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Title: STUDY OF THE FUNCTION AND TARGET GENES OF THE MIR-17-92 CLUSTER IN MLL-ASSOCIATED LEUKEMIA
Creator: Wiley, Anissa
Date: 2011-04-19, 2011-05
Description: MicroRNAs (miRs) have recently been found to be important regulatory agents in cancers. More specifically, the miR-17-92 cluster, which is...
Show moreMicroRNAs (miRs) have recently been found to be important regulatory agents in cancers. More specifically, the miR-17-92 cluster, which is overexpressed in acute leukemias with fusions of the mixed lineage leukemia (MLL) gene, such as MLL-AF9 and MLL-ELL, has been shown to affect regulation of normal genes in human acute leukemias. Studies show that the miR-17-92 cluster targets genes in acute leukemias that play roles in cell differentiation and proliferation. To study this, a luciferase reporter assay was performed showing that miR-17-92 targets transforming growth factor beta induced (TGF!I) and suppressor of zeste 12 (SUZ12). Studies have shown TGF!I to be downregulated in MLL fusion acute leukemias and SUZ12 to have an inverse correlation of expression with miR-17-92. Both genes are important in the regulation of cell differentiation and proliferation. Furthermore, previous studies have shown that the miR- 17-92 cluster partially blocks cell differentiation and induces cell proliferation, and when combined with MLL fusions, these effects are amplified. To further validate the results of these studies, a colony forming assay was performed confirming that a knock-in version of the miR-17-92 cluster promotes formation of colonies of non-differentiating cells, especially in the presence of MLL-AF9 and MLL-ELL. These studies implicate the miR- 17-92 cluster as an important regulator in acute leukemias with MLL fusions given its effect on cell differentiation and proliferation.
M.S. in Biology, May 2011
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Title: EFFECTS OF ENGINEERING USING BACTERIAL HEMOGLOBIN ON THE ABILITY OF ETHANOLOGENIC E. COLI TO FERMENT LIGNOCELLULOSIC HYRDOLYSATE TO ETHANOL
Creator: Kunkel, Stephanie
Date: 2011-05-03, 2011-05
Description: E. coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol has been further engineered in our laboratory using...
Show moreE. coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol has been further engineered in our laboratory using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The resulting strain (TS3) expresses VHb at a moderate level under microaerobic conditions in shake flasks and produces more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the carbon source. In the work reported here, this research was extended, focusing particularly on fermentation of lignocellulosic hydrolysate. The increased ability of the VHb-bearing strain to produce ethanol from pure glucose or xylose was confirmed. A VHb-correlated increase in ethanol production was also noted for lignocellulosic hydrolysate produced in our laboratory, but not for ligncellulosic hydrolysate prepared by the National Laboratory for Renewable Energy (NREL). Experiments were performed at several hydrolysate levels for both prepartories, in an attempt to determine the cause of the difference. One possibility is differences in levels of toxic hydrolysis byproducts in the two preparations as well as increased sensitivity to these toxins due to VHb expression.
M.S. in Biology, May 2011
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Title: SUBCLONING AND ACTIVITY EXAMINATION OF VARIANT VITREOSCILLA HEMOGLOBINS PRODUCED BY RANDOM MUTAGENESIS
Creator: Raba, Daniel A.
Date: 2015, 2015-05
Description: A comparison of mutant versus wild type versions of the Vitreoscilla hemoglobin gene (vgb) were the focus of this study. E. coli DH5a cells...
Show moreA comparison of mutant versus wild type versions of the Vitreoscilla hemoglobin gene (vgb) were the focus of this study. E. coli DH5a cells were transformed with recombinant pUC plasmids containing either the wild type or one of a series of mutated versions of the vgb gene. The study was conducted in two pmis. In the first part, eight strains in total were tested (one plasmid-free negative control, one vector-only (pUC18) negative control, one wild type positive control (pUC8: 16), and five mutants (pUC vgb M1-4 and pUC18 vgb M3)) and comparisons were made to reveal growth advantage or possible increased Vitreoscilla hemoglobin protein (VHb) expression due to vgb gene mutation. Three different growth assays were carried out to compare growth rates among the eight strains under different oxygen concentration conditions: (1) Luria-Bertani (LB) medium, aerobic growth assay, (2) Terrific Broth (TB) medium, low-oxygen growth assay, and (3) TB medium, microaerobic growth assay. Additionally, a series of carbon monoxide (CO) difference spectra were run to quantify functioning VHb protein concentrations in the different strains grown under each of the aforementioned oxygen concentration conditions. In the second part of the study, the two vgb gene mutants that exhibited the highest growth rates and the wild type version were subcloned into the vector pUC8, transformed into E. coli DH5a cells, and then compared using the CO difference spectrum assay. Five E. coli DH5a strains were tested (one plasmid-free negative control, one vector-only negative control (pUC8), one vgb wild type positive control (pUC8 vgb WT), and two vgb mutants (pUC8 vgb M1 and M3)). Transfmmation of plasmids containing the mutated or unmutated version of the vgb gene was verified through E.Z.N.A. plasmid miniprep with gel electrophoresis and additionally through growth on selective medium containing ampicillin (Amp) [50 micrograms (ug)/ milliliter (ml)]. Contrary to the results obtained by our Australian collaborators, our growth assays and CO difference spectra revealed no growth advantage or increased expression of functioning VHb protein due to any of the vgb mutations.
M.S. in Biology, May 2015
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Title: CLONING AND CHARACTERIZATION OF THE SNTA: NNOS: DYSTROPHIN INTERACTION SITES
Creator: Shi, Chenyue
Date: 2014, 2014-07
Description: Duchenne Muscular Dystrophy (DMD) is a lethal genetic disease caused by malfunction of the protein dystrophin. The main function of dystrophin...
Show moreDuchenne Muscular Dystrophy (DMD) is a lethal genetic disease caused by malfunction of the protein dystrophin. The main function of dystrophin is to connect the sarcolemma to the machinery of muscle function via specific binding domains near the C-terminus and N-terminus. These binding domains are connected by a large central rod region that had previously thought to have been biologically inert, but has recently been shown to contain binding sites for various biological partners, including a specific rod region that binds to nNOS, which is an important molecule in modulating vasodilation and increasing blood flow by relax vessel. Removal of this nNOS binding eliminates NO signaling during exercise, causing exercise-related ischemia leading to muscle damage. A major therapeutic strategy being studied to treat DMD is exon skipping, which results in modifications to the dystrophin protein. However, it remains unclear exactly where the binding sites on dystrophin protein and nNOS are, or how they interact with each other. This project is embarked upon more fully characterize the D1617 binding site on the nNOS PDZ domain. Previous studies show that, in neural tissue, PDZnNOS binds to two other proteins (PSD95 and N-methyl-D-aspartate receptor, NMDAR) forming a ternary complex in which the nNOS: PSD95 is a typical PDZ type interaction, but in which the nNOS :NMDAR binding occurred in an unusual fashion, through a unique “finger” region present in PDZnNOS but not in most other PDZ domains. Interestingly, in muscle cells, nNOS also interacts with two proteins, dystrophin and syntrophin (SNTA). Using this as an analogy, we hypothesize that in this case, the binding may also involve both the canonical and finger regions of PDZnNOS, one binding to each protein. We are testing this by constructing PDZnNOS variants with specific amino acid changes designed to xi disrupt each of these interactions (canonical and finger region) independently, and will then examine the impact on both the PDZSNTA and dystrophin D1617 interactions. It is hoped fully understanding the dystrophin nNOS interaction will allow therapies that require modification to dystrophin to conduct these modifications in a way that retains nNOS signaling required for proper muscle function.
M.S. in Cell and Molecular Biology, July 2014
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Title: THE LENGTH-TENSION CURVE OF AND LATTICE SPACING CHANGES IN SKINNED FLIGHT MUSCLE IN MANDUCA SEXTA
Creator: Shaoshuai, Chen
Date: 2016, 2016-05
Description: The synchronous indirect flight muscle (DLM1) of the Hawk moth, Manduca Sexta, has similarities to cardiac striated muscle in its...
Show moreThe synchronous indirect flight muscle (DLM1) of the Hawk moth, Manduca Sexta, has similarities to cardiac striated muscle in its physiological properties. In particular, both operate in vivo on the so-called ascending limb of the length-tension curve. The length-tension curve is a classical experiment to study the physiological properties of muscle. The length tension curve of DLM1 of Manduca sexta has previously been reported with a descending limb that is steeper than what would be expected given likely dimensions for the thick and thin filaments. Excessive rundown of the muscle preparations is a likely cause of these observations. Factors caused muscle rundown are many, such as the time allowed for the muscle to relax, the time for muscle spends contracting or the time spent on sarcomere length adjusting. Insights into the factors mentioned above were obtained by conducting a series of experiments designed to systematically explore the causes of rundown. These included Constant Sarcomere Length Experiments, Reciprocating Stretch Experiments and Back to SL 3.25 μm Experiment. Then a modified protocol for carrying out the length-tension experiment was developed based on these findings. A new length-tension curve was plotted and shows a shape closer to what might be expected theoretically. The force went to zero at about SL 5.7μm. This result is constant with other measurements of the length of the myofilaments. Finally, the lattice spacing experiment was carried out to figure out how the interfilament lattice spacing changes across the SL growing. Result shows that the lattice spacing did not change much over the plateau region of the length-tension curve (SL 2.9- 3.1) but increased substantially as SL decreases further in the ascending limb of the length tension curve.
M.S. in Biology, May 2016
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Title: STRUCTURAL ANALYSIS OF MUTANT TYPES OF VITREOSCILLA HEMOGLOBIN
Creator: Bai, Dongyang
Date: 2016, 2016-07
Description: The aerobic, Gram-negative bacterium Vitreoscilla spp. produces an oxygen-storage protein, which is a hemoglobin. Vitreoscilla hemoglobin (VHb...
Show moreThe aerobic, Gram-negative bacterium Vitreoscilla spp. produces an oxygen-storage protein, which is a hemoglobin. Vitreoscilla hemoglobin (VHb) was the first microbial hemoglobin to be characterized. Like mammalian hemoglobins, it can be described in terms of the distal (ligand-binding) and proximal sides of the heme group. VHb is regarded as a model to study the structure and function of hemoglobin. After the crystal structure of wild-type VHb was obtained, along with several mutation studies on its distal site, it was proposed that residue Tyr29 might play a vital role in ligand binding. In a recent study, a Tyr29Ala mutant displayed functional properties similar to those of the wild-type protein, evidently because the space in the ligand-binding domain occupied by the aromatic ring of Tyr29 in wild type is occupied by the aliphatic ring of Pro54 residue in the mutant which results in a similar ring structure at the ligand-binding domain. This project is aimed to characterize structural changes when Pro54 is mutated to Ala, either with or without the accompanying mutation of Tyr29 to Ala. In an earlier effort, the Y29A/P54A and P54A mutant vgb gene was constructed, but mutant proteins could not be crystallized. In the current study, a hexahistidine tag was added to the C terminus of VHb to aid in protein purification. The Y29A/P54A and P54A mutant protein was purified by affinity chromatography, ion-exchange chromatography and hydrophobic interaction chromatography. The purity of the protein was determined on SDS-PAGE. Circular dichroism data indicated that both Y29A and Y29A/P54A mutants’ α-helix content decreased significantly. Thermal denaturation studies suggest that there are no major changes in the Tm for the Y29A/P54A mutant, and a slight increase for the P54A mutant.
M.S. in Biology, July 2016
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Title: EFFECT OF METABOLIC INHIBITION ON THE GROWTH AND BIOFILM PRODUCTION OF VIBRIO CHOLERAE AND PSEUDOMONAS AERUGINOSA
Creator: Bunn, Dakota C.
Date: 2017, 2017-05
Description: V. cholerae is a gastrointestinal pathogen which causes extreme watery diarrhea and results in over 120,000 deaths per year worldwide. It is...
Show moreV. cholerae is a gastrointestinal pathogen which causes extreme watery diarrhea and results in over 120,000 deaths per year worldwide. It is especially prevalent in developing countries that lack proper water treatment and in areas struck by natural disasters such as hurricanes. P. aeruginosa is an opportunistic pathogen that is ubiquitous in nature, and increasingly found in hospitals burn wards, sinks, catheters and other surgical equipment. Both bacteria are developing increased antibiotic resistance through several mechanisms, with one of the most common ones being the formation of a complex exopolysaccharide matrix known as a biofilm. In this study, using metabolic inhibition, we determined that Na+-NQR is essential for the growth of V. cholerae and P. aeruginosa in both nutrient rich and physiological conditions. We were also able to confirm that inhibition of this enzyme, in both growth conditions, resulted in decreased biofilm production, subsequently eliminating one of the main mechanisms for antibiotic resistance of these bacteria.
M.S. in Biology, May 2017
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Title: GENETIC MODIFICATION OF PAENIBACILLUS UTILIZING VITREOSCILLA HEMOGLOBIN TO IMPROVE BIODESULFURIZATION
Creator: Bai, Yu
Date: 2015, 2015-05
Description: Biodesulfurization (BDS) is a type of microbial biodegradation involving the removal of organic sulfur compounds from fossil fuels that is...
Show moreBiodesulfurization (BDS) is a type of microbial biodegradation involving the removal of organic sulfur compounds from fossil fuels that is accomplished by utilizing the metabolism of certain microorganisms (McFarland et al., 1999). In past decades, many related genetic modifications or transformations have been made to improve biodesulfurization. In this study, Vitreoscilla hemoglobin (VHb) was used to enhance the ability of sulfur metabolism and growth in Paenibacillus naphthalenovorans. Paenibacillus naphthalenovorans wild type (32O-Y) is a thermophilic bacterium isolated in our lab that has also been transformed with recombinant plasmid pnW33N-vgb (producing strain 32O-Y[pNW33N-vgb]). This plasmid encodes both VHb and chloramphenicol (Chl) resistance. The Chl resistance gene provides a method to distinguish from wild type and select for transformed 32O-Y[pNW33N-vgb]. Presence of the plasmid in the transformed strain was verified utilizing PCR. Both 32O-Y and 32O-Y[pNW33N vgb] grew in minimal medium (CDM) that contained dibenzothiophene (DBT) as the sole sulfur source (while Chl was added to 32O-Y[pNW33N-vgb] cultures). DBT is a common organic sulfur-containing molecule in petroleum. It is metabolized by the dsz pathway, present in a number of soil bacteria, which produces 2-hydroxybiphenol (2-HBP) as the final product. Growth (OD600mm) of 32O-Y and 32O-Y[pNW33N-vgb] at 37°C, 45°C, 50°C were compared. The desulfurization activities of wild type and transformed Paenibacillus naphthalenovorans strains were monitored by the Gibbs assay, which measures the concentration of 2-HBP through absorbance at 610 nm. The results demonstrate that the expression of VHb in strain 32O-Y[pNW33N-vgb] correlated with increased growth, especially at 37°C. The desulfurization activities of 32O-Y[pNW33N-vgb] at various temperatures were also improved, compared to untransformed 32O-Y, as VHb was expressed. Efficiency of the VHb-related improvements at the three temperatures was calculated as the ratio between maximum 2-HBP produced and OD600nm at this point. The efficiency values indicated that when compared to 32O-Y, 32O-Y[pNW33N-vgb] had maximum increased productivity at 37°C and 50°C, and minimum at 45°C.
M.S. in Biology, May 2015
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Title: CHARACTERIZING THE ROLE OF PCL1 IN YEAST PHEROMONE RECEPTOR POLARIZATION
Creator: Pai, Chih-yu
Date: 2015, 2015-05
Description: Chemotropism, or directed cell growth in response to a chemical gradient, plays a vital role in many processes, including pollen tube...
Show moreChemotropism, or directed cell growth in response to a chemical gradient, plays a vital role in many processes, including pollen tube formation, axon guidance, angiogenesis, and fungal infections. One of the best-studied models of eukaryotic directional sensing is Saccharomyces cerevisiae, also known as budding yeast. Chemotropic growth relies on both pheromone receptor and its associated G-protein. In vegetative yeast cells, the pheromone receptor is uniformly distributed on the plasma membrane. Upon receptor activation, G~y is released from Ga and G~ is subsequently phosphorylated at multiple residues. Free G~y signals to the nucleus via a MAPK cascade that causes G1 cell cycle arrest and induction of mating specific genes. In addition, G~y recruits various polarity proteins that orient actin-directed secretion to a specific growth site, and newly synthesized receptors are delivered to it. This pheromone-induced receptor polarization appears as "crescents" where the mating projection later forms. However, how yeast cell interpret the external signals and establish the chemotropic growth site remains unknown. Here, we characterized the role of Pcl1, which is a cyclin for the CDK Ph085, and which has been implicated in polarization during budding. It is known that G~ phosphorylation plays a role in receptor polarization and chemotropism. Because G~ has a consensus phosphorylation motif for Ph085, and because G~ and Pcl1 showed a genetic interaction, we hypothesized that Ph085-Pcl1 regulates receptor polarity through G~ phosphorylation. Our results showed Pcl1 localized towards mating projection during mating response. Moreover, Pcl1 and G~ exhibit a direct interaction in response to pheromone. Lastly Ph085 inactivation also appears to affect receptor polarity. Hence, our data are consistent with the hypothesis that Pho85-Pcl1 facilitates receptor polarity establishment by phosphorylating G~.
M.S. in Biology, May 2015
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Title: Efforts to Obtain Desulfurization Competent Cultures from Environmental Samples with Increased Desulfurization Activity
Creator: Davaadelger, Batzaya
Date: 2011-04-18, 2011-05
Description: In this study, environmental samples were collected from different places to obtain various desulfurization competent cultures with increased...
Show moreIn this study, environmental samples were collected from different places to obtain various desulfurization competent cultures with increased desulfurization activity. In order to obtain the cultures the soil samples were inoculated in enriched medium and minimal medium containing DBT as the sole source of sulfur. The cultures grown in minimal medium with DBT were transferred multiple times with increasing temperatures, where as the cultures grown in enriched media were grown only one cycle at 37 degrees. After a certain growth period the cells were isolated and DNA was extracted. Efforts to amplify dsz genes from the obtained cultures with universal dszABC primers were mostly unsuccessful. DNA from bacterial strains isolated from the environmental samples were amplified with universal primers and amplicons were cloned. Samples #2, #6, #17, #21, #32 were amplified successfully but sequencing analyses showed little homology with dszABC genes. DNA from samples #32*, 32*Y and 32*W was isolated with Power Soil kit and PCR analysis showed a 3 kb amplicon. Sequencing analyses, however, showed less homology with dszABC and revealed homology instead with proteins such as those involved in Fe/S biogenesis and thiol:disulphide interchange. In addition, alternate universal primers were designed and PCR analyses were made with R .erythropolis IGTS8 and sample #32*. Alternate primer combination #3 (A1 and C1) and combination #8 (B1 and C2) amplified expected bands with IGTS8 DNA. Combination #8 amplified a 1 kb band from sample #32* DNA. Cultures #32*, #32*Y and #32*W were selected due to their ability to grow in minimal medium with DBT as high as 55 ˚C for multiple transfers. These isolated cultures from a soil sample from the Chicago area are able to catabolize dibenzothiophene (DBT) to 2-hydroxybiphenyl (2-HBP), which was detected by the Gibbs assay.
M.S. in Biology, May 2011
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Title: DEGUELIN AS A THERAPEUTIC DRUG FOR TRIPLE NEGATIVE AND METASTATIC BREAST CANCER
Creator: Kalra, Amit
Date: 2011-12-13, 2011-12
Description: Breast cancer is a second leading cause of cancer related deaths. 15-20 % of women with Breast cancer are diagnosed with Triple negative...
Show moreBreast cancer is a second leading cause of cancer related deaths. 15-20 % of women with Breast cancer are diagnosed with Triple negative breast cancer (ER, PR, and HER2 –ve) which is the most aggressive type showing higher incidence of recurrence at the local as well as distant sites. Conventional chemotherapy, which shows high levels of toxicity, is the only option available for triple negative breast cancer as hormonal therapy does not work. Thus new drugs which could successfully inhibit the growth of triple negative breast cancer along with the inhibition of metastasis are highly desired. Deguelin, originally isolated from an African plant Mundulea sericea is shown to be effective in skin, lung, breast and colon cancer, effect is thought to be mediated by downregulation of the PI3K/AKT pathway. However, its effect on triple negative breast cancer in vitro and in vivo has not been evaluated. There are few in vivo experimental models to study the effect of novel drugs for the therapy of TNBCs. Murine 4T1 mammary breast cancer cell model is so far the most appropriate experimental model to study efficacy of new drugs on not only tumor growth but also on metastasis to different visceral organs including lungs. This model is relevant and mimics stage IV breast cancer in women. We evaluated the effects of Deguelin on triple negative and highly metastatic breast cancer cell line MDA MB 231 and also In vivo in the 4T1 breast cancer model. We also evaluated effect of Deguelin in combination with low dose (IC30) Taxol. Deguelin treatment alone inhibited the growth of MDA MB 231 breast cancer cells in a time and dose dependent manner causing S-G2 arrest. Deguelin induced apoptosis and inhibited cell proliferation. Deguelin also induced changes in the cell shape and subcellular distribution of the cytoskeletal (F-actin and Tubulin) and cell-cell attachment protein beta-catenin. Growth inhibitory effect was enhanced when Deguelin was given along with low dose Taxol. In vivo, Deguelin (2 or 6mg/kg body weight) administered daily for 21 days significantly (P<0.05) inhibited growth of 4T1 cells transplanted s.c. in to 4-6 weeks old female BALB/c mice. Interestingly, as compared to vehicle only, Deguelin treatment also significantly (P<0.02) reduced the number of metastatic lesions from intravenously injected 4T1 cells. Western blot analysis of several key signaling proteins in MDA MB 231 cells suggested that Deguelin (250nM) reduces PI3K, pAKT, and NF-κB protein levels. Immunohistochemical studies in 4T1 xenografts and metastatic lung lesions obtained from vehicle and Deguelin treated animals suggested that Deguelin reduces pAKT, COX2 and HIF-1 alpha, major key players involved in angiogenesis, cell proliferation and metastasis. In conclusion, our results show that Deguelin has growth inhibitory effect on TNBC cell lines; it inhibits in vivo and in vitro growth of murine mammary cancer cell line. Deguelin has anti metastatic activity in 4T1 experimental model. Anti proliferative and anti metastatic effects of Deguelin are mediated through down regulation of pAKT, COX2 and HIF-1 alpha. Deguelin alone or in combination to Taxol showed promising results and could be further developed as potential drugs for the treatment of triple negative breast cancer.
M.S. in Biology, December 2011
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Title: Elastic Proteins in the Flight Muscle of Manduca Sexta
Creator: Yuan, Chen-ching
Date: 2012-05-01, 2012-05
Description: Unlike the asynchronous flight muscles of Lethocerus or Drosophila, the flight muscles (DLM1) of the Hawkmoth Manduca sexta are synchronous,...
Show moreUnlike the asynchronous flight muscles of Lethocerus or Drosophila, the flight muscles (DLM1) of the Hawkmoth Manduca sexta are synchronous, requiring a neural spike for each contraction. While the asynchronous muscles can only extend a few percent, Manduca flight muscle can reversibly extend 50% in vitro and 9% in vivo. Furthermore, the dorsal and ventral regions of the DLM1 have different power output presumably due to the temperature gradient across from the cooler dorsal to the warmer ventral subunits. Together with the observation that length-tension curves of Manduca flight muscles resemble mammalian cardiac muscle, these observations suggest that Manduca muscle might be a useful model system to study some aspects of cardiac muscle contractility. The detailed protein composition of Manduca flight muscle is not known. Here we aimed to identify proteins which might be responsible for the unique properties of Manduca muscle. We used 1% vertical SDS-agarose gel electrophoresis (VAGE) to separate the high molecular weight proteins in Manduca, Lethocerus, and Drosophila flight muscle and bovine left ventricular myocardium. The Manduca samples showed two bands around 2MDa and 1.6MDa, smaller than the two titin isoforms in bovine cardiac muscle, but larger than the largest Lethocerus proteins. Projectin and Kettin are elastic proteins found in Lethocerus and Drosophila with sequence homologies to vertebrate titin. Using western blots, the Manduca sample showed two bands cross-reacting with projectin antibodies at ~800 kDa and ~1030 kDa. Ventral (warmer) DLM1 subunits expressed higher level of both projectin isoform. The 1030 kDa projectin is the predominant expressed isoform in both dorsal and ventral subunits. Kettin antibodies cross-reacted to bands at the same position in both Lethocerus and Manduca. We also used western blots from 4-15% PAGE gels to detect a flightin –cross reacting band at around 23kDa in Lethocerus and 30 kDa in Manduca. Flightin is a thick filament associated protein that presumably helps filament assembly and stability. However, toponin I and T antibodies did not cross-react with similar molecular weight proteins from Manduca flight muscle. Thus, Manduca flight muscle has not only proteins homologous to Lethocerus projectin, kettin, flightin, but also several unknown high molecular weight proteins which might play a role in stabilizing sarcomere structure and give the muscle its unique mechanical properties.
M.S. in Biology, May 2012
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Title: A TIME COURSE STUDY OF FIBRONECTIN MATRIX ASSEMBLY ON SURFACES ACTIVATED WITH CELL AND FIBRONECTIN BINDING DOMAINS
Creator: Chiang, Chun-yi
Date: 2011-07, 2011-07
Description: Fibronectin mediates cell adhesion, migration and initiates extracellular matrix assembly. Therefore, it plays critical roles on wound healing...
Show moreFibronectin mediates cell adhesion, migration and initiates extracellular matrix assembly. Therefore, it plays critical roles on wound healing processes. The purpose of the study is to elucidate the effects of the combination of two functional domains, III1-2 and III9-10 subunits, on the fibronectin matrix assembly. In the present study, NIH 3T3 cells were cultured on the functionalized polyurethane (0.5 μM III9-10 and the mixture with 0.5 μM III9-10 and 0.5 μM III1-2). Functionalization enabled the formation of the covalent linkages between the coverslip polyurethane and the recombinant proteins. Surface characterization was examined by ELISA. NIH3T3 cells were plated on the functionalized coverslips and harvested after 3, 6, 12, and 24 hours culture. Total fibronectin and extracellular matrix fibronectin which is insoluble in deoxycholic acid (DOC) were analyzed through western blotting. The quantification results showed that the mixture groups tended to have higher total fibronectin than the III9-10 alone groups at most time point (3, 6, and 12 hours). Especially obvious differences of DOC-insoluble fibronectin between the mixture group and III9-10 alone group were found in the early stage (3 hour) of the fibronectin matrix assembly. Immunostaining images also showed that fiber-like FN had been generated after three hours culture in the mixture group but FN still looked faint in III9-10 group. Longer and larger FN fibrils were identified in the mixture groups than in III9-10 groups at all time points. In conclusion, III1-2 enhances fibronectin matrix assembly when present with III9-10 through increase the rate of the formation of DOC-insoluble fibronectin in 24 hours culture.
M.S. in Biology, July 2011
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