MicroRNAs (miRNAs) are 20-22 nucleotide non-coding RNAs that can play important roles in developmental transitions by post-transcriptional regulation of mRNA translation and stability. We profiled miRNA expression in mouse thymocytes, mature T cells, and activated T cells, and identified distinctive patterns of miRNA expression during development, maturation, and activation of T cells. The miR-128 and miR-181 miRNA families are expressed at significantly higher levels in thymocytes. Examining the expression levels of these microRNAs in more detail, we observed that the expression pattern of these microRNA families distinguishes cells committed to lymphoid lineages from cells committed to myeloid lineages during normal mouse hematopoiesis. Extending this work to human malignancies, we determine that high miR-128 expression distinguishes lymphoid precursor derived malignancies from myeloid precursor derived malignancies. Little information is available regarding miRNA expression early after CD8 T cell activation. We demonstrate dynamic miRNA expression during early CD8 T cell activation, including the repression of miR-150, miR-181a, miR-26, miR-29 and miR-30, and the induction of miR-155, miR-31, miR-146, and the miR-17-92 cluster. We show that miR-31 is induced by calcium/Calcineurin signaling during acute CD8 T cell activation, and demonstrate elevated miR-31 expression in regulatory and memory T cell populations. We identify miR-31 targets in primary CD8 T cells and propose a model where miR-31 induction primes CD8 T cells for activation by promoting T cell survival, activation, and proliferation. Activation induced miRNA expression patterns are also found in some human malignancies. Chronic lymphocytic leukemia is typically thought to be a disease of resting lymphocytes. However, we demonstrate an activated B cell miRNA expression signature in CLL. Similarities in miRNA expression between activated B cells and CLL cells include high expression of miR-34a, miR-155, and miR-342-3p and low expression of miR-103, miR-181a and miR-181b. Additionally, we show that decreased levels of miR-29c and miR-223 in CLL are negative prognostic markers associated with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the expression level of individual miRNAs may predict clinical course in CLL.

Plant microRNAs have been shown to have important roles in regulating diverse processes ranging from reproductive development to stress response. In the first two chapters, I focus on miRNA diversity in Aquilegia studying both anciently evolved broadly conserved and rapidly evolving species specific miRNAs. In chapter one, I utilize Aquilegia's critical phylogenetic position between the well developed models Arabidopsis thaliana and Oryza sativa to study the evolution of ancient miRNAs across the angiosperms. In chapter two, I utilize smallRNA high-throughput sequencing to annotate Aquilegia specific miRNAs and, in the process, uncover the novel regulation of a floral homeotic gene by an Aquilegia-specific miRNA. In chapter three, I look at the tissue specific development of miRNA regulation in the bioenergetically relevant model organism Populus trichocarpa. High-throughput smallRNA sequencing from four diverse tissue sets including leaves, xylem, mechanically treated xylem, and pooled vegetative and reproductive tissues were analyzed, revealing a total of 155 previously unannotated miRNAs, most of which are P. trichocarpa specific. Expanding on my work with the petal identity pathway, I turned a broader analysis of Aquilegia petal spurs. Petal spurs are the distinguishing characteristic of Aquilegia and are argued to be a key innovation in the adaptive radiation of the genus. In the fourth chapter, I explore the cellular basis of extreme spur length diversity in the genus and find that a single parameter, cell shape, can explain this morphological range. Next, I seek to describe the cellular patterns that give rise to a spur primoridia from an initially flat laminar petal and find that spur initiation is characterized by concentrated, prolonged, and oriented cell divisions. Inspired by this quantitative analysis of growth, chapter five looks at the mechanisms of shape change in cucumber tendrils. I find that anisotropic contraction of a multi-layered gelatinous fiber ribbon explains coiling in cucumbers. Surprisingly, we discover that tendrils display twistless-overwinding when pulled and exhibit an unforeseen force-extension response as a result. These results provide the design basis for twistless springs with tunable mechanical responses and serve as a clear example of how the biological systems can inspire applied mechanical designs.

Li-Fraumeni Syndrome (LFS) is a rare cancer predisposition syndrome, typically involving germline mutations in the TP53 gene. Despite the high penetrance of TP53 mutations, LFS patients display striking phenotypic differences, suggesting the presence of secondary risk loci. To date, all genetic modifiers in LFS have been shown to map to either TP53 or its principal negative regulator, Mdm2. Given this strong association, we set out to interrogate the contribution of a recently-described miRNA regulator of the p53-MDM2 loop, called miR-605. We hypothesized that, if functional, the miR-605 gene and its variant (rs2043556) could impact cancer risk in TP53 mutation carriers. Consistent with this proposition, the variant allele of miR-605 was associated with a significant acceleration in tumor onset and caused a decrease in the processing efficiency of its host miRNA. We also demonstrate that miR-605 overexpression activates the MAPK pathway and leads to tumor suppression in TP53 mutant cell lines.

Li-Fraumeni Syndrome (LFS) is a rare cancer predisposition syndrome, typically involving germline mutations in the TP53 gene. Despite the high penetrance of TP53 mutations, LFS patients display striking phenotypic differences, suggesting the presence of secondary risk loci. To date, all genetic modifiers in LFS have been shown to map to either TP53 or its principal negative regulator, Mdm2. Given this strong association, we set out to interrogate the contribution of a recently-described miRNA regulator of the p53-MDM2 loop, called miR-605. We hypothesized that, if functional, the miR-605 gene and its variant (rs2043556) could impact cancer risk in TP53 mutation carriers. Consistent with this proposition, the variant allele of miR-605 was associated with a significant acceleration in tumor onset and caused a decrease in the processing efficiency of its host miRNA. We also demonstrate that miR-605 overexpression activates the MAPK pathway and leads to tumor suppression in TP53 mutant cell lines.

The Apelin-APJ system is transiently up-regulated in murine models of cardiac dysfunction. We have previously shown that Apelin-deficient mice subjected to aortic constriction suffer from severe fibrosis. In turn, we hypothesized that Apelin deficiency will also exaggerate the fibrosis phenotype post experimental myocardial infarction, associated with changes in fibroblast cell activity. Apelin-deficient and wildtype mice were randomly subjected to sham operation or left coronary artery ligation. Apelin deficiency worsened cardiac functionality, enhanced fibrosis-related gene expression and morphology, and enhanced vimentin intermediate filament expression, which may be involved in increasing fibroblast proliferation. MicroRNA target prediction softwares predict that apelin and vimentin 3 ’UTRs are potential targets of microRNA-378 regulation, and were confirmed with Luciferase reporter assays and western blot. Apelin up-regulation may be a useful strategy for attenuating unfavorable fibrosis through down-regulating vimentin-mediated adverse fibroblast activity. MicroRNA-378 regulation may be partly responsible for changes in apelin and vimentin expression.

No description available.

Blood vessels deliver nutrients and oxygen to tissues. Importantly, the functions and growth of blood vessels are commonly altered in disease. The inside of all blood vessels are lined with endothelium, a thin specialized layer of endothelial cells that separate the blood from other tissues. This thesis deals with the identification and functional characterization of proteins and microRNAs that have key roles as modulators of growth factor signaling and directed cell migration of endothelial cells and other vascular cells. A previously uncharacterized protein of the exocyst complex, Exocyst complex component 3-like 2 (ExoC3L2) was identified and shown to be highly expressed in endothelial cells of sprouting vessels. Suppression of ExoC3L2 resulted in reduced VEGF-A signaling together with reduced chemotaxis in response to VEGF-A gradients. VEGF-A-signaling via its receptor VEGFR-2 is thus modulated by the exocyst complex and ExoC3L2. Expression profiling of highly vascularized tissues were used to identify several microRNAs selectively expressed in blood vessels. miR-145, targeting the transcription factor Fli1, was shown to be expressed in pericytes and mural cells. Elevated levels of miR-145 reduced chemotaxis of both endothelial cells and fibroblasts in response to growth factor gradients. miR-145 depletion in fibroblasts was shown to inhibit chemotaxis in response to PDGF-BB. The guanine nucleotide exchange factor FGD5 was shown to be selectively expressed in endothelial cells and to regulate Cdc42 activity. FGD5 was shown to regulate the turnover of activated VEGF-receptors. Suppression of FGD5 impaired endothelial cell chemotaxis, suggesting that FGD5 is required for efficient and sustained VEGF-A signaling. Inactivation of RhoD, a regulator of endosomal trafficking, resulted in an increased pool of acetylated and stable microtubules. Knockdown of RhoD in human fibroblasts resulted in a loss of cell polarity. A link between PDGFR-β and RhoD was implicated by the finding that PDGF-BB was shown to trigger formation of GTP-bound RhoD. Chemotaxis towards PDGF-BB was severely inhibited in cells with reduced RhoD expression, suggesting a role for RhoD in chemotaxis via its regulation of microtubule dynamics.

The Apelin-APJ system is transiently up-regulated in murine models of cardiac dysfunction. We have previously shown that Apelin-deficient mice subjected to aortic constriction suffer from severe fibrosis. In turn, we hypothesized that Apelin deficiency will also exaggerate the fibrosis phenotype post experimental myocardial infarction, associated with changes in fibroblast cell activity. Apelin-deficient and wildtype mice were randomly subjected to sham operation or left coronary artery ligation. Apelin deficiency worsened cardiac functionality, enhanced fibrosis-related gene expression and morphology, and enhanced vimentin intermediate filament expression, which may be involved in increasing fibroblast proliferation. MicroRNA target prediction softwares predict that apelin and vimentin 3 ’UTRs are potential targets of microRNA-378 regulation, and were confirmed with Luciferase reporter assays and western blot. Apelin up-regulation may be a useful strategy for attenuating unfavorable fibrosis through down-regulating vimentin-mediated adverse fibroblast activity. MicroRNA-378 regulation may be partly responsible for changes in apelin and vimentin expression.

miRNAs are short, non-coding RNA molecules that regulate gene expression posttranscriptionally through inhibition of translation and/or mRNA degradation. Mammalian development is a complex series of developmental transitions, which relies on accurate spatial and temporal regulation of gene expression and we are interested in the role that miRNAs may play in these developmental transitions. An initial objective was to establish which, if any, miRNAs were dynamically regulated in a cell model of an early developmental transition, and to establish whether differential expression of any particular miRNA played a functional role in this developmental process. Having established a role for specific miRNAs, further objectives were to assess the reliability of current miRNA-mRNA target identification procedures and to assess the general role of miRNAs in cellular differentiation. In order to explore the roles of miRNAs during an early developmental transition, an embryonic stem (ES) cell model of trophectoderm differentiation was used. In this model system the expression of the key ES cell regulatory gene, Oct4, can be conditionally repressed, which induces the ES cells to differentiate down the trophectoderm lineage. The expression of microRNAs was profiled in this model system by cloning and sequencing of small RNAs. This approach identified miRNAs that were dynamically regulated during differentiation. The expression patterns of differentially regulated miRNAs were confirmed by miRNA northern analysis. The miRNA profiling data showed that mmu-miR-294 and mmu-mir-295 are expressed at similar levels in ES cells and differentiated cells, which disagrees with previous reports that these miRNAs are ES cell specific. Several of the miRNAs with higher expression levels in differentiated cells are encoded within a placental-enriched polycomb group gene, Sfmbt2, suggesting an important role for these miRNAs in extraembryonic development. One of the miRNAs that was expressed at higher levels in ES cells than in differentiated cells, mmu-miR-92a, was shown to play a role in regulation of cell proliferation. Three current methods of identifying miRNA targets were assessed. A sequencebased method using the web-based utility miRecords, which amalgamates results from numerous target prediction databases, was used to generate lists of potential targets of the Sfmbt2 miRNA cluster and of mmu-miR-92a. Amalgamating results from multiple target prediction programs may improve the likelihood that the predicted targets are real. Exemplifying this, the single mmu-miR-92a target that was predicted by six different target prediction programs had been previously experimentally verified. An experimental method of identifying direct miRNA targets, PAR-CLIP, was investigated but proved technically limiting for routine use in the laboratory. A proteome-based experimental method for identifying potential miRNA targets, called SILAC, was successfully used to identify proteins that were differentially expressed in the cell model of trophectoderm differentiation. Differential expression of two of these proteins, CTBP2 and CKB, was confirmed by western analysis. miRecords was then used to assess whether the differentially expressed proteins were likely to be targets of the differentially expressed miRNAs that had been identified in the miRNA profiling analysis. The general role of miRNAs in cell differentiation was investigated using a cell line that does not express miRNAs. This ES cell line is deficient for the miRNAprocessing enzyme DGCR8, which results in loss of expression of mature miRNAs in these cells. Compared to wild type ES cells, miRNA-deficient ES cells expressed normal levels of the ES cell marker genes Oct4 and Sox2 but elevated levels of Nanog. In contrast to wild type ES cells, miRNA-deficient ES cells did not upregulate the mesoderm marker gene Brachyury during embryoid body differentiation and showed reduced upregulation of the endoderm marker gene Gata6. These findings suggest that miRNAs are not required for maintenance of pluripotency, but are essential for proper ES cell differentiation. The results presented in this thesis show that miRNAs are dynamically expressed during a mammalian developmental transition and are involved in regulating early developmental processes. We believe that miRNAs act as an additional level of genetic regulation to ensure canalisation during embryonic development.

<p>The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro. </p><p>The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence. </p><p>Major results of the dissertation are as follows. Culture conditions of 100 &#956;g/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.</p> / Dissertation