Cell and Developmental Biology Commons^™

Expression Of Artemia Lea Proteins In Drosophila Melanogaster Cells Using Multicistronic Vector Constructs, Kazi Nazrul Islam

Masters Theses

In nature several organisms exhibit anhydrobiosis, the outstanding feature to survive in extreme desiccation by entering into a state of dormancy known as diapause. The cyst of the brine shrimp Artemia franciscana shows anhydrobiosis by entering into a diapause phase. Previous studies showed a correlation between anhydrobiosis and expression of highly hydrophilic polypeptides termed late embryogenesis abundant (LEA) proteins. However, the precise molecular mechanisms of LEA proteins are still unknown. The presence of multiple LEA proteins in Artemia suggests that some of them might work together. Here, I aimed to express different combination of two LEA proteins from Artemia franciscana …

Impact Of Transcription Factors On Fibroblast Specific Gene Silencing In Somatic Cell Hybrids, Tania Ray

Masters Theses

As totipotent cells differentiate to specific cell types in multicellular organisms, certain sets of genes are turned off (gene silencing/gene extinction) while others are turned on (gene activation). Cell type specific gene silencing and gene activation is the basis of cell differentiation. Somatic cell hybrids which are produced by fusing different types of somatic cells from two different tissues have been used as a valuable resource to discern the phenomenon of lineage- specific gene extinction. Identification of regulatory factors that mediate gene extinction in hybrids are key to understanding regulatory mechanisms that govern cell differentiation in a multicellular organism. Transcription …

One Fish, Two Fish, Red Fish, Blue Fish: The Analysis Of The Specter Cell Cycle Mutant In Zebrafish, Tetiana Petrachkova

Masters Theses

Cell division is controlled by genes that regulate the cell cycle. Here we show that the zebrafish specter (spr) mutant is mutation in the cyclin B1 gene, a gene necessary for the G2 to M transition of the cell cycle.

The spr mutation mapped to the cyclin B1 gene. Sequencing showed a transition (C139→T) that caused a nonsense mutation in exon 2 of the cyclin B1 gene. In situ hybridization of cyclin B1 revealed that the mRNA is absent in the mutant embryo by gastrulation. We found that the earliest visible mutant phenotype was a darkening of the head caused …

Progressive Decline Of Testicular Function In Leptinreceptor Deficient Obese Mice, Jennifer Long

Masters Theses

The incidence of obesity and metabolic syndrome among the human population is increasing, but the effects on male fertility are unresolved. Leptin insensitivity appears to show a consistent relationship between obesity and fertility problems in men. Genetically modified mice lacking the leptin hormone (ob/ob) or leptin receptor (db/db) are reported to be subfertile and/or infertile but there is only minimal information about testicular function (sperm and hormone production) and the progression of infertility. Therefore, this study was designed to investigate testicular function using a leptin-receptor-deficient obese mouse model (the POUND mouse; Charles River). Testicular sperm production is reduced in POUND …

The Role Of Creg1 And Pitx1 As Master Regulators Of Liver Function, Anit Shah

Masters Theses

Development in mammals requires a complex array of positive and negative regulatory signals and responses. The liver is a major organ that has been studied extensively to understand underlying genetic processes responsible for specification, establishment and maintenance of tissue identity. Hepatoma and hepatoma variant cell lines have been used as a model to understand genetic networks responsible for liver function. Whole genome analysis of these cell types has identified candidate genes that might serve as master regulators of liver identity. In this study, the role of two candidate genes - cellular repressor of E1A stimulated gene (CREG1) and paired-liked homeodomain-1 …

The Effects Of Solutes In The Cryopreservation Of Adherent Neuroblastoma (Neuro-2a) Cells, Trisha L. Bailey

Masters Theses

A simple method to cryopreserve adherent monolayers of neuronal cells is currently not available, but the development of this technique could facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. However, complex tissues of some exceptional animals survive freezing in nature. These animals are known to accumulate several small molecular weight solutes prior to freezing. Following a similar strategy, we investigated the effects of osmolytes such as trehalose, proline, and sucrose as additives to the traditional cryoprotectant dimethyl sulfoxide (Me2SO) in modulating the cryopreservation outcome of mouse neuroblastoma (Neuro-2a) cells. Neuro-2a cells adhered to …

Bridging In Vi Tro & In S Il Ico Techniques: Uncovering The Structural Characteristics And Light-Dependent Modifications Of The Light-Response Btb Proteins In Arabidopsis Thaliana, Nicholas Reitz

Masters Theses

The conjugation of the NEDD8 protein has been found to cause a wide range of changes to the overall function of a protein. The addition of NEDD8 to a conserved lysine on Cul proteins has been found to provide Cul with increased flexibility, while the transcription factor protein p53 was prevented from binding to DNA after NEDD8 conjugation. Using Arabidopsis thaliana, we investigated the potential conjugation of NEDD8 to the AtLRB1 and AtLRB2 proteins, which are both members of Cul3-RING E3 Ubiquitin Ligase complexes, using both in vitro and in silico techniques. The AtLRBs are negative regulators of the red …

Characterization Of Histidine Decarboxylase In Drosophila Using An Internal Flag Epitope, Maxwell Mianecki

Masters Theses

Histamine is a neurotransmitter in arthropods and is responsible for synaptic transmission in vision, mechanosensation, temperature sensing and sleep cycle in Drosophila. Histamine is synthesized by the enzyme histidine decarboxylase (HDC). While histamine is detectable within tissues using current immunofluorescent labeling techniques, immunological approaches have not been successful for HDC itself, with both direct antibodies and terminal epitope tags determined to be ineffective. In order to avoid loss of the epitope tag through putative N-­‐ and C-­‐terminal proteolytic cleavage, known to occur for HDC in other organisms, an internal epitope tag that does not disrupt enzyme function was utilized. A …

Vaccinia Virus Flll Mediated Expedition Of Tanapoxvirus Replication In Cell Culture, Yih Wen Goh

Masters Theses

Tanapoxvirus (TPV) produces large but slow-forming plaques as opposed to vaccinia virus (VACV) that forms similar large plaques but more rapidly. A number of genes were identified in VACV, inclucding FllL, A33R, A34R and A36R that contribute to the regulation of virus release and dissemination, and are particularly responsible for the induction of actin tails. Among them, TPV lacks homologs of the FllL and A36R genes. F11Lmediated inhibition of RhoA-mDia signaling was shown to enhance the microtubules dynamics and modulates the cortical actin that assisted in the release of progeny virus from infected cells. To understand the possible effects of …

The Effect Of Drying On Desiccation Tolerance And Late Embryogenesis Abundant Protein Gene Expression In Immature Seeds Of Phalaenopsis Amabilis, Huijing Zhu

Masters Theses

Orchidaceae as the most diverse family of flowering plants are endangered due to losing habitats or destruction of their unique symbiotic living style. Long-term seed storage banks can be a solution for threatened plant species but the stored seeds must tolerate extreme drying and cold. This ability is acquired during the last stage (maturation drying) of seed development and is correlated with a decline in water content and expression of the Late Embryogenesis Abundant (LEA) protein genes. Our goal is to investigate if premature orchid seeds can be artificially induced to become desiccation tolerant and the potential role of LEA …

Dephosphorylation Of Iqg1 By Cdc14 Temporally Regulates Actin Ring Formation, Daniel Patrick Miller

Masters Theses

"Cytokinesis is the final step in cell division when the cell separates the cytoplasm by contracting a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential scaffolding protein in budding yeast (S. cerevisiae) required for actin recruitment to, and contraction of, the actomyosin ring. Actin is recruited by the calponin homology domain (CHD) in anaphase after Iqg1 is localized to the bud neck. Consensus sites for the cyclin-dependent kinase (CDK) Cdc28 were identified flanking the CHD. This led us to the hypothesis that phosphorylation of Iqg1 by Cdc28 negatively regulates actin …

Identification Of Actin Cytoskeletal Protein Interactions With The Fission Yeast Scaffold, Mid1, Marian Testori

Masters Theses

Cancer is a disease of uncontrolled cell division. Cell division in eukaryotic cells occurs by the formation of a contractile ring, predominantly composed of filamentous actin (F-actin). During mitosis, actin filaments polymerize to form rings at the medial plane of the cell that constrict causing the cell membrane to pinch and divide. Proteins that regulate this process are essential because dysregulation can lead to uninhibited cell growth and division. In fission yeast, the protein Mid1 functions as a scaffold to recruit regulatory proteins required for actin filament formation and simultaneously anchors the contractile ring at the cell division site. The …

Freeze Tolerance Conferred By Transgenic Expression Of Late Embryogenesis Abundant Protein In Insect Cells, Alyssa N. Walser

Masters Theses

Anhydrobiotic organisms such as the brine shrimp Artemia franciscana have developed strategies to survive and thrive under conditions of limited water availability. One strategy is the accumulation of late embryo genesis abundant (LEA) proteins. Cells of Drosophila melanogaster (Kc167), which had previously been genetically modified to express a group 1 LEA protein (LEA1.3) found in A. franciscana, were used to test the impact of water stress due to freezing to -80°C. Two different freezing devices were used to determine the impact of the freezing protocol on the viability of cells after freezing and thawing. The first method used a 12-well …

Molecular Mechanisms Involved In Anhydrobiosis Of Insect Cells, Diyagama Arachchi Ralalage Dilini Sewwandi Samarajeewa

Masters Theses

Animals possessing tolerance to extreme water stress are termed anhydrobiotes. Many desiccation tolerant organisms respond to water stress by intracellular accumulation of selected sugars such as trehalose and larger macromolecules such as Late Embryogenesis Abundant (LEA) proteins and thereby maintain the cell viability. Evidence indicates that the presence of trehalose and Late Embryogenesis Abundant (LEA) proteins may work synergistically to confer cellular protection during drying in eukaryotic cells. We evaluated any increase in cellular desiccation tolerance by expressing different LEA proteins in a non-desiccation tolerant insect cell line Drosophila melanogaster (fruit fly) (Kc167 cells) in the presence of trehalose.

Transgenic …

Early Arrest Mutant Ogre: A Defect In Cytokineis Caused By A Mutation In The Gene Racgap1, April Wicklund

Masters Theses

The early arrest mutant ogre, isolated in the Tübingen screen, has a block in cytokinesis (Kane et al., 1996). Here I use genetic and molecular techniques to demonstrate that the ogre phenotype is caused by a lesion in the gene encoding Racgap1. I performed fine mapping studies using microsatellite markers to a 0.15 cM region containing the ogre locus. DNA sequence data revealed a nonsense mutation in the Racgap1 gene of ogre mutants, and in situ hybridization experiments showed a loss of racgap1 mRNA at later stages of development, likely by nonsense mediated decay. This evidence supports the abolishment …

Catalytic Engineering Of The Flagellin Protein, Alexandra M. Haase

Masters Theses

Flagellin is the protein monomer that comprises the bacterial flagella for most bacteria, including Salmonella typhimurium. This protein has attracted attention for protein engineering because it is exported out of the cell, polymerized into stable fibers, is produced in large quantities and is relatively simple to purify. Using rational design with computer modeling, potential active sites in the flagellin protein structure were modeled after the human carbonic anhydrase II tetrahedral zinc binding site. In total, three locations were selected as potential active sites and the necessary mutations were successfully introduced. Flagella formation for the flagellin variants was demonstrated through …

Generation Of Tanapoxvirus Ankyrin Repeat Gene Deletion Mutant For Evaluating Its Role In Cell Tropism, Yogesh Suryawanshi

Masters Theses

The host range of poxviruses is highly regulated and is limited to certain hosts. A number of genes in different poxviruses have been identified that contribute to cell tropism. The ankyrin (ANK) repeat genes encode for proteins characterized by the repetitive motifs in its amino acid sequence. These genes have been found to play a role in determining the ability of poxviruses to replicate in certain cell types. Tanapoxvirus (TPV) Kenya genome analysis indicated the presence of six ANK repeat genes of which T146R, T147R and T148R are present in the terminal part of the TPV-Kenya genome. To understand the …

Studies On Enzyme Functional Evolution In The Sabath Multigene Family Using Phylogenetic And Biochemical Approaches, Ruiqi Huang

Masters Theses

Gene duplication is believed to be the major source for providing genetic materials for the innovation and diversification of protein functions; natural selection and/or neutral drift then works on these genetic materials to guide their evolutionary directions. Here, I used the salicylic acid/benzoic acid/theobromine (SABATH) multigene family to study how natural selection acted on duplicated genes to prompt functional diversification. Members in this family methylate plant secondary metabolites by transferring the methyl group from S-adenosyl-L-methionine (SAM) to the carboxyl group or ring nitrogen of the substrates. In the Apocynaceae and Solanaceae lineage of this family, I documented three putative gene …

Desiccation Tolerance In Insect Cells, John Marcus Anderson

Masters Theses

In anhydrobiotic animals a reoccurring strategy to survive water stress involves the accumulation of low molecular weight solutes (e.g., trehalose) and highly hydrophilic macromolecules [e.g., late embryogenesis abundant (LEA) proteins]. We utilized two different water-stress models (spin-drying, and hyperosmotic dehydration) to investigate the impact of transgenic expression of a trehalose transporter [BT003466 (TRET)] and of a group 1 LEA protein [ACX81198 variant (LEA1)] on membrane integrity in cell lines from Spodoptera frugiperda (Sf-21) and Drosophila melanogaster (Kc167), respectively. Kc167 cells were challenged with sucrose or trehalose concentrations ranging from 0 to 200 mM supplemented in standard culture medium. Significant inhibition …

Ancient Mitochondrial Dna From Pre-Historic Southeastern Europe: The Presence Of East Eurasian Haplogroups Provides Evidence Of Interactions With South Siberians Across The Central Asian Steppe Belt, Jeremy R. Newton

Masters Theses

Studies of mitochondrial DNA (mtDNA) polymorphism have provided valuable insights for understanding patterns of human migration and interaction. The ability to recover ancient mtDNA sequence data from post-mortem bone and tissue samples allows us to view snapshots of historic gene pools firsthand, provided that great care is taken to prevent sample contamination. In this study, we analyzed the DNA sequence of the first hypervariable segment (HVSI) of the mtDNA control region, as well as a portion of the coding region, in 14 individuals from three collective burials from the Neolithic Dnieper-Donetz culture and three individuals from Bronze Age Kurgan burials, …

Inhibition Of Cytochrome P-450 But Not Neutrophil Depletion Prevents The Pulmonary Toxicity Associated With 1-Nitronaphthalene, Steven Michael Green

Masters Theses

1-Nitronaphthalene (1-NN) is an environmental contaminant that has been shown to cause pulmonary toxicity in rodents. To examine the contribution of cytochrome P-450 to the metabolism and subsequent toxicity of 1-NN, rats were pretreated with chloramphenicol, a suicide substrate of P-450. To determine if inflammation plays a role in 1-NN toxicity, rats were pretreated with cyclophosphamide to deplete neutrophils. In both experiments, morphological analysis of the lower airways was performed with light and electron microscopy. A second group of animals was examined for biochemical indicators of inflammation and cell damage. The parameters examined were cell content, protein content and lactate …

Histology Of Dichromatic And Seasonal Color Change Of The Cranial Region Of Callagur Borneoensis, Kevin William Matson

Masters Theses

Male Callagur borneoensis exihibit sexual dichromatism and seasonal coloration which is rare among turtles. In the breeding season male's heads are colored white with a scarlet stripe from the occiput to the tip of the snout. After the breeding season they change to drab charcoal-gray to black with a dull orange-yellow stripe. Females and juveniles are a drab brown throughout the year.

This study was performed to determine the mechanisms of this color change at the histological level. Histological preparation of the head revealed a significant increase in vascular tissue just below the epidermis of the stripe area with increased …

The Structure And Fate Of Germinal Plasm In The Asiatic Fire-Bellied Frog, Bombina Orientalis (Anura-Discoglossidae), Ted W. Odom

Masters Theses

Lehman's polychrome stain was used to follow the fate of germinal plasm in early development of Bombina orientalis embryos. Islets of germinal plasm approximately 6 um in diameter were observed in the vegetal pole region of the 2-cell embryo. By the 4-cell stage, some of these islets had coalesced to form larger ones so that two size classes of islets were present (approximately 7.5 and 19.5 um in diameter).

As cleavage continued to the 16-cell stage, the islets were moved further up the cleavage furrow and were always observed to remain in close association with the cleavage membrane. The islets …

A Histochemical Study Of Tooth Development In The Chinese Hamster (Cricetulus Griseus), Robert B. Daszkiewicz

Masters Theses

The normal morphological development of the Chinese hamster (Cricetulus griseus) during the last week of gestation was studied. A review of the literature reveals no distinct study of tooth development in this species. An identical gestation period is found in the mouse (Mus musculus) making direct comparison between the embryos of the same age of the two species feasible. During this investigation, it was found that morphological tooth development in the Chinese hamster lagged behind that of the mouse by about 2 days. On day 14 of gestation in the Chinese hamster, the oral epithelium is …

Cytology Of Chaetomidium Fimeti, David D. Kimmel

Masters Theses

No abstract provided.

Cytological Investigations Of Eremosphaera Viridis De Bary, Richard Allen Valentino

Masters Theses

Eremosphaera viridis de Bary was studied with light microscope techniques to determine the method of cytokinesis and to explain the ontogeny of the membranous ring separating the mature daughter cells in the intact sporangium.

It is proposed that cytokinesis in this organism is phylogenetically advanced and that the separation of mature daughter cells involves the sequential deposition of two cell walls. The first wall develops centripetally by the addition of membrane subunits. The resulting cleavage furrow is oriented by transverse microtubules found along the cell division plane. The newly formed walls separate the two daughter cells and give rise to …

A Cytogenetic Study Of Xx/Xy Chimerism And Other Anomalies Of Bos Taurus In A Sampled Dairy Herd, Ferne M. Rogers

Masters Theses

The establishment of normal chromosomal patterns for a wide range of plants and animals has led to the recognition of abnormalities as well as their relationship to phenotypic irregularities. Research dealing with the expression of human karyotype is of prime interest; but, closely rivaling that is the genetic study of domestic animals which are of economic importance to man.

One observed anomaly which has come under scrutiny by cytogeneticists is that of the female member of a heterogeneous bovine twin pair, the freemartin. This is a sterile animal with internal morphology showing varying degrees of intersex. Cytogenetic studies reveal XX/XY …

The Effect Of Caffeine On Pisum Sativum With Emphasis On Meiosis, Terese M. Mikottis

Masters Theses

No abstract provided.

The Morphology And Cytology Of Preussia Vulgare (Corda) Cain, Marcial Antonio Pastor-Corrales

Masters Theses

No abstract provided.

Studies On Muropeptide Amino Acids Of Rhizobium Japonicum, Mohammed Alauddin

Masters Theses

The isolation of Rhizobium japonicum cell walls was performed by ultrasonication and the enzymatic digestion with DNase, RNase and pronase separately. Crude cell walls were washed with M NaCl and 5% SDS and were isolated by 20,000 g centrifugation. Muropeptide amino acids were freed by hydrolysis at 121C for 10 minutes in 6N HCl.

Detection of Rhizobium japonicum murein layer amino acids was done by 2-dimensional instant thin layer chromatography on silica gel plates. The solvents found most favorable, were n-butanol: acetic acid: water and n-propanol: 34% ammonium hydroxide. Amino acid spots were developed by spraying ninhydrin on the chromatograms, …