Biology

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Open Access
Elucidating Cell Fate Regulation of the Bone Marrow Vascular Niche Through Development of Engineered Extracellular Matrices
(2023-03-28) Pagnani, Adriana; Sachlos, Eleftherios; Haas, Tara
Haematopoietic stem and progenitor cells (HSPCs) are crucial to the curative treatment of a variety of leukaemias and autoimmune diseases. However, their rapid differentiation and exhaustion within 72 hours ex vivo poses a barrier to efficient treatment. Here, we have developed a culture method that mimics that of the in vivo haematopoietic vascular niche in order to extend viability and maintain multipotency of HSPCs. The two-factor approach combined the paracrine support of endothelial cells as well as the structural and chemical support of an engineered two-dimensional scaffold comprised of several extracellular matrix (ECM) components. HSPCs that were co-cultured on ECM-mimicking scaffolds maintained higher viability in culture for up to seven days in comparison to solo-culture counterparts, and functional tests revealed an improvement in colony formation potential. Continued development of the culture system and understanding of the molecular pathways at play can lead to improved patient outcomes after transplantation.
Open Access
Characterization of RYamide neuropeptides and their Receptor in the Disease Vector Mosquito, Aedes aegypti
(2023-03-28) Luong, Thomas Duy Nguyen; Paluzzi, Jean-Paul
Insects utilize neuropeptides to fine-tune their physiological systems, including the tissues and organs involved in excretion, respiration, reproduction, and metabolism. The focus of this study is on the recently identified RYamide insect neuropeptides, named for the arginine-tyrosine amidated C-terminus. Here, the molecular basis will be investigated to propose physiological relevance in the disease vector, Aedes aegypti. Utilizing a cell-based functional assay, the endogenous RYamide receptor (RYa-R) was deorphanized and shown to have high specificity to the two mosquito RYamide peptides, with little to no activation by structurally-distinct neuropeptides. RYa-1 immunolocalization was found in the central nervous system, midgut, pyloric valve, ileum, rectal papillae, and surprisingly the seminal vesicles. RYamide and its receptor transcript analysis revealed potential regulatory effects of the digestive, reproductive, and excretory systems, along with a neuromodulatory role within the central nervous system. Further research is needed to confirm the physiological relevance of this novel neuropeptide family.
Open Access
Examination of Endoplasmic Reticulum Stress, the Unfolded Protein Response and Autophagy in Iron Overload-Induced Insulin Resistance
(2023-03-28) Nguyen, Khang Ngoc Yen; Sweeney, Gary
Identification of new mechanisms mediating insulin sensitivity is important to allow validation of corresponding therapeutic targets. In this study, I first used a cellular model of skeletal muscle cell iron overload and found endoplasmic reticulum (ER) stress and insulin resistance occurred after iron treatment. Insulin sensitivity was assessed using cells engineered to express an Akt biosensor, based on nuclear Foxo localization, as well as western blotting for insulin signaling proteins. Use of salubrinal to elevate eIF2a phosphorylation and promote the unfolded protein response (UPR) attenuated iron-induced insulin resistance. Salubrinal induced autophagy flux and its beneficial effects on insulin sensitivity were not observed in autophagy-deficient cells generated by overexpressing a dominant-negative Atg5 mutant or via knockout of ATG7. This indicated the beneficial effect of salubrinal-induced UPR activation was autophagy-dependent. I translated these observations to an animal model of systemic iron overload-induced skeletal muscle insulin resistance. Administration of salubrinal as pre-treatment enhanced eIF2a phosphorylation and induced autophagy in skeletal muscle. This attenuated insulin resistance upon systemic iron administration. Together, my results show that salubrinal elicited an eIF2a-autophagy axis leading to improved skeletal muscle insulin sensitivity both in vitro and in mice.
Open Access
Development of Exposure Biomarkers for the Honey Bee (Apis mellifera): Neonicotinoids Versus Traditional Pesticides
(2023-03-28) Jamieson, Aidan Robert; Zayed, Amro
Pollination is a vital ecosystem service crucial for reproduction of flowering plants, including agricultural crops. The western honey bee, Apis mellifera, is the most used managed pollinator worldwide. The use and overuse of agrochemicals is hypothesized to have played a role in increasing rates of colony mortality in Canada and globally. The identity of stressors affecting a colony is difficult to discern; information critical for diagnosing and managing honey bee colony health. Here, I explored the potential of using gene expression profiles as diagnostic biomarkers for exposure to various agrochemicals in honey bees. I found genes differentially expressed unique to each stressor, which could be putative biomarkers for specific agrochemical exposure. I found genes common between pesticides, which could be a putative general agrochemical stress signal. My research indicates that gene expression profiles can be an excellent tool for discovering stressor-specific biomarkers and diagnosing stressors found in honey bee colonies.
Open Access
Heterogeneity Exhibited in Cells that Escape from Drug-Induced Senescence
(2023-03-28) Miller, David Jacob; Benchimol, Samuel
Maintaining genomic integrity is key in the prevention of cancer. To limit the accumulation of mutations over time and stop damaged cells from continuing to proliferate and further gain subsequent mutations, intrinsic terminal cellular programs are activated. Senescence, an evolutionarily conserved stress state that traditionally has been viewed as an irreversible state of cell cycle arrest, acts as a tumour suppressive barrier. However, senescence acts as a double-edged sword, as senescent cells accumulate and contribute to age-associated diseases by altering tissue microenvironments and disrupting tissue homeostasis. Atrophied telomeres, mitotic errors, diminished DNA damage response and loss of nuclear envelope integrity all contribute to the increased rate of the formation of senescent cells during aging. In this study, I observed an age-associated accumulation of cytoplasmic DNA, in the form of micronuclei and chromatin bridges. I extended these finding to rare segmental accelerated aging diseases such as classical and non-classical Hutchinson-Gilford progeria syndrome, and Dyskeratosis Congentia. I further reveal that age-associated micronuclei and chromatin bridges undergo DNA damage and are recognized by cGAS that likely initiates an innate immune inflammatory response. My findings, along with recent reports showing chromosomal instability in senescent cells, led me to hypothesize that senescence may be reversible. To address this question, I utilized a drug-induced senescence model in BJ fibroblast and leukemia cells. Performing cloning experiments and monitoring single cells, I show that escape from a drug-induced senescent-like state occurs in a rare sub-population of cells for both transformed and normal cells. The escaped population displayed phenotypic heterogeneity, with respect to drug-response, gene expression, rate of proliferation and duration of arrest. RNA-sequencing highlighted the extensive transcriptional and metabolic reprogramming senescent cells undergo. Inhibition of lysosome function with FDA approved drug, chloroquine, specifically kills some senescent leukemia cells. By stratifying acute myeloid leukemia patients based on expression of senescent-associated gene signatures, I show that senescent gene signatures are associated with poorer overall survival. My findings provide evidence challenging the previous notion that senescence is a permanent state of cell cycle arrest and may be a mechanism which cancer cells exploit to survive therapy.
Open Access
Processing of Non-Coding RNA by Mlp1 in Tetrahymena thermophila
(2023-03-28) Kerkhofs, Kyra; Bayfield, Mark
RNA species are commonly transcribed as precursors and require post-transcriptional processing to become functional mature RNA transcripts. This includes the abundant cytoplasmic transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) that function in messenger RNA (mRNA) translation. Other non-coding RNAs also require extensive processing and assembly into ribonucleoproteins (RNPs), including small nuclear RNAs (snRNAs) that function in pre-mRNA splicing and small nucleolar RNAs (snoRNAs) involved in the processing and post-transcriptional modification of non-coding RNAs. The ciliate Tetrahymena thermophila is a highly studied eukaryotic model organism, however, many of its RNA processing pathways remain unexplored. In this work, we use molecular biology, biochemistry, cell biology and bioinformatic techniques to investigate the role of a novel La protein, Mlp1, in pre-tRNA, snRNP and snoRNP biogenesis. Unlike previously studied genuine La proteins, Mlp1 lacks an RNA-binding domain typically required for high-affinity binding of uridylate-tailed La target RNAs. We confirm that Mlp1 performs typical La protein functions, including uridylate-dependent preferential binding of pre-tRNAs and RNA chaperone activity to promote processing and maturation of misfolded nascent pre-tRNAs. However, in contrast to pre-tRNA processing in other eukaryotes, depletion of Mlp1 results in 3′-trailer stabilization instead of rapid trimming by 3′-exonucleases, indicating that Mlp1 is linked to a fundamentally different mechanism of tRNA processing in Tetrahymena thermophila. We also find that Mlp1 associates with mature snRNAs lacking the typical high affinity uridylate binding site and a core protein component of the snRNP. More specifically, Mlp1 interacts with the U4/U6 di-snRNP complex, which is formed during splicing, and Mlp1 depletion affects assembly of this complex. Mlp1 depletion also results in diminished splicing efficiency of pre-mRNAs, further supporting a functional role for Mlp1 in splicing in this system. Lastly, we show that Mlp1 associates with all previously annotated snoRNAs which function as guide RNAs in post-transcriptional modification of non-coding RNAs. We use this as a metric to predict novel snoRNAs in Tetrahymena thermophila and validate expression. Our work demonstrates new roles for the variant genuine La protein Mlp1 in the biogenesis of non-coding RNAs critical for the translation of proteins.
Open Access
Mesostoma ehrenbergii spermatocytes provide an unusual and exciting model to investigate chromosome movement, cleavage furrows and tethers during cell division
(2023-03-28) Arch, Eleni; Forer, Arthur H.
For my PhD thesis I studied and tracked chromosome movement in primary spermatocytes from a species of aquatic flatworm, the Mesostoma ehrenbergii, with the hope to better understand what determines the force behind these intricate and highly co-ordinated movements during meiosis. Canonical theories of chromosome movement state that microtubules are the main producers of force for chromosome movement in the meiotic spindle, however my research has shown chromosomes can not only move but can move even faster in the absence of microtubules. I also tracked the cleavage furrow and showed it was able to simultaneously move and ingress in the absence of microtubules. I worked on determining how the force is produced by using a laser microbeam to sever various components in the cell and by adding various myosin and actin inhibiting and enhancing drugs to see how chromosome movement may be altered. In addition, I discovered elastic tethers are present in Mesostoma during metaphase and anaphase and my research on functionally disabling tethers suggests they play a role in coordinating or maintaining non-random chromosome movement in these cells. In sum, my PhD research has added evidence to the non-microtubule model of chromosome movement by showing actin and myosin is involved in movement and that tethers may play a role in coordinating or maintaining non-random chromosome movement in these cells.
Open Access
Understanding the evolutionary origin and ancestral composition of honey bee (Apis mellifera) populations.
(2023-03-28) Dogantzis, Kathleen Andrea; Zayed, Amro
The honey bee, Apis mellifera, is arguably the most important managed pollinator globally. Yet despite its economic and ecological importance, there are still several unknowns regarding the species ancestral origin and ancestral complexity. Understanding the genetic composition of native and managed honey bee colonies is imperative for resolving the species life history and elucidating how ancestry may inform management strategies. In this dissertation, I take a deep dive into the evolutionary origins of Apis mellifera and learn how ancestral complexity has shaped the composition of contemporary populations. In Chapter two, I settle a long-standing debate about the ancestral origins of the species. I find that Apis mellifea diverged out of Western Asia via at least three colonization routes, which resulted in the evolution of at least seven genetically distinct lineages. Interesting, I find that these lineages were able to adapt to their current distribution by repeated selection among a core set of genes. In Chapter three, I take a closer look at the genetic complexity of managed Canadian honey bees by estimating the ancestral composition of colonies using the genomic dataset from Chapter two. I find that patterns of ancestry differ between Canadian provinces, and that admixture correlates strongly with levels of genetic diversity. Interestingly, I find that genomic intervals with elevated levels of admixture segregate non-randomly in the genome and are associated with genes related to parasite and xenobiotic tolerance. Though admixture may bear advantages for managed colonies, admixture among honey bee is not always valued. In Chapter four and five I make use of the ancestral composition of invasive Africanized honey bees to develop assays to identify and track populations. This was achieved using machine learning models to choose the most informative single nucleotide polymorphisms (Chapter 4) and insertion-deletion (Chapter 5) markers that best delineate Africanized genetics from managed European colonies. My research addresses many gaps in our understanding of honey bee origins and ancestral complexity.
Open Access
The TAZ Protein Interactome in Striated Muscle
(2023-03-28) Kelebeev, Jonathan; McDermott, John Charles
Hippo signalling is a prominent regulator of cell proliferation, differentiation, and death. Previously, we characterized the Hippo transcriptional effector TAZ as a repressor of the myogenic differentiation program. Without DNA-binding ability, TAZ function exclusively depends on its protein:protein interaction network. This prompted us to undertake a proteomic-based study aimed at identifying the TAZ interactome in striated muscle cells. Using a novel GFP-Nanotrap based affinity purification approach coupled with LC-MS/MS protein identification, we document a comprehensive list of known and novel TAZ interactome components in myogenic cells. TAZ interacting proteins include components of Hippo signalling: TEAD1-4, LATS1, 14-3-3 proteins. Epigenetic regulators were also represented: NuRD complex, FACT complex, and SWI/SNF complex. We focused on characterizing the TAZ interaction with the Wnt co-repressor TLE3 in myogenic cells. In myogenic cells, TAZ and TLE3 interact and co-localize within the nucleus. Functionally, TAZ and TLE3 repress β-catenin activation, as indicated by the Wnt-responsive TOP FLASH reporter gene system. Depletion of TAZ reduced the degree of TLE3-mediated repression of β-catenin activation. TAZ and TLE3 repressed MyoD-driven activation of the myogenin promoter. Overall, these data demonstrate a role for a TAZ/TLE3 complex in repressing the myogenic differentiation machinery having implications for muscle development and regeneration.
Open Access
Characterization of the Zebrafish Panx1a Phosphorylation Profile and Interactome in the Regulation of the Channel Function and Trafficking
(2023-03-28) Timonina, Ksenia; Zoidl, Georg R.
Pannexin-1 (Panx1) is an integral membrane protein that forms heptameric channels, involved in the communication between the intracellular and the extracellular environment. Its primary function is to passage small molecules, such as ATP, in and out of the cell, which can trigger vital cellular responses like cell death. Panx1s have been implicated in various diseases, including epilepsy, ischemia, and inflammation. They also play roles in the sensory processing systems, such as vision, hearing, olfaction, and taste. Although it is clear that Panx1s are vital to the function of biological processes, the exact mechanism of how these channels operate and what activates/inactivates them is still under investigation. Using the zebrafish orthologues of Panx1, we explore the structural properties responsible for the trafficking of the channel while maintaining a focus on the phospho-proteomics and the kinase-dependent signaling involving Panx1. We identified a critical residue highly conserved in the third transmembrane domain, Y205 of the zebrafish Panx1a protein, which plays a role in the stabilization of the protein, likely through aromatic-aromatic interactions. Our results show that this residue is not subject to phosphorylation but is vital for the localization of the channel at the cell surface. Using protein purification techniques combined with mass spectrometry analysis, we identified several phosphorylated residues in the cytoplasmic loop of Panx1a and explored the potential interacting kinases of the protein. We studied the interactions between Panx1a with two kinases: ERK2 and CaMKIIa. Our results indicated that a strong interaction exists with CaMKIIa, which plays a role in the trafficking dynamics of Panx1a to the cell membrane. The overexpression of CaMKIIa led to an accumulation of Panx1a in the intracellular compartments. We investigated the effects of various CaMKIIa functional and structural mutants on expression and interaction with Panx1a. We found that the constitutively active T286D CaMKIIa mutant leads to a partial rescue in Panx1a expression, suggesting that Panx1a requires phosphorylation by CaMKIIa for proper cell membrane localization. Together, the research in this thesis provides novel findings that contribute to the phospho-proteomic exploration of Panx1 channel activity and regulation.
Open Access
Characterization of the Functional Roles of the E3 Ligase HUWE1 in the Regulation of Poly [ADP-ribose] Polymerase 1 (PARP-1) under DNA Damage
(2023-03-28) Huang, Song; Sheng, Yi
The Poly (ADP-ribose) polymerase 1 (PARP-1) is responsible for synthesis of polymeric ADP-ribose chain on substrate proteins associated with DNA damage repair. PARP-1 overexpression has been associated with rapid proliferation and metastasis in cancers. Ubiquitylation is a protein post-translational modification and the key bioprocess for the regulation of PARP-1 functionally. Recently, ubiquitin E3 ligase HUWE1 (HECT, UBA and WWE domain containing 1) was reported to be involved in DNA damage response. HUWE1 also possesses a functional WWE domain, which is a structural motif to recognize the ADP-ribose unit of PAR polymers. In this study, the functional roles of HUWE1 in the regulation of PARP-1 were characterized. This study reveals (1) HUWE1 could bind to cellular PAR polymers through its WWE domain. (2) As PARP-1 is modified through auto-PARylation in the cell, HUWE1 could interact with the PARylated PARP-1 via WWE domain. (3) HUWE1 could regulate PARP1 stability through ubiquitination. This study provides mechanistic insight on the regulation and the function of these two proteins in both biological and pathological conditions.
Open Access
Characterization of Microrna Expression Profiles and Role of Nodal-Related Genes in Zebrafish Ovarian Follicles
(2023-03-28) Zayed, Yara Amer; Peng, Chun
Zebrafish is a valuable model to study the biology of reproduction as the processes that regulate follicle development and oocyte maturation are conserved among vertebrates. In zebrafish, early vitellogenic (stage IIIa) ovarian follicles are maturationally incompetent while mid-late vitellogenic (stage IIIb) follicles are able to undergo oocyte maturation in response to maturation-inducing hormone signals. Signaling molecules derived from the ovary, such as microRNAs (miRNAs) and growth factors, are important in controlling ovarian function. To determine whether miRNAs may play a role in maturation competency acquisition, we characterized miRNA expression profiles in follicular cells isolated from stage IIIa and IIIb follicles. Bioinformatics analysis uncovered 214 known, 31 conserved novel and 44 novel miRNAs, of which 24 miRNAs were significantly regulated between stage IIIa and IIIb follicular cells. In addition, gene enrichment and pathway analyses of the predicted targets of the significantly regulated miRNAs supported the involvement of several key signaling pathways in regulating ovarian function. We then investigated the role of Nodal, a member of the transforming growth factor-β family, in regulating zebrafish ovarian function. We used real-time PCR to detect the zebrafish Nodal orthologs, nodal-related (ndr1) and ndr2 and found that they were expressed in ovarian follicles at all stages of development. We also detected the mRNAs for Nodal signaling components in follicular cells of vitellogenic follicles. Recombinant human Nodal activated Smad3, CREB, and ERK, and inhibited cell proliferation in ovarian follicular primary cell cultures. The mRNA levels of cyp17a1, hsd3b2 and paqr8 were increased in response to Nodal treatment. Subsequently, we used CRISPR/Cas9 technology to generate ndr1 and ndr2 null mutants, which caused severe defects in early development. To overcome this lethality in vivo, we developed a fluorescently-labeled, Doxycycline-inducible CRISPR-ON system that expresses single or multiplexed sgRNAs to knockout ndr1, ndr2, and ndr3. Activation of the system induced gene editing in the designated genomic loci. Our findings suggest that miRNAs and Nodal play a role in zebrafish follicles. The CRISPR-ON system will facilitate further investigating the roles of miRNAs and Nodal in adult zebrafish in vivo.
Open Access
Characterization and functional deorphanization of CCHamides and their receptors in the yellow fever mosquito, Aedes aegypti
(2022-12-14) Tan, Jinghan; Paluzzi, Jean-Paul
Studying the neuroendocrine system of mosquitoes, which transmit various pathogens leading to human diseases, allows us to better understand their physiology. Novel CCHamide neuropeptides, CCHa1 and CCHa2, and their associated receptors, CCHa1R and CCHa2R, were recently identified across insects, but they have not been studied in mosquitoes. This study aims to quantify and localize expression of CCHamides and CCHa2R to determine their physiological role in the mosquito, Aedes aegypti. Results revealed adults express the most CCHamide transcripts while CCHa2R transcript was enriched in pupa. The midgut was found to be the primary source for both CCHamide transcripts while higher CCHa2R transcript abundance was detected in the hindgut and the reproductive system. Both CCHamide receptors, the CCHa1R and CCHa2R, were deorphanized, revealing CCHamides exhibited the strongest ligand activity. CCHa2 and CCHa2R transcripts were unresponsive to either sucrose ingestion or starvation stress while CCHa1 and CCHa2 transcript levels responded to a blood-meal.
Open Access
Localization and Interaction of Circadian Clock and TOR Pathway Components in Neurospora Crassa
(2022-12-14) Eskandari, Rosa; Lakin-Thomas, Patricia
Circadian (daily) rhythmicity is an attribute of almost all eukaryotic cells and some prokaryotes. Our lab utilizes the filamentous fungus Neurospora crassa as a model organism to study the molecular basis of rhythmicity. Models for the endogenous oscillators that regulate circadian rhythms in eukaryotes are primarily based on a small number of ‘‘clock genes’’ operating as transcription/translation feedback loops (TTFL). Still, rhythms can be observed, when TTFLs are nonoperating. Understanding the mechanism of rhythmicity operating outside of TTFLs is the key unresolved problem in circadian biology. Our lab has identified two genes in Neurospora crassa required for this TTFL-less rhythmicity, vta, and gtr2. Both are components of the TOR (Target of Rapamycin) nutrient-sensing pathway preserved in eukaryotes. Coimmunoprecipitation and mass spectrometry found TOR pathway components, including GTR2 (homologous to yeast Gtr2 and RAG C/D in mammals) as binding partners of VTA (homologous to yeast EGO1 and mammalian LAMTOR 1). In this thesis I report reciprocal co-IP with GTR2 confirming VTA as a binding partner. I also report that the expression of GTR2 protein is rhythmic, and VTA is required for GTR2 rhythmicity. FRQ protein, central to the clock TTFL, is rhythmic in the presence of GTR2 but dampened in the absence of GTR2. I also report my research regarding the subcellular localization of VTA and GTR2 in different nutritional states and the presence and absence of TOR pathway inhibitors. Kog1/Raptor is a regulator for TOR1 activity in yeast and mammals, and I report the localization of KOG1 in Neurospora crassa. My data indicate that GTR2-RFP localizes in filamentous structures identified as vacuolar membranes but is cytoplasmic in the absence of VTA. KOG1-GFP looks similar to GTR2-RFP. In the presence of arginine, KOG-1 is localized around the vacuole. Starvation using no glucose media looks similar to the vta ko condition in the GTR2 strain. In KOG-1GFP, there is the presence of KOG1 P bodies around the edges of the vacuole. The latter effect is similar to the yeast homolog kog-1 protein behavior in the absence of glucose. In the presence of TOR pathway inhibitors, Torin I and Torin II, there is an abnormal structural morphology in both cases. It is indicated that there is a destruction of the vacuolar structure and internal hyphal network in inhibitors that moves the fluorescence signal to the outer plasma membrane. These results support the connection between the mutual components of the clock and TOR pathway and the essential interaction to maintain the proper function of the signaling pathway and the circadian clock. As we have previously reported, the TTFL cannot explain the whole circadian clock mechanism. An independent oscillator may be linked to the TTFL to help maintain its function. This link is identified as the TOR pathway, a significant and nutrient-sensitive pathway conserved in eukaryotes. The circadian clock is also conserved in almost all organisms. Our results established a network between the control of metabolism through the TOR pathway and the circadian clock.
Open Access
Urbanization Effects on Wild Bee Communities and Their Plant Interactions
(2022-12-14) Ayers, Anthony Caleb; Rehan, Sandra
Urbanization has contributed greatly to the diminishment of habitat that supports diverse bee communities. Despite generally having negative effects on bees broadly, bees exhibit differential responses to local and landscape features associated with urbanization. As a result, cities possess the potential to harbor unique bee communities; however, determining which bees are vulnerable in such landscapes is essential to developing appropriate conservation initiatives that promote biodiversity in urban spaces. This study reviews the current known effects of urbanization on bees while recognizing knowledge gaps that require further research attention. Additionally, this study samples twenty-nine sites along an urban gradient to investigate the effects of urbanization on bee community structure and plant interactions. Bee communities were dominated by key functional traits and were found to be influenced by percent impervious surface and tree cover. Bee-plant interactions revealed the genus Solidago and other plant genera to be particularly attractive to various bee species.
Open Access
Molecular Mechanisms Underlying Regulation and Function of Neuronal Gap Junction Proteins: Connexin 36 and Connexin 27.5.
(2022-12-14) Kotova, Anna Yuriyivna; Zoidl, Georg R.
Electrical synapses, known as gap junctions, are critical to neuronal synchronization and signal transmission. Gap junctions are composed of two docked hemichannels, each consisting of single protein subunits called connexins. Regulation and function of connexins are vital for the plasticity of the electrical synapses. Connexin regulation is dependent on their interacting partners as they affect turnover and channel properties. Here we explored the functional relevance of the interaction between mouse Connexin 36 (Cx36), major neuronal connexin, and mouse Caveolin-1 (Cav-1) in the Neuro 2a cell line. Cav-1 is known to mediate the endocytosis of membrane proteins, therefore its role in trafficking of Cx36 was explored. Together results showed that Cx36/Cav-1 interaction selects for the rapid calcium and caveolin dependent endocytosis and is critical for the internalization of Cx36. Another means for regulating connexin function is through their ability to oligomerize with different connexin isoforms to form heteromeric and heterotypic channels. The oligomerization capabilities between zebrafish Cx35b, an orthologue of mouse Cx36, and a novel zebrafish connexin, Cx27.5, were explored in Neuro 2a cells. The co-localization of Cx36/35b and Cx27.5 was also investigated in the zebrafish retina, as its high organization allows to study the gap junction connections between different cell types. Data showed that oligomerization of Cx35b and Cx27.5 led to a formation of distinct channels which potentially allow for specialized intercellular connection between different cell types. Cx27.5 is a novel connexin, and its functional relevance remains unknown. Previously reported expression in the zebrafish retina and co-localization with Cx36/35b further reinforced the hypothesis that the Cx27.5 function could be critical for visual processing. The protein and mRNA expression profile of Cx27.5 showed high expression in the brain and retina, specifically, the inner plexiform layer. By employing a Cx27.5 knock-out zebrafish line, the role of Cx27.5 in visual processing was investigated. The results proved Cx27.5 to be an essential player in the signal transmission and network connectivity of the zebrafish retina, as well as the perception of directional motion. In summary, the findings in this thesis describe the multifaced molecular mechanisms that underlie the function and regulation of neuronal connexins.
Open Access
INDIRECT EFFECTS OF LOW AMPLITUDE ARVICOLINE CYCLES ON DAILY NEST SURVIVAL OF DUNLIN (CALIDRIS ALPINA HUDSONIA) IN A RAPIDLY CHANGING CLIMATE
(2022-12-14) Hong, Milly; McKinnon, Laura
The Alternative Prey Hypothesis (APH) states that predators switch to relatively more abundant prey when their main prey is scarce. Arctic lemming population cycles may indirectly affect predation risk on alternative prey such as shorebird nests as they share predators. I examined the indirect effects of arvicoline rodent cycles on Dunlin (Calidris alpina hudsonia) reproduction in Churchill, Manitoba. Using 10 years of field data, the study suggests collared lemming (Dicrostonyx richardsoni) cycles did not influence Dunlin nest success. Meadow vole (Microtus pennsylvanicus) cycles had an interactive effect with arctic fox (Vulpes lagopus) and red fox (Vulpes vulpes) abundance, indirectly affecting Dunlin nest success. North Atlantic Oscillations had a positive effect on Dunlin nest success. The results suggest that subarctic ecosystems are more complex than the High Arctic with multispecies trophic dynamics that can be used to predict the changing landscapes of the Arctic as the boreal forest expands northwards.
Open Access
Characterization of KPAP, a Novel Pokeweed Antiviral Protein Isoform
(2022-12-14) De La Selle, Fernand Jean; Hudak, Katalin A.
Phytolacca americana is a perennial plant that harbors the pokeweed antiviral protein (PAP) genes which belong to the N-glycosidase family. These proteins are called ribosome inactivating proteins (RIPs) which remove an adenine base from the sarcin/ricin loop (SRL) of the 28S rRNA in eukaryotes. This depurination results in protein translation inhibition leading to a concentration-dependent cytotoxic consequence. Many RIPs in the plant kingdom are being discovered with new genomic sequencing technologies but, many of them remain uncharacterized. Recently, our lab discovered a new RIP called Novel PAP/KPAP in the sequenced pokeweed genome. KPAP transcript levels did not behave the same way as other isoforms under phytohormone stimulated biotic stress, however, they showed a slight decrease in drought. In this study, I provide preliminary groundwork on the KPAP gene model, expression in abiotic stresses, protein structure, depurination activity and potential binding arrangement on the SRL. My results show the presence of predicted upstream open reading frames (uORFs) in the long leader intron, perhaps belonging to retrotransposons. The enzymatic activity recorded from KPAP in an in vitro translation assay resembles the activity of PAP-I. Moreover, the exclusive expression of KPAP in leaves compared to other isoforms and its downregulation in drought may indicate a function related to photosynthesis, carbon metabolism, plant growth or leaf expansion. Structurally the predicted protein was docked in silico to an adenine base and showed a similar disposition of the active site compared to PAP-I. Lastly, KPAP was docked in silico to the E.coli SRL and residue interactions are discussed in the context of other RIPs. This early research shows KPAP’s similarities and differences compared to the other isoforms, however, much remains to be learned from this isoform and RIPs in general.
Open Access
Identification of the Pokeweed Antiviral Protein Interactome by Co-Immunoprecipitation-Mass Spectrometry
(2022-12-14) Chivers, Jennifer Anne; Hudak, Katalin A.
Ribosome-inactivating proteins (RIPs) are produced primarily by plants and are named for their enzymatic ability to depurinate ribosomal RNA. RIPs have been shown to have antiviral, antifungal, and antibacterial activity in vitro and when expressed transgenically. They are therefore of interest for their potential in human health, as both pathogenic agents and therapeutics, as well as in agriculture, to confer disease resistance in transgenic crops. However, little is known about the biological function of RIPs in their native context. Phytolacca americana, the American pokeweed, produces a RIP called pokeweed antiviral protein (PAP). The objective of this work is to investigate the role of PAP by mapping out the PAP-protein interactome; this will elucidate PAP’s function by implicating the processes in which it is involved. Co-immunoprecipitation coupled with mass spectrometry was used to identify PAP protein interactors in pokeweed. Results identified protein interactions with diverse cellular functions in both the extracellular matrix, where PAP is primarily localized, and the cytoplasm, where the ribosomal target resides. One interactor was identified as a probable extracellular cysteine protease (paCP1); since this protein class has known roles in plant defense, paCP1 was chosen for further validation of its interaction with PAP using reverse co-IP. Differential expression and in silico promoter analysis demonstrated PAP and paCP1 co-expression in response to jasmonic acid, supporting the role of this interaction in defense. This work represents the first protein interactome mapping for a RIP; identification of PAP interactors in plant cells contributes to understanding PAP function and will aid in characterizing the biological role of RIPs in general.
Open Access
Oxalate Handling and Transporter Expression in Poecilia Latipinna in Response to Environmental Salinity and Antibiotic Exposure
(2022-12-14) Rajan, Felicia Vimala; Bucking, Carol
Little is known about oxalate transport in fish, however mammalian models suggest the involvement of solute carrier family transporters (SLC), specifically SLC26A3 and SLC26A6. To study the effects of salinity and bacteria on intestinal oxalate excretion, sailfin mollies (Poecilia latipinna) were acclimated to freshwater or seawater (for either 14 or 28 days) and seawater for 14 days with broad-range antibiotic treatment. Initial acclimation to seawater increased the plasma oxalate concentration but homeostasis was recovered by 28 days. Kidney oxalate concentrations were unaffected by salinity, however the concentration in the anterior intestine increased in seawater while it decreased in the posterior intestine. SLC26A3 expression in both the anterior and posterior intestine was downregulated in response to seawater acclimation, while SLC26A6 expression was upregulated after seawater acclimation for 14 days. Antibiotics reduced the expression of SLC26A6 and SLC26A3 in both intestinal sections and altered the concentrations of oxalate along the intestine.