Melatonin is an indolamine hormone presumably synthesized by retinal photoreceptors, and may act as a paracrine signal of darkness within the retina. Previous studies have suggested that melatonin, acting through specific receptors, may be involved in cyclic retinal functions such as photoreceptor outer segment disc shedding and phagocytosis, and modulation of neurotransmitter release in the inner retina. The goal of this study was to determine if melatonin receptor mRNA is expressed in the neural retina and retinal pigment epithelium (RPE) of Xenopus laevis. Sheets of RPE, devoid of contaminating cells, were obtained from Xenopus eyes, and epithelial cultures were subsequently established on microporous membrane filters in a defined medium. Total RNA was isolated from whole brain, neural retina, fresh RPE sheets, and cultured RPE cells. RNA expression of the three known Xenopus melatonin receptor subtypes (MEL1A, 1B, and 1C) was determined by reverse- transcription/polymerase chain reaction (RT/PCR) amplification, followed by Southern hybridization with RNA probes. PCR-amplified cDNA encoding melatonin receptor subtypes 1B and 1C, but not 1A, were detected in reverse-transcribed RNA obtained from brain, neural retina and RPE. RPE cells grown in culture for two weeks also demonstrated 1B and 1C receptor RNA expression. This study suggests that RNA encoding the 1B and 1C melatonin receptor subtypes is expressed in the neural retina and RPE of Xenopus retina, and the expression persists in RPE cells when grown in culture. The expression of melatonin receptor RNA in the RPE may reflect a regulatory role for melatonin in some diurnal events that occur in this tissue, such as phagocytosis of photoreceptor outer segment membranes, and intracellular migration of pigment granules.

CK2α is a serine-threonine kinase that is involved in a large number of biological processes, including embryonic development, cancer, and cell proliferation. Recently, it has been found that mutations in CK2α results in a developmental condition known as Okur-Chung neurodevelopmental syndrome (OCNDS). This disorder commonly results in intellectual disability, congenital heart defects (CHDs), gross motor delay, and facial abnormalities. CK2α inhibition has so far primarily been studied in mice, through methods such as knockout, gene floxing, and CRISPR/Cas9 mutations. In this thesis, we provide a proof of principle that chemical inhibition of CK2 in Xenopus laevis embryos can induce a phenocopy similar to the heart phenotype of the CK2α knockout mouse model, and demonstrate the potential of Xenopus laevis as an animal model to study molecular mechanisms that may underlie OCNDS. Here we carefully examined whole embryos, sections stained with multiple antibodies, sections stained with hematoxylin and eosin, and assessment of proliferation and apoptosis rates. The phenotypes observed in the Xenopus laevis model were analyzed and compared to both the CK2α knockout mouse model and OCNDS patients. Results found commonalities among facial features, heart deformities, and muscle patterning between the animal models, which overlapped heavily with patient symptoms. Thus, this work has established Xenopus laevis treated with chemical inhibitors as an appropriate animal model for further characterization of the mechanisms that may underlie OCNDS. / 2023-11-18T00:00:00Z

Thesis advisor: Laura Anne Lowery / Thesis advisor: Sarah McMenamin / Development of the central nervous system (CNS) is a complex process that requires the proper function of many genes in order for neurons to proliferate and divide, differentiate, and subsequently migrate long distances to form connections with one another. Abnormalities in any one of these cellular processes can lead to detrimental developmental defects. Growing evidence suggests that genetic mutations caused by rare copy number variants (CNVs) are associated with neurodevelopmental disorders including intellectual disabilities (ID), Autism spectrum disorder (ASD), and schizophrenia. Additionally, these pathogenic CNVs are characterized by extensive phenotypic heterogeneity, as affected individuals often present with microcephaly, craniofacial and heart defects, growth retardation, and seizures. Despite their strong association as risk factors towards neurodevelopmental disorders, the developmental role of individual CNV-affected genes and disrupted cellular mechanisms underlying these mutations remains poorly understood. Moreover, it is unclear as to how the affected genes both individually and combinatorially contribute to the phenotypes associated with pathogenic CNVs. Thus, in this thesis, we explore the functional basis of phenotypic variability of pathogenic CNVs linked to neurodevelopmental disorders. In particular, we focus on the 3q29 deletion and 16p12.1 deletion, to provide insight towards the convergent cellular, molecular, and developmental mechanisms associated with decreased dosage of the affected gene homologs using two complementary model systems, Xenopus laevis and Drosophila melanogaster. First, we examine the role of individual homologs of several CNV-affected genes at chromosome 3q29 and their interactions towards cellular processes underlying the deletion. We find that multiple 3q29-affected genes, including NCBP2, DLG1, FBXO45, PIGZ, and BDH1, contribute to disruptions in apoptosis and cell cycle pathways, leading to neuronal and developmental defects. We then expand further upon this work by discerning the individual contribution of four CNV-affected genes at chromosome 16p12.1, POLR3E, MOSMO, UQCRC2, and CDR2, towards neurodevelopment and craniofacial morphogenesis. We demonstrate that several of these genes affect multiple phenotypic domains during neurodevelopment leading to brain size alterations, abnormal neuronal morphology, and cellular proliferation defects. We then explore their functions during vertebrate craniofacial morphogenesis and demonstrate that some 16p12.1-affected genes are enriched in migratory neural crest, and contribute to early craniofacial patterning and formation of cartilaginous tissue structures. Together, these data are the first to suggest that signature neurodevelopmental phenotypes demonstrated in the 3q29 deletion and 16p12.1 deletion may stem from convergent cellular mechanisms including aberrations in neuronal proliferation, apoptosis and cell cycle regulation, and neural crest cell development. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19142号 / 生博第325号 / 新制||生||43(附属図書館) / 32093 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 豊島 文子, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Climate change has caused an increase in extreme rain events and flooding in certain regions across the globe. During rain events, water flows over impervious surfaces structures such as roads and sidewalks, picking up contaminants such as metals, fertilizers and other nutrients, and various organics that which may impact organisms in such as streams, river, and lakes. Previous work has found significant differences in survival of organisms that were exposed to contaminated stormwater runoff. This study investigated stormwater chemistry parameters at collection sites on the East Tennessee State University campus. Sites were selected based on the extent of human interaction and traffic in the areas. Additionally, acute toxicity of stormwater samples was investigated through 48-h bioassays with the cladoceran, Daphnia magna. In September and November 2022, water chemistry and toxicity analyses were conducted across multiple rain events and over a six-hour time course of an individual rain event. For each of the events and the time course, chlorophyll levels, specific conductivity, pH, temperature, and dissolved oxygen were measured. No statistical difference between the water chemistry parameters between sampling sites or between rain events were observed. Additionally, no significant differences in 48-h survival of D. magna were detected between sampling locations or during the single event time course study. These data suggest that there were no pollutant surges at the collection sites and that D. manga survival was not affected by the contaminants.

Monoclonal antibodies are powerful and versatile tools that enable the study of proteins in diverse contexts. They are often utilized to assist with identification of subcellular localization and characterization of the function of target proteins of interest. However, because there can be considerable sequence diversity between orthologous proteins in Xenopus and mammals, antibodies produced against mouse or human proteins often do not recognize Xenopus counterparts. To address this issue, we refined existing mouse monoclonal antibody production protocols to generate antibodies against Xenopus proteins of interest. Here, we describe several approaches for the generation of useful mouse anti-Xenopus antibodies to multiple Xenopus proteins and their validation in various experimental approaches. These novel antibodies are now available to the research community through the Developmental Study Hybridoma Bank (DSHB).

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