidazole-based drugs, nitric oxide detoxification

The TATA-binding protein (TBP) is a key regulator of eukaryotic transcription initiation. The TBP homolog from Giardia intestinalis (gTBP) is highly divergent among all TBPs; notably lacking three of the four phenylalanine residues to unwind double- stranded DNA. I show that gTBP preferentially binds to single-stranded DNA (ssDNA) in two modes based on sequence and protein concentration. The proposed A mode likely represents multimeric binding of gTBP to ssDNA with four or more consecutive guanine bases. The B mode involves monomeric binding utilizing the structural properties of the ssDNA. To demonstrate this, I developed a novel technique using base stacking energy potentials to approximate the per-nucleotide flexibility of ssDNA. I also attempted to create a polynomial regression model to predict binding; however, further work is required to improve accuracy. Overall, this thesis presents a new perspective on eukaryotic transcription regulation based on the discovery of unconventional binding between gTBP and ssDNA.

Author Keywords: computer modelling, DNA binding protein, DNA structure, DNA transcription, general transcription factor (GTF), parasite

Frog virus 3 (FV3) belongs to the genus Ranavirus within the Iridoviridae family.Its 105,903 base genome encodes 98 open reading frames (ORFs), including ORF 97R, a putative apoptosis regulator sharing 31% structural similarity with the anti-apoptotic Bcl- 2 family protein, myeloid cell leukemia 1 (Mcl-1). 97R contains a BH1 domain, implicated in apoptosis regulation, and a predicted C-terminal transmembrane domain, which acts as a membrane-anchoring domain, localizing 97R to the ER membrane. To study its role in host cell modulation, 97R was cloned into a vector and transfected into HeLa cells. Immunofluorescence revealed a time-dependent decrease in Protein Disulfide Isomerase (PDI) in 97R-transfected cells. Immunoprecipitation and western blotting revealed that 97R interacts with Prohibitin 1 (PHB1), a host protein involved in apoptosis regulation. This research provides insight into the novel functional role of 97R in host cells, enhancing our understanding of how FV3 may manipulate its host.

Author Keywords: Bcl-2 protein family, frog virus 3, Iridoviridae, ORF 97R, Protein-protein interactions, Ranavirus

Cytokinins (CKs) are a pervasive group of growth-promoting signaling molecules spanning every kingdom of life. Their roles are best known in plants, where they act as phytohormones controlling nearly all aspects of plant growth and development. CKs continue to be detected in new organisms, posing questions about their roles in such widespread forms of life. The research presented in this thesis, therefore, investigated CK dynamics in a non-plant system using the simple eukaryotic model, Dictyostelium discoideum. Prior to this thesis, CKs were established as key intercellular signals necessary for proper development of D. discoideum – specifically in the induction of sporulation and maintenance of spore dormancy. However, there were no documented roles of CKs prior to the late stages of multicellular development. Comprehensive mass spectrometric screening for CKs detected six novel CK forms during all stages of D. discoideum growth and development. Based on these findings, a model was proposed that mapped CK biosynthesis in D. discoideum. The CK profiles indicate that there are differing dominant CK forms during vegetative growth and early development compared to those detected during late multicellular development. This led to the hypothesis that CKs have different roles during the distinctive life cycle stages of D. discoideum. This hypothesis was tested by generating knockout and overexpression strains of the key, primary CK biosynthesis gene, iptA, to investigate potential expanded roles for CKs during growth and the early stages of D. discoideum development. iptA-deficiency resulted in cytokinesis defects and both iptA-deficiency and overexpression caused altered mitochondrial morphology, dysregulated TCA cycle and amino acid metabolism, as well as increased levels of the energy metabolite, AMP. These combined phenotypes were suggestive of mitochondrial-associated dysfunction in vegetative growth and provided the first evidence of expanded roles of CKs during the D. discoideum life cycle. This was the first metabolomics-based evidence of CKs influencing mitochondrial function in D. discoideum. Lastly, a key CK-activating enzyme was functionally characterized, DdLOG, and additional CK biosynthesis enzymes were identified for future examination. Together, the findings of this thesis provide insights into: CK biosynthesis in a non-plant associated model; new roles for CKs during the D. discoideum life cycle; and CK interactions with mitochondria. The methods established as part of this thesis can be used as a foundation for characterizing further CK biosynthesis enzymes and as a guide for detecting subtle sub-cellular phenotypes related to CK metabolism in D. discoideum and other CK-producing organisms.

Author Keywords: cytokinin biosynthesis, cytokinins, Dictyostelium discoideum, IptA, mass spectrometry, mitochondria

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of fatal neurodegenerative disorders that primarily affect children. Several subtypes of NCLs have been reported, each being caused by a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene; this results in aberrant lysosome function and the accumulation of lipoprotein aggregates (known as ceroid lipofuscin) within cells. Several innate cellular pathways exist to alleviate the stress caused by the buildup of aggregates. The endoplasmic reticulum (ER) is an essential organelle in this process because it is responsible for maintaining cellular homeostasis through protein production, quality control, and regulating several signalling pathways. The unfolded protein response (UPR) consists of several conserved pathways devoted to attenuating ER stress caused by an accumulation of misfolded proteins or aggregates; at the center of this stress response is GRP78, a molecular chaperone that binds to misfolded proteins to facilitate proper folding. The social amoeba Dictyostelium discoideum is an excellent model system for studying NCLs as it encodes more CLN-like proteins when compared to other classical model organisms (e.g., yeast, worm, fruit fly). In this study, D. discoideum was used to elucidate the effects of ER stress and build an understanding of how cells cope with increased stress. Beyond this, ER stress in D. discoideum models for CLN3 disease and CLN5 disease were evaluated. First and foremost, during the induction of ER stress by tunicamycin, there was an increase in intracellular and extracellular amounts of Grp78 accompanied by an increase in stress-related changes to the ER. Furthermore, models of CLN3 disease and CLN5 disease displayed increased amounts of Grp78 as well as a disrupted ER morphology. Interestingly, wildtype D. discoideum, AX3 cells, treated with tunicamycin displayed a similarly disrupted ER when compared to CLN models. Finally, when subjected to tunicamycin-induced ER stress, these NCL models displayed a trend towards increased Grp78 amounts, however, these cells appear to have a reduced sensitivity to tunicamycin-induced stress compared to wild-type cells. In summary, this study highlights D. discoideum as a model for studying ER stress through the conserved role of Grp78 in the stress response and concludes that an aberrant ER stress underlies the pathology of the NCLs.

Author Keywords: Batten disease, Dictyostelium discoideum, ER stress, GRP78, neuronal ceroid lipofuscinoses (NCLs)

Access to human blood for forensic research and training in bloodstain pattern analysis (BPA) can be difficult due to many ethical, safety and cost concerns. Mammalian blood alternatives can be sourced, especially from local and willing abattoirs, but some concerns remain, and the added difficulties of high variation and species-specific differences in cellular components pose other issues. Therefore, synthetic alternatives to human blood provide practical options for the BPA community. This thesis explores the use of alginate hydrogels as a base material for forensic blood substitute (FBS) development. Hydrogels are first explored as a suitable environment for DNA stability and functionality and compared to other polymer systems. The ability of DNA to remain intact while undergoing electrospray ionization (ESI) is also investigated. The FBS design focuses on mimicking the fluid properties and genetic capabilities of whole human blood – a material not developed in FBSs previously. ESI was used to develop microparticles (MPs) that serve as cellular components of human blood (the red blood cells – RBCs, and white blood cells – WBCs). The microparticles were ionically crosslinked using calcium to provide small MPs (RBCs) or covalently crosslinked with functional DNA to provide larger WBC-like functional particles. The integration of these novel MPs into alginate-based materials is optimized and their use in BPA scenarios is explored. The FBS is tested in BPA scenarios of dripping experiments, impact patterns, and the ability to extract and amplify the contained DNA. In addition, the stability (or shelf-life) of the FBS was also assessed. The FBS exhibited similar spreading ratios to blood and demonstrated feasibility in use for impact angle (a) determination and impact pattern creation. Importantly, the DNA contained within the FBS could be processed with analogous protocols used in DNA evidence processing, enhancing its applicability to BPA research and training.

Author Keywords: Alginate hydrogels, Bloodstain pattern analysis, Electrospray ionization, Forensic blood substitutes, Forensic materials, Synthetic DNA design