Molecular biology

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

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

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 neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of inherited neurodegenerative lysosomal storage disorders. CLN7 disease is a subtype of NCL that is caused by mutations in the MFSD8 gene. MFSD8 encodes a lysosomal transmembrane protein that is predicted to play a role in transporting small substrates across membranes. However, little is known about its role and substrate specificity. Previous work identified an ortholog of human MFSD8 in the social amoeba Dictyostelium discoideum and reported its localization to endocytic compartments. In this study, the effects of mfsd8 loss during Dictyostelium growth and multicellular development were further characterized. Dictyostelium mfsd8- cells displayed increased rates of proliferation and pinocytosis in liquid media. During growth, loss of mfsd8 altered lysosomal enzymatic activities and reduced the intracellular and extracellular levels of autocrine proliferation repressor A. mfsd8- cells grown on a lawn of bacteria formed plaques in a shorter period of time compared to WT cells, providing additional support for the enhanced growth of mfsd8- cells. Upon starvation, the aggregation of mfsd8- cells was delayed, and mfsd8- cells formed more mounds that were smaller in size, which may be attributed to the reduced cell-substrate adhesion and altered lysosomal enzymatic activities observed for mfsd8- cells. Following aggregation, tipped mound formation was delayed, however, loss of mfsd8 did not affect the timing of slug/finger and fruiting body formation. Additionally, slug migration was reduced in mfsd8- cells. These aberrant phenotypes, excluding fruiting body formation, were effectively or partially rescued when Mfsd8-GFP was introduced into mfsd8- cells. Overall, these results show that Mfsd8 plays a role in regulating growth and developmental processes in Dictyostelium via lysosomal-associated functions.

Author Keywords: CLN7, Dictyostelium discoideum, Lysosomes, MFSD8, Neuronal Ceroid Lipofuscinoses

In Ontario, the dominant cash crop rotations consist of soybean (SB), which is a leguminous crop grown in rotation with maize (MZ) and winter wheat (WW). In addition to these crops, some farmers integrate cover crops (CC) into crop rotation, especially during the fallow period and winter seasons, to reduce nitrogen (N) losses via nitrate (NO3-) leaching and emission of N2 and the greenhouse gas nitrous oxide (N2O). This thesis focused on understanding the impact of crop phases in a MZ-(SB-WW)-CC rotation on the abundance of N-cycling bacterial communities that mediate nitrification and denitrification pathways. In addition, the influence of CCs on soil cytokinin (CK) profiles, which are plant growth-promoting hormones, were studied in a greenhouse trial to assess their potential impacts when integrating CCs into crop rotations. In particular, the relationship between traditional soil health parameters and the soil CK profiles was studied to understand how CKs might reflect biotic interactions and soil vitality. Results indicate N fertilizer application mono ammonium phosphate (MAP) and starter N:P: K (24:6:24) during WW planting in fall largely supported nitrifying bacterial communities (amoA) and potentially contributed to NO3- leaching. Management of MZ, which included spring-applied MAP resulted in larger denitrifying (nirK) bacterial communities, increasing the potential risk of N-loss via emission of dinitrogen gas (N2) and greenhouse gas N2O. However, CC soils had significantly lower nirK than MZ, reflecting the importance of strong and deep root systems of CCs, which have a higher ability to scavenge the substrates for denitrifying communities (NO3-). This highlights the importance of growing CCs in reducing the potential risk for N-loss via leaching and denitrification. Additionally, in the greenhouse trial, the ability of CCs to affect CK was detected, highlighting the importance of integrating CC in crop rotations. This is particularly noteworthy, given that total CK profiles showed strong associations with traditional soil health parameters such as labile or active carbon and soil microbial community diversity. It was concluded that total soil CK can be used as a novel and dynamic soil health measure. Future research on quantifying N2O fluxes and levels of NO3- in leachates would provide a more precise understanding of the impact of different crop rotation phases on N-dynamics in these fields. Further studies on single or combined measures of soil CKs are warranted to develop its potential as a practical and effective soil health parameter.

Author Keywords: Cover crops, Crop rotations, Cytokinin hormone, Nitrogen Cycle, qPCR, Soil health

Fungal pathogens adapt to environmental changes faster than their hosts, due in part to their adaptive mechanisms exhibited in response to stress. Ustilago maydis was used to investigate potential natural antisense transcript (NAT) RNA-mediated mechanisms that enhance fungal adaptation to stress. Of the 349 NATs conserved amongst U. maydis and two related smut fungi, five NATs were identified as having altered transcript levels in response to multiple stress conditions. Subsequently, antisense transcript expression vectors were created for select NATs and transformed into U. maydis haploid cells. When exposed to stress conditions, two antisense expressing mutant strains exhibited alterations in growth. RT-qPCR analysis of mRNA complementary to expressed NATs revealed no significant change in mRNA levels, which suggests NAT expression may influence stress response through dsRNA formation or other RNA mediated mechanisms. These results establish a basis for further investigations into the connection between NATs and the stress response of fungi.

Author Keywords: natural antisense transcripts, non-coding RNAs, stress response, Ustilago maydis

Heavy metal pollution threatens human and ecosystem health. E. gracilis was investigated for its potential use in bioremediation due to its tolerance for heavy metals and ability to sequester them from the environment. E. gracilis can remove metals by producing metal binding compounds enriched in sulfur and nitrogen. In this thesis, E. gracilis cultures that were pretreated with elevated levels of sulfur or nitrogen had increased tolerance to CdCl2 compared to non-pretreated cultures. RNA-sequencing revealed that both pretreatments led to transcript level changes and that exposure to CdCl2 led to further transcript level changes. Gene ontology (GO) enrichment analysis reflected changes in nitrogen and sulfur metabolism as well as physiological processes related to metal binding. The data from this thesis revealed important transcription level changes that occur when E. gracilis is challenged with CdCl2 and helps us understand how organisms adapt to heavy metal pollution in the environment.

Author Keywords: bioremediation, Cadmium, Euglena gracilis, GO-enrichment, metal-binding, RNA-Sequencing

Giardia intestinalis is a waterborne enteric parasite that lacks mitochondria and the capacity for heme biosynthesis. Despite this, Giardia encodes several heme proteins, including four cytochrome b5 isotypes (gCYTB5-I – IV) of unknown function. The aim of this thesis is to gain insight into the function of the Giardia cytochrome b5 isotype III (gCYTB5-III) that is found in the nucleus, as first reported by our laboratory using immunofluorescence microscopy experiments with an isotype-III specific antibody. Nuclear localization of isotype-III is supported by two of my experiments: i) immunoblot analysis of crude cytoplasmic and nuclear enriched fractions of Giardia trophozoites; ii) association of gCYTB5-III with the insoluble fraction of Giardia lysates crosslinked with formaldehyde is reversed by DNase I treatment. To gain an understanding of the possible roles of gCYTB5-III, I performed immunoprecipitation (IP) experiments on lysates from Giardia trophozoites to identify its protein partners. Mass spectroscopy analysis of the immunoprecipitate identified proteins localized to the nucleus (RNA polymerase, DNA topoisomerase, histones, and histone modifying enzymes). Intriguingly, over 40% of the known mitosomal proteome, which functions in iron-sulfur (Fe-S) cluster assembly was also associated with gCYTB5-III. One of these proteins, the flavoenzyme GiOR-1, has been shown to mediate electron transfer from NADPH to recombinant gCYTB5-III. These IP results provide evidence that GiOR-1 and gCYTB5-III interact in vivo, and furthermore, suggest that some proteins in the mitosome could interact with those in the nucleus. I also found that DNA stress, caused by low concentrations of formaldehyde (0.1 – 0.2%) resulted in the increased expression of gCYTB5-III. Collectively these findings suggest a role of gCYTB5-III in Giardia's response to DNA stress and perhaps the formation of Fe/S clusters.

Author Keywords: cluster, cytochrome, heme, iron, mitosome, nuclear