<p>Down syndrome (DS) is a genetic condition caused by the triplication of human chromosome  21 and presents with many phenotypes including decreased brain size, hypocellularity in the brain,  and assorted ocular phenotypes. Some of the ocular phenotypes seen are increased risk of cataracts,  accommodation difficulties, increased risk of refractive errors, and increased retinal thickness. The  Ts65Dn mouse model of DS is a classically used mouse model as it presents a number of  phenotypes also seen in those with DS. Some of these phenotypes include decreased brain volume,  abnormal synaptic plasticity, and ocular phenotypes. These ocular phenotypes include decreased  visual acuity, cataracts, and increased retinal thickness. The Ts65Dn mouse model is trisomic for <em>Dyrk1a</em>, a gene of interest in DS research. We hypothesize that there will be a genotypic and sex effect of retinal thickness and retinal ganglion cell (RGC) count at postnatal day 15 in the Ts65Dn  mouse model of DS. Retinal slices were taken from male and female trisomic and euploid Ts65Dn  mice at P15 and fluorescently labeled for RGCs and bipolar cells via immunohistochemistry. The  retinas were measured for total retinal thickness and RNA-binding protein (RBPMS) positive cells in the RGC layer were counted. There was no genotypic or sex effect when comparing retinal  thickness in trisomic mice as compared to euploid mice. There was a genotypic effect of RBPMS  positive cell count in which the trisomic mice had a higher number of RBPMS positive cells than  euploid mice. Increased retinal thickness along with increased RGC number have both been  implicated with decreased apoptosis in the retina. In the Ts65Dn mouse model along with in  individuals with DS, this could be due to an increase in DYRK1A protein levels reducing apoptosis.  In future studies, determining DYRK1A’s influence in retinal thickness and RGC number could  result in a treatment for overactive <em>DYRK1A</em> that could normalize retinal thickness and RGC  number in those with DS.</p>

No description available.

A key question in developmental biology is how form is generated. The model species Arabidopsis thaliana produces simple leaves with marginal outgrowths termed serrations. Serration development in A. thaliana requires both the transcription factor CUP-SHAPED COTYLEDON2 (CUC2) and the auxin efflux facilitator PIN-FORMED1 (PIN1), which regulates polar auxin transport by forming convergence points (Hay et al., 2006; Nikovics et al., 2006; Scarpella et al., 2006). In Chapter 3, I investigate how CUC2, PIN1 and auxin interact to control serration development. I demonstrate that CUC2 promotes PIN1 convergence point and auxin activity foci formation along the margin of the leaf, whilst high auxin activity represses CUC2 expression. Furthermore, interspersed peaks of CUC2 and auxin activity pattern serration development along the proximo-distal axis of the leaf. Thus, auxin, PIN1 and CUC2 form a negative feedback loop that patterns serration development. CUC genes and PIN1 are required for leaflet development in Cardamine hirsuta (Barkoulas et al., 2008; Blein et al., 2008), a close relative of A. thaliana that produces compound leaves subdivided into units termed leaflets. However, it is unclear how CUC and PIN1 interact to control leaflet development. In Chapter 4, I demonstrate that similar to A. thaliana, CUC genes promote PIN1 convergence point and auxin activity foci formation at the C. hirsuta leaf margin, whilst high auxin activity represses CUC2 expression. These genetic interactions likely create interspersed peaks of CUC2 and auxin activity that pattern leaflet development. Thus, the same negative feedback loop between CUC, PIN1 and auxin patterns both leaflet development in C. hirsuta and serration development in A. thaliana. KNOTTED1-LIKE HOMEOBOX (KNOX) genes are expressed in C. hirsuta leaves, and interact with ChCUC and PIN1 in positive and negative feedback loops, respectively, to control leaflet development (Barkoulas et al., 2008; Blein et al., 2008). KNOX genes are not expressed in A. thaliana leaves, but deeply lobed margins reminiscent of leaflets develop in association with ectopic KNOX expression in leaves (Chuck et al., 1996; Hay et al., 2006). However, it is unclear whether regulatory interactions of PIN1, CUC and KNOX which occur in C. hirsuta leaflets are employed during KNOX-induced lobe development in A. thaliana. In Chapter 5, I demonstrate that CUC2 and polar auxin transport are required for ectopic KNOX expression. Conversely, I show that KNOX misexpression up-regulates CUC2 expression in A. thaliana leaves. Thus, interactions between KNOX, CUC and PIN1 that occur in leaflet development in C. hirsuta also occur in association with KNOX-induced lobe development in A. thaliana. In addition to investigating the regulatory interactions between known components of leaf development pathways, I sought to identify novel genes that mediate CUC2-dependent serration development in A. thaliana. In Chapter 6, I identify a suppressor of the smooth margin phenotype of cuc2 leaves that partially restores PIN1 localisation in the absence of functional CUC2. Finally, in the General Discussion I evaluate how interlinking feedback loops between CUC, KNOX and auxin pattern serration and leaflet development. I then discuss why interlinking feedback loops may have been deployed to control outgrowths in both plant and animal systems.

Nós investigamos o papel da variação genética do gene RALDH2 e as doenças cardíacas congênitas (DCCs). Seis SNPs foram utilizados em um estudo de TDT. Testes de associação foram desenvolvidos e tanto os marcadores testados quanto os haplótipos analisados não mostraram associação com a doença. Análise do polimorfismo A151G indica que a variante produz mudanças substanciais na estrutura do RNAm. Esta variante está localizada em um exonic splicing enhancer (ESE). Estudos funcionais de splicing não mostraram impacto significante desta variante sobre a alteração do splicing do gene. Este estudo foi aplicado à outra mutação (G151T) encontrada no exon 4 durante o sequenciamento do gene RALDH2 e mostrou aumento no sinal de splicing. Nós encontramos mais quatro mutações: rs34645259 (5\'UTR), T157G (exon 4), rs4646626 (exon 9) e rs35251510 (exon 11). Em resumo, não foi encontrada associação entre DCCs e variações genéticas no gene RALDH2. As mutações encontradas deverão ser analisadas funcionalmente de forma a definir seu papel na perturbação da via do AR em humanos. / The aim of the study was to investigate the role of genetic variation in the RALDH2 locus and congenital heart disease. Six different SNPs were analyzed in 101 patient-parents trios in a TDT study. None of the markers displayed any association with CHD. No single haplotype was associated with an increased risk of CHD. Analysis of the A151G polymorphism indicated that the variant produced substantial changes in mRNA structure. This variant is also localized in a putative exonic splicing enhancer (ESE). Functional splicing studies failed to reveal a significant impact of this variant and gene splicing. This methodology was applied to another mutation (G151T) found in exon 4 during the sequencing of RALDH2 gene and an increase in splicing signal was observed. We found four mutations more: rs34645259, T157G (exon 4), rs4646626 and rs35251510. In summary, no association between CHD and genetic variation at the RALDH2 locus in humans was found. Potential functional genetic variants should be further studied in order to define their real role in RA pathway disturbances.

FGFR 3 is one of a family of four high affinity receptors for FGF ligands. Activating mutations in FGFR 3 result in skeletal dysplasias that vary in severity from undetectable to neonatal lethal. Mice with congenital deficiency of FGFR3 develop severe kyphosis and skeletal overgrowth. FGFR3 is also expressed in calvarial pre-osteoblasts, osteoblast and articular chondrocytes, although it biological role in these cells remains undefined. By changing the genetic background of the Fgfr3-/- mice we were able to extend their lifespan and examine its impact on post-natal skeletal growth. To investigate the implication of FGFR 3 in post-natal cartilage and bone metabolism we used a combination of imaging, classic histology, molecular biology and biomechanical testing. The results demonstrated that the synovial joints of young adult Fgfr3-/- mice revealed a progressive deterioration, loss of the joint space width and changes in the subchondral bone. These alterations were accompanied by an increase of cartilage matrix degradation. Increased aggrecan and collagen type II degradation products, generated by MMPs were detected with DIAPEN and COL2-3/4C antibodies. Increased collagen type X, cellular hypertrophy and loss of proteoglycan at the articular surface were also demonstrated. A novel micro-mechanical indentation protocol revealed that the humeral heads of Fgfr3-/- mice were less stiff than those of wild type littermates. On the other hand, young adult Fgfr3-/- mice are osteopenic due to reduced cortical bone thickness and defective trabecular bone mineralization. The reduction in mineralized bone and lack of trabecular connectivity observed by micro-computed tomography were confirmed by histological and histomorphometric analyses, which revealed a significant decrease in calcein labeling of mineralizing surfaces and a significant increase in osteoid in the long bones of 4-month-old Fgfr3-/- mice. Primary cultures of adherent bone marrow-derived cells from Fgfr3-/- mice expressed markers of differentiated osteoblasts but developed fewer mineralized nodules than Fgfr3+/+ cultures of the same age. These data point to a major role for FGFR3 signaling in development and homeostatic maintenance of cartilage and bone post-natally and identify FGFR3 as a potential target for intervention in degenerative disorders of cartilage, osteopenia and those associated with defective bone mineralization.

abstract: At the interface of developmental biology and evolutionary biology, the very criteria of scientific knowledge are up for grabs. A central issue is the status of evolutionary genetics models, which some argue cannot coherently be used with complex gene regulatory network (GRN) models to explain the same evolutionary phenomena. Despite those claims, many researchers use evolutionary genetics models jointly with GRN models to study evolutionary phenomena. How do those researchers deploy those two kinds of models so that they are consistent and compatible with each other? To address that question, this dissertation closely examines, dissects, and compares two recent research projects in which researchers jointly use the two kinds of models. To identify, select, reconstruct, describe, and compare those cases, I use methods from the empirical social sciences, such as digital corpus analysis, content analysis, and structured case analysis. From those analyses, I infer three primary conclusions about projects of the kind studied. First, they employ an implicit concept of gene that enables the joint use of both kinds of models. Second, they pursue more epistemic aims besides mechanistic explanation of phenomena. Third, they don’t work to create and export broad synthesized theories. Rather, they focus on phenomena too complex to be understood by a common general theory, they distinguish parts of the phenomena, and they apply models from different theories to the different parts. For such projects, seemingly incompatible models are synthesized largely through mediated representations of complex phenomena. The dissertation closes by proposing how developmental evolution, a field traditionally focused on macroevolution, might fruitfully expand its research agenda to include projects that study microevolution. / Dissertation/Thesis / Doctoral Dissertation Biology 2017

Nós investigamos o papel da variação genética do gene RALDH2 e as doenças cardíacas congênitas (DCCs). Seis SNPs foram utilizados em um estudo de TDT. Testes de associação foram desenvolvidos e tanto os marcadores testados quanto os haplótipos analisados não mostraram associação com a doença. Análise do polimorfismo A151G indica que a variante produz mudanças substanciais na estrutura do RNAm. Esta variante está localizada em um exonic splicing enhancer (ESE). Estudos funcionais de splicing não mostraram impacto significante desta variante sobre a alteração do splicing do gene. Este estudo foi aplicado à outra mutação (G151T) encontrada no exon 4 durante o sequenciamento do gene RALDH2 e mostrou aumento no sinal de splicing. Nós encontramos mais quatro mutações: rs34645259 (5\'UTR), T157G (exon 4), rs4646626 (exon 9) e rs35251510 (exon 11). Em resumo, não foi encontrada associação entre DCCs e variações genéticas no gene RALDH2. As mutações encontradas deverão ser analisadas funcionalmente de forma a definir seu papel na perturbação da via do AR em humanos. / The aim of the study was to investigate the role of genetic variation in the RALDH2 locus and congenital heart disease. Six different SNPs were analyzed in 101 patient-parents trios in a TDT study. None of the markers displayed any association with CHD. No single haplotype was associated with an increased risk of CHD. Analysis of the A151G polymorphism indicated that the variant produced substantial changes in mRNA structure. This variant is also localized in a putative exonic splicing enhancer (ESE). Functional splicing studies failed to reveal a significant impact of this variant and gene splicing. This methodology was applied to another mutation (G151T) found in exon 4 during the sequencing of RALDH2 gene and an increase in splicing signal was observed. We found four mutations more: rs34645259, T157G (exon 4), rs4646626 and rs35251510. In summary, no association between CHD and genetic variation at the RALDH2 locus in humans was found. Potential functional genetic variants should be further studied in order to define their real role in RA pathway disturbances.

Tillers are vegetative branches found in grasses, which develop in early stages of plant life. Located at the base of the central stalk, tillers have agronomical importance by increasing seed production with fewer tillers, or providing alternative forms of biofuel with more tillers. As grains have typically decreased tiller number while undergoing domestication, we explored wild and domesticated strains of varying grains by doing a morphological analysis on tiller development. This thesis shows how the decrease of tillers through in domestication cereals shows diversity not only across maize, Sorghum, and Setaria, but also between lines of maize and Setaria species. To do so, we first measured axillary bud growth across these grasses and compared bud initiation, growth, dormancy and outgrowth. While maize inbred B73 demonstrated a tiller dormancy pattern by initiating buds, growing buds and then bud dormancy we measured growth in Sorghum and Setaria to compare and found that although Sorghum patterns dormancy similar to maize, Setaria had more than one way tiller suppression not previously expected. We look further at Setaria buds with a statistical analysis of tiller origin and bud frequency in a wild strain and two domesticated strains of Setaria. Furthermore we performed Scanning Electron Microscopy (SEM) to have a clear understanding of bud initiation or lack of initiation in Setaria italica (B100) comparing it to its wild ancestor Setaria viridis. Because of the diversity in Setaria, we re-visited maize tiller domestication by taking bud measurements, performing SEMs and counting bud frequency on other strains of inbred maize. We found that maize also shows diversity in its patterning of tiller domestication. These results demonstrate that there is diversity in the patterns in which tiller domestication has occurred. This diversity is shown here through differences in tiller bud decisions to initiate or not initiate, or to have axillary buds go dormant post-initiation. Furthermore this variance is shown through differences in bud frequency counts, growth measurements, SEMs, and where tiller branches originate across the grains of maize, Sorghum and Setaria.

No description available.

<p>  </p> <p>Down syndrome (DS) is caused by the partial or complete trisomy of human chromosome 21 (Hsa21) and can result in skeletal deficits, including lower bone mineral density (BMD) and increased risk of fracture and osteoporosis or osteopenia earlier than the general population. Mouse models of DS have been developed to understand the genetic mechanisms resulting in these phenotypes, but models differ due to the complex genetic nature of DS and differing genome structures between humans and mice. Ts65Dn mice have been a popular model of DS as they contain ~50% of Hsa21 orthologous genes on a freely segregating minichromosome, but there is speculation that the phenotypes are exaggerated by non-Hsa21 orthologous trisomic genes also present. To address this issue, the Ts66Yah mouse model was developed to remove the non-Hsa21 orthologous trisomic genes. In this study, male and female Ts66Yah mouse femurs were evaluated during bone accrual and peak bone mass to investigate structural differences using micro-computed tomography. Additionally, the role of trisomic <em>Dyrk1a</em>, a Hsa21 gene previously linked to bone deficits in Ts65Dn mice, was evaluated through genetic and pharmacological means in Ts66Yah femurs at postnatal day 36. Ts66Yah mice were found to have little or no trabecular deficits at any age evaluated, but sex-dependent cortical deficits were present at all ages investigated. Reducing <em>Dyrk1a</em> copy number in Ts66Yah mice significantly improved cortical deficits but did not return cortical bone to euploid levels. Pharmacological treatment with DYRK1A inhibitor L21 was confounded by multiple variables, making it difficult to draw conclusions about DYRK1A inhibition in this manner. Overall, these results indicate trabecular deficits associated with Ts65Dn mice may be due to the non-Hsa21 orthologous trisomic genes, and more Hsa21 orthologous trisomic genes are necessary to produce trabecular deficits in DS model mice. As more mouse models of DS are developed, multiple models need to be assessed to accurately define DS-associated phenotypes and test potential treatments.</p>