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De, SantanuProtein 14-3-3 (YWHA) isoforms and their roles in regulating mouse oocyte maturation
PHD, Kent State University, 2014, College of Arts and Sciences / Department of Biological Sciences
The 14-3-3 (YWHA) is a highly conserved, ubiquitously expressed protein family regulating important cellular processes including cell cycle. This work, for the first time, explored the differential expression and roles of 14-3-3 isoforms during mouse oocyte maturation. All seven mammalian 14-3-3 isoforms were identified in mouse eggs and ovarian follicular cells including oocytes, by Western blotting. Immunocytochemical and immunohistochemical staining confirmed the presence of all 14-3-3 isoforms in oocytes, eggs and ovarian follicles with characteristic similarities and differences in their distributions. Mammalian oocytes are arrested at meiosis prophase I by an inhibitory phosphorylation on Cyclin-Dependent Kinase I (CDK1), released by CDC25B phosphatase which is bound and inactivated in phosphorylated form by 14-3-3 in oocyte cytoplasm. Here, in situ Proximity Ligation Assays (PLA) revealed that all 14-3-3 isoforms interact with CDC25B in oocytes, with reduced interactions in eggs. Phosphorylation of CDC25B at Ser-149 was found to be reduced in eggs compared to oocytes. Microinjection of a translation-blocking morpholino oligonucleotide against 14-3-3eta mRNA caused germinal vesicle breakdown in significantly higher percentage of oocytes compared to oocytes injected with morpholinos targeting other 14-3-3 isoforms. Thus, interaction of 14-3-3eta with CDC25B is required for maintaining prophase I arrest in oocytes. Protein 14-3-3eta was observed to accumulate and co-localize with alpha-tubulin at both meiosis I and II spindles during mouse oocyte maturation in vivo as well as in vitro. It interacts directly with alpha-tubulin with an accumulation of the interactions at meiotic spindles, detected by in situ PLA. In a significant 76% of mouse oocytes microinjected with the morpholino against 14-3-3eta mRNA, meiotic spindles were deformed or absent with clumped chromosomes, no accumulation of 14-3-3eta and no polar body formation. All control eggs showed normal, bipolar spindles with accumulation of 14-3-3eta. Therefore, 14-3-3eta is essential for normal meiotic spindle formation during in vitro maturation of mouse oocytes, in part by interacting with alpha-tubulin, to regulate the assembly of microtubules. These studies reveal 14-3-3 isoform-specific interactions with key proteins involved in mouse oocyte maturation, such as CDC25B and alpha-tubulin. The results help to elucidate the roles of 14-3-3 proteins in mammalian oogenesis and reproductive development.

Committee:

Douglas Kline (Advisor); Derek Damron (Committee Member); Colleen Novak (Committee Member); Werner Geldenhuys (Committee Member); Jennifer Marcinkiewicz (Other); Nicola Brasch (Other)

Subjects:

Animal Sciences; Biology; Biomedical Research; Cellular Biology; Developmental Biology; Molecular Biology; Organismal Biology; Physiology

Keywords:

14-3-3; YWHA; spindle; CDC25B; phosphorylation; interaction; mouse; ovary; oocyte; egg; oocyte maturation

Machemer-Noonan, Katja MarleneInterplay between unusual MYB transcription factors and their role in cell size regulation in plants
Doctor of Philosophy, The Ohio State University, 2014, Molecular Genetics
The study of transcription factors involved in the regulation of cellular processes such as cell expansion and cell division or metabolic pathways such as biosynthesis of phenylpropanoids presents a valuable and effective approach to improve the quality of agriculturally important crops. We have identified SlFSB1 (Solanum lycopersicum Fruit SANT/MYB Binding protein1, FSB1) as a MYB transcription factor that physically interacts with two additional MYBs, SlFSM1, previously associated with tomato fruit-specific expression, and SlMYBI, a DNA-binding protein with two non-canonical MYB domains. Both proteins compete for binding to the SlFSB1 MYB domain, and these interactions can be confirmed in Arabidopsis, suggesting a similar mode of action. RNA-Seq analysis of plants ectopically expressing SlFSM1 suggests the involvement of this complex in several pathways resulting in development of smaller fruits when compared to the wild type. The interplay between the three MYBs is thought to be involved in the regulation of differential cell expansion, as overexpression of SlFSM1 results in a significant reduction of cell expansion in the tomato of cells with the highest expansion potential. Tomato fruit size is a major agronomical trait, and understanding the contribution of the studied MYBs will be greatly beneficial. Additionally, as modification of lignocellulosic biomass is becoming progressively more imperative in the generation of cost-efficient biofuels, the control of phenylpropanoid biosynthesis plays an important role in the achievement of this goal. We identified two R2R3-MYB transcription factors, ZmMYB40 and ZmMYB95, which potentially present a way of fine-tuning lignin composition in maize cell walls, in combinatorial gene expression control with two negative regulators of the phenylpropanoid pathway, ZmMYB31 and ZmMYB42. Maize plants harboring RNAi constructs against ZmMYB40 display statistically lower expression for a number of pathway genes, further supporting this hypothesis. Further, RNA-Seq of rice plants over-expressing the rice homolog of ZmMYB31 elucidates its transcriptional effect beyond those targets already identified in maize, expanding its possible impact.

Committee:

Erich Grotewold (Advisor); Venkat Gopalan (Committee Member); David Mackey (Committee Member); Iris Meier (Committee Member); Rivka Barg (Committee Member)

Subjects:

Agricultural Engineering; Alternative Energy; Bioinformatics; Biology; Botany; Cellular Biology; Developmental Biology; Horticulture; Molecular Biology; Plant Biology; Plant Sciences

Drummond, Christopher AnsonRegulation of Ecdysone 20-Monooxygenase Activity in the Tobacco Hornworm, Manduca sexta and the Apparent Occurrence of this Activity in Ascaris suum (Nematoda)
Doctor of Philosophy (Ph.D.), Bowling Green State University, 2011, Biological Sciences
At specific intervals, increased concentrations of two steroid hormones, i.e., ecdysone (E) and 20-hydroxyecdysone (20E), elicit developmental changes in arthropods. Conversion of E to the active molting hormone, 20E, in the tobacco hornworm Manduca sexta is catalyzed by the cytochrome P450-containing ecdysone 20-monooxygenase system (E20M). During embryogenesis, M. sexta E20M activity increased for the first 72 hours at which time it peaked and subsequently significantly declined. The increased activity coincided with the increase of free ecdysteroids and the progression of two embryonic molts. In midgut tissue of fifth instar M. sexta, decreases in second messenger 3’,5’cyclic guanosine monophosphate (cGMP) concentration inhibited day five E20M activity, but increases in cGMP concentration restored E20M activity. Midgut cGMP content peaked on day five of the instar in concert with the highest level of E20M activity observed. Molecular studies with midgut tissue demonstrated that the E agonist RH-5849 elicited increases in E20M (the shade gene) expression. In the presence of two guanylate cyclase inhibitors, E20M expression significantly increased. Inhibition remediation by pharmacological means resulted in significantly decreased shade expression. While it is unclear as to where cGMP exerts its effects on E20M activity, the data indicated that the second messenger affected the level of transcription, translation, or enzyme activity either individually or in some combination. Interestingly, E20M activity also was found to be affected by six synthesized anthraquinones suggesting that these compounds can serve to disrupt M. sexta development. Lastly, E20M-like activity was observed in female Ascaris suum (Nematoda) both in muscle and reproductive tissue. Although E20M localization in muscle was unclear, in reproductive tissue E20M activity resided mainly with microsomes. This work provides a number of important insights into the regulation of M. sexta E20M during development, a role of cGMP in these events, and the possible occurrence of E20M in the parasitic nematode Ascaris suum.

Committee:

Carmen Fioravanti, Ph.D. (Committee Chair); Daniel Pavuk, Ph.D. (Committee Member); Lee Meserve, Ph.D. (Committee Member); Scott Rogers, Ph.D. (Committee Member); Hangfeng Chen, Ph.D. (Committee Member)

Subjects:

Agriculture; Animal Sciences; Animals; Biochemistry; Biology; Cellular Biology; Developmental Biology; Endocrinology; Entomology; Organismal Biology; Parasitology; Physiology

Keywords:

Ecdysone 20-monooxygenase; Manduca sexta; second messengers; cyclic guanosine monophosphate; ecdysteroidogenesis; steroids; ecdysone; 20-hydroxyecdysone; endocrinology

Cooper, Sharon Roseδ-Protocadherin Function: From Molecular Adhesion Properties to Brain Circuitry
Doctor of Philosophy, The Ohio State University, 2017, Molecular, Cellular and Developmental Biology
Selective cell-to-cell adhesion is essential for normal development of the vertebrate brain, contributing to coordinated cell movements, regional partitioning and synapse formation. Members of the cadherin superfamily mediate calcium-dependent cell adhesion, and selective adhesion by various family members is thought to contribute to the development of neural circuitry. Members of the δ-protocadherin subfamily of cadherins are differentially expressed in the vertebrate nervous system and have been implicated in a range of neurodevelopmental disorders: schizophrenia, mental retardation, and epilepsy. However, little is known about how the δ-protocadherins contribute to the development of the nervous system, nor how this development is disrupted in the disease state. Here I focus on one member of the δ-protocadherin family, protocadherin-19 (pcdh19), since it has the clearest link to a neurodevelopmental disease, being the second most clinically relevant gene in epilepsy. Using pcdh19 transgenic zebrafish, we observed columnar modules of pcdh19-expresing cells in the optic tectum. In the absence of Pcdh19, the columnar organization is disrupted and visually guided behaviors are impaired. Furthermore, similar columns were observed in pcdh1a transgenic zebrafish, located both in the tectum and in other brain regions. This suggests protocadherin defined columns may be a theme of neural development. Our X-ray crystal structure of Pcdh19 reveals the adhesion interface for Pcdh19 and infers the molecular consequences of epilepsy causing mutations. We found several epilepsy causing mutations were located at the interface and disrupted adhesion, which further validated the interface and revealed a possible biochemical cause of Pcdh19 dysfunction. Furthermore, sequence alignments of other δ-protocadherins with Pcdh19 suggest that this interface may be relevant to the entire δ-protocadherin subfamily. We used the information gained about Pcdh19 to design PCDH19-FE mutations in the genome of zebrafish for comparing the circuitry of embryos with wild-type pcdh19, non-adhesive pcdh19 or without pcdh19. The combination of in vitro adhesion studies and in vivo brain imaging analysis provides a more comprehensive understanding of protocadherin-19 function, and suggests a broader role for the δ-protocadherin family in differential adhesion during brain development.

Committee:

James Jontes (Advisor); Marcos Sotomayor (Advisor); Heithem El-Hodiri (Committee Member); Sharon Amacher (Committee Member)

Subjects:

Biochemistry; Biology; Biomedical Research; Biophysics; Cellular Biology; Developmental Biology; Molecular Biology; Neurosciences

Keywords:

epilepsy; EFMR; Pcdh19; protocadherins; cadherins; calcium binding; adhesion; neurons; neural circuitry; neural development; brain development; neuroscience; biophysics; structural biology; zebrafish; superior colliculus; optic tectum; TALEN

Yuan, WenjueSas4 N terminal as a potential binding probe for tubulin-GDP
Master of Science, University of Toledo, 2014, Biology (Cell-Molecular Biology)
As a conserved organelle, the centrosome and its regulated biogenesis is required for cilium formation, accurate cell division, and genome integrity (Nigg and Raff, 2009). Centrosomes consist of a centriole pair surrounded by an amorphous protein network known as pericentriolar material or PCM (Nigg and Raff, 2009). PCM assembly is a tightly regulated step during centrosome biogenesis that determines the size and capability of centrosomes (Conduit et al., 2010; Kirkham et al., 2003; Piehl et al., 2004). During PCM assembly, Sas4, one of the PCM proteins activated by tubulin in GDP bound conformation, provides a scaffold for preassembled cytoplasmic complexes to be tethered to the centrosome. Tubulin directly binds to Sas4; together, they are components of cytoplasmic complexes of centrosomal proteins (Gopalakrishnan et al., 2011; Hung et al., 2004). On the basis of the guanine nucleotide bound state, tubulin can act as a molecular switch in PCM recruitment. While tubulin-GTP prevents Sas4 from forming protein complexes, tubulin-GDP promotes it (Gopalakrishnan et al., 2012). Since tubulin-GDP, instead of tubulin-GTP, promotes formation of centrosomal protein complex recruited by Sas4, it is interesting to note that in a normal, healthy cell, the proportion of GTP: GDP in cytoplasm is near 100:1 (Gamberucci et al., 1994). Thus, in this study, we hypothesize that in order to guarantee proper PCM assembly, Sas4 specifically binds to tubulin-GDP. In other words, Sas4 could be utilized as a binding probe specifically for tubulin-GDP conformation. Our research focuses on in vitro experiments that identify Sas4-tubulin interaction and the specificity under different guanine nucleotides. A number of in vitro biochemistry assays were carried out in order to test Sas4-Tubulin interaction in different guanine nucleotide statuses. Co-Immunoprecipitation, Pulldown assay, Native PAGE-Far Western and Far Western Dot-Blot methods were utilized to test our hypothesis. The results are consistent with our hypothesis that Sas4 specifically recognizes tubulin-GDP, and it is further confirmed by the fact that tandem-Sas4, a recombinant protein derived from our newly built plasmid, specifically increased the binding strength to tubulin-GDP by at least 50%. Our results further confirmed that Sas4N specifically binds tubulin-GDP and function as a core in scaffolding centrosomal protein in the maturation of centrosome. Meanwhile , it also give us a potential tool to utilize Sas4, which specifically binds to tubulin-GDP, to gain more details of insights on the function of free tubulin-GDP as molecule switch instead of a building block in biological process like microtubule dynamic instability

Committee:

Tomer Reiss (Committee Chair); John Plenefisch (Committee Member); Song-Tao Liu (Committee Member); William Taylor (Committee Member)

Subjects:

Biochemistry; Biology; Biomedical Engineering; Biomedical Research; Cellular Biology; Developmental Biology; Health Sciences; Molecular Biology; Molecular Chemistry; Molecules

Keywords:

centrosome; Sas4; tubulin; GTP; GDP; GMPCPP

Shintaku, Jonathan KenjiAlternative NF-κB Regulation of Skeletal Muscle Oxidative Metabolism
Doctor of Philosophy, The Ohio State University, 2016, Molecular, Cellular and Developmental Biology
Skeletal muscle facilitates movement through contraction, which requires substantial energy production. Therefore, proper metabolism is essential for muscle homeostasis. Interestingly, muscle fibers with more oxidative metabolism are more resistant to atrophy caused by chronic inflammation and cancer cachexia. Understanding how metabolism is regulated in muscle homeostasis may reveal mechanisms of disease pathogenesis and therapeutic intervention. NF-κB signaling regulates a wide array of skeletal muscle processes. For example, aberrant classical NF-κB activation in skeletal muscle has been directly linked to atrophy, inflammation, and cancer progression. However, less is known about the role of alternative NF-κB in skeletal muscle homeostasis. Evidence suggests that alternative NF-κB directly stimulates transcription of PGC-1β, a master regulator of mitochondrial biogenesis, and thereby regulates skeletal muscle oxidative metabolism. Here, we sought to understand in vivo the role of alternative NF-κB in skeletal muscle physiology, skeletal muscle homeostasis, and overall metabolic health. We therefore generated a transgenic mouse expressing the alternative NF-κB mediator IKKα in skeletal muscle. We find that alternative NF-κB is sufficient to induce slow Type I myofiber specification, without causing muscle atrophy. Furthermore, IKKα transgenic mice were significantly more active than their wild type littermates, suggesting healthier, more fatigue resistant skeletal muscles. This in vivo evidence reinforces the distinction between classical and alternative NF-κB signaling, suggests a role for alternative NF-κB in promoting slow Type I myofibers, and provides a potential tool to study the link between fiber type and atrophy resistance. In addition to alternative NF-κB, we also studied muscle homeostasis in the context of the transcription factor MyoD. MyoD is a key regulator of skeletal myogenesis that directs contractile protein synthesis, but whether this transcription factor also regulates skeletal muscle metabolism, and particularly aerobic respiration, has not been explored. In a genome-wide bioinformatics analysis of skeletal muscle cells, we unexpectedly observed that MyoD directly binds to numerous metabolic genes, including those associated with mitochondrial biogenesis, fatty acid oxidation, and the electron transport chain. Results in cultured cells and adult skeletal muscle confirmed that MyoD regulates oxidative metabolism through multiple transcriptional targets, including PGC-1β. We find that PGC-1β gene expression is cooperatively regulated by MyoD and the alternative NF-κB signaling pathway. Bioinformatics evidence suggests that this cooperativity between MyoD and alternative NF-κB extends to other metabolic genes as well. Together, these data uncover a novel role for MyoD in regulating the metabolic oxidative capacity of mature skeletal muscle to ensure that a sufficient energy pool is available to support muscle contraction. Collectively, this dissertation highlights novel regulatory mechanisms uniquely structured to support the fundamental metabolic needs of skeletal muscle.

Committee:

Denis Guttridge, PhD (Advisor); Martha Belury, PhD RD (Committee Member); Jeffrey Parvin, MD PhD (Committee Member); Qianben Wang, PhD (Committee Member)

Subjects:

Bioinformatics; Biology; Biomedical Research; Cellular Biology; Developmental Biology; Genetics; Molecular Biology

Keywords:

skeletal muscle; oxidative metabolism; alternative NF-kappaB; PGC-1beta

Mitra, SayantanArabidopsis Cohesin proteins: WAPL, CTF7 and PHD finger proteins: MMDL1, MMDL2 are essential for proper meiosis, gamete development and plant growth
Master of Science, Miami University, 2017, Cell, Molecular and Structural Biology (CMSB)
The cohesin complex is an essential set of proteins required for sister chromatid cohesion, which enables the proper segregation of chromosomes during mitosis and meiosis. In this study, we characterized the epistatic interaction between WAPL and CTF7, two proteins involved in the control of cohesion in Arabidopsis thaliana, Atctf7 homozygous mutant plants display stunted vegetative growth and complete sterility. Inactivation of both copies of Arabidopsis WAPL in Atwapl1.1wapl2 plants results in no significant growth defects, but causes reduced fertility. Co-inactivation of both WAPL and CTF7 in Atwapl1.1wapl2ctf7 plants rescues the ctf7 vegetative growth defects and partially restores fertility. Atwapl1.1wapl2ctf7 plants display higher levels of fertility than Atctf7 plants. While inactivation of WAPL had a profound effect on ctf7 associated defects, CTF7 inactivation, interestingly, had little impact on the Wapl phenotype. In this study, we further characterized two genes in Arabidopsis thaliana: AtMMDL1 and AtMMDL2 that contain a plant homeo domain. Plants containing heterozygous mutations in either MMDL1 (Atmmdl1+/-) or MMDL2 (Atmmdl2+/-) as well as double heterozygous mutations (Atmmdl1mmdl2+/-) resulted in reduced male and female fertility. In contrast, plants homozygous for mutations in either MMDL1 or MMDL2 resembled wild type, but the double homozygous mutation (Atmmdl1mmdl2-/-) resulted in lethality.

Committee:

Christopher Makaroff (Advisor); Richard Edelmann (Committee Member); Eileen Bridge (Committee Member)

Subjects:

Biology; Cellular Biology; Molecular Biology; Plant Biology; Pollen

Keywords:

meiosis, mitosis, DNA repair, chromatin condensation, sister chromatid cohesion, cohesin complex, chromosome segregation, wings apart like, chromosome transmission fidelity 7, plant homeo domain, male meiosis death 1, male sterility 1

Chen, DongxingThe Essential Role of Jab1 in Skeletogenesis During Mouse Development
Master of Sciences, Case Western Reserve University, 2011, Pathology
Jab1, also named Csn5, is an essential subunit of COP9 signalosome. The role of Jab1 in skeletogenesis is poorly understood. Our study in two conditional knock-out mouse models shows that deletions of Jab1 at successive steps of skeletogenesis lead to severe developmental defects. Deletion of Jab1 in differentiated chondrocytes using Col2a1-Cre leads to lethal chondrodysplasia with abnormal cell differentiation, increased apoptosis, and altered cell cycle progression. Furthermore, Jab1 appears to be a novel inhibitor of canonical BMP signaling via repressing Smad1/5 expression during chondrocyte differentiation. BMP signaling is critical for bone and cartilage formation and BMP is also used in clinical treatments for bone regeneration. Thus, our study provides important insights into the link between Jab1 and BMP in cartilage formation. Moreover, deletion of Jab1 in limb mesenchyme using Prx1-Cre leads to severe limb truncation with increased apoptosis. Thus, our study underscores the essential role of Jab1 in skeletal formation in vivo.

Committee:

James M. Anderson, PhD; MD (Committee Chair); Edward M. Greenfield, PhD (Committee Member); Guang Zhou, PhD (Advisor)

Subjects:

Animal Sciences; Biology; Biomedical Research; Cellular Biology; Developmental Biology; Genetics; Molecular Biology; Pathology; Physiology

Keywords:

Jab1; Skeletogenesis; Mouse

Adhikari, BinayaUnderstanding natural expression of cytoplasmic male sterility in flowering plants using a wildflower Lobelia siphilitica L. (Campanulaceae)
PHD, Kent State University, 2018, College of Arts and Sciences / Department of Biological Sciences
ADHIKARI, BINAYA, Ph. D., August 2018 BIOLOGY Understanding natural expression of cytoplasmic male sterility in flowering plants using a wildflower Lobelia siphilitica L. (Campanulaceae) Dissertation Advisor: Andrea L. Case, Ph. D. Cytoplasmic male sterility (CMS) is an interesting form of cytonuclear incompatibility found in flowering plants. Plants are male-sterile (or female) when mitochondrial CMS genes are expressed, but can produce viable pollen (hermaphrodites) if matching nuclear restorer genes suppress them. Because both CMS genes and their restorer genes are common in angiosperms, female plants are relatively rare. However, female plants have evolved hundreds of times, being found in about a third of plant families, creating questions about why the expression of male sterility is rare but taxonomically widespread. In theory, females should be found wherever multiple unique CMS genes are maintained within populations, either by natural selection or frequent invasion by new CMS genes. Empirical tests of these predictions are limited. Here, I evaluated CMS expression in Lobelia siphilitica (Campanulaceae). Populations of this species range from 0–100% female across eastern North America, making it useful for studying the evolution of female plants. I investigated whether and how variation in female frequency among populations relates to patterns of cytoplasmic gene sequence, mitochondrial gene expression and sex expression in controlled matings. I found that mitochondrial genomes of L. siphilitica evolve rapidly and show high diversity in populations with many females. Crossing experiments showed that high mitochondrial diversity reflected high CMS gene diversity within populations. Furthermore, some CMS types were widespread across populations, consistent with long-term maintenance by natural selection. Further research should study mitochondrial genomes to characterize CMS genes and understand if specific mitochondrial genomes have unique CMS genes.

Committee:

Andrea Case (Advisor); Oscar Rocha (Committee Member); Christina Caruso (Committee Member); Srinivasan Vijayaraghavan (Committee Member); Soumitra Basu (Committee Member); John Portman (Committee Member)

Subjects:

Biology; Botany; Ecology; Evolution and Development; Genetics; Molecular Biology; Organismal Biology; Plant Biology; Plant Sciences

Keywords:

Lobelia siphilitica, Cytoplasmic male sterility, CMS, mitochondrial genome, plastid genome, recombination, cytonuclear interaction, LD, cytoplasmic discordance, crossing experiment, Northern blotting

Horowitz, Evan Richard KoppDnmt1 Expression is Required for Lens Epithelial Cell Survival
Master of Science, Miami University, 2015, Cell, Molecular and Structural Biology (CMSB)
Epigenetic mechanisms involving DNA methylation play an integral role in regulating gene expression during the process of cellular differentiation. The catalytic function of DNA methyltransferase 1 (DNMT1) is to maintain methylation marks through subsequent rounds of DNA replication by methylating corresponding cytosines on the newly synthesized strand. To determine the role of Dnmt1 in lens development, Dnmt1 was deleted from the lens lineage by crossing mice carrying a Cre-dependent deletion allele of Dnmt1 (Dnmt1L) to either Le-Cre or MLR10 transgenic mice. Dnmt1 deletion in Le-Cre;Dnmt1L/L lenses led to the loss of lens epithelial cells due to increased apoptosis. MLR10;Dnmt1L/L lenses contained lens epithelial cells that escaped deletion, which partially rescued the lens phenotype. In summary, removal of Dnmt1 from the lens (mediated by Le-Cre) results in loss of the proliferating population of lens cells followed by lens degeneration thereby suggesting that DNMT1 is required for lens epithelial cell survival.

Committee:

Michael Robinson, Dr. (Advisor); Katia Del Rio-Tsonis, Dr. (Committee Member); Andor Kiss, Dr. (Committee Member)

Subjects:

Biology; Cellular Biology; Developmental Biology; Molecular Biology

Keywords:

DNA Methylation; Dnmt1; Epigenetics; Lens Development;

Ostler, Jeffery BrentCharacterization of Pol IV and Pol V-Dependent Non-Coding RNAs Derived from a Geminivirus Genome
Doctor of Philosophy, The Ohio State University, 2017, Molecular Genetics
RNA-directed DNA methylation (RdDM) plays a key role in transcriptionally silencing both transposons and DNA viruses in many organisms. In plants, siRNAs guide ARGONAUTE (AGO)-containing silencing complexes to target DNA, which results in repressive histone and DNA methylation and subsequent heterochromatin formation. This silencing is critical for genome stability and antiviral defense. In canonical, or Pol IVRdDM, RNA Polymerase IV (Pol IV) generates non-coding RNA that is rendered doublestranded and processed into 24 nt siRNAs, which guide AGO4 to RNA Polymerase V scaffold transcripts. While regions of Pol IV and Pol V activity have been identified, significant information about their primary transcripts is lacking, including transcription initiation sites and possible promoter regions, 5' and 3' modifications, and whether the transcripts undergo splicing. In this work, we describe the characterization of Pol IV and Pol V-dependent non-coding transcripts generated from the intergenic region of Beet curly top virus. Additionally, we have identified non-coding transcripts that are Pol IV and Pol V independent, suggesting a role for Pol II in RdDM.

Committee:

David Bisaro, Ph.D. (Advisor); Deborah Parris, Ph.D. (Committee Member); Richard Slotkin, Ph.D. (Committee Member); Feng Qu, Ph.D. (Committee Member)

Subjects:

Biology; Cellular Biology; Genetics; Molecular Biology; Plant Biology; Plant Pathology; Virology

Keywords:

Arabidopsis; RNA-directed DNA methylation; RdDM; RNA polymerase IV; Pol IV; RNA polymerase V; Pol V; Geminivirus; Beet curly top virus; BCTV

Ball, Hope CMetabolic Activity in a Non-Model System: Leptin and Lipolysis in Bowhead (Balaena Mysticetus) and Beluga (Delphinapterus Leucas) Whale
Doctor of Philosophy, University of Akron, 2013, Integrated Bioscience
The discovery of leptin provided a long-anticipated feedback mechanism in vertebrates linking metabolic requirements with adipose deposits. Encoded by the obese (ob) gene and demonstrating a highly conserved primary amino acid sequence across vertebrates, the role of leptin in a plethora of interconnected systems is well documented. Effects of hibernation, migration and temporal influences and sex-specific variations are also well-known in mammals. Migrating or hibernating species frequently require large seasonal changes in overall adipose stores. Temporal leptin resistance in these species is a natural adaptation; allowing them to overcome normal physiological effects of leptin, which result in a reduction in appetite and an increase in metabolic rate. In these instances, adipose stores are increased in preparation for seasonal energetic demands and serve as excellent examples of the allostatic model of leptin function in action. Overall adipose stores increase without an expected correlated increase in leptin titer and mRNA expression; possibly through modulation of leptin receptor isoforms or modifications to other downstream signaling processes. Bowhead and beluga whales possess life histories which mimic that of other seasonally migrating mammals. In the study population, bi-annual migrations each occur between summering grounds in the Beaufort Sea and Bering Sea wintering grounds. Additionally, bowhead whales represent a physiological extreme in terms of adipose deposition. Here, variations in leptin transcript expression and that of two associated genes (leptin receptor and leptin-receptor gene-related product) were examined to understand and detect seasonal and ontogenetic variation and assess the effects on cetacean physiology. Correlations to leptin protein titer were also assessed. Additionally, variation in expression of two lipolysis-associated genes adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) were examined and detected seasonal and ontogenetic differences in utilization of adipose deposits in these marine mammals.

Committee:

Robert Joel Duff, Dr. (Advisor); Richard L Londraville, Dr. (Committee Member); J.G.M. Thewissen, Dr. (Committee Member); Qin Liu, Dr. (Committee Member); Chrys Wesdemiotis, Dr. (Committee Member)

Subjects:

Biology; Cellular Biology; Developmental Biology; Experiments; Histology; Molecular Biology; Morphology; Physiology; Zoology

Keywords:

leptin, lipolysis, qPCR, OB-RGRP, leptin receptor, ATGL, HSL, histology, marine mammal, bowhead whale

Gallina, DonikaThe Role of Glucocorticoid Receptor-signaling and Wnt-signaling in Avian Retinal Regeneration
Doctor of Philosophy, The Ohio State University, 2015, Molecular, Cellular and Developmental Biology
Sight-threatening diseases of the eye are prevalent across the world and result in a progressive loss of visual acuity that often culminates in blindness. These ocular diseases are caused by the degeneration and death of neurons in the retina, the neurosensory tissue of the eye. A promising possible treatment for retinal diseases is to stimulate neuronal regeneration from the Muller glia. Muller glia regenerate the retina in cold blooded vertebrates, but fail to do so mammalian retinas. To achieve retinal regeneration, Muller glia must de-differentiate into Muller glia-derived progenitor cells (MGPCs), proliferate, differentiate into neurons, and functionally integrate into neuronal circuits. Identification of the signaling pathways that influence the reprogramming of Muller glia into MGPCs is key to harnessing the potential of these cells to regenerate the retina. This dissertation focuses on understanding the role Glucocorticoid Receptor (GCR)- and Wnt-signaling pathways, in regulating proliferative, regenerative and neuroprotective properties of Muller glia in the avian retina in vivo. In the first part of this dissertation, we analyze the impact that the GCR- signaling pathway has on the MGPCs in the presence and absence of damage. The primary amino acid sequence of GCR and its expression by Muller glia is highly conserved across vertebrate species, including chickens, mice, guinea pigs, dogs and humans. We find that, in damaged retinas, activation of GCR-signaling suppresses the formation MGPCs via inhibition of MAPK-signaling, and inhibition of GCR-signaling stimulates the formation of proliferating MGPCs. In undamaged retina, FGF2/MAPK-signaling stimulates the formation of MGPCs, and activation of GCR-signaling reduces the number of proliferating MGPCs in FGF2 treated retinas. We also find that inhibition of the GCR-signaling enhances the neuronal differentiation of MGPC-derived cells in damaged retina. The second part of this dissertation describes the impact that Wnt- signaling pathway has on the MGPCs in the presence and absence of damage. We find dynamic changes in retinal levels of the components and readouts of the Wnt pathway. In damaged retinas, inhibition of Wnt-signaling suppresses the formation of MGPCs, and activation of Wnt-signaling stimulates the formation of proliferating MGPCs. Further, in undamaged retina, FGF2/MAPK-signaling stimulates the formation of MGPCs by activating a signaling network that includes Wnt/ß-catenin, and inhibition of Wnt-signaling reduces the number of proliferating MGPCs in FGF2 treated retinas. In the third part of this dissertation, we examine how signaling through GCR influences the survival of neurons. We find that activation of GCR signaling suppresses the reactivity of microglia/macrophages and inhibits the loss of retinal neurons from excitotoxicity, protects ganglion cells from colchicine-induced death, inhibits the loss of photoreceptors in retinal detachment, and inhibits the formation of FGF2/NMDA-induced retina detachments. Taken together, the data presented in this dissertation implicates GCR-signaling not only as an important pathway in neuro-protection, but as a novel pathway that significantly impacts retinal regeneration from MGPCs, and furthers the understanding of how Wnt-signaling influences the formation of MGPCs. These findings will help to guide the development of therapeutic approaches for vision preservation in the future.

Committee:

Andy Fischer (Advisor)

Subjects:

Biology; Cellular Biology; Developmental Biology; Molecular Biology; Neurosciences

Keywords:

Retinal Regeneration; Muller glia; Muller glia derived progenitor cells; Glucocorticoid Receptor signaling; Wnt signaling; neuroprotection

Kershner, LeahRACK1 regulates point contact formation and local translation in neuronal growth cones
PHD, Kent State University, 2018, College of Arts and Sciences / Department of Biological Sciences
In the developing nervous system, select mRNAs are transported to and locally translated within axonal growth cones. Formation of appropriate connectivity in the developing nervous system depends on local translation within axonal growth cones, but the molecular mechanisms underlying this process are not well understood. We have previously shown that local translation of β-actin mRNA within growth cones is necessary for appropriate axon guidance and is dependent on receptor for activated C kinase 1 (RACK1), a multi-functional ribosomal scaffolding protein that can interact with several signaling molecules concurrently. In response to stimulation with brain-derived neurotrophic factor (BDNF), phosphorylation of RACK1 facilitates the local translation of β-actin mRNA. Here, we show that RACK1 is required for the formation of point contacts, which are growth cone adhesion sites that link the extracellular matrix to the intracellular cytoskeleton and regulate growth cone motility. In line with this, RACK1 is also required for axon outgrowth, growth cone spreading, and axon guidance. Therefore, RACK1 is vital for functional aspects of neuronal development. We also find that RACK1 localizes to point contacts, which suggests that local translation may be regulated at these adhesion sites that are important for axonal pathfinding. Thus, we investigated whether local translation occurs at point contacts. First, we examined the location of components of the local translation complex relative to point contacts under both basal and growth factor stimulated conditions. Indeed, both β-actin mRNA and RACK1 colocalize with point contacts, and this colocalization increases following BDNF stimulation, suggesting that local translation is regulated at point contacts. RACK1 is also required for the BDNF-induced increase of β-actin mRNA at point contacts. Furthermore, use of fluorescent translation reporters demonstrates that point contacts are sites of β-actin mRNA local translation. Taken together, these data are the first to show that point contacts are a targeted site of local translation within growth cones, and that RACK1 is critical to the formation of point contacts, the local translation process, and appropriate neural development. Thus, this study makes the important finding that local translation and cell adhesion are vital co-regulators of neural development. These data provide new insight into how and where local translation is regulated within growth cones, and thereby leads to appropriate connectivity formation in the developing nervous system.

Committee:

Kristy Welshhans, Ph.D. (Advisor); Wilson Chung, Ph.D. (Committee Member); Sean Veney, Ph.D. (Committee Member); Soumitra Basu, Ph.D. (Committee Member); Douglas Delahanty, Ph.D. (Committee Member)

Subjects:

Biology; Cellular Biology; Developmental Biology; Molecular Biology; Neurosciences

Keywords:

growth cone; local translation; axon guidance; RACK1; point contact; neural development

Ryan, Sean F.Intraspecific Variation in Natal Plant Secondary Chemistry Leads to Plasticity in Lepidopteran Oviposition Behavior
Master of Science (MS), Bowling Green State University, 2011, Biological Sciences
Environmentally-induced behavioral plasticity, either through learning or experience, is likely to influence insect chemosensory preferences. Previous behavioral studies using insect herbivores as model systems have shown that interspecific variation in plant secondary chemistry can induce behavioral changes in oviposition preferences. However, what has remained untested in these studies is whether female oviposition preferences may be influenced by intraspecific variation in the natal host plant secondary chemistry. Using genetically modified lines of Arabidopsis thaliana with variable profiles of compounds known to act as oviposition stimulants (i.e., glucosinolate hydrolysis products), the prediction that natal exposure to these genotypes would affect the oviposition behavior of two major insect pests of crucifer plants was tested. Specifically, choice oviposition assays were performed using both naïve and experienced females of the specialist Plutella xylostella and the generalist Trichoplusia ni, and three genetic lines of A. thaliana expressing variation in glucosinolate hydrolysis products found in this plant species. These lines included the wild type Col-0 that mainly produces isothiocyanates in its rosette leaves, a transgenic line producing mainly nitriles (i.e., 35S:ESP), and the double myrosinase knockout tgg1 tgg2, which produces virtually no hydrolysis products. In addition to oviposition preferences, the effects of plant genetic lines on insect fitness-related traits were assessed (i.e., insect development, growth, survival and fecundity). Overall, intraspecific variation in natal genotype plant chemistry led to changes in the oviposition behavior of P. xylostella and T. ni. However, the magnitude of the effect of natal experiences on oviposition preferences was dependent on the species and significant only for P. xylostella. In addition, for both species, experience led to oviposition choices that would have maximized fitness of the offspring. This study, for the first time, demonstrates that intraspecific variation in natal plant chemistry can affect insect oviposition preferences, and suggest that pre-dispersal experiences with different plant genotypes may play an important role in the ecology, evolution and pest management of phytophagous insects.

Committee:

Gabriela Bidart-Bouzat, PhD (Advisor); Juan Bouzat, PhD (Committee Member); Dan Pavuk, PhD (Committee Member)

Subjects:

Agriculture; Behavioral Sciences; Biology; Botany; Conservation; Developmental Biology; Ecology; Entomology; Neurobiology; Organismal Biology; Plant Biology; Plant Sciences; Wildlife Conservation; Wildlife Management; Zoology

Keywords:

intraspecific; Arabidopsis; oviposition; plasticity; Plutella xylostella, Trichoplusia ni, Natal Habitat Preference Induction; cabbage looper, diamondback moth, GMO; Hopkins Host Selection Principle

Liang, YanIdentification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation
Doctor of Philosophy (PhD), Ohio University, 2012, Molecular and Cellular Biology (Arts and Sciences)
Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline-rich glycoproteins (HRGPs) that are frequently characterized by the presence of the repetitive dipeptide motifs [Ala-Hyp] or [Hyp-Ala]. Hydroxyproline (Hyp) residues in the dipeptide motifs are sites for the attachment of arabinogalactan (AG) sugar side chains. Fucose residues are found in some dicot AGPs, and AGP fucosylation is developmentally regulated. AGP galactosyltransferase (GalT) and fucosyltransferase (FUT) activities were investigated in three ways in this study. First, an in vitro AGP GalT assay was developed, which used permeabilized microsomal membranes from tobacco (Nicotiana tobacum) or Arabidopsis (Arabidopsis thaliana) suspension-cultured cells as the enzyme source and UDP-[14C] Gal as the sugar donor. Two model AGP peptides, [Ala-Hyp]7 or [AO]7 and deglycosylated [Ala-Hyp]51 or d[AO]51, were used as substrate acceptors. Product analysis indicated that the [AO]7:GalT assay and the d[AO]51:GalT assay mainly detected GalT activities that added the first and the second Gal residues in the Hyp-AG side chain. Examination of the Hyp:GalT activity using various acceptor substrates, including two extensin sequences and a [AP]7 peptide, indicated this activity was specific for Hyp dipeptide motifs in AGPs. The [AO]7:GalT and d[AO]51:GalT activities were localized to the endomembrane system of Arabidopsis suspension cultured cells following sucrose density gradient centrifugation. Second, five candidate AGP GALT genes (GALT1: At1g26810; GALT3: At3g06440; GALT4: At1g27120; GALT5: At1g74800; GALT6: At5g62620) were expressed in Pichia pastoris and tobacco suspension-cultured cells and tested for AGP GalT activity using the [AO]7:GalT and d[AO]51:GalT assay systems. Preliminary results indicate that heterologously expressed GalT3, GalT5 and GalT6 have AGP GalT activities. Furthermore, GalT3 was shown to be Golgi localized in a tobacco leaf expression system. Third, functions of the FUT4 and FUT6 genes were investigated using Arabidopsis fut4, fut6 and fut4/fut6 mutant plants. Biochemical analysis indicated that FUT4 was required for fucosylation of leaf AGPs, while both FUT4 and FUT6 contributed to fucosylation of root AGPs. In addition, glycome profiling indicated that fucosylated AGPs may regulate intermolecular interactions between AGPs and other wall components. Finally, a model of HRGP biosynthesis is proposed which highlights the glycosyltransferases involved in this process.

Committee:

Allan Showalter, PhD (Advisor); Ahmed Faik, PhD (Committee Member); Marcia Kieliszewski, PhD (Committee Member); Sarah Wyatt, PhD (Committee Member)

Subjects:

Biochemistry; Biology; Cellular Biology; Molecular Biology; Physiology; Plant Biology

Keywords:

Arabinogalactan-proteins; galactosyltransferase; fucosyltransferase; hydroxyproline-rich glycoproteins; plant cell walls

Yuvaraj, PadhmavathyRole of Smad4 in the Morphological and Migratory properties of Mouse Trophoblast stem cells
MS, Kent State University, 2011, College of Arts and Sciences / School of Biomedical Sciences

PADHMAVATHY YUVARAJ, M.S. AUGUST 2011* BIOMEDICAL SCIENCES

ROLE OF SMAD4 IN MORPHOLOGICAL AND MIGRATORY PROPERTIES OF MOUSE TROPHOBLAST STEM CELLS (50 pp.)

Director of Thesis: Yijing Chen

Abstract

The mouse trophoblast stem (TS) cells give rise to the fetal portion of the placenta that serve structural and endocrine functions important for maintaining a successful pregnancy. TS cells are derived from the trophectoderm layer of the blastocyst, which differentiates into various trophoblast lineages. This process can be recapitulated in cultured TS cells, making them a useful model system for studying the extra-embryonic lineages in vitro.

In addition to the Fibroblast growth factor signaling, Transforming growth factor (TGF-β) signaling and Nodal/Activin signaling are involved in the functioning of TS cells. Smad4 is the central mediator of many TGF-β superfamily ligand initiated signaling events. Its role in TS cells and the extra-embryonic lineage development, however, is not well understood. Smad4 null embryos die around embryonic day 6.5-7.5 from a variety of developmental defects, some of which affect the extra-embryonic lineage. To understand the role of Smad4 in the extra-embryonic lineage, Smad4 knockout (KO) TS cell lines were derived from mutant mouse blastocysts. Smad4 null TS cell colonies have an abnormal shape and thickness compared to the wild-type TS cell colonies that typically are flat and epithelial in nature. Analysis using transmission through dye microscopy shows that Smad4 KO TS cells are at least two times taller than the wild-type cells. Under normal culture conditions, Smad4 KO TS cells undergo a spontaneous morphological transition and acquire a mesenchymal like morphology. Transwell and wound-healing assays were performed to study the migratory behavior of these cells. Smad4 KO TS cells displayed increased motility compared to wild-type TS cells. Time-lapse imaging studies demonstrate that the wild-type TS cells migrated in a coordinated fashion as an epithelial sheet, whereas the Smad4 KO TS cells fail to maintain their epithelial architecture and frequently migrate as individual cells. Abnormal cell-cell junctions formed between Smad4 null TS cells were revealed with immunofluorescent staining of the adherens junction protein E-cadherin and the tight junction protein ZO-1. There were also changes in localization of N-cadherin and β-catenin, proteins associated with epithelial and mesenchymal transition (EMT). Increased invasiveness and increased expression of EMT markers were observed in TS cells lacking Smad4. In the absence of self-renewing factors, the Smad4 KO TS cells show increased differentiation compared to wild-type TS cells. All these results show that Smad4 is required for regulating EMT and differentiation during embryonic development.

Committee:

Yijing Chen, PhD (Advisor); Srinivasan Vijayaraghavan, PhD (Committee Member); Gail Fraizer, PhD (Committee Member)

Subjects:

Biochemistry; Biology; Biomedical Research; Cellular Biology; Developmental Biology; Molecular Biology; Oncology

Keywords:

Smad4; morphology; migration; EMT; Trophoblast stem cells

Sinha, NilamMammal specific protein phosphatase isoform, PPP1CC2, is essential for sperm function and male fertility
PHD, Kent State University, 2012, College of Arts and Sciences / Department of Biological Sciences

Four isoforms of phospho-protein phosphatase 1 (PPP1), PPP1CA (α), PPP1CB (β), PPP1CC1 (γ1), PPP1CC2 (γ2) are derived from three genes in mammals (Ppp1ca, Ppp1cb, Ppp1cc). PPP1CC1 and PPP1CC2 are differentially spliced products of the Ppp1cc gene. All four isoforms are virtually identical (1-298 aa, approximately 90% identity) except at their extreme C-termini. While PPP1CC1, PPP1CA and PPP1CB are ubiquitous, PPP1CC2 is expressed largely in testis and is the sole PPP1 isoform present in spermatozoa. We have previously shown that sperm motility is inversely related to the activity of the protein phosphatase PPP1CC2. Furthermore, targeted disruption of Ppp1cc gene in mice, which eliminates both PPP1CC isoforms, results in male infertility due to lack of spermatogenesis suggesting an indispensible role for the proteins in spermatogenesis and sperm development. Mutant female mice are fertile and normal, presumably since PPP1CA or PPP1CB can substitute for the loss of PPP1CC isoforms.

The goal of goal of this dissertation work was to identify the role of PPP1CC isoforms, PPP1CC1 and PPP1CC2 in spermatogenesis and male fertility. We wanted to determine if any one or both of the two PP1 isoforms are essential for spermatogenesis and sperm function. To this end we created transgenic mice lines eTg-G2 (γ2) and pTg-eG2 (γ2) expressing PPP1CC2 driven by either the endogenous promoter of Ppp1cc gene and by the testis specific Pgk2 promoter respectively. Based on in-silico and promoter assay studies a genomic fragment spanning 2.6 kb upstream of the transcription start site was identified as putative endogenous promoter and was thus subsequently used in these experiments. In addition, we also generated a third transgenic mouse line eTg-G1 (γ1) where PPP1CC1 transgene expression was driven by the same endogenous promoter fragment. Parallel to this, we used the Cre-Lox approach to generate germ cell conditional Ppp1cc gene knocdown mice to determine the somatic and germ cell requirement for PPP1CC1 and PPP1CC2.

Our analysis of transgenic line eTg-G2 show that Ppp1cc gene promoter is regulated in testis manner and transgene expressed under its control show wild-type pattern of testis predominant PPP1CC2 expression. We have identified putative testis specific factors like Spz1, A-Myb which in combination with Sp1 might regulate the promoter in a testis manner. As expected Pgk2-driven expression of PPP1CC2 was also testis-specific. More importantly, we conclude from our study of the eTg-G2 and the pTg-G2 transgenic lines for the first time that expression of PPP1CC2 alone, via either promoter, is able not only to restore normal spermatogenesis, but the fertility of Ppp1cc null mice as well, provided that transgenic PPP1CC2 expression in testis reaches at least a lower threshold level equivalent to approximately 50% of its expression by a Ppp1cc +/- male. We conclude that the endogenous Ppp1cc promoter normally functions in the testes to maintain a sufficient level of PPP1CC2 expression for normal spermatogenesis to occur, and that production of spermatozoa capable of fertilization in vivo can take place in the complete absence of PPP1CC1 expression. We also conclude from our analysis of the germ cell conditional knockout mice that a reduction in testis levels of PPP1CC2 results in reduced in sperm numbers, increased proportion of abnormal sperm and consequently male infertility. It also demonstrated for the first time the dispensability of Ppp1cc gene in testicular sertoli cells and pre-meiotic germ cells. From the study of eTg-G1 transgenic lines we conclude that PPP1CC1 when forced to express in developing germ cells though able to partially restore spermatogenesis cannot restore fertility due to lack of normal sperm morphogenesis and motility. However further studies are in progress to confirm this observation.

A potential implication of my dissertation work is that one of the causes of infertility in men could be low expression or activity levels of PPP1CC2 in testis and spermatozoa. Thus PPP1CC2 activity/levels could be used as clinical diagnostic marker to assess male fertility.

Committee:

Srinivasan Vijayaraghavan, PhD (Committee Chair); Douglas W. Kline, PhD (Committee Member); Yijing Chen, PhD (Committee Member); Roger B. Gregory, PhD (Committee Member); Soumitra Basu, PhD (Other)

Subjects:

Biology; Biomedical Research; Cellular Biology; Developmental Biology; Molecular Biology

Keywords:

Male fertility; promoter; spermatogenesis; phosphatases; transgenic mice; molecular genetics; serine/threonine phosphatse; expression

Sears, James CooperFoxO Regulates Microtubule Dynamics and Polarity to Promote Dendrite Branching in Drosophila Sensory Neurons
Doctor of Philosophy, Case Western Reserve University, 2017, Neurosciences
The morphology of dendrites is a critical determinant of neuronal function. Microtubules are crucial structural and functional components in dendrites, yet the molecular mechanisms and pathways that regulate dendrite development are unclear. Previous work from our lab showed that FoxO regulates microtubule dynamics in axons at the neuromuscular junction. Therefore, we tested if FoxO regulates microtubules and morphology in the dendritic arborization (da) system. We found that FoxO is required for normal morphology in all four classes of da neurons. FoxO is necessary not only for branching, but also for initiation and maintenance of terminal branches. Furthermore, overexpression of FoxO is sufficient to promote increased branching in multiple cell types. Consistent with findings in motor axons, FoxO limits the distribution of stable microtubules. When we tested for microtubule dynamics, we found that FoxO is necessary and sufficient to promote anterograde microtubule polymerization. Similar to other studies in the system, loss of branching seen in foxO nulls correlates with a decreased nociceptive response. Taken together, our results identify FoxO as a regulator of dendritic arborization and neuronal function. Furthermore, our data support the hypothesis that FoxO promotes dendrite branching by promoting anterograde microtubule polymerization. This conclusion leads to testable hypotheses for how FoxO regulates microtubules and morphology of dendrites.

Committee:

Heather Broihier, PhD (Advisor); David Katz, PhD (Committee Chair); Jerry Silver, PhD (Committee Member); Helen Salz, PhD (Committee Member)

Subjects:

Biology; Biomedical Research; Cellular Biology; Developmental Biology; Genetics; Molecular Biology; Neurosciences

Keywords:

FoxO; Dendrites; Drosophila; EB1; Microtubules; Microtubule Polarity; Neurodevelopment; Development

Evans, Joshua R.Transcriptional Regulation of Select Light-Harvesting Genes during Photoacclimation in Lympha mucosa gen. et sp. prov. (Batrachospermales, Rhodophyta)
Master of Science (MS), Ohio University, 2017, Plant Biology (Arts and Sciences)
The strictly freshwater red algal order Batrachospermales has undergone numerous taxonomic rearrangements in the recent past to rectify the paraphyly of its largest genus Batrachospermum. These systematic investigations have led to the description of new genera and species as well as re-circumscription of some taxa. Specimens collected from two locations in southeastern USA were initially identified as being allied to Batrachospermum sensu lato, but could not be assigned to any previously described species. Comparison of DNA sequence data for two gene regions and morphology with other batrachospermalean taxa resulted in the proposal of a new monospecific genus Lympha mucosa gen. et sp. prov. to accommodate these specimens. A phylogeny of L. mucosa showed it is sister to the genus Volatus, but has morphological similarities with Batrachospermum sections Turfosa and Virescentia. This new taxon adds to the freshwater red algal diversity of southeastern USA, a region already known for biodiversity and high endemism of the aquatic flora and fauna. Lympha mucosa occurs in open and shaded sites of temperate streams and is abundant during summer months. Although most freshwater red algal taxa are considered shade-adapted, many species exhibit differences in photosynthetic rates and characteristics that indicate they have a much greater ability to acclimate to higher irradiances. Specimens of L. mucosa were collected from open (sun-acclimated) or shaded (shade-acclimated) sites and were exposed to low (<20 µmol photon m-2 s-1) or high (220 µmol photon m-2 s-1) for 72h in controlled conditions to examine photoacclimation. To observe regulation for this process at the transcriptional level, the L. mucosa plastid genome was assembled to provide sequence data for photosynthetic genes involved with light harvesting machinery. Of the six light-harvesting genes selected, two involved with photosystem I and one involved with phycoerythrin synthesis were downregulated at high light. This is the first evidence of transcriptional regulation as a potential mechanism for acclimation to varying irradiances in a freshwater red alga.

Committee:

Morgan Vis (Advisor); Sarah Wyatt (Committee Member); Harvey Ballard (Committee Member)

Subjects:

Aquatic Sciences; Bioinformatics; Biology; Biostatistics; Ecology; Evolution and Development; Experiments; Freshwater Ecology; Genetics; Molecular Biology; Organismal Biology; Plant Biology; Plant Sciences; Systematic

Keywords:

photoacclimation; red algae; gene expression; aquatic biology; phylogeny; photosynthesis

Zhang, YingxiaoGenetic Engineering of Rubber Producing Dandelions
Doctor of Philosophy, The Ohio State University, 2016, Horticulture and Crop Science
Natural rubber (cis-1, 4-polyisoprene) is a biopolymer of significance used in both manufacturing and our daily lives. Unfortunately, the current rubber production system, based on the Para rubber tree (Hevea brasiliensis), is unsustainable due to increasing costs of manual latex collection, competition with other cash crops, and the pervasive threat of South American Leaf Blight, a fatal fungal pathogen. It is imperative to develop alternative rubber-producing crops. Rubber dandelion (Taraxacum kok-saghyz, TK) and Taraxacum brevicorniculatum (TB) are dandelion species which produce rubber in roots and have several desirable agronomic characteristics. TK is currently under development as an alternative rubber producing crop while TB is a model species for rubber biosynthesis. TK domestication will inevitably involve the introduction of novel traits through breeding or genetic modifications. To develop tools to monitor the potential gene flow between TK and its ubiquitous weedy relative, common dandelion (Taraxacum officinale, TO), chloroplast genomes have been sequenced for TK, TB and TO and chloroplast and nuclear species-specific markers have been developed and validated. The genomic and marker resources generated here provide a molecular tool kit for germplasm identification and gene flow studies. To advance crop improvement efforts by biotechnology, a rapid and hormone-free Agrobacterium rhizogenes-mediated transformation system was developed for TK and TB. By using root fragments as explants, non-composite transgenic plants were obtained within 8 weeks and the average transformation efficiency for TK and TB was 24.7% and 15.7%, respectively. Protocols developed here were used to transform TK and TB with rubber biosynthesis genes. The rate-limiting enzyme in the mevalonate pathway (MVA pathway), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), was introduced into TK and TB. Six genes encoding the entire MVA pathway were introduced into TK and the corresponding enzymes were located to chloroplast. Transgenic plants generated here will be used for metabolic analysis to understand genetic regulation of rubber biosynthesis. Due to the rapid development of novel biotechnologies, precise gene manipulation methods were also developed. A fast pipeline was developed to apply genome editing to TK using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9). In parallel, preliminary attempts were made to manipulate the plastid genome using plastid engineering. Overall, this research will facilitate biogenesis studies, as well as domestication and commercialization of rubber producing dandelions.

Committee:

Katrina Cornish, Dr. (Advisor); Joshua Blakeslee, Dr. (Advisor); John Cardina, Dr. (Committee Member); Feng Qu, Dr. (Committee Member)

Subjects:

Bioinformatics; Biology; Cellular Biology; Ecology; Horticulture; Molecular Biology; Plant Biology; Plant Sciences

Keywords:

natural rubber; rubber-producing dandelions; Taraxacum kok-saghyz; Taraxacum brevicorniculatum; Taraxacum officinale; chloroplast genome; species-specific molecular markers; genetic engineering; CRISPR genome editing; plastid engineering

Broderick, Shaun RobertPollination-Induced Gene Changes That Lead to Senescence in Petunia × hybrida
Doctor of Philosophy, The Ohio State University, 2014, Horticulture and Crop Science
Flower longevity is a genetically programmed event that ends in flower senescence. Flowers can last from several hours to several months, based on flower type and environmental factors. For many flowers, particularly those that are ethylene-sensitive, longevity is greatly reduced after pollination. Cellular components are disassembled and nutrients are remobilized during senescence, which reduces the net energy expenditures of floral structures. The goal of this research is to identify the genes that can be targeted to extent shelf life by inhibiting pollination-induced senescence. Identifying and characterizing regulatory shelf-life genes will enable breeders to incorporate specific alleles that improve post production quality into ethylene-sensitive crops. Petunia × hybrida is particularly amenable to flower longevity studies because of its large floral organs, predictable flower senescence timing, and importance in the greenhouse industry. A general approach to gene functional analysis involves reducing gene expression and observing the resulting phenotype. Viruses, such as tobacco rattle virus (TRV), can be used to induce gene silencing in plants like petunia. We optimized several parameters that improved virus-induced gene silencing (VIGS) in petunia by increasing the consistency and efficiency of silencing. They included applying inocula to wounded apical meristems, growing petunias at temperatures of 20 °C day/18 °C night, utilizing the cultivar `Picobella Blue’, and inoculating plants at three or four weeks after sowing. As a control for VIGS experiments, an empty vector is frequently used, but severe TRV symptoms often lead to death in petunia. We developed a control construct, which contained a fragment of the green florescent protein. This construct eliminated all severe viral symptoms and served as a better control. This optimized protocol and control construct enabled us to silence many genes and screen for phenotypic results within a few months. To identify candidate, pollination-associated genes for VIGS analysis, RNA-sequencing (RNA-seq) libraries were developed from RNA extracted from pollinated and unpollinated petunia corollas at 12, 18, and 24 h after flower opening. The libraries were sequenced and an expressed sequence tag (EST) library of 33K contigs was generated, which represents the largest petunia corolla transcriptome to date. Differential gene expression analysis and a weighted gene co-expression network analysis (WGCNA) were performed to identify pollination-associated genes. Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment provided a biological overview of the gene networks and molecular pathways associated with flower senescence, which included cellular catabolic processes and pathways such as the Regulation of autophagy, Plant hormone signal transduction, and Sucrose & starch metabolism. With this molecular information, better targets can be selected for further studies. Fifteen differentially expressed, pollination-associated genes were phenotypically characterized using VIGS for alterations in flower longevity. Petunias inoculated with a VIGS vector containing a putative EIN3-binding F-box protein (EBF) resulted in accelerated senescence. Two additional PhEBFs were identified from the assembled RNA-seq transcripts. PhEBF2b and PhEIL expression increased sooner in plants inoculated with the CHS-EBF2b construct than in the CHS control. We hypothesize that this work will lead to improved post production quality in floricultural crops.

Committee:

Michelle Jones (Advisor); Feng Qu (Committee Member); Eric Stockinger (Committee Member); Esther van der Knaap (Committee Member)

Subjects:

Bioinformatics; Biology; Cellular Biology; Horticulture; Molecular Biology; Plant Biology; Plant Pathology; Plant Sciences; Pollen; Virology

Keywords:

VIGS; petunia; RNA-seq; transcriptome; WGCNA; KEGG; gene ontology; pollination; autophagy; calcium; tobacco rattle virus; viral symptoms; lesions; necrosis; ethylene; EIN3-binding F-box protein; EIL; EIN3; style; corolla; petal; pollen; empty vector

Jeon, Young-JunThe Roles of Non-Coding RNAs in Solid Tumors
Doctor of Philosophy, The Ohio State University, 2015, Molecular, Cellular and Developmental Biology
In this study, I showed that chronic exposure to subtoxic concentrations of TRAIL results in acquired resistance. This resistance was associated with the increase in miR-21, miR-30c and miR-100 expression which target tumor suppressor genes fundamental in the response to TRAIL.

Committee:

Croce Carlo (Advisor); Thomas Schmittgen (Committee Member); Patrick Nana-Sinkam (Committee Member); Yuri pekarsky (Committee Member)

Subjects:

Biology; Biomedical Research; Cellular Biology; Molecular Biology

Keywords:

Non-coding RNAs Tumorigenesis Solid Tumors

Wu, QiongThe Role of Lipid Raft-Translocation of Prohibitin in Regulation of Akt and Raf-Protected Apoptosis of HaCaT Cells upon Ultraviolet B Irradiation
Doctor of Philosophy (PhD), Ohio University, 2013, Chemistry and Biochemistry (Arts and Sciences)
Modification of major lipid raft components, such as cholesterol and ceramide, plays a role in regulation of Ultraviolet B light (UVB)-induced apoptosis. Upon UVB irradiation, an alternation of major lipid raft components can lead to the recruitment/activation of raft-associated proteins and initiation of downstream apoptotic signaling pathways. To identify potential regulators of UVB-induced apoptosis, we used two-dimensional gel electrophoresis and mass spectrometry fingerprint analysis to identify proteins that are altered in the rafts after UVB irradiation. Our data shows that levels of several proteins, including prohibitin (PHB), were changed in lipid rafts after UVB irradiation. PHB plays a critical role in many cell functions, such as cell-cycle control, cell proliferation, senescence, development, apoptosis, and tumor suppression. However, until now, no report has identified a role for PHB in mediating apoptosis upon UVB irradiation. We now demonstrate that while total and cell surface PHB expression was not changed, the protein was enriched in lipid rafts after UVB irradiation. Reduced expression of PHB using siRNA knockdown resulted in an increase in apoptosis after UVB irradiation. To reveal the detailed mechanism for PHB-mediated apoptosis upon UVB irradiation, we evaluate whether PHB is involved in regulation of Akt and Raf activity as well as oxidative stress in UVB-irradiated HaCaT cells. Our data shows that upon UVB irradiation PHB is translocated to the lipid rafts, which is correlated with a release of both Akt and Raf from membrane. Overexpression of Akt and/or Raf impedes UVB-induced lipid rafts translocation of PHB. Immunoprecipitation analysis indicates that UVB alters the interactions among PHB, Akt, and Raf. Reduced expression of PHB leads to a decreased phosphorylation of Akt and ERK, as well as a decreased activity of Akt, and an increased apoptosis of the cells after UVB irradiation. In addition, our data shows that the effect of LNAC on UVB-mediated apoptosis and proliferation is independent of PHB knockdown, suggesting an alternative pathway independent of UVB-induced oxidative stress. These results suggested that PHB regulates UVB-induced apoptosis of keratinocytes via a mechanism that involves detachment from Akt and Raf on the plasma membrane, and sequentially lipid rafts translocation.

Committee:

Shiyong Wu (Advisor); Marcia Kieliszewski (Committee Member); Hao Chen (Committee Member); Monica Burdick (Committee Member)

Subjects:

Biochemistry; Biology; Cellular Biology; Molecular Biology

Keywords:

ultraviolet B; lipid raft; prohibitin; apoptosis

Blasiman, Julia LThe Effects of Cannabinoids on Regeneration Rates and Potential Matrix Metalloproteinase and Collagenase Levels in Planaria (Dugesia tigrina)
BS, Kent State University, 2013, College of Arts and Sciences / Department of Biological Sciences
Planaria are free-living flatworms that are well known for their regenerative abilities, though the exact mechanisms for this process remain unknown. Several studies have shown that cannabinoids have the potential to alter the behavior of Planaria. These two pieces of information raise the question of whether or not cannabinoid agonists and antagonists also have the potential to affect the rate at which Planaria regenerate as well as affect the levels of potential collagenases and matrix metalloproteinases (MMPs) that may be critical to tissue remodeling. In regard to regeneration rates, it was found that cannabinoid agonist Arachidonyl-2'-chloroethylamide (ACEA) and cannabinoid antagonist Rimonabant did not affect regeneration rates or enzyme levels at the 1&#xb5;M concentration level to a statistically significant degree. There was a significant amount of variation between individual organisms. Therefore, further experimentation with a clonal strain of Planaria along with higher dosages of the test chemicals may help clarify the effects cannabinoids have on planarian regeneration and enzyme levels, which will help provide new insight on the potential mechanisms behind planarian regeneration.

Committee:

Robert Hamilton, IV (Advisor); Paul Bagavandoss (Committee Member); Leslie Heaphy (Committee Member); Christine Kauth (Committee Member)

Subjects:

Biology; Cellular Biology; Organismal Biology; Zoology

Keywords:

Dugesia tigrina, Planaria, regeneration, cannabinoid, ACEA, Rimonabant, matrix metalloproteinase, collagenase

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