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Tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are added to polyurethane foams in a variety of industrial and consumer products to prevent flame ignition. The gradual release of these flame retardants (FRs) from such products leads to contamination of various abiotic and biotic media, including wild birds. Recent studies demonstrated endocrine-disrupting effects of TCPP and TDCPP, including alteration of circulating thyroid hormone (TH) levels. The TH-pathway is essential for normal growth and development in birds. There are limited data on the toxicological effects of TCPP and TDCPP in avian species and, prior to this work, no study has examined their effects in avian embryos. This M.Sc. thesis investigates the developmental, molecular and biochemical effects of TCPP and TDCPP in chicken (Gallus gallus domesticus) embryos via egg injection studies. TCPP delayed pipping at doses ≥9.24 μg/g, both TCPP and TDCPP reduced embryo growth at the highest dose (51.6 μg TCPP/g and 45 μg TDCPP/g), and TDCPP decreased free plasma thyroxine and gallbladder size at 7.64 μg/g and 45 μg/g, respectively. Real-time reverse transcription polymerase chain reaction was used to measure changes in mRNA levels of hepatic genes that were responsive to these FRs in a previous in vitro study. TCPP dysregulated the expression of TH-responsive genes and xenobiotic metabolizing enzymes (cytochrome P450s; CYPs), whereas TDCPP only affected CYPs. Less than 1% of the administered TCPP or TDCPP was detected in egg contents following 19 days of incubation, indicating extensive metabolism of the parent compounds. DNA microarrays were used to perform a global transcriptional analysis on liver samples from embryos that exhibited adverse effects following TDCPP injection. 47 differentially expressed genes were identified at the 45 μg/g dose. Functional analysis revealed that immune function and lipid and steroid metabolism were major targets of TDCPP toxicity and indicated a state of cholestatic liver/biliary fibrosis. Since the TH-pathway is a key regulator of metabolic homeostasis, its disruption early in development is a potential cause of the observed adverse effects. This thesis demonstrates, for the first time, developmental and endocrine-disrupting effects of TCPP and TDCPP in an avian species and attempts to link phenotypic changes to molecular-level disruptions in hopes to improve the understanding of their modes of action.

Thyroid hormones (THs) are important hormones involved in developmental processes, including foetal brain maturation. THs are also involved in the maintenance of homeostasis. One in three people in Canada are considered to have some form of thyroid disorder. One reason for the high level of thyroid disorders may be the increasing amount of anthropogenic chemicals released into the environment that affect normal hormone action. Amphibian metamorphosis is completely dependent on TH and provides a model to study such chemicals. This thesis uses the Rana catesbeiana tadpole as a model to study potential TH disrupting chemicals by developing a novel screening assay called the cultured tail fin biopsy assay, or the “C-fin” assay. The C-fin assay uses tail biopsies from premetamorphic tadpoles, Taylor-Kollros stage VI-VIII. The biopsies are cultured in serum-free media along with the test chemical for 48 hours. QPCR is used to measure the mRNA steady-state levels of selected gene transcripts. Two TH-responsive gene transcripts were measured: the up-regulated gene transcript, thyroid hormone receptor β (TRβ) and the down-regulated gene transcript, Rana larval keratin type I (RLKI). Heat-shock protein 30 (HSP30) and catalase (CAT) were used as indicators of cellular stress. Another model system used in this thesis is rat pituitary cells, or GH3 cells. QPCR was used to measure the mRNA steady-state levels of three TH-responsive genes growth hormone (GH), deiodinase I (DIOI), and prolactin (PRL); heat-shock protein 70 (HSP70) was used as an indicator of cellular stress. Nanoparticles, used in various consumer products, were one class of chemicals examined. Using the C-fin assay, nanosilver and quantum dots (QDs) caused perturbations in TH-signalling and also showed signs of cellular stress. There was no overt toxicity observed as was determined by the normalizer, house-keeping gene transcript, ribosomal protein L8. The GH3 cells also detected TH disrupting effects by both nanosilver and QDs; however, nanosilver did not appear to cause cellular stress whereas QDs did. Nitrate and nitrite, major waterway contaminants, were also examined and there were no TH-perturbations observed using the C-fin assay. Finally, two antimicrobials used in many consumer products, triclocarban (TCC), triclosan (TCS) and its metabolite, methyl-TCS (mTCS) were examined using both the C-fin assay and GH3 cells. Both the C-fin assay and the GH3 cells determined mTCS to be more potent than TCS in disrupting TH action. TCC also caused perturbations in TH-signalling as well as causing a significant amount of cellular stress. Overall the C-fin assay and the GH3 cells proved to be excellent models in studying the potential disruptors of the TH axis. The C-fin assay and GH3 cells detected novel TH disruptors and gave further insight into already known disruptors of the TH axis.

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.

国立情報学研究所で電子化したコンテンツを使用している。

国立情報学研究所で電子化したコンテンツを使用している。

Messenger RNA (mRNA) posttranscriptional regulation has been implicated in the development and/or progression of several diseases including many types of cancer, rheumatoid arthritis, vascular disease, and Alzheimer's disease. Differential regulation of Androgen Receptor (AR) mRNA has been associated specifically with prostate cancer progression. In this thesis, molecular beacons were developed to allow for the detection of the expression and localization of AR mRNA in live prostate cancer cells. These beacons were then applied as a tool for studying how AR mRNA regulation is involved in prostate cancer growth and advancement. Two AR mRNA targeted beacons were designed and tested in solution and in live cells to determine their functionality. The beacon-based approach for AR mRNA detection was then optimized through the use of the two beacons in tandem and alteration of their backbone chemistry. A series of validation tests were performed on these beacons, including testing their abilities to: 1) produce a feasible localization pattern, 2) discriminate between AR positive (AR+) and AR negative (AR-) prostate cancer cell lines and 3) follow stimulus-induced changes in AR mRNA expression. Based on these results, a dual chimeric beacon approach was selected to determine the role of AR mRNA regulation in two systems that represent important stages in prostate cancer growth and progression: 1) hormone stimulation of androgen-dependent prostate cancer cells and 2) progression of androgen-dependent prostate cancer cells to the androgen-independent state. Our results suggest that changes in AR mRNA expression, organization, and localization may be indicative of molecular mechanisms involved in these critical transitions associated with prostate cancer progression. Taken together, this work provides a feasibility study for visualizing changes in AR mRNA state as a diagnostic measure for evaluating the aggressiveness of the disease and demonstrates the possible utility of therapeutically targeting AR mRNA regulation in order to prevent prostate cancer advancement.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of proteins intitially characterised in the late 1980’s by their presence in complexes that form on nascent RNA transcripts. This definition was purely operational, and was based on protein isolation techniques available at that time. Since then, the tendency to refer to and view the hnRNPs as a protein family has become increasingly prevalent, although there has been no systematic sequence- or structure-based study of their evolutionary history. While the hnRNPs share some structural characteristics (modular structure, presence of RNA-binding domains) and functional properties (binding to RNA, involvement in multiple steps of RNA processing), these criteria also apply to other types of RNA binding proteins (RBPs), such as the SR and ELAV families of proteins. Thus, we have adopted a more methodical and rigorous approach to the classification of hnRNPs and other RBPs, through the phylogenetic analysis of their sequences and domains. Besides establishing phylogenetic relationships and simplifying nomenclature, studying the evolutionary divergence of the hnRNPs is important for understanding their functional features. The hnRNP A/B subfamily is comprised of paralogues A1, A2/B1, A3 and A0, which exhibit a high level of similarity at both the sequence and structural level. While they are often treated as functional homologues, they are not functionally identical. Hence, we undertook a detailed comparison of their sequences, and found that the introduction of novel splicing signals or mutation of existing sequence elements has led to changes in alternative splicing patterns between the paralogues, which may affect the regulation of their expression and their RNA binding properties. In addition, we also investigated species-specific alternative splicing of the hnRNPs A/B, which has significant implications for the interpretation of current research, since different research groups tend to use different model organisms in their experiments. Hence, exploration of the sequence divergence of the hnRNPs A/B has provided some clues as to how their functional differences arose, and also highlighted the need to take species-specific splicing into consideration. Alternative splicing can create functional variation not only between paralogues, but also between splice variants. hnRNP A2/B1, which has a well-established role in mRNA trafficking in neuronal cells, has four spliceoforms. In order to study the contribution of each isoform to this process, we investigated isoform-specific variations in intracellular localisation, and expression in different developmental stages and species. We found that in rat, minor isoform A2b was the predominant isoform in the cytoplasm, and may be the key player in mRNA trafficking. These findings demonstrate the importance of considering individual isoforms (including those expressed in low abundance) when studying the function of alternatively spliced proteins, especially when the function is restricted to a particular subcellular compartment. In addition to its cytoplasmic role in mRNA trafficking, hnRNP A2/B1, and the other hnRNPs A/B, have multiple nuclear functions, including packaging of nascent transcripts, nuclear export of mRNA, regulation of alternative splicing and telomere maintenance. These processes take place in discrete regions within the nucleus, and thus we examined the subnuclear distribution of the hnRNPs A/B. We found that hnRNP A1 had a localisation pattern distinct from that of A2/B1 and A3, and that these patterns were spatially and temporally regulated. Hence, the evolutionary divergence of the hnRNPs A/B has affected the localisation, expression and splicing patterns of these proteins, which we have examined at multiple levels, including comparisons across all hnRNPs, within the hnRNP A/B paralogues, and between the hnRNP A2/B1 splice variants. As the hnRNPs A/B are involved in almost every step in RNA processing, this functional diversity has significant implications for transcriptomic complexity. Furthermore, our findings highlight the importance of taking species- and isoform-specific differences into account when investigating protein function. In conclusion, this study of the hnRNPs A/B provides a conceptual framework for exploring the relationships between sequence, structural and functional divergence, which may be applicable to protein families in general.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of proteins intitially characterised in the late 1980’s by their presence in complexes that form on nascent RNA transcripts. This definition was purely operational, and was based on protein isolation techniques available at that time. Since then, the tendency to refer to and view the hnRNPs as a protein family has become increasingly prevalent, although there has been no systematic sequence- or structure-based study of their evolutionary history. While the hnRNPs share some structural characteristics (modular structure, presence of RNA-binding domains) and functional properties (binding to RNA, involvement in multiple steps of RNA processing), these criteria also apply to other types of RNA binding proteins (RBPs), such as the SR and ELAV families of proteins. Thus, we have adopted a more methodical and rigorous approach to the classification of hnRNPs and other RBPs, through the phylogenetic analysis of their sequences and domains. Besides establishing phylogenetic relationships and simplifying nomenclature, studying the evolutionary divergence of the hnRNPs is important for understanding their functional features. The hnRNP A/B subfamily is comprised of paralogues A1, A2/B1, A3 and A0, which exhibit a high level of similarity at both the sequence and structural level. While they are often treated as functional homologues, they are not functionally identical. Hence, we undertook a detailed comparison of their sequences, and found that the introduction of novel splicing signals or mutation of existing sequence elements has led to changes in alternative splicing patterns between the paralogues, which may affect the regulation of their expression and their RNA binding properties. In addition, we also investigated species-specific alternative splicing of the hnRNPs A/B, which has significant implications for the interpretation of current research, since different research groups tend to use different model organisms in their experiments. Hence, exploration of the sequence divergence of the hnRNPs A/B has provided some clues as to how their functional differences arose, and also highlighted the need to take species-specific splicing into consideration. Alternative splicing can create functional variation not only between paralogues, but also between splice variants. hnRNP A2/B1, which has a well-established role in mRNA trafficking in neuronal cells, has four spliceoforms. In order to study the contribution of each isoform to this process, we investigated isoform-specific variations in intracellular localisation, and expression in different developmental stages and species. We found that in rat, minor isoform A2b was the predominant isoform in the cytoplasm, and may be the key player in mRNA trafficking. These findings demonstrate the importance of considering individual isoforms (including those expressed in low abundance) when studying the function of alternatively spliced proteins, especially when the function is restricted to a particular subcellular compartment. In addition to its cytoplasmic role in mRNA trafficking, hnRNP A2/B1, and the other hnRNPs A/B, have multiple nuclear functions, including packaging of nascent transcripts, nuclear export of mRNA, regulation of alternative splicing and telomere maintenance. These processes take place in discrete regions within the nucleus, and thus we examined the subnuclear distribution of the hnRNPs A/B. We found that hnRNP A1 had a localisation pattern distinct from that of A2/B1 and A3, and that these patterns were spatially and temporally regulated. Hence, the evolutionary divergence of the hnRNPs A/B has affected the localisation, expression and splicing patterns of these proteins, which we have examined at multiple levels, including comparisons across all hnRNPs, within the hnRNP A/B paralogues, and between the hnRNP A2/B1 splice variants. As the hnRNPs A/B are involved in almost every step in RNA processing, this functional diversity has significant implications for transcriptomic complexity. Furthermore, our findings highlight the importance of taking species- and isoform-specific differences into account when investigating protein function. In conclusion, this study of the hnRNPs A/B provides a conceptual framework for exploring the relationships between sequence, structural and functional divergence, which may be applicable to protein families in general.