Biology

Permanent URI for this collectionhttps://uwspace.uwaterloo.ca/handle/10012/9938

This is the collection for the University of Waterloo's Department of Biology.

Research outputs are organized by type (eg. Master Thesis, Article, Conference Paper).

Waterloo faculty, students, and staff can contact us or visit the UWSpace guide to learn more about depositing their research.

Browse

Now showing 1 - 20 of 620

1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) DEAMINASE GENES IN RHIZOBIA: ISOLATION AND CHARACTERIZATION
(University of Waterloo, 2007-05-14T13:14:45Z) Duan, Jin
A collection of 233 putative Rhizobia strains from 30 different sites across Saskatchewan, Canada was assayed for ACC deaminase activity, with 27 of the strains displaying activity. When all 27 strains were characterized based on 16S rRNA gene sequences, it was noted that 26 strains are Rhizobium leguminosarum and one strain is Rhizobium gallicum. PCR was used to rapidly isolate ACC deaminase structural genes from the above mentioned 27 strains; 17 of them have 99% identities when compared with the previously characterized ACC deaminase structural gene (acdS) from Rhizobium leguminosarum bv. viciae 128C53K, whereas the other 10 strains are 83% identical compared to the acdS of R. leguminosarum bv. viciae 128C53K. Southern hybridization showed that each strain has only one ACC deaminase gene. Using inverse PCR, the region upstream of the ACC deaminase structural genes was characterized for all 17 strains and shown to encode a leucine responsive regulatory protein. The results are discussed in the context of a previously proposed model for the regulation of bacterial ACC deaminase and facilitates an elaboration of the role of ACC deaminase in nodulation and nitrogen fixation.
A 30-Year Study of Impacts, Recovery, and Development of Critical Effect Sizes for Endocrine Disruption in White Sucker (Catostomus commersonii) Exposed to Bleached-Kraft Pulp Mill Effluent at Jackfish Bay, Ontario, Canada
(Frontiers, 2021-04-22) Ussery, Erin J.; McMaster, Mark E.; Servos, Mark. R.; Miller, David H.; Munkittrick, Kelly R.
Jackfish Bay is an isolated bay on the north shore of Lake Superior, Canada that has received effluent from a large bleached-kraft pulp mill since the 1940s. Studies conducted in the late 1980s found evidence of reductions in sex steroid hormone levels in multiple fish species living in the Bay, and increased growth, condition and relative liver weights, with a reduction in internal fat storage, reduced gonadal sizes, delayed sexual maturation, and altered levels of circulating sex steroid hormones in white sucker (Catostomus commersonii). These early studies provided some of the first pieces of evidence of endocrine disruption in wild animals. Studies on white sucker have continued at Jackfish Bay, monitoring fish health after the installation of secondary waste treatment (1989), changes in the pulp bleaching process (1990s), during facility maintenance shutdowns and during a series of facility closures associated with changing ownership (2000s), and were carried through to 2019 resulting in a 30-year study of fish health impacts, endocrine disruption, chemical exposure, and ecosystem recovery. The objective of the present study was to summarize and understand more than 75 physiological, endocrine, chemical and whole organism endpoints that have been studied providing important context for the complexity of endocrine responses, species differences, and challenges with extrapolation. Differences in body size, liver size, gonad size and condition persist, although changes in liver and gonad indices are much smaller than in the early years. Population modeling of the initial reproductive alterations predicted a 30% reduction in the population size, however with improvements over the last couple of decades those population impacts improved considerably. Reflection on these 30 years of detailed studies, on environmental conditions, physiological, and whole organism endpoints, gives insight into the complexity of endocrine responses to environmental change and mitigation.
Age Determination and Growth of Rainbow Darter (Etheostoma caeruleum) in the Grand River, Ontario
(University of Waterloo, 2016-05-31) Crichton, Alexandra
The accurate determination and validation of age is an important tool in fisheries management. Age profiles allow insight into population dynamics, mortality rates and growth rates, which are important factors in many biomonitoring programs, including the Canadian Environmental Effects Monitoring (EEM) program. Many monitoring studies in the Grand River, Ontario have focused on the impact of municipal wastewater effluent (MWWE) on fish health. Much of the research has been directed at understanding the effects of MWWE on responses across levels of biological organization. The rainbow darter (Etheostoma caeruleum), a small-bodied, benthic fish found throughout the Grand River watershed has been used as a sentinel species in many of these studies. Although changes in somatic indices (e.g. condition, gonad somatic indices) have been included in previous studies, methods to age rainbow darters would provide additional tools to explore impacts at the population level. The objective of the current study was to develop a method to accurately age rainbow darter, validated by use of marginal increment analysis (MIA) and edge analysis (EA) and to characterize growth of male and female rainbow darter at a relatively unimpacted site on the Grand River. Rainbow darter were collected from the Grand River at West Montrose on a monthly basis (May 2014 - June 2015). Size (length, weight) and gonad/liver weight were recorded, and left and right sagittal otoliths were collected. Length-frequency distributions were constructed for the darter population in July and October 2014 to assess population structures. Darters spawn in the Grand River in late April-early May and young-of-the-year (YOY) darters reached a catchable size, using backpack electro-shockers, by July. A distinct YOY cohort was apparent in the July length-frequency distribution; YOY ranged in length from 1.2–2.5 cm. By October the length-frequency distribution demonstrated that the YOY had started to merge into the other age classes. Direct age determination (using sagittal otoliths) of a subset of the October collections supports that YOY fish are no longer a distinct cohort on the length-frequency distribution, and have assimilated into the rest of the population by this time of year. Direct age determination of fish at this time of the year is therefore necessary to separate age classes. Examination of rainbow darter otoliths collected monthly was used to validate the use of this structure for accurate age estimation. MIA showed that one annulus was formed per year on sagittal otoliths, and that summer (opaque) growth zone formation began in early summer. EA was able to identify the timing of both summer growth zone and annulus (translucent zone) formation. Summer growth zone formed as early as April, with all fish exhibiting growth by July. Annulus formation was noted in some fish in September, and in all fish by November. Size-at-age data resulting from the October length-frequency subsampled fish showed differences between male and female rainbow darter. Young fish, both male and female, grow quickly in the first two years (ages 0+ and 1+) and exhibit similar mean length and weight-at-age. Beginning at age 2+ and in each older age group, male rainbow darter become significantly longer and heavier at age compared to females. Additionally, male fish continued to increase significantly in weight each year, with no apparent decrease in weight gain, whereas females did not gain weight significantly after the age of 2+. Estimated von Bertalanffy growth curves for male and female length-at-age relationships further emphasize the difference in male and female growth beginning at age 2+. Furthermore, this model predicted male maximum length to be greater than that of female fish (male: Linf=7.42; female: Linf=6.48). Liver and gonadosomatic indices collected each month indicate increased energy allocation into liver and gonad development in female fish for reproductive purposes, which may account for the difference in male and female size (length/weight) in older age cohorts. This study has contributed to our understanding of the aging and growth of a small-bodied fish species that is widespread in North America. An accurate and reliable method to age rainbow darter was validated and the knowledge necessary for the addition of growth into biomonitoring studies was established for using rainbow darter as a sentinel species. The ability to accurately estimate age in rainbow darter provides the opportunity to assess growth as an additional population level endpoint in ongoing studies in the Grand River and in other watersheds that are experiencing environmental change.
Aging affects circadian clock and metabolism and modulates timing of medication
(Elsevier, 2021-04) Sadria, Mehrshad; Layton, Anita T.
Aging is associated with impairments in the circadian rhythms, and with energy deregulation that affects multiple metabolic pathways. The goal of this study is to unravel the complex interactions among aging, metabolism, and the circadian clock. We seek to identify key factors that inform the liver circadian clock of cellular energy status and to reveal the mechanisms by which variations in food intake may disrupt the clock. To address these questions, we develop a comprehensive mathematical model that represents the circadian pathway in the mouse liver, together with the insulin/IGF-1 pathway, mTORC1, AMPK, NAD+, and the NAD+ -consuming factor SIRT1. The model is age-specific and can simulate the liver of a young mouse or an aged mouse. Simulation results suggest that the reduced NAD+ and SIRT1 bioavailability may explain the shortened circadian period in aged rodents. Importantly, the model identifies the dosing schedules for maximizing the efficacy of anti-aging medications.
Algal priming mediates the effects of light and nutrients on organic matter processing: insights from artificial and natural streams
(University of Waterloo, 2023-10-31) Pinks, Lauryn
Environmental factors such as light and nutrients may play important roles in determining detrital decomposition through their effects on primary production. Thus, human activities that alter the availabilities of light and nutrients may have direct consequences on organic matter (OM) processing and nutrient cycling in freshwater ecosystems. I compared heterotrophic function (OM processing and respiration) between two P levels (5 µg P L-1 and 51 µg P L-1) and two shade levels (0% and 80% shade) in natural streams, as well as 3 P levels (10 µg P L-1, 50 µg P L-1 and 100 µg P L-1) and 4 shade levels (0%, 50%, 80% and 100%) in artificial streams using the cotton-strip assay (CSA). Data from these experiments show a negative association between algal abundance (GPP and chlorophyll a) and recalcitrant OM (ROM) processing, implying a negative priming effect. Light was an important driver of negative priming which disproportionately affected ROM decomposition at lower P treatments over time. Overall, there were limited interactive effects between light and nutrient availability; rather, ROM processing was positively associated with P availability at all light levels while it was negatively associated with light availability at all P levels. There was no evidence to support positive priming (i.e., algal stimulation of ROM processing) in either experiment. Overall, results from this study illustrate the importance of considering light levels and nutrient availability when considering long term C and nutrient budgets in freshwater ecosystems.
AlignDx: Enabling Automated, Cloud-Based Workflows for Streamlined Bioinformatic-Focused Pathogen Surveillance
(University of Waterloo, 2023-07-13) Hunjan, Manjot
The rising trends in infectious disease burden, alongside the recent COVID-19 pandemic, underline the need for effective public health disease mitigation strategies like pathogen surveillance. Improvements to surveillance systems can be realized by incorporating a variety of surveillance data sources such as comprehensive genomics and simpler point-of-care approaches. In this thesis, a novel bioinformatic-focused surveillance platform is presented for executing scientific workflows in cloud-based environments. The platform in question, AlignDx, addresses gaps in available surveillance systems via its modular component-based design providing security, workflow management, summary reports and data archiving. Two workflows were created and tested using this platform. First, a metagenomics next-generation sequencing workflow was developed for human pathogenic virus surveillance. Using a clinical nasopharyngeal RNA-seq test dataset, the workflow performed well in classification of severe acute respiratory syndrome coronavirus 2. Also, a lateral flow assay workflow was developed for mass automated point-of-care pathogen surveillance. Using an original test dataset of serially diluted LFA images, under controlled lighting, the workflow performed well in correctly classifying tests according to their manually curated results. Overall, the AlignDx platform is an effective system for automated surveillance applications and its constituent workflows are flexible and primed for further development.
Alternative splicing of Lymnaea Cav3 and NALCN ion channel genes serves to alter biophysical properties, membrane expression, and ion selectivity
(University of Waterloo, 2012-08-30T15:19:56Z) Senatore, Adriano
Evidence is presented that Lymnaea contains homologues for mammalian Cav3 and NALCN 4-domain ion channels, which retain key amino acid sequence motifs that differentiate these channels from other 4-domain types. Molecular cloning and heterologous expression of the first invertebrate Cav3 channel cDNA from Lymnaea confirms that it indeed is a true homologue to mammalian Cav3 channels, retaining some hallmark biophysical and pharmacological features1. Interestingly, the Lymnaea Cav3 channel gene also exhibits alternative splicing that is conserved with mammalian Cav3.1 and Cav3.2 channels, with homologous exons 8b in the I-II linker (Cav3.1) and 25c in the III-IV linker (Cav3.1 and Cav3.2), that can selectively be included or omitted from the full length channel. We show that the developmental and spatial expression patterns of these splice variants are remarkably conserved, and that these splice variants produce analogous changes in membrane localization and biophysical properties when channels are expressed in HEK-293T cells. The Lymnaea Cav3 channel gene also undergoes alternative splicing in the domain II P-loop, with mutually exclusive exons 12A and 12B that code for a large portion of the P-loop just upstream of the selectivity filter. Such splicing is a novel discovery that is not conserved with vertebrates or any other deuterostome animal, all of which only contain 12A homologues of exon 12. However, protostome animals including Lymnaea stagnalis, Drosophila melanogaster, and C. elegans all have mutually exclusive 12A and 12B exons in their Cav3 channel genes. Evidence is presented that exon 12A is likely the ancestral exon for the domain II P-loop, and that alternate exon 12B evolved later. Furthermore, although the two Lymnaea variants possess the same selectivity filter motifs characteristic for Cav3 channels (i.e. EEDD), they exhibit dramatic differences in calcium vs. sodium selectivity, without significant differences in biophysical properties. This is the first account of alternative splicing used to modulate ion selectivity in a Cav3 channel homologue, and given that calcium is such an important electrogenic signaling molecule, these alterations are expected to have profound physiological implications. Amazingly, Lymnaea NALCN was also found to undergo alternative splicing in the domain II P-loop, but in this case, the entire P-loop is replaced by mutually exclusive exons 15a and 15b such that the selectivity filter is converted from the proposed non-selective sodium-permeable configuration (15b/EKEE; EEKE in mammals, nematodes and insects), to a calcium channel-like pore (15a/EEEE). Thorough phylogenetic analysis reveals that NALCN is extremely unconventional, in that alternative splicing has frequently and independently evolved to alter the selectivity filter in domains II or III, in multiple animal clades. Furthermore, the ancestral NALCN channel most likely contained an EEEE pore. This work brings into question NALCN’s proposed role as a major leak sodium conductance that depolarizes neurons to help set the resting membrane potential, since some species possess only an EEEE variant, and based on homology to other 4-domain ion channels, this should render the channel calcium-selective. Unfortunately, heterologous expression and electrophysiological characterization of the two Lymnaea NALCN isoforms was unsuccessful, corroborating with others the inability to record NALCN ionic currents in heterologous systems.
Amelioration of Plant Drought Stress by Plant Growth-Promoting Bacteria
(University of Waterloo, 2017-04-28) Eman, Alsharif
Plant growth-promoting bacteria (PGPB) that contain the enzyme (1-aminocyclopropane-1-carboxylate) ACC deaminase protect a variety of plants against damage from various biotic and abiotic stresses. Here, the ability of bacterial strains Pseudomonas sp. UW4 (a rhizospheric PGPB) and Pseudomonas fluorescens YsS6 (an endophytic PGPB) and their respective (1-aminocyclopropane-1-carboxylate) ACC deaminase minus mutants, to ameliorate the effects of drought stress on canola (Brassica campestris) seedlings was assessed. Initially, biochemical assays were used to estimate the levels of a number of the plant growth-promoting activities encoded within each of the four above-mentioned strains. These activities include: ACC deaminase activity, indole-3-acetic acid (IAA) production, siderophore synthesis and the trehalose concentration produced. Subsequently, the effect of the four bacterial strains on canola seedlings grown in the greenhouse in potting soil and subjected to different levels of drought stress (0, 6, 8, and 10 days) was measured. In all experiments, measurements were taken of shoot fresh weight, shoot dry weight, shoot protein concentration, leaf chlorophyll concentration, and shoot trehalose concentration. The results are discussed in terms of a previously developed model of PGPB functioning in the facilitation of plant growth.
Ammonia-oxidizing archaea and complete ammonia-oxidizing bacteria in a municipal wastewater treatment plant
(University of Waterloo, 2018-09-25) Spasov, Emilie
High levels of ammonia in aquatic environments can lead to eutrophication, oxygen depletion, and toxicity to aquatic animals. An important goal of municipal wastewater treatment plants (WWTPs) is to remove ammonia from human waste before the treated effluent is released into receiving waters. Nitrification, the oxidation of ammonia to nitrate via nitrite, is a process that removes ammonia, and it is mediated solely by microorganisms. Historically it was thought that the two enzymatic steps of nitrification were carried out by distinct groups of microorganisms. The first step, aerobic ammonia oxidation, was long-known to be carried out by ammonia-oxidizing bacteria (AOB), and more recently the ammonia-oxidizing archaea (AOA) were discovered as contributors to the same process. The second step, nitrite oxidation, is carried out by nitrite-oxidizing bacteria (NOB), which are phylogenetically distinct from the AOB. However, certain species of Nitrospira are now recognized as being capable of catalyzing both steps of nitrification in the process of complete ammonia oxidation (comammox). Due to the importance of nitrifiers to the functionality of WWTPs, this research aimed to better understand microorganisms and processes associated with ammonia oxidation within the tertiary treatment system biofilm of a municipal WWTP. The Guelph WWTP has four trains of rotating biological contactors (RBCs) that are each composed of eight individual RBC stages. As water flows from RBC 1 to 8, the ammonia concentration decreases due to the activity of ammonia oxidizers. Because of relatively low ammonia concentrations entering the RBCs, compared to secondary treatment aeration basins, and their fixed-film design, these RBCs present a valuable opportunity to study novel nitrifiers, such as the AOA and comammox bacteria. The group I.1b AOA representative Candidatus Nitrosocosmicus exaquare was previously enriched from the RBCs. Initial enrichment cultivation and genome analysis data indicated that ammonia oxidation by this archaeon was stimulated by the addition of organic carbon sources, such as pyruvate and succinate. For other AOA, pyruvate stimulation is likely a result of hydrogen peroxide detoxification, not mixotrophy. Conversely, Ca. N. exaquare may already be capable of detoxifying hydrogen peroxide without pyruvate because it possesses a gene for catalase. Additionally, it possesses dicarboxylate transporters. This suggests that Ca. N. exaquare may be able to use pyruvate and succinate mixotrophically. To test this hypothesis, incubation experiments without organic carbon, and with organic carbon sources or catalase were established. Both succinate and pyruvate did not consistently or significantly stimulate ammonia oxidation over controls with repeated incubations. Catalase was also not stimulatory to nitrite production, indicating that Ca. N. exaquare does not require exogenous catalase, and the pyruvate may be used instead as a carbon source. Evidence for mixotrophy was inconclusive, and further work with a pure culture of Ca. N. exaquare, and experiments with labelled organic carbon, would help determine if this AOA species is capable of mixotrophy. Due to their recent discovery, comammox bacteria had not previously been reported in the Guelph WWTP, and little is known about their abundances in WWTPs. Using a combination of metagenomics and quantitative PCR, the abundance and diversity of comammox bacteria in the RBCs were explored over two sampling years. Taxonomic profiling of all microorganisms revealed that Nitrospira spp. dominated the RBC microbial community. Functional profiling indicated that comammox-associated Nitrospira represented the most abundant group of ammonia-oxidizing microorganisms. The diversity of comammox bacteria was also high, with multiple populations present. A cluster of comammox Nitrospira was also phylogenetically distinct from cultivated comammox species and these taxa represent ideal targets for future cultivation efforts. Genetic evidence that RBC Nitrospira are capable of complete ammonia oxidation includes Nitrospira metagenome assembled genomes that contain genes for both ammonia oxidation (e.g. amoA) and nitrite oxidation (e.g. nxrB). The results indicate that comammox bacteria co-exist with AOA and AOB, as well as with strict nitrite-oxidizing Nitrospira. The ammonia oxidizers displayed distinct patterns in relative abundance. In 2010 samples, AOA-associated amoA and 16S rRNA genes were higher in abundance in RBC 8 than RBC 1, but the opposite pattern was observed for 2016 samples. The relative abundance of AOB amoA genes was lower for 2016 samples than 2010 samples, suggesting a change in the environmental conditions of the RBCs. The relative abundance patterns of comammox Nitrospira amoA genes were consistent across the two years. This study indicated that the environmental factors that govern AOA, AOB, and comammox bacteria differ, suggesting that these groups occupy distinct niches within this wastewater biofilm environment. This thesis research is an important step towards understanding the nitrifying microorganisms in the Guelph RBCs. Although Ca. N. exaquare possesses the ability to deal with hydrogen peroxide stress, this research did not conclusively address organic carbon stimulation and assimilation. This thesis work showed that comammox bacteria were the dominant ammonia oxidizers in the Guelph RBCs, and this is the first study showing that comammox bacteria can be the dominant nitrifiers in a WWTP. This research paves the way for future studies on the microbial ecology of nitrification in engineered and aquatic environments.
Ammonia-oxidizing archaea in engineered biofiltration systems
(University of Waterloo, 2016-12-21) Sauder, Laura
Ammonia is a nitrogenous metabolic waste product that is produced by all animal life. High concentrations of ammonia are toxic to animals and may result in algal blooms and eutrophication in aquatic environments. To prevent negative impacts on animal and environmental health, water treatment systems use biological filters to host populations of nitrifying microorganisms that oxidize ammonia to nitrite and subsequently to nitrate. Ammonia-oxidizing archaea (AOA) outnumber ammonia-oxidizing bacteria (AOB) in many terrestrial and aquatic environments, but few studies have characterized AOA in engineered environments, despite the importance of these systems for ecosystem health. This thesis research examined two types of nitrifying biofiltration systems, including freshwater aquaria and fixed-film reactors from a municipal wastewater treatment plant (WWTP), to investigate the abundance, diversity, activity, and ecology of AOA in freshwater engineered systems. Although nitrification is the primary function of aquarium biofilters, few studies have investigated the microorganisms responsible for this process in aquaria. Based on quantitative PCR (qPCR) for ammonia monooxygenase (amoA) and 16S rRNA genes of Bacteria and Thaumarchaeota, AOA were numerically dominant in 23 of 27 freshwater biofilters, and contributed all detectable amoA genes in 12 of these biofilters. AOA also outnumbered AOB in five of eight sampled marine aquarium biofilters. For freshwater aquaria, the proportion of amoA genes from AOA relative to AOB was inversely correlated with ammonia concentration, suggesting an adaptation to low ammonia conditions. Composite clone libraries of AOA amoA genes revealed distinct freshwater and saltwater clusters, as well as mixed clusters containing both freshwater and saltwater amoA gene sequences. From one analyzed freshwater aquarium biofilter that demonstrated a high archaeal abundance, AOA representative Candidatus Nitrosotenius aquariensis was enriched in laboratory culture. Ca. N. aquariensis oxidized ammonia stoichiometrically to nitrite with a concomitant increase in thaumarchaeotal cells. Ca. N. aquariensis has a generation time of 34.9 hours, is mesophilic with an optimal growth temperature of 33ᵒC, and can tolerate up to 3 mM NH4Cl. Transmission electron microscopy (TEM) revealed that Ca. N. aquariensis cells are rod-shaped with a diameter of ~0.4 µm and lengths ranging from 0.6-3.6 µm. In addition, these cells possess paracrystalline S-layers and up to five appendages per cell. Phylogenetically, Ca. N. aquariensis belongs to the Group I.1a Thaumarchaeota, and clusters with environmental sequences from freshwater aquarium biofilters, aquaculture systems, and wastewater treatment plants (WWTPs). The complete genome sequence is 1.70 Mbp, and encodes genes involved in ammonia oxidation, urea hydrolysis, and bicarbonate assimilation. Several genes encoding flagella synthesis and chemotaxis were identified in the genome, as well as genes associated with S-layer production, defense, and protein glycosylation. Incubations of aquarium filter biomass revealed that PTIO is strongly inhibitory of ammonia oxidation, suggesting an in situ role for Ca. N. aquariensis-like AOA in freshwater aquarium biofilters. AOA have been detected in WWTPs based on targeted gene sequences, but contributions of AOA to ammonia oxidation in WWTPs remain unclear. In this thesis, ammonia-oxidizing populations in nitrifying rotating biological contactors (RBCs) from a municipal WWTP were investigated. Individual RBC stages are arranged in series, with nitrification at each stage contributing to an ammonia concentration gradient along the flowpath. Quantitative PCR for thaumarchaeotal amoA and 16S rRNA genes in RBC biofilm samples demonstrated that AOA abundance increased as ammonia decreased across the RBC flowpath. In addition, a negative correlation (R2=0.51) existed between ammonia concentration of RBC-associated water samples and the relative abundance of AOA amoA genes detected in corresponding biofilm samples. A single AOA population was detected in the RBC biofilms and this phylotype shared low 16S rRNA and amoA gene homologies with existing AOA cultures and enrichments. From RBC biofilm, Ca. Nitrosocosmicus exaquare was enriched in laboratory culture. Ca. N. exaquare oxidizes ammonia to nitrite stoichiometrically and assimilates bicarbonate, as demonstrated by microautoradiography. The 2.99 Mbp genome of Ca. N. exaquare encodes pathways for ammonia oxidation, bicarbonate fixation, and urea transport and hydrolysis. Despite assimilating inorganic carbon, the ammonia-oxidizing activity of Ca. N. exaquare is greatly stimulated in enrichment culture by the addition of organic compounds, especially malate and succinate. Ca. N. exaquare cells are coccoid and large in comparison to all other cultured AOA, with a diameter of approximately 1-2 µm. Phylogenetically, Ca. N. exaquare belongs to the Group I.1b Thaumarchaeota, clustering in the Nitrososphaera sister cluster, which includes most other environmental AOA sequences from municipal and industrial WWTPs. Incubations of WWTP biofilm demonstrated partial inhibition of ammonia-oxidizing activity by PTIO, suggesting that Ca. N. exaquare-like AOA contribute to nitrification in situ. Interestingly, CARD-FISH-microautoradiography revealed no incorporation of bicarbonate by Ca. N. exaquare-like AOA in actively nitrifying biofilms, suggesting that these cells may assimilate non-bicarbonate carbon sources. In natural and engineered environments, differential inhibitors are important for assessing the relative contributions of microbial groups to biogeochemical processes. For example, PTIO is a nitric oxide scavenger used for the specific inhibition of nitrification by AOA. This research investigated four alternative nitric oxide scavengers for their ability to differentially inhibit AOA and AOB in comparison to PTIO. Caffeic acid, curcumin, methylene blue hydrate, and trolox all demonstrated differential inhibition on laboratory cultures of AOA and AOB, providing support for the proposed role of nitric oxide as a key intermediate in the thaumarchaeotal ammonia oxidation pathway. Overall, this research demonstrated that AOA were abundant in aquarium biofilters and nitrifying RBCs, and that they contributed to ammonia-oxidizing activity in sampled biofilm environments. Niche partitioning of AOA and AOB was observed based on environmental ammonia concentrations, with AOA adapted to low ammonia conditions. Moreover, the enrichment cultures and genome sequences of novel AOA representatives provide insight into the ecophysiology of AOA originating from engineered systems.
Analysis of Cyanobacteria and its Abundance in the Turkey Lakes Watershed using Molecular Techniques
(University of Waterloo, 2023-12-21) Krishna, Anjali
Toxic cyanobacterial blooms continue to pose a threat to the quality and safety of drinking water globally by producing toxins and forming dense surface blooms. Forested watersheds naturally provide high quality drinking water to various communities but are threatened by bloom events that are increasing due to warming climates and anthropogenic land use. Monitoring programs utilized in drinking water sources are required to adapt to the changing intensity and frequency of these blooms where observation of cyanobacterial composition and abundance may vary based on sampling efforts. However, due to the variation and adaptability of these organisms spatially and temporally, cyanobacteria are often overlooked if surface blooms are not visualized, where these organisms may be present and abundant throughout the water column at different depths and vary throughout the day. The undetected organisms may release potent toxins that are threats to drinking water security if left untreated. The harmful toxic blooms comprise of cyclic hepatotoxins, involved in causing severe liver damage and affecting human and aquatic health. The aim of this study was to identify and quantify the cyanobacterial community composition and abundance of potential toxin producing genes in an oligotrophic northern forested watershed (Turkey Lakes Watershed, Ontario, Canada). To evaluate the composition and abundance, water samples were collected from Little Turkey Lake in May, June, July, and August 2022 at integrated and varying depths to determine variability over a summer season and at different timepoints in a single day. Microbial DNA was extracted from the water samples for 16S rRNA gene sequencing where data was obtained for bioinformatic and phylogenetic analyses. Extracted samples underwent quantitative PCR analysis for identification of gene copy numbers of cyanobacteria and potential microcystin producing organisms. With the extension of the ice-free season through warmer temperatures, and changes in environmental parameters, cyanobacteria and potential cyanotoxin producers appeared as early as May in this oligotrophic lake system. Peak abundances of cyanobacterial and potential cyanotoxin producing gene copy numbers were observed in the months of July and August, without visible blooms during sampling. Cyanobacterial composition had variability between the months, days, and timepoints when sampling, demonstrating the importance of consisting monitoring and sampling efforts due to the changing composition and abundance. Variation was observed among the depths within the water column, where integrated sampling provided a snapshot of the water system and can be useful for efficient analysis of the system, but multiple depth sampling is more representative of the community composition and abundance of cyanobacteria. This illustrates that monitoring protocols for drinking water sources require evaluation for the appropriate sampling protocol, timepoints, and location of the water column as each water system is unique. This research provides insight into cyanobacterial emergence in earlier summer months in an oligotrophic water system. It is applicable for the development of monitoring and drinking water treatment protocols for toxin-producing cyanobacteria, where analysis of the full water column is required with consistent sampling and integrated sampling is efficient, especially when there is an absence of a visible surface bloom. The inclusion of molecular characterization (amplicon sequencing and qPCR) is a valuable tool that can be cost efficient and effective ways to analyze samples. This research can then be expanded to other toxins and secondary metabolites produced by cyanobacteria.
Analysis of heat shock protein 30 gene expression and function in Xenopus laevis A6 kidney epithelial cells
(University of Waterloo, 2014-04-30) Khan, Saad
Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. The present study examined the pattern of accumulation of HSP30 and HSP70 in cells recovering from heat shock as well as the effect of proteasome inhibition on cytoplasmic/nuclear and endoplasmic reticulum (ER) molecular chaperone accumulation, large multimeric HSP30 complexes, stress granule and aggresome formation in Xenopus laevis A6 kidney epithelial cells. Initial immunoblot analysis revealed the presence of elevated levels of HSP30 after 72 h of recovery. However, the relative levels of HSP70 declined to near control levels after 24 h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24 h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. To verify if proteasome inhibition in A6 cells induced the formation of similar HSP30 granules, immunoblot and immunocytochemical analyses were performed. MG132, celastrol and withaferin A enhanced ubiquitinated proteins, inhibited chymotrypsin-like activity of the proteasome and induced the accumulation of cytoplasmic/nuclear HSPs, HSP30 and HSP70 as well as ER chaperones, BiP and GRP94 and heme oxygenase-1. Northern blot experiments determined that proteasome inhibitors induced an accumulation in hsp30, hsp70 and bip mRNA but not eIF1α. The final part of this study demonstrated that treatment of A6 cells with proteasome inhibitors or sodium arsenite or cadmium chloride induced HSP30 multimeric complex formation primarily in the cytoplasm. Moreover, these stressors also induced the formation of RNA stress granules, pre-stalled translational complexes, which were detected via TIA1 and polyA binding protein (PABP), which are known stress granule markers. These stress granules, however, did not co-localize with large HSP30 multimeric complexes. In comparison, proteasome inhibition or treatment with sodium arsenite or cadmium chloride also induced the formation of aggresome-like structures, which are proteinaceous inclusion bodies formed as a result of an abundance of aggregated protein. Aggresome formation was identified by monitoring the presence of vimentin and γ-tubulin, both of which are cytoskeletal proteins and serve as markers of aggresome detection. Aggresome formation, which was also verified using the ProteoStat assay, co-localized with large HSP30 multimeric complexes. Co-immunoprecipitation experiments revealed that HSP30 associated with γ-tubulin and β-actin in cells treated with proteasome inhibitors or sodium arsenite or cadmium chloride suggesting a possible role in aggresome formation. In conclusion, this study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation. Finally, proteasome inhibition, sodium arsenite and cadmium chloride treatment of A6 cells induced cytoplasmic/nuclear and ER chaperones as well as resulting in the formation stress granules and aggresome-like structures which associated with large HSP30 multimeric complexes.
Analysis of heat shock-, sodium arsenite- and proteasome inhibitor-induced heat shock protein gene expression in Xenopus laevis
(University of Waterloo, 2009-05-01T19:33:53Z) Young, Jordan T.F.
Previous studies have focused on the effect of individual stressors on hsp gene expression in eukaryotic organisms. In the present study, I examined the effect of concurrent low doses of sodium arsenite and mild heat shock temperatures on the expression of hsp30 and hsp70 genes in Xenopus laevis A6 kidney epithelial cells. Northern hybridization and western blot analysis revealed that exposure of A6 cells to 1-10 μM sodium arsenite at a mild heat shock temperature of 30˚C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 μM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26˚C with larger responses at 28 and 30 ˚C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 μM ) and heat shock (30 ˚C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 ˚C. Acquired thermotolerance was not observed with cells treated with the two mild stresses individually. It is likely that the enhanced accumulation of HSPs under these conditions permits the organism to cope with multiple environmental stresses encountered in their natural aquatic habitat. Previous studies have shown that inhibiting the activity of the proteasome also leads to the accumulation of damaged or unfolded proteins within the cell. In the second phase of this study, I report that inhibition of proteasome activity by the inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and lactacystin induced the accumulation of HSP30 and HSP70 as well as their respective mRNAs. The accumulation of HSP30 and HSP70 in A6 cells recovering from MG132 exposure was still relatively high 24 h after treatment and it decreased substantially after 48 h. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. HSP30 localization in A6 cells was primarily in the cytoplasm as revealed by immunocytochemistry. In some A6 cells treated with higher concentrations of MG132 and lactacystin, HSP30 was also found to localize in relatively large cytoplasmic foci. In some MG132-treated cells, HSP30 staining was substantially depleted in the cytoplasmic regions surrounding these foci. The activation of HSF1 may be involved in MG132-induced hsp gene expression in A6 cells since KNK437 inhibited the accumulation of HSP30 and HSP70. Lastly, MG132 treatment also conferred a state of thermotolerance in A6 cells such that they were able to survive a subsequent thermal challenge. Analysis of this phenomenon is important given the fact that impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.
Analysis of Immune System and Stress Parameters of Oncorhynchus mykiss in Response to Juvenile Thermal Preconditioning or Thermal-Based Parental Selection
(University of Waterloo, 2021-09-20) Guo, Huming
Higher water temperatures and pathogens are both significant factors that negatively affect the welfare of teleost fish. In aquaculture, compared to natural populations, these problems are especially exacerbated, as the animals have relatively limited mobility, and the higher density promotes faster spread of infectious diseases. Because of the potential harm these stressors can inflict, methods that can limit the damage of these stressors are particularly valuable. Two of the methods that have been explored as ways to improve the thermotolerance of aquacultured teleosts are juvenile-stage thermal preconditioning and thermotolerance-based parental selection. These methods have thus far demonstrated potentially effective improvements in the thermotolerance of animals, yet their potential effects on the immune system have not been explored, even though the heat-shock response and immune response are interconnected. In the first experiment, juvenile-stage thermal preconditioned rainbow trout (Oncorhynchus mykiss) were subject to a secondary thermal challenge in which animals were collected and sampled at the time of lost equilibrium. The effects of preconditioning were assessed on spleen and gill transcript profiles by quantifying IL-1β, IL-6, TNF-α, IFN-1, β2m, MH class I, HSP70, and HSC70 transcripts via qPCR, as well as assessment of the general stress response by measuring plasma cortisol levels. No changes in CTmax were observed between the preconditioned and control cohorts upon the second challenge. IL-1β and IL-6 transcripts were generally upregulated with increased temperature of the secondary thermal challenge, whereas IFN-1 transcripts were upregulated in the spleen, but downregulated in the gills, along with MH class I. The juvenile thermal preconditioning produced a series of changes in transcript levels for IL-1β, TNF-α, IFN-1, and HSP70, but the dynamics of these differences were inconsistent. Otherwise, plasma cortisol levels were lower in the pre-conditioned animals. In the second experiment, rainbow trout offspring of crosses raised from thermally selected parents were co-stimulated with a thermal and pathogenic challenge. Animals held at different temperatures were injected with an inactivated Vibrio anguillarum vaccine, and spleen tissue was sampled at 6-, 24-, and 96-hours post-injection. Relative transcript analysis of splenic IL-1β, IFN-γ, tapasin, and IL-10 was conducted with qPCR. All four transcripts were upregulated in response to the vaccine at 6- and 24-hours post-injection, and IL-10 was upregulated at 96-hours post-injection. The vaccinated cohorts held at a higher temperature presented evidence of an accelerated regulation profile compared to the vaccinated cohort held at a lower control temperature. The thermal selection of parents had a few significant changes across the assessed transcripts, but no consistent trends between transcripts. In general, these two methods were able to induce significant changes in immune transcript expression at higher temperatures both with and without immune stimulation. However, the biological significance of these changes will require further analysis to understand. If these thermotolerance-based methods can confer additional protection to the immune response, the revelation will be relevant to a global aquaculture industry that is constantly struggling with environmental and pathogenic pressures.
Analysis of premature loss of the extraembryonic Amnioserosa in Drosophila morphogenetic mutants
(University of Waterloo, 2009-01-23T19:24:11Z) Chaudhary, Roopali
During Drosophila embryogenesis, an extra-embryonic tissue, known as the amnioserosa (AS), is required for the morphogenetic processes of germ band retraction (GBR) and dorsal closure (DC). Being extra-embryonic, the AS is not part of the embryo proper but is eliminated via programmed cell death (PCD) in the late stages of embryogenesis. Programmed elimination of the AS during normal development can be prevented by directly inhibiting apoptosis, either through the deletion of the pro-apoptotic genes hid, grim and reaper, or through the expression of the pan-caspase inhibitor P-35. PCD in the AS can also be prevented by indirect inhibition of apoptosis via inactivation of autophagy, either through activation of the InR/PI3K pathway, or through activation of the Ras signalling pathway. The timing of AS elimination is critical to development as mutants associated with premature AS loss fail in GBR. To better characterize this premature AS death, a detailed phenotypic analysis of the AS behaviour in the GBR mutant hindsight (hnt) was performed. Direct inactivation of apoptosis failed to rescue the GBR defects in hnt mutant, though the premature AS death was completely rescued. Inactivation of autophagy, however, rescued AS cell behaviour and contacts during GBR, with partial rescue of the GBR defects in the hnt mutant. The nature of premature AS loss is characterized as a possible model for anoikis, a form of cell death that is triggered through reduced cell-cell or cell- matrix contact.
Analysis of Programmed Cell Death in the Amnioserosa, an Extra-embryonic Epithelium in Drosophila melanogaster
(University of Waterloo, 2008-01-24T15:56:37Z) Mohseni, Nilufar
The amnioserosa (AS) is an epithelium that plays major roles in two crucial morphogenetic processes during Drosophila embryogenesis: Germ Band Retraction (GBR) and Dorsal Closure (DC). The AS is extraembryonic and as such, it does not contribute to the mature embryo but is eliminated during development by programmed cell death. In this thesis, a comprehensive investigation of the timing and characteristics of the AS death and degeneration is performed. It is demonstrated that AS elimination occurs in two phases: “cell extrusion” during DC, embryonic stages 12 to 14, and “tissue dissociation” following DC, embryonic stages 15 to 16. Ten percent of AS cells are eliminated during phase one while the remaining ninety percent are removed during phase two. It is found that both cell extrusion and tissue dissociation are absent in apoptotic defective backgrounds, as well as in genetic backgrounds associated with increased class I phosphoinositide 3-kinase (PI3K) activity, a key regulator of autophagy. It is also found that extrusion is enhanced two-fold in embryos expressing the pro-apoptotic reaper gene product, and that tissue dissociation also accelerates in this background. Interestingly, our observations suggest that the activation of caspase cascade is not complete until AS cells have lost apical contacts with neighboring cells. Shortly after the loss of apical contact, an apoptotic morphology including membrane blebbing, cell fragmentation, and macrophage engulfment is readily observed. Measurements of the rate of DC demonstrate that this process is protracted in backgrounds lacking extrusion, leading to the conclusion that extrusion contributes towards generating adequate AS tension required for normal DC rates. Overall, our data suggest that phase one extrusion and phase two dissociation are manifestations of the same cellular event and that both are caspase dependent. It is also demonstrated that autophagy is a key component of AS death that acts upstream of apoptosis. Strikingly, our results lead to the suggestion that autophagy may function to trigger apoptosis during the programmed elimination of this extra-embryonic tissue.
Analysis of Temporal Changes in Estrogenic Compounds Released from Municipal Wastewater Treatment Plants
(University of Waterloo, 2019-09-26) Srikanthan, Nivetha
Wastewater treatment plants (WWTPs) are traditionally designed to target the removal of contaminants such as total suspended solids, phosphorous, biological oxygen demand, and ammonia. Recent changes to the Federal Wastewater Systems Effluent Regulations (WSER) in Canada require all WWTPs to be operating with secondary treatment or equivalent by 2021. Upgrades being implemented at WWTPs across the country will improve the quality of final effluent discharged into the receiving waters. However, over the past several years, contaminants of emerging concern such as pharmaceuticals, personal care products, and endocrine disrupting compounds have become widely prevalent in wastewater. These compounds are not monitored or targets for removal in Canada causing them to be routinely discharged into surface waters. The Grand River watershed is the largest watershed in southern Ontario and receives effluent discharge from 30 WWTPs. Several studies have been conducted in the Grand River to assess the impacts of effluent discharge on fish found in the river. The two largest WWTPs are the Kitchener and Waterloo WWTPs, both of which having recently undergone upgrades to improve nitrification processes and improve the overall effluent quality. Studies linked effluent from the plant’s pre-upgrade, to several adverse impacts on fish, such as severe cases of intersex and altered hormone production. Upgrades at the Kitchener WWTP were shown to reduce these impacts on fish. Effluent from both Waterloo and Kitchener have been collected and analyzed for pharmaceuticals and estrogens since before the upgrades providing the unique opportunity to evaluate the change in effluent quality and composition over time. In addition to the Kitchener and Waterloo WWTPs, nine secondary WWTPs across southern Ontario were studied to compare the composition of influent and effluent as well as evaluate the apparent removal of various pharmaceuticals and estrogens. Despite all the plants being classified as having secondary level treatment there was a considerable amount of variability in their ability to treat the incoming influent. Pharmaceuticals of interest were ibuprofen, naproxen, carbamazepine, and venlafaxine because of their different behaviour during treatment. Ibuprofen and naproxen were significantly reduced at all plants, with an increased reduction at plants achieving better nitrification. Carbamazepine and venlafaxine are recalcitrant and remained untreated. Of the estrogens measured, estriol was significantly reduced across all plants while 17α-ethinylestradiol had no difference post treatment. Estrone and 17β-estradiol were both reduced to varying degrees and were more influenced by external factors such as treatment type and biotransformation. Although there was compound specific variability, the total estrogenicity was significantly reduced post treatment at all plants except those with poor nitrification. Through the analysis of the pharmaceuticals and estrogens as well as nutrient data, nitrification was related to the apparent removal of these non-target compounds (although a direct relationship cannot be established). This correlates with the findings at the Kitchener and Waterloo WWTPs. With the introduction of nitrification at both plants there was a decrease in ammonia concentrations, improved treatment of ibuprofen, naproxen, estrone, and estradiol. There was also a decrease in the total estrogenicity of the effluent discharged from the plants. While venlafaxine, carbamazepine and ethinylestradiol concentrations remained unchanged post upgrades. Understanding the composition and concentration of contaminants in influent and effluent can provide insight into treatment processes influencing the removal and biotransformation of these compounds. This information is important when deciding on the regulation of these contaminants in effluent discharge. Chemical analysis of these compounds is also critical in developing relationships between contaminant exposure to impacts found in the Grand River. This data can aid in validating predictive models linking contaminants to specific biological endpoints.
Analysis of the structural determinants for voltage-dependent G protein modulation of synaptic Cav2 channels
(University of Waterloo, 2008-08-28T20:19:51Z) Huang, Xuan Jay
Specialized voltage-gated calcium channels in the Cav2 channel class (such as Cav2.2, N-type) mediate neurotransmitter release from presynaptic nerve terminals. Cav2.2 channels are exquisitely sensitive to inhibition by G protein-coupled receptors. The ubiquitous form of G protein modulation is a fast, membrane delimited, voltage-dependent form of regulation, which is relieved by strong depolarizations. LCav2, an invertebrate homolog from the pulmonate snail Lymnaea stagnalis, serves a similar function as a mediator of transmitter release in the nervous system. To examine the G protein modulation capacity in invertebrates, LCav2 was cloned to a bicistronic expression vector pIRES2-EGFP and expressed in HEK293T cells. Although LCav2 was almost indistinguishable from mammalian Cav2.2 in biophysical characteristics observed in vitro, snail LCav2 channel lacked the property of voltage dependent G protein modulation. The structural elements essential for the voltage sensitivity to G protein modulation were explored by swapping the N-terminus and I-II linker regions of rCav2.2 channels into LCav2 calcium channels. Functional comparisons were also made using both mammalian and invertebrate homologs of G protein beta subunits, Gβ1. Neither the N-terminus or I-II linker region of Cav2.2 alone, nor the invertebrate G protein beta subunit was sufficient for voltage-dependent G protein modulation. Further analyses using chimeric channels and G protein subunits will be required to find the minimal structural determinants for voltage-dependent G protein modulation.
Analytical Approach for Modeling of Multi Well CO2 Injection
(Elsevier, 2016-11-14) Robinson, Morgan; Leonenko, Yuri
Disposal of carbon dioxide (CO2) into underground geological formations is considered a viable strategy for the mitigation of global warming. It aims to reduce greenhouse gases emitted from point sources such as power plants. In order to select and evaluate a potential storage formation, many reservoir properties such as porosity, permeability, lateral and vertical extents, and a variety of residual fluid properties are considered. Injection design, which includes the placement of injectors and their flow rates, should be chosen to optimize injection capacity. One of the most important considerations to be addressed during design stage of sequestration is evaluation of pressure behaviour inside the reservoir during and after injection as the sequestered CO2 increases the pressure within the formation. In order to maintain the integrity of the reservoir the pressure needs to be maintained below the fracture pressure, typically at least 10% below. Thus evaluation of reservoir pressure is essential to ensure the reservoir remains under the maximum allowable pressure while sequestering the maximum amount of CO2 for long term storage in the reservoir. The optimal injection rates within a multi well injection site occur when the bottom-hole pressure at each injection site is at the maximum allowable pressure. In this study we present an analytical approach for modeling the pressure evolution during multi well CO2 injection into saline aquifers and using the model modify the injection rates to optimize injection capacity within a formation. We show that this optimization procedure significantly increases the capacity of formation as opposed to using the same injection rate at each wellbore.
Antimicrobial Peptide Daptomycin and its Inhibition by Pulmonary Surfactant: Biophysical Studies using Model Membrane Systems
(University of Waterloo, 2017-04-25) Lee, Brenda
Daptomycin is a lipopeptide antibiotic that is clinically used to treat severe infections caused by Gram-positive bacteria. It is highly potent against resistant strains of bacteria such as methicillin-resistant Staphylococcus aureus. However, in cases of community-acquired pneumonia (a leading cause of death worldwide), daptomycin is somehow inhibited by lung surfactant and therefore unable to exert its bactericidal activity against Streptococcus pneumoniae, the primary cause of this disease. This thesis presents the successful development of lipid model systems to mimic the lipid composition of S. pneumoniae bacterial membranes, human cell membranes, and both synthetic and natural lung surfactant. Experiments were performed that help to elucidate the basis for daptomycin’s inhibition by lung surfactant, culminating in a new, detailed model of daptomycin sequestration that summarizes the findings from these studies. Daptomycin is believed to be sequestered by lung surfactant and has been shown to insert into this surfactant. Fluorescence spectroscopy experiments were used to test the interaction of daptomycin with different lipid model membranes in the presence of calcium. The results provided strong evidence that daptomycin is sequestered by lung surfactant and that daptomycin has a similar affinity for both lung surfactant and bacterial membrane, suggesting these two entities play a competitive role in the binding of daptomycin. Increased emission spectra for daptomycin and bacterial membranes at higher concentrations of calcium suggest that calcium may remove an inhibited late step of daptomycin pore formation that has previously been shown. Using Langmuir-Blodgett monolayer techniques, studies were performed on how daptomycin affects monolayer properties. Compression isotherms provided data on monolayer compressibility, and it was found that daptomycin and calcium reduce the compressibility of lung surfactant monolayers, possibly improving its function. Constant-area insertion assays provided additional data that verified daptomycin’s avid binding to lung surfactant at low calcium concentrations. Scanning probe microscopy techniques were employed to obtain atomic force microscopy and Kelvin probe force microscopy images for monolayers in air. In the presence of daptomycin and calcium, the lung surfactant monolayers exhibited multilayer formation and increased electrical surface potential. Atomic force microscopy images taken of model lipid bilayers in liquid show multi-bilayer formation for the lung surfactant bilayers in the presence of daptomycin and calcium. This provides further evidence that daptomycin and calcium induce multilayer formation in lung surfactant. These findings allowed for the development of a novel model of daptomycin inhibition by lung surfactant. In the presence of physiological levels of calcium, daptomycin binds to lung surfactant and is sequestered. This binding causes a decrease in lung surfactant compressibility, allowing it to easily form multilayers that effectively reinforce the sequestration of daptomycin. The lipid models, methods, and experimental protocols developed in this thesis will help foster future studies in the field of membrane biophysics.

Browse

Browsing Biology by Title