Dissertations / Theses on the topic 'Science biological sciences biology molecular biology'
To see the other types of publications on this topic, follow the link: Science biological sciences biology molecular biology.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Science biological sciences biology molecular biology.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Oikonomou, Eftychia. "Molecular biology and biochemistry of brain tumours." Thesis, Aston University, 2004. http://publications.aston.ac.uk/11021/.
Full textAbstract:
Elucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the b-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine/paracrine role of CCK in the control of growth and/or functioning of gliomas, meningiomas and pituitary tumours. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between b-catenin gene alterations and craniopharyngiomas of the adamantinomatous type.
2
Lohman, Kenton L. "Isolation and Characterization of Temperature-sensitive Protein Synthesis Mutants of Escherichia Coli by Directed Mutagenesis of the Defective Bacteriophage Lambda Fus2." Digital Commons @ East Tennessee State University, 1985. https://dc.etsu.edu/etd/2722.
Full textAbstract:
Mutagenesis of the defective transducing bacteriophage lambda fus2 was used to isolate a collection of temperature-sensitive mutants of E. coli in the major ribosomal protein gene cluster. Four mutants were examined in detail. Two of the mutants were resistant to the ribosomal antibiotics neamine and spectinomycin. Another mutant was defective in 50S ribosomal subunit assembly at 42(DEGREES)C. The 30S subunit proteins S17 and S19 were changed in two different mutants. Each protein migrated as a more basic species in two-dimensional gels of ribosomal proteins. Ribosomes from each of the four mutants examined showed a temperature-dependent reduction in translational activity in cell-free assays. The kinetic assays showed declines in both the rate and extent of translation at three temperatures. Ribosomes from three of the four mutants were also found to have an increased rate of heat inactivation at 45(DEGREES)C compared to control particles. Mixed subunit assays idendtified a t.s. subunit in each mutant. A defect in reassociation at high temperature was found for the subunits from one mutant. Another mutant showed significantly high levels of misreading at 32(DEGREES)C and 42(DEGREES)C. Two mutants showed a decreased ability to bind 14C-phenylalanine tRNA at the two temperatures tested. The increased efficiency and utility of this mutagenesis method for the isolation of protein synthesis mutants is discussed.
3
Chaharmahali, Pegah M. "Calcium homeostasis in Pichia pastoris." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/179.
Full textAbstract:
Pichia pastoris is a methylotrophic yeast that has been used widely in biological and industrial researches. P. pastoris is able to produce from milligram to gram quantities of protein. In this study, we examined the effects of calcium and magnesium on growth, heterologous protein expression, and calcium homeostasis in P. pastoris . The divalent cations calcium and magnesium are responsible for diverse roles in in the function of the cells in eukaryotes. Although it is known that calcium is responsible for many biological functions in eukaryotes, there is a limited understanding of the calcium homeostasis in P. pastoris . In this study, we found that addition of calcium and magnesium to yeast extract peptone dextrose (YPD) does not increase the cell growth of wild type P. pastoris . However, the original concentrations of calcium and magnesium in YPD were critical to cells, as removing calcium and magnesium using EGTA and EDTA, respectively, decreased the cell growth of wild type P. pastoris . Changes to cytoplasmic calcium concentration in P. pastoris was studied using the fluorescent calcium sensitive dye, indo-1 (10 μM) and fluorescent spectrophotometer. The intracellular calcium concentration increased with addition of calcium chloride, magnesium chloride, and phosphate buffered saline (PBS). PBS (standard 1x) induced a significantly higher increase in intracellular calcium concentration compared to inductions with calcium chloride (1.2 mM). Our results showed that the addition of calcium and magnesium into the growth medium did not increase the expression of alcohol oxidase-1 driven β-galactosidase expression. However, calcium and magnesium may still play crucial roles in protein expression as EDTA and EGTA caused an increase in β-galactosidase expression as indicated by higher β-galactosidase activity. Our findings suggest that EDTA and EGTA may also increase the expression of other heterologous proteins in P. pastoris .
4
Pacheco, Ryan John. "Characterication of aggregate gland silk factor 1." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/186.
Full textAbstract:
Spider silk is a high performance fiber with extraordinary mechanical properties, including high tensile strength and toughness. Due to these outstanding material properties, scientists are rapidly pursuing the production of synthetic spider silks for a variety of different applications. In these studies, we characterize the aggregate gland specific factor 1 (AgSF1) from the black widow spider, Latrodectus hesperus. After the development of an anti-AgSF1 polyclonal antiserum, we demonstrate by western blot analyses that the AgSF1 protein is highly expressed in the aggregate gland and the protein is localized to the connection joints of cobweaver webs. We also overexpress and purify two different recombinant AgSF1 fusion proteins, named AgSF1G and AgSF1G+GXPXP. These recombinant proteins encompass different regions within the AgSF1 amino acid sequence. Using wet-spinning methodology we also demonstrate that these proteins can be spun into synthetic silk fibers. Mechanical studies and ultrastructure analyses of the synthetic fibers reveal tensile strengths and toughness values that are below natural dragline silk fibers. Secondary structural analyses of the AgSF1 recombinant proteins in solution using circular dichoism reveal the N-terminal region of AgSF1 is alpha helical in nature. Collectively, these studies advance our understanding of silk proteins that are expressed in the aggregate gland and support that these proteins play an important role in prey capture in cobweavers.
5
Chu, Wei. "Mouse Mast Cell Proteases: Induction, Molecular Cloning, and Characterization." Digital Commons @ East Tennessee State University, 1991. https://dc.etsu.edu/etd/2656.
Full textAbstract:
Tryptase, a mast cell-specific serine protease with trypsin-like specificity, has been identified in a mouse mast cell line (ABFTL-6) based on it's enzymatic activity, inhibition properties, and cross-reactivity to a human mast cell tryptase antibody. The effects of fibroblast-conditioned medium and sodium butyrate on ABFTL-6 mast cell differentiation and tryptase expression have been examined. ABFTL-6 mouse mast cells undergo phenotypic changes upon culturing in media supplemented with fibroblast-conditioned media at 50% or 1 mM sodium butyrate. The induced cells increased in size, had larger and more metachromatic cytoplasmic granules, and increased their total cellular protein about four-fold. Tryptase activity increased 13- and 6-fold upon fibroblast-conditioned media and butyrate induction, respectively. However, tryptase antigen levels increased dramatically from 2.3 $\mu$g/10$\sp6$ uninduced cells to 125 (54-fold) and 75 (33-fold) $\mu$g/10$\sp6$ cells induced with fibroblast-conditioned media or butyrate, respectively. A cDNA library was constructed in $\lambda$gt10 from ABFTL-6 cell poly(A)$\sp+$ RNA, and screened with dog mast cell tryptase and rat mast cell chymase cDNAs. Clones encoding two distinct tryptases (mouse tryptases I and II), a chymase (mouse chymase I) and a novel carboxyl terminal chymase (mouse chymase II) were isolated and sequenced. Mouse tryptases I and II have 75% and 70% sequence identity at the nucleotide and amino acid levels, respectively. The deduced amino acid sequence for the mature active enzyme for each mouse tryptase contains 245 residues and all the characteristics of a serine protease. Asp is found in the substrate binding pockets, consistent with a trypsin-like specificity for Arg-X and Lys-X bonds. It is predicted that tryptases are synthesized with prepropeptides, requiring signal peptidase processing and removal of a three amino acid propeptide for activation. Mouse chymase I consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. An Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine protease.
6
Yelpaala, Yuora. "The characterization of cysteine protease 4 and superoxide dismutase 6 in Trichomonas vaginalis." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/189.
Full textAbstract:
Pathogenesis attributable to infection with Trichomonas vaginalis , the causative agent of Trichomoniasis, is largely unknown although cysteine proteases have been implicated. This paper investigated the role of cysteine protease 4 (CP4) in T. vaginalis through characterization and expression of CP4. T. vaginalis strains showed differential protein and mRNA expression, although it was unclear which CP4 variants (other than TVAG_355480 and TVAG_467970) were recognized by the CP4 antibody for the protein studies. Iron did not regulate expression of TVAG_355480 and TVAG_467970 at the transcriptional but possibly at the post-transcriptional level. Our results also suggested that processing of recombinant TVAG_467970 occurred through cleavage by proteins rather than autocatalytic processing. Finally, endogenous CP4 was localized to vesicles though it was unclear which CP4 variant was recognized and was co-localized with HA-tagged VAMP1/2. Localization of HA-tagged TVAG_467970 proved problematic when co-staining with anti-HA and anti-CP4, so further localization studies need to be optimized. This paper also examined the role of iron superoxide dismutase 6 (TvFeSOD6) in resistance to metronidazole, the current drug used for Trichomoniasis infections. Of the two types of resistance, aerobic resistance may occur due to a high concentration of intracellular oxygen which can outcompete metronidazole for electrons or can re-oxidize reduced metronidazole to its inactive form. We determined that there was differential expression of TvFeSODs in T. vaginalis strains with varying levels of resistance although this may not correlate with the degree of resistance. Our results also showed that an increase in ectopic TvFeSOD6 in MSA1121 led to an augmentation in SOD activity and in resistance under aerobic conditions due to the possible role of TvFeSOD6 in also contributing to a higher intracellular oxygen concentration in MSA1121 (which is already sensitive to oxygen), leading to an increase in resistance.
7
Winkler, Wade C. "RNA elements required for T box antitermination." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1381251178.
Full text
8
Buchanan, Fritz G. "Endogenous Alkylglycerol Functions As a Mediator of Protein Kinase C Activity and Cell Proliferation." Digital Commons @ East Tennessee State University, 1997. https://dc.etsu.edu/etd/2885.
Full textAbstract:
To explore the possibility that 1-O-alkyl-sn-glycerol (alkylglycerol) may serve a regulatory role in the control of cell proliferation or PKC activity, we examined the ability of alkylglycerol to influence PKC activity and subcellular distribution as well as the ability of alkylglycerol to effect cell proliferation. MDCK cells grown to confluence show a loss of PKC activity associated with the membrane, as reported in fibroblasts. Preconfluent cultures of MDCK cells have a high level of PKC activity associated with the membrane. However, treatment of preconfluent cultures with alkylglycerol causes a reduction of PKC activity. A similar inhibition was observed with alkylglycerol when cells were treated with TPA, an activator of PKC. To confirm that alkylglycerol was exerting an effect directly on PKC, alkylglycerol was shown to inhibit PKC activity in vitro in a dose dependent manner. Since PKC exists as a family of closely related isozymes, we have determined the effects of growth arrest and alkylglycerol treatment on PKC $\rm\alpha,\ \epsilon,\ and\ \zeta$ (expressed in MDCK cells). The active forms of PKC $\alpha$ and $\epsilon$ are lost early in the growth of MDCK cells during the endogenous accumulation of alkylglycerol and synthetic alkylglycerol inhibits the membrane form of PKC $\alpha$ and $\epsilon.$ However, alkylglycerol inhibits the TPA induced translocation of PKC $\alpha$ but not $\epsilon$ suggesting a differential inhibition among these isoforms. Neither TPA or alkylglycerol had any effects on the distribution of PKC $\zeta.$ To examine the effect of alkylglycerol on cell proliferation, Swiss 3T3 cells were used. GLC analysis shows that 3T3 cells accumulate alkylglycerol in a similar manner as MDCK cells. Since this accumulation occurs just prior to cell growth arrest, the effects of alkylglycerol on preconfluent cells was observed. Preconfluent cultures of 3T3 cells were treated with alkylglycerol on day 1 of growth. After 8 days of culture, the treated group showed a slower growth rate and saturation density. Furthermore, after these cells were reseeded in the absence of alkylglycerol, the original growth rate and saturation density returned. Thus alkylglycerol induces a decrease in cell proliferation without causing any detrimental effects. Similarly, alkylglycerol was found to inhibit the induction of mitogenesis by TPA (a PKC dependent pathway) and these effects were shown not to be stereospecific. To further investigate the effect of alkylglycerol on cell proliferation, the content of the monoglycerides in ras-transformed cells was analyzed. These cells have lost contact dependent growth arrest indicating a disruption of cell growth regulation. We observed a massive increase in the content of alkylglycerol during the culture of ras transformed cells. This increase is 3 fold higher than MDCK or 3T3 cells. This raises the possibility that alkylglycerol may be the end result of an increased number of cell-cell contacts. We have observed an increase in the accumulation of alkylglycerol in normal and ras-transformed cells. This accumulation is accompanied by a decrease in PKC activity and alkylglycerol was shown to be a potent in vitro inhibitor of PKC. Similarly, alkylglycerol was shown to inhibit PKC $\alpha$ under stimulation by TPA. Alkylgylcerol is a inhibitor of the TPA induced induction of mitogenesis and slows the growth rate of proliferating cultures of 3T3 cells. These results indicate that the endogenous ether-linked glycerolipid, alkylglycerol, is a regulator of cell proliferation through its inhibitory effects on protein kinase C. (Abstract shortened by UMI.)
9
Burnette-Vick, Bonnie A. "Characterization of Two Temperature-sensitive Mutants of Escherichia Coli Exhibiting an Altered L22 Ribosomal Protein." Digital Commons @ East Tennessee State University, 1991. https://dc.etsu.edu/etd/2645.
Full textAbstract:
Analysis of E. coli strains SK1047 and SK1048 have shown them to be temperature-sensitive, protein-synthesis deficient. An alteration in ribosomal protein L22 was detected in both strains using two dimensional gel electrophoresis. Protein L22 was purified from both strains by reversed phase high performance liquid chromatography and from two dimensional electrophoretic gels. Purified ribosomal protein L22 was labeled by reductive methylation and used in 23S RNA binding assays with and without ribosomal protein L4. At the permissive temperature, protein L22 from SK1047 bound less efficiently than the control while protein L22 from SK1048 bound as efficiently as the control. At the restrictive temperature, both forms of mutant protein L22 bound less efficiently than the control. In both mutants, temperature sensitivity was mapped to the chromosomal region containing the rplV gene for ribosomal protein L22 using bacteriophage P1 transduction and bacteriophage $\lambda$ complementation. The wild type rplV gene subcloned into plasmid pLF1.0 was also shown to complement temperature sensitivity. The partial diploid nature of strains complemented by $\lambda$fus2 and plasmid pLF1.0 was verified when both wild type and mutant protein L22 were found on two dimensional gels. Reisolation of protein L22 from gels of $\lambda$fus2 complemented cells showed that both forms of protein L22 were in equal proportion irrespective of growth temperature. Reisolation of protein L22 from gels of plasmid pLF1.0 complemented cells showed that incorporation of the mutant protein exceeded the control protein at the permissive temperature; while the reverse was seen at the restrictive temperature. Temperature-shift experiments were conducted on complemented mutant cells to determine the effect of increased gene dosage on the coordinated regulation of ribosomal protein synthesis. Mutants complemented with $\lambda$fus2 exhibited normal cell growth, indicating that regulation was not effected. Cells transformed with plasmid pLF1.0 exhibited a reduction in growth possibly due to the disruption of balanced synthesis. The wild type and both mutant rplV genes were amplified using polymerase chain reaction and the PCR product was sequenced using primer extension. Sequencing of DNA from both mutants revealed the codon CGC for the amino acid arginine at position 8 in the protein chain was mutated to the TGC codon for the amino acid cysteine. The wild type ribosomal protein L22 contains no cysteine residues. The mutation was confirmed by testing control and mutant protein L22 for the presence of sulfhydryls using 4,4$\sp\prime$-dithiodipyridine. Ribosomal protein L22 isolated from both mutant strains was found to contain one cysteine sulfhydryl group.
10
Chittum, Harold S. "A Molecular Basis for Erythromycin Sensitivity and Resistance in Escherichia Coli." Digital Commons @ East Tennessee State University, 1993. https://dc.etsu.edu/etd/2655.
Full textAbstract:
The effect of erythromycin on the 50S ribosomal subunit during cell growth has been extensively investigated. Sucrose density gradient analysis of ribosomes formed in the presence and absence of the drug revealed a 50S specific assembly defect is partially responsible for erythromycin's inhibitory effects on wild type cells. Examination of two erythromycin-resistant mutants of E. coli (N281 and N282) revealed that mutant N281 (L22 mutant) but not N282 (L4 mutant) was assembly defective in the presence of the drug, although only at much higher drug concentrations (300 ug/ml vs. 75 ug/ml for wild type cells). The altered genes from each mutant have been isolated and sequenced. The L22 mutant was found to contain a 9 bp deletion which eliminated codons 82-84, and the sequence Met-Lys-Arg from the protein. The L4 mutant had an A to G transition mutation in codon 63 resulting in a Lys to Glu change in the protein. Complementation of each mutant by their respective wild type genes resulted in an increased sensitivity to the drug in the partial diploid strains. Two other macrolide antibiotics (oleandomycin and spiramycin) were also examined but revealed no apparent assembly effect on wild type cells. The MLS antibiotics also appeared to be unable to effect assembly. However, the erythromycin derivative azithromycin showed a similar effect on assembly to that of the parent compound although clarithromycin (another erythromycin derivative) did not. These results suggest erythromycin and azithromycin effect assembly through ribosomal protein L4 and to a lesser extent through protein L22.
11
Boismenu, Richard. "A molecular approach for elucidating biological functions of alpha-fetoprotein." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41049.
Full textAbstract:
Alpha-fetoprotein (AFP) is a major plasma protein in the fetus and newborn. Extensive studies in both human and murine systems defined a selective down-regulatory role for AFP on certain types of immune reactions in vitro. Direct evidence was obtained in experimental models of autoimmune disease showing that mouse and human AFP can, in a predictable fashion, influence in vivo immune parameters. However, attempts to define the molecular basis of the immunoregulatory functions of AFP have until now been handicapped by the lack of an appropriate consistent source for pure AFP in quantities suitable for detailed investigations. The aim of my thesis work was to generate such a source to elucidate structure-function relationships in AFP. Human and mouse AFP cDNAs were cloned and expressed at high-levels in Escherichia coli. In addition, we also overexpressed truncated versions of the mouse AFP gene that code for the first third and two third of the molecule. Optimum fractional abundance for an individual rAFP protein had to be determined empirically and was found to be influenced by several parameters including the expression vector, the bacterial strain, as well as by the induction and harvesting protocols. It was determined that rAFPs were largely present as insoluble aggregates involving intermolecular disulphide bonds. We devised very specific procedures to recover pure and soluble forms of rAFP proteins produced in certain preselected Escherichia coli strains.
12
Mehrpouyan, Majid. "A Temperature-sensitive Mutant of Escherichia Coli Affected in the Alpha Subunit of RNA Polymerase." Digital Commons @ East Tennessee State University, 1990. https://dc.etsu.edu/etd/2728.
Full textAbstract:
A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44$\sp\circ$C. The thermostability at 44$\sp\circ$C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation. A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about four fold at 37$\sp\circ$C. The role of the alpha dimer in the structure and function of RNA polymerase is discussed. In addition during the course of this study a new procedure for the purification of E. coli RNA polymerase was developed. This method is rapid, convenient, and useful for the preparation of enzyme from 1-5 grams of cells in two days. The ease and speed of this method allowed the rapid characterization of the mutant enzyme. This system should also find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.
13
Lu, Weiqun. "Application of molecular biological techniques to the identification of cyanobacteria." Thesis, Liverpool John Moores University, 1998. http://researchonline.ljmu.ac.uk/4966/.
Full text
14
Kumar, Shalini. "Advanced Techniques in Microbial and Molecular Biology: Laboratory Procedures for a Graduate Level Course." Thesis, University of North Texas, 1998. https://digital.library.unt.edu/ark:/67531/metadc935668/.
Full textAbstract:
Advanced laboratory techniques for Microbial and Molecular Biology at the graduate level are presented in this thesis. The procedures for the laboratory experiments are set forth in detail. This laboratory is conducted as two parts, each by a different professor. Part 1 covers the experiments conducted by Dr G.A.O. Donovan. These experiments include an introduction, staining procedures, biochemical reactions, mutagenesis experiment, essays,. preparation and analysis of plasmid DNA and various other topics. Part 2 covers the experiments conducted by Dr. Daniel Kunz and includes various topics like media preparation, phenotyping strains, conjugative transfer of plasmids, SDS-PAGE, induction and measurement of enzyme and transposon mutagenesis
15
Posey, Eugenia L. "The Influence of a Human Repetitive Dna on Genome Stability." Digital Commons @ East Tennessee State University, 1998. https://dc.etsu.edu/etd/2960.
Full textAbstract:
A uniquely human interspersed repetitive DNA sequence family, the L2Hs, are highly polymorphic in human genomes. Several features of interspersed repeated DNA may contribute to the instability observed. Certain motifs (direct repeats, palindromes, and inverted repeats) comprising L2Hs elements may adopt unusual secondary structures such as cruciforms or hairpins. These motifs have been associated with features of genome instability in recombination, insertions and deletions. The L2Hs elements also are AT-rich (76%) compared to the bulk of human DNA (52%). That their dynamic nature (i.e. polymorphisms) may arise from recombination, insertions and deletions has led to the hypothesis that the L2Hs element is intrinsically dynamic and may influence the stability of the surrounding genome. Thus, the stability of the L2Hs element was tested in a bacterial model system. A cloned 0.6 kb L2Hs element forms non-B-form structures in recombinant plasmids pN6 and pN2, which differ only in insert orientation. Instability of pN6 and pN2 plasmids was observed in serial propagation studies in which E. coli cells containing the plasmids were cultured every 24 hours for 28 days. The vector plasmid pTZ19U, as control, was found to be stable in all passages while the two L2Hs recombinants developed deletions of the L2Hs insert as well as adjacent vector sequences. The isolated deletion mutants have been characterized via restriction cleavage studies and sequencing to map the boundaries of the deletions. Direct repeats and potential stem-loop structures have been discovered at or within close proximity to the deletion boundaries. The data demonstrate that the L2Hs recombinants' unusual sequence features with potential for non-B-form secondary structures, influence genome stability via their involvement in generating errors during DNA replication and DNA repair.
16
Matala, Erik John. "Molecular and biological characterization of human immunodeficiency virus type 1 envelope gp120 associated with maternal-fetal transmission." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/298765.
Full textAbstract:
Vertical transmission of HIV-1 represents a major, global health concern with particular regard to developing areas of the world, and perinatally acquired infections account for the majority of all pediatric HIV-1 cases. Maternal-fetal transmission of HIV-1 occurs at three stages: prepartum, intrapartum, and post-partum, however the mechanism of this transmission is not yet clearly defined. Additionally, the rate of transmission is estimated at ∼30%, leaving ∼70% of infected mothers who do not transmit to their infants. While several maternal factors including viral load, clinical stage of the mother, low CD4 counts, recent infection, and the maternal immune response to infection have been implicated in transmission, the viral factors which may influence transmission are not known. In this study, we have investigated the molecular and biological properties associated with the env V3 region and the entire env from both transmitting and non-transmitting mothers. Our results show that the minor genotypes of the mothers' heterogeneous viral populations are transmitted to their infants, the biological properties associated with the transmitted variants' V3 regions appear to be macrophage tropic (R5) and non-syncytium inducing, and the transmitted variants are initially maintained in the infants with the same properties. In addition, the molecular analyses of the env of non-transmitting mothers, including the variable regions V1-V2, V3, and V4-V5 were found to be significantly more homogeneous than that of transmitting mothers, suggesting that a limited heterogeneity within an infected mother may prevent vertical transmission. Since effective therapies should target the specific properties of transmitted variants, these results provide significant insight into the molecular mechanisms of maternal-fetal transmission, aiding the development of effective therapies for prevention of transmission and treatment of disease.
17
Ratnasinghe, Duminda D. "Unusual Structure of a Human Middle Repetitive DNA." Digital Commons @ East Tennessee State University, 1993. https://dc.etsu.edu/etd/2767.
Full textAbstract:
The L2Hs sequences are a polymorphic, interspersed, middle repetitive DNA family unique to human genomes. Genomic fingerprinting indicates that these DNAs vary from one individual to another and between tissues of the same individual. Sequence analysis reveals that they are AT-rich (76%) and contain many unusual sequence arrangements (palindromes, inverted and direct repeats). These sequence properties confer on the L2Hs elements the potential to fold into non-B-form structures, a characteristic of recombination hot spots. To test this hypothesis carbodiimide, osmium tetroxide and S$\sb1$ nuclease were used as single-strand specific probes to study a recombinant plasmid, pN6.4.39, containing a single L2Hs segment. Different forms of the plasmid substrate were analyzed, including linear molecules and circular forms of low, intermediate and high superhelical densities. In addition, plasmid DNA in growing E. coli cells were analyzed. Modified plasmid DNA was analyzed by primer extension in a sequencing-type reaction format. These studies demonstrate that the L2Hs sequences: (1) assume non-B-form structures both in vitro and in vivo, (2) map to predicted cruciform structures, (3) behave as C-type extrusion sequences, and (4) that these unusual DNA structures are dependent on plasmid superhelicity.
18
Kehoe, Katelin E. "Microarray analysis of Trichomonas vaginalis strains T1 and G3: Identifying genes that may contribute to virulence and metronidazole resistance." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/182.
Full textAbstract:
Trichomonas vaginalis is a protozoan parasite responsible for causing nearly eight million cases of Trichomoniasis every year in the United States. Trichomoniasis is often an asymptomatic infection, but in some cases it does lead to mild clinical manifestions. Trichomoniasis is easily treated with a single dose of Metronidazole. Drug resistance is not common, but it is on the rise. In addition to increasing rates of drug resistance, Trichomoniasis also poses a public health threat as it has been shown to increase the risk of HIV transmission. In order to combat this emerging public health threat, we must better understand the mechanism by which metronidazole exerts its action on T. vaginalis , as well as how the parasite has responded by evolving mechanisms of drug resistance on a molecular level. In order to investigate these questions, a microarray analysis of two distinct strains of T. vaginalis , one being more virulent and slightly less metronidazole sensitive, was performed. This allowed the identification of several genes that may play a role in virulence and drug susceptibility. Once these genes were identified, their differential regulation was further confirmed by Northern Blot analysis. One of these genes, Thioredoxin Reductase (TrxR) was then cloned and transfected into T. vaginalis . After confirming expression of the HA-tagged TrxR, the cell line was then used to determine the effect of over-expression of the gene on drug sensitivity. The metronidazole IC 50 for this cell line was compared to wild type cells. Additionally, immunostaining of the transfected cells was performed to determine the localization of the HA-tagged thioredoxin reductase. The results of this investigation provide further support for the role of TrxR in metronidazole activation as well as metronidazole sensitivity. Additionally, several other genes identified as differentially regulated may play a role in virulence, and should be targeted for further investigation.
19
Chuang, Tyler Casey. "Characterization of a family of cysteine rich proteins and development of a MaSp1 derived miniature fibroin." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/180.
Full textAbstract:
Spider silk displays a unique balance of high tensile strength and extensibility, making it one of the toughest materials on the planet. Dragline silk, also known as the lifeline of the spider, represents one of the best studied fiber types and many labs are attempting to produce synthetic dragline silk fibers for commercial applications. In these studies, we develop a minifibroin for expression studies in bacteria. Using recombinant DNA methodology and protein expression studies, we develop a natural minifibroin that contains the highly conserved N- and C-terminal domains, along with several internal block repeats of MaSp1. We also characterize a family of small cysteine-rich proteins (CRPs) and demonstrate that these factors are present within the spinning dope of the major ampullate gland using MS analysis. Biochemical studies and characterization of one of the family members, CRP1, demonstrate that this factor can self-polymerize into higher molecular weight complexes under oxidizing conditions, but can be converted into a monomeric species under reducing conditions. Self-polymerization of CRP1 is also shown to be independent of pH and salt concentration, two important chemical cues that help fibroin aggregation. Overall, our data demonstrate that the polymerization state of CRP1 is dependent upon redox state, suggesting that the redox environment during fiber extrusion may help regulate the oligomerization of CRP molecules during dragline silk production.
20
Aravena, Andrés. "Probabilistic and constraint based modelling to determine regulation events from heterogeneous biological data." Phd thesis, Université Rennes 1, 2013. http://tel.archives-ouvertes.fr/tel-00922346.
Full textAbstract:
Cette thèse propose une méthode pour construire des réseaux de régulation causales réalistes, qui a une taux de faux positifs inférieur aux méthodes traditionnelles. Cette approche consiste à intégrer des informa- tions hétérogènes à partir de deux types de prédictions de réseau pour déterminer une explication causale du gène observé co-expression. Ce processus d'intégration se modélise comme un problème d'optimisation combinatoire, de complexité NP-difficile. Nous introduisons une approche heuristique pour déterminer une solution approchée en un temps d'exécution pratique. Notre évaluation montre que, pour l'espèce modèle E. coli, le réseau de régulation résultant de l'application de cette méthode a une précision supérieure à celle construite avec des outils traditionnels. La bactérie Acidithiobacillus ferrooxidans présente des défis particu- liers pour la détermination expérimentale de son réseau de régulation. En utilisant les outils que nous avons développés, nous proposons un réseau de régulation putatif et analysons la pertinence de ces régulateurs centraux. Il s'agit de la quatrième contribution de cette thèse. Dans une deuxième partie de cette thèse, nous explorons la façon dont ces relations réglementaires se manifestent, en développant une méthode pour compléter un réseau de signalisation lié à la maladie d'Alzheimer. Enfin, nous abordons le problème ma- thématique de la conception de la sonde de puces à ADN. Nous concluons que, pour prévoir pleinement les dynamiques d'hybridation, nous avons besoin d' une fonction de l'énergie modifiée pour les structures secondaires des molécules d'ADN attaché surface et proposons un schéma pour la détermination de cette fonction.
21
Yun, Jeongfill. "Biochemical analysis of a potential drug target in the human protozoal pathogen Trichomonas vaginalis." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/192.
Full textAbstract:
Trichomonas vaginalis carries out unique mode of carbohydrate decarboxylation by an intracellular compartment, hydrogenosome. It was suggested that enzymes exclusively associated with hydrogenosome were responsible for activating metronidazole. This provoked researchers to target hydrogensosomal enzymes such as pyruvate: ferredoxin oxioreductase and ferredoxin, to treat trichomoniasis caused by T. vaginalis. Recent studies have shown alternative pathways responsible for activating metronidazole without the involvement of hydrogenosomal enzymes. This pathway requires the participation of thioredoxin and thioredoxin reductase. These enzymes are essential for T.vaginalis' survival as it is responsible for maintaining cell's redox system, as well as many other cellular processes. Targeting thioredoxin and thioredoxin reductase could be a novel drug target to treat metronidazole-resistant T. vaginalis. In this study, recombinant T. vaginalis TrxR and T. vaginalis Trx were produced to test its activity against known TrxR inhibitor, auranofin, using DTNB assay. Cell lysates of metronidazole susceptible and metronidazole resistant lines were also tested to see its activity against auranofin. Auranofin derivatives that were effective on E. histolytica recombinant TrxR were also tested to compare their effects on T. vaginalis recombinant TrxR. The results showed that auranofin effectively inhibits recombinant TvTrxR. Aurnofin derivatives were also shown to have different effects on inhibiting the activity of recombinant TvTrxR. It is known that auranofin also targets mammalian TrxR. With the degree of different inhibitions observed between auranofin derivatives, it opened the possibility of developing an auranofin derivative that can specifically targets thioredoxin reductase of parasitic protozoan, T. vaginalis.
22
Tau, Kimberly R. "Molecular and Cellular Analysis of Chlamydia Trachomatis: Persistence and Reactivation." Digital Commons @ East Tennessee State University, 1992. https://dc.etsu.edu/etd/2804.
Full textAbstract:
Chlamydia trachomatis (CT) is the most prevalent sexually-transmitted infection in the United States. It has been suggested that CT infections can become latent. This has not been substantiated. CT persistence was examined at the molecular and cellular level in vitro and in vivo. Penicillin treatment of CT in vitro results in abnormal inclusions and reduced recovery of infectious CT. Penicillin did not inhibit initial stages of infection, but did downregulate CT rRNA levels after 25 hours post-inoculation (p.i.). DNA amplification was employed to differentiate between a resolved infection and a persistent one. Utilizing a primer pair that amplified a 144 bp fragment in the CT MOMP gene, CT-persistently-infected McCoy cells maintained in penicillin medium were examined. Though undetectable by other assay methods, these cells harbored the CT genome for 18 passages. Removal of penicillin 1, 3 or 6 passages p.i. and subsequent cultivation in permissive medium resulted in "recovery" to productive infection. Removal of penicillin at later passages resulted in low level inclusion formation but no infectious progeny. Penicillin treatment in vitro resulted in a persistent infection undetectable by most methods. Female C$\sb3$H/HeNCRL mice were inoculated with CT intrauterinely and intravaginally in two separate experiments. In one, CT infection was established in untreated and Depo-Provera (DP)-pretreated mice. DP pretreatment enhanced vaginal shedding of infectious CT. A negative vaginal culture did not correlate with elimination of CT from the upper tract. In the second, penicillin therapy did not halt vaginal CT shedding, however, it reduced frequency of recurrent vaginal CT shedding. To examine reactivation, culture-negative mice ($\geq$2 successive vaginal cultures) were injected with cortisone-acetate (CA) or DP; mice from same subpopulation injected with saline served as controls. Transient vaginal CT shedding was reactivated in penicillin-treated mice (14% CA-injected), and in unmedicated mice (28% CA-injected, 33% DP-injected). Saline injection did not reactivate vaginal CT shedding. At time of sacrifice (16 or 22 weeks p.i.) no infectious CT was detected in upper tract tissues, although tissue damage was observed in most mice (70-71%). It is unknown if these mice harbored a persistent infection undetectable by culture. Further work utilizing molecular techniques is needed to resolve this question.
23
Ahmed, Mohammed Ismail. "Morphological, ecological and molecular examination of the seacucumber species along the Red Sea coast of Egypt and Gulf of Aqaba : with the investigation of the possibility of using DNA barcoding technique as a standard method for seacucumber ID." Thesis, University of Hull, 2009. http://hydra.hull.ac.uk/resources/hull:2413.
Full textAbstract:
In this study the ecology, biology and classification of the sea cucumber species of the Red Sea coast of Egypt and Gulf of Aqaba were examined in order to resolve some of the long standing question on the identification and classification of sea cucumber.The introduction of the new barcoding technique as a tool for sea cucumber identification was also tested in this study in order to assess its accuracy and potential of the technique in identifying sea cucumber individuals. A total of 18 different (species) of sea cucumber were collected from the Egyptian coast and examined for both morphological and molecular characteristics. One new species of sea cucumber were identified from the Egyptian coast of the Red Sea (Actinopyga.sp.nov). Cryptic species complex were also identified for the Holothuria atra population in the Red Sea using the molecular analysis of the mitochondrial COI gene.In this study another experiment were conducted in order to identify sea cucumber species from cooked or dried materials using the molecular techniques. As well as testing the possibility of using the DNA barcoding technique in order to identify badly/long period preserved museum specimens in order to try to identify the unknown specimens in the natural history museums around the globe.The use of the molecular DNA barcoding technique proves to be a good reliable method for sea cucumber ID; the technique was capable of resolving some of the standing taxonomic problems including the Holothuria fuscogilva /Holothuria nobilis species complex and the Pearsonthuria graeffei. The results also raised some questions about the classification of the genus Bohadchia and the Actinopyga crassa species in the Red Sea.
24
Lu, Daniel Kee. "The Rad51 family of proteins: Interactions, vitamin D, and implications in head and neck cancer." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/191.
Full textAbstract:
Protection of the genome from carcinogenic consequences of DNA double-strand breaks (DSBs) is accomplished through the pathways of non-homologous end-joining (NHEJ) or homologous recombinational repair. Five human proteins with homology to Rad51 known as the Rad51 paralogs, Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3, whose loss of function in cell lines leads to high chromosomal instability. Previous studies have shown Rad51C participate in two paralog protein complexes, one containing Rad51B, Rad51C, Rad51D and XRCC2 (BCDX2) and the other containing only Rad51C and XRCC3 (CX3). However, the only structural data available is the crystal structure of RecA, the bacterial homolog, the determination of the N-terminus of human Rad51 by NMR, and the crystal structure of Pyroccocus furious Rad51. Currently the Alvinlla pompejana Rad51C has been cloned, expressed and is currently being crystallized in the Tainer laboratory (UC Berkeley) since the human Rad51C protein has proven too difficult to be utilized. To test functional association of Hs Rad51B and Hs XRCC3 to Ap Rad51C. The human proteins were heterologously expressed in Pichia pastoris and the other expressed in E. coli. The proteins were extracted and interaction was tested through co-immunoprecipitation. Initial results depict weak binding or an unstable interaction between Hs Rad51B and Ap Rad51C. Hs XRCC3 and Ap Rad51C interaction remains unclear and requires further testing. Additionally, we have utilized a cellular model of HNSCC to identify whether the down-regulation of Rad51 after application of VD 3 is concomitant with the down-regulation of NBS1. NBS1 is a DNA repair protein involved in both pathways of DNA double-strand break repair, non-homologous end-joining and homologous recombinational repair. It has recently been demonstrated that NBS1 binds to Rad51 aiding in its localization to sites of DNA damage. VD 3 is a potential chemopreventive agent in the treatment of head and neck cancer. For the in vitro model Rad51 and NBS1 protein were both extracted from SCC25 and MCF-7 cancer cell lines were treated with 100 nM of VD 3 . For the in vivo model hamsters cheek pouch tissue sections with VD 3 treated and DMBA over the course of 14 weeks were used. Rad51 and NBS1 staining is restricted to the nuclei of the basal cell layer of the epithelium in VD 3 treated animals as compared to untreated controls where staining is evident throughout the dysplastic epithelium and is not restricted to nuclei. Unlike the western blot data of Rad51 that shows similar downregulation as the immunocytochemistry, the western blot analysis of NBS1 is unclear. However, the immunocytochemistry suggests that NBS1 is also downregulated by VD 3 in vivo, and therefore, it may be implied that both the HRR and NHEJ pathways are involved in the cellular effects of VD 3 in HNSCC.
25
Hanson-Smith, Victor 1981. "Error and Uncertainty in Computational Phylogenetics." Thesis, University of Oregon, 2011. http://hdl.handle.net/1794/12151.
Full textAbstract:
xi, 119 p. : ill. (some col.)The evolutionary history of protein families can be difficult to study because necessary ancestral molecules are often unavailable for direct observation. As an alternative, the field of computational phylogenetics has developed statistical methods to infer the evolutionary relationships among extant molecular sequences and their ancestral sequences. Typically, the methods of computational phylogenetic inference and ancestral sequence reconstruction are combined with other non-computational techniques in a larger analysis pipeline to study the inferred forms and functions of ancient molecules. Two big problems surrounding this analysis pipeline are computational error and statistical uncertainty. In this dissertation, I use simulations and analysis of empirical systems to show that phylogenetic error can be reduced by using an alternative search heuristic. I then use similar methods to reveal the relationship between phylogenetic uncertainty and the accuracy of ancestral sequence reconstruction. Finally, I provide a case-study of a molecular machine in yeast, to demonstrate all stages of the analysis pipeline. This dissertation includes previously published co-authored material.
Committee in charge: John Conery， Chair； Daniel Lowd， Member； Sara Douglas， Member； Joseph W. Thornton， Outside Member
26
Kilaru, Aruna, Pamela Tamura, Giorgis Isaac, Ruth Welti, Barney J. Venables, Edith Seier, and Kent D. Chapman. "Lipidomic Analysis of N-Acylphosphatidylethanolamine Molecular Species in Arabidopsis Suggests Feedback Regulation by N-Acylethanolamines." Digital Commons @ East Tennessee State University, 2012. https://dc.etsu.edu/etsu-works/4755.
Full textAbstract:
N-Acylphosphatidylethanolamine (NAPE) and its hydrolysis product, N-acylethanolamine (NAE), are minor but ubiquitous lipids in multicellular eukaryotes. Various physiological processes are severely affected by altering the expression of fatty acid amide hydrolase (FAAH), an NAE-hydrolyzing enzyme. To determine the effect of altered FAAH activity on NAPE molecular species composition, NAE metabolism, and general membrane lipid metabolism, quantitative profiles of NAPEs, NAEs, galactolipids, and major and minor phospholipids for FAAH mutants of Arabidopsis were determined. The NAPE molecular species content was dramatically affected by reduced FAAH activity and elevated NAE content in faah knockouts, increasing by as much as 36-fold, far more than the NAE content, suggesting negative feedback regulation of phospholipase D-mediated NAPE hydrolysis by NAE. The N-acyl composition of NAPE remained similar to that of NAE, suggesting that the NAPE precursor pool largely determines NAE composition. Exogenous NAE 12:0 treatment elevated endogenous polyunsaturated NAE and NAPE levels in seedlings; NAE levels were increased more in faah knockouts than in wild-type or FAAH overexpressors. Treated seedlings with elevated NAE and NAPE levels showed impaired growth and reduced galactolipid synthesis by the “prokaryotic” (i.e., plastidic), but not the “eukaryotic” (i.e., extraplastidic), pathway. Overall, our data provide new insights into the regulation of NAPE–NAE metabolism and coordination of membrane lipid metabolism and seedling development.
27
Guziolowski, Carito. "Analysis of Large-Scale Biological Networks with Constraint-Based Approaches over Static Models." Phd thesis, Université Rennes 1, 2010. http://tel.archives-ouvertes.fr/tel-00541903.
Full text
28
Carr, Brandon W. "Characterization of zebrafish zipper-interacting protein kinase." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/178.
Full textAbstract:
Zipper-Interacting Protein Kinase (ZIPK) is a known modulator of actin-myosin contractility in vertebrate species. Interestingly, rodent and mouse ZIPK has undergone a divergence in regulation in comparison to other vertebrate orthologs including human. Whereas the human ortholog of ZIPK requires phosphorylation of residues TT299/300 for nuclear exit, rodents and mouse require interaction with another protein termed PAR-4. In this project we completed several experiments to examine zebrafish ZIPK in development and its effect on acto-myosin contractility. It was found that zebrafish ZIPK was expressed ubiquitously in maternal stages. In zygotic stages, ZIPK expression dropped dramatically and localized to the anterior portions of the embryo. Zebrafish and human ZIPK, but not rodent ZIPK were able to increase stress fiber formation and myosin light chain-2 (MLC-2) phosphorylation in vitro. Human and zebrafish ZIPK underwent nucleocytoplasmic shuttling without PAR-4 interaction, unlike rodent ZIPK, which required PAR-4 for nuclear exit. Unlike human ZIPK, zebrafish ZIPK TT299/300AA mutants were able to undergo shuttling. Similar to human ZIPK, catalytic mutations to zebrafish ZIPK abolished or dramatically reduced activity. Through these experiments we were able to show human and zebrafish ZIPK homologs function and are regulated similarly, while the rodent ZIPK was much more unique. Although the exhibited phenotypes were similar between human and zebrafish ZIPK orthologs, the mechanism of regulation is not completely conserved.
29
Lin, Albert. "Characterizing small molecular weight proteins from Latrodectus hesperus dragline and tubuliform silks." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/184.
Full textAbstract:
Spiders produce a diverse number of silk proteins that are well-known for their extraordinary mechanical and biological properties. Dragline silk has been the most prominent focus of research because of its exceptional high tensile strength and extensibility. In our research, we have focused on the characterization of small molecular weight proteins found within dragline and tubuliform silks. Within the black widow spider, Lactrodectus hesperus, these proteins have been named Cysteine-Rich Protein (CRP) and determined to be a family of five individual proteins. The small protein identified within the tubuliform silks has been named Egg Case Protein 3 (ECP-3). In this study, recombinant expression of ECP-3 in the pET-19b-SUMO vector was to facilitate purification and development of an immunological reagent. Using western blot analysis, we have demonstrated that ECP-3 is efficiently expressed in bacteria. We also investigated CRP1 protein and its ability to bind MaSp1 components using pull down assays to determine potential interactions. No substantial biochemical evidence was produced to demonstrate protein-protein interactions between the two. Additionally, we show that using RT-PCR analysis from mRNAs collected from the major ampullate gland that transcript levels for CRP-family members from non-silked and a silked spider are different. CRP2 and CRP4 mRNA levels were shown to increase upon silking. Overall, the major findings of this thesis involved characterizing the ECP-3 protein found within tubuliform silks as well as determining the expression patterns for CRP-family members.
30
Dornbush, Padraick J. "Compound discovery and expression of a putative cathepsin D-like protease in Trichomonas vaginalis." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/181.
Full textAbstract:
Trichomonas vaginalis is a sexually-transmitted parasite that is the causative agent in the disease trichomoniasis. Resistance to the only FDA-approved medication to this disease, metronidazole, has been on the increase giving rise to the need for finding targets for new inhibitors to exploit. New inhibitors can target enzymes such as 4-coumarate:CoA ligase and S-adenosylhomocysteine hydrolase. Another potential target is a cathepsin D-like protease found in T. vaginalis . This aspartic protease in humans is responsible for degrading proteins in the lysosome, and degrading hemoglobin in P. falciparum as the homologue plasmepsin. Searching the gene database, only one cathepsin-D like protease was discovered throughout the organism's genome. Utilizing RT-PCR, this gene is found to be expressed in two different strains of the organism. Transfection of an epitope-tagged version of this cathepsin D-like protease into T. vaginalis was accomplished, and subsequent immunofluorescence of this tagged version shows it to be localized in intracellular compartments, which can be colocalized using the SNARE and VAMP proteins found in T. vaginalis .
31
Snowden, Kimberley Cathryn. "The molecular response of wheat roots to aluminium stress." Thesis, University of Auckland, 1994. http://hdl.handle.net/2292/1967.
Full textAbstract:
Aluminium (Al) toxicity to plants is a significant problem, limiting agricultural production in up to 40% of the world's arable soils. In spite of a large amount of research, there is still no consensus on the physiological mechanisms of Al toxicity in plants. In addition, very little is known about the molecular response of plants to Al stress. This body of research was aimed at identifying the changes in gene expression that occurred in the root tips of plants that had been stressed with Al. A cDNA library made from the root tips of Al-treated wheat (Triticum aestivum L., cultivar Warigal) plants was differentially screened to identify clones whose expression was induced by Al stress. Seven cDNA clones, representing five different genes were identified as being induced in the presence of Al. Initial sequencing and northern analysis revealed that none of the clones isolated were full-length, and that some contained multiple cloning adaptors at their 5' ends. A new cDNA library was then constructed from the root tips of Al-treated Warigal plants, and homologues to each of the original five genes were isolated. These five clones were named wali1 to wali5 (for wheat aluminium induced). Northern analysis showed that wali1, -3 and -5 were induced 24 to 96 h after Al treatment, and their expression declined when the Al was removed. wali4 had a similar pattern of expression with a transient increase in expression also observed after 0.5 h of Al stress. Each of these four genes was induced by inhibitory concentrations of Al in two wheat cultivars - Warigal, an Al-sensitive cultivar, and Waalt, an Al-tolerant cultivar, - and also in two inbred lines of wheat, RR (Al-tolerant) and SS (Al-sensitive). The fifth gene (wali2) had a bimodal pattern of induction, and was induced by Al only in the Al-sensitive Warigal and the Al-tolerant RR. The nucleotide sequence of each of the wali clones was determined, and the databases were searched for homologous sequences. Wali1 was found to be homologous to a group of metallothionein-like proteins (MLPs) from plants, and wali4 was homologous to phenylalanine ammonia-lyase (PAL). wali3 and wali5 encode related, cysteine-rich proteins with homology to Bowman-Birk proteinase inhibitors, and wali2 encodes a novel protein with a repeating motif of cysteine amino acids. The induction of the wali genes was investigated in response to a number of other stresses through northern analysis. The expression of wali1, -3, -4 and -5 was induced in root tips of wheat after 2 d treatments with toxic levels of all other metals tested (Cd, Fe, Zn, Cu, Ga, In and La). The expression levels of wali1, -3, -4, and -5 also increased in the root tips of plants grown in the presence of low levels of Ca (10μM). The transcript levels of wali1, -3 and -5 increased in wounded leaf and root tissue, whereas the transcript levels of wali4 increased only in wounded leaves. The expression of wali2 was greatly reduced by low concentrations of Ca, and showed no induction, or a variable response with most of the other treatments. The site of expression of wali1, -2, -3 and -5 in root tips (and wali1 also in leaf tissue) was identified using in situ hybridisation. Wali1 was expressed predominantly in the meristematic tissue of the root tip, while wali3 and wali5 were expressed predominantly in the cortical tissue of the root. wali2 expression was detected primarily in the epidermis and root cap. Some changes in the site of expression of these genes were evident in the roots of Al-treated plants. In leaf tissue, wali1 expression was found in the mesophyl1 layer of cells. The coding sequences for wali1,-2,-3 and -5 were each cloned into the bacterial expression vector pGEX-2T. The resultant fusion proteins between glutathione S-transferase (GST) and the walis were then successfully purified from E coli. Antibodies were made to the wali1-GST fusion protein and purified by immunoaffinity chromatography. However, when used in western analysis, no specific bands corresponding to the native wali1 protein were identified. The wali2-GST protein was used in a south-western procedure to determine if the protein was capable of binding DNA, but no DNA binding to this protein was detected under the conditions tested. The wali3 and wali5 fusion proteins were tested in proteinase inhibitor assays, where no inhibition of either trypsin or chymotrypsin was detected. It is possible that the native wali3 and wali5 proteins may not function as proteinase inhibitors, or that the lack of activity detected for the fusion proteins may be due to incorrect folding or processing in the bacterial system. This research constitutes the first identification of plant genes whose expression is increased by Al stress. The genes identified are also induced in response to other environmental and nutrient stresses, indicating that they form part of the plant's general response to stress.Appendix 4 restricted.
32
Podivinsky, Ellen. "Molecular studies on actinidin, a cysteine protease from kiwifruit." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/2001.
Full textAbstract:
Research in this thesis describes the characterisation of mRNA sequences coding for actinidin, a cysteine protease found in abundance in the fruit of kiwifruit (Actinidia deliciosa). The first step in the characterisation required the isolation of mRNA from ripe kiwifruit tissue. The suitability of a number of RNA extraction procedures was investigated. The method finally adopted differed from that used for unripe fruit tissue, and was chosen as a result of the nature of the polysaccharide that contaminated nucleic acids prepared from extracts of kiwifruit fruit tissue. RNA extracted from ripe fruit was used to synthesis a partial cDNA library and clones for actinidin were isolated. A number of these cDNA clones were sequenced; three clones were almost full-length. The actinidin cDNA clones obtained fall into two broad sequence classes. The majority of them encode acidic proteins (pI˜4.7), with 97% homology to the published amino acid sequence of actinidin. The second class encode basic proteins (pI˜8.1), with 83% homology to the published amino acid sequence of actinidin. Both classes of actinidin cDNA sequence encode zymogens, which contain N- and C-terminal extensions not present in the mature form of the enzyme. The N-terminal extension of both sequence classes includes a putative signal peptide. Northern hybridization analysis was used to investigate the tissue specificity of actinidin mRNA expression, and the expression of mRNA for the two actinidin sequence classes during fruit ripening. Both actinidin sequence classes were expressed differentially during the latter stages of kiwifruit fruit development and through post-harvest fruit ripening. The expression of both sequence classes increased from just prior to fruit maturity through ripening and reached a maximum as fruit attained the stage of 'eating' ripeness. The level of expression of the sequences encoding acidic actinidin reached a plateau at this point, while the expression of the sequence encoding basic actinidin appeared to decrease slightly as fruit continued to ripen. The sequences encoding acidic actinidin were expressed during ripening at a much higher level than those encoding basic actinidin. No actinidin mRNA was detected in other tissues except for very low levels of the acidic form in kiwifruit leaf, and low levels of the basic form in senescing petals. A full-length, acidic, actinidin cDNA sequence was introduced into tobacco (Nicotiana tabacum) plants via Agrobacterium tumefaciens-mediated transformation. Using the binary vector pGA643, the sequence was introduced in both the sense and antisense orientation relative to the cauliflower mosaic virus 35S promoter and transgenic plants were obtained for both sequence orientations. The presence of the T-DNA cassette (containing the actinidin sequence) in the plant genomes was determined using PCR analysis, and confirmed by Southern hybridization. A number of the transgenic plants contained multiple insertions of the actinidin sequence, and most plants contained at least one intact copy of the T-DNA cassette. The transcription of the introduced actinidin sequence was investigated by Northern hybridization analysis. All of the plants containing actinidin in the sense orientation, and some of those incorporating the antisense construct, transcribed the actinidin sequence. Attempts to detect actinidin protein in the transgenic plants were unsuccessful. Acidic actinidin was identified as one of the most abundant bands in the total protein profile from ripe kiwifruit fruit tissue. The identity of the protein was confirmed by N-terminal sequence analysis. The electrophoretic mobility of actinidin, both in the total cell homogenate and when partially purified, suggested that the first step in post-translational processing of the zymogen may be the removal of the N-terminal extension. Actinidin was also partially purified and used to raise antibodies. Poor specificity of the antibody for actinidin led to preliminary evidence for the glycosylation of actinidin.
33
Mosaliganti, Kishore Rao. "Microscopy Image Analysis Algorithms for Biological Microstructure Characterization." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1211390127.
Full text
34
Gill, Lyndell R. "Moraxella (Branhamella) Catarrhalis: A Molecular Epidemiology Study." Digital Commons @ East Tennessee State University, 1995. https://dc.etsu.edu/etd/2684.
Full textAbstract:
Moraxella (Branhamella) catarrhalis is the third-most-frequently isolated microorganism associated with acute exacerbations of chronic bronchitis in patients during their stay at the Mountain Home VA Medical Center (MHVAMC). In order to develop a practical, epidemiologically-meaningful typing method for M. (B.) catarrhalis, we tested two methods based on analysis of chromosomal DNA for typeability, reproducibility, and ability to differentiate between unrelated strains (discriminatory power, D). M. (B.) catarrhalis isolants from MHVAMC from 7/1/87-6/30/88 were grown overnight in broth and embedded in agarose. DNA was isolated by standard methods. The DNA was subjected to: (1) restriction endonuclease digestion (with either Bgl II or Pme I) followed by pulsed-field gel electrophoresis (PFGE) and (2) restriction endonuclease digestion (with Hae III), followed by horizontal gel electrophoresis, Southern transfer and hybridization with a M. (B.) catarrhalis-specific DNA probe (M46). Reliable and reproducible patterns were produced from 144 of 159 isolants (91%) using Hae III, 155 of 159 (97%) using Pme I, and all isolants using Bgl II. Three clusters of isolants, Groups A (n = 18), B (n = 18), and C (n = 12) were detected. Within each group, isolants were identical by all typing methods tested. Chart review revealed no apparent epidemiologic link for Group A, while in Group B, 16 of 18 patients were housed on two wards, and in Group C, all cases occurred within two months, suggesting epidemiologic links within Groups B and C. Comparisons of results from isolants from various wards and isolants from outpatients were used to determine D of each method. Digestion with Pme I followed by PFGE was the most discriminating technique (D = 0.978) followed by Bgl II with PFGE (D = 0.962), then M46 probe hybridization (D = 0.929). The restriction endonucleases Pme I and Bgl II were highly discriminating and useful in the epidemiologic typing of M. (B.) catarrhalis. While useful, the M46 probe following Hae III digestion was not as discriminating.
35
Weaver, Jun Eon. "Characterizing phenotypes of Pichia pastoris mutants that show enhanced secretion of recombinant proteins." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/188.
Full textAbstract:
In effort to understand and isolate genes that are associated with protein secretion, the Lin-Cereghino laboratory at University of the Pacific created mutant strains of Pichia pastoris using the restriction enzyme mediated integration method. The mutants exhibited an unusual ability to supersecrete beta-galactosidase, due to the effects of a randomly disrupted gene by pREMI-Z. To learn more about the novel effects of the gene disruption, nine beta-galactosidase supersecreters ( bgs ) have been characterized for their phenotypes such as growth rate, cell wall integrity, and ability to produce and secrete various types of recombinant proteins. The mutants showed various population doubling times, which ranged from 1.7 to 2.4 hours. Generally, the mutants with severely diminished growth rates had much lower secretion of the reporter proteins. The mutants also showed different levels of cell wall (osmotic) defect, indicated by moderate to severe leakage of alkaline phosphatase from the vacuole. It was revealed that the cell wall defect was not necessarily associated with increased protein secretion, which suggests that the cell wall may not be a limiting barrier for the secretion of most reporter proteins. The result of the reporter study suggests that the secretion phenotypes of bgs mutants were protein specific and likely to be dependent upon the structure of the secreted protein rather than the size.
36
Spiers, Andrew J. (Andrew Julian). "Molecular and genetic analysis of RepA from the P307 RepFIB replicon." Thesis, University of Auckland, 1992. http://hdl.handle.net/2292/2044.
Full textAbstract:
The work in this Thesis concerns the replication control system of the P307 plasmid RepFIB replicon. The basic replicon occupies ≈ 1.6kb of DNA and contains a single large open reading frame (repA) flanked on either side by a series DNA repeat elements. The organisational structure of fie replicon has placed RepFIB into the Step function class of replicons. The placement of RepFIB within this group, as well as a strong homology between RepFIB and mini-P1, has resulted in a series of predictions concerning the control elements of RepFIB replication. The aim of this work was to test some of these predictions and to characterise the fundamental control elements utilised by the replicon. This Thesis describes three different active promoter elements found embedded within the repeat elements flanking repA. Although the functional significance of two of the promoter is unknown (orip and EFp), the third is responsible for the expression of RepA and has been designated 'repAp'. All three promoters are sensitive to RepA in trans, demonstrating that repA is autoregulated and that RepA is a DNA-binding protein capable of recognising copies of the repeat elements. RepA DNA-binding has also been demonstrated in vitro using a modification of the Western analysis technique (referred to a 'Western-DNA') in order to complement the in vivo experimental results. Although the coding region of repA had been determined in earlier work, the identification of the translational start codon was uncertain. This uncertainty has been resolved by limited N-terminal sequence analysis of a RepA:β-galactosidase fusion protein which has demonstrated that translation begins from a CTG codon located upstream of the predicted start sites. Finally, a series of genetic experiments have been used to determine the functional significance of RepA binding to the repeat elements. The repeat group upstream of repA are involved in autoregulation and also form part of the origin of replication, whilst the downstream repeats appear to be involved in the sensing and setting of plasmid copy number. Although the work presented in this Thesis does not directly test the applicability for RepFIB of various control models proposed to explain the behaviour of Step function replicons, the nature and type of control elements identified in RepFIB support the placement of RepFIB within the Step function class. As a result of this work, it is clear that RepFIB is confronted by the same kind of control paradox faced by replicons such as mini-P1 and mini-F. All three replicons use autoregulation and titration to control the supply of initiator protein required for replication. However, concurrent autoregulation and titration appear to be incompatible in current control models, and the identification of both mechanisms in these replicons has lead to a control paradox. Some of the results presented here suggest potentially valuable avenues of future research which may help resolve the paradox faced by RepFIB and other Step function replicons.
37
Zhou, Yinhan. "Expression and functional characterization of the recombinant spider protein GW2 in yeast Pichia pastoris." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/193.
Full textAbstract:
The chairperson of the candidate's dissertation committee is responsible for securing the signature of each committee member and the grade, which she/he wishes to assign, to be entered in the appropriate spaces below. Most dissertations are graded on a pass (P) or no credit (NC) basis. The grades assigned need not be the same for all committee members. The exact title of the dissertation must appear in the space indicated for that purpose. The undersigned confirm that we have reviewed this document and examined the student regarding its content. We agree that this document conforms to acceptable standards of scholarly presentation in scope and quality and that the attainments of this student are such that we recommend the conferral of
38
Choi, Eva. "Detection And Characterization of Insecticide Resistance Mechanisms in Culex Tarsalis." Scholarly Commons, 2016. https://scholarlycommons.pacific.edu/uop_etds/167.
Full textAbstract:
Insecticide resistance in disease-transmitting arthropods has become a serious hindrance for successful vector control. Mosquitoes, in particular, are notorious vectors of potentially deadly diseases like malaria, dengue fever, and West Nile virus. Anopheles gambiae and Culex quinquefasciatus are just two examples of mosquito vectors that possess genetic mutations (denoted kdr and ace-1 ) and/or enhanced detoxifying enzymes (oxidases, esterases, and glutathione-s-transferases) that confer insecticide resistance. Culex tarsalis, a primary vector for West Nile virus among other arboviruses in Northern California, is a target for insecticide application and is under constant insecticide pressure, making it likely to adapt resistance mechanisms like kdr or ace-1 or increased detoxifying enzymes. Culex tarsalis adult females were collected from Yolo and Sutter counties. A bottle bioassay was completed to determine prevalence of resistance to Sumithrin (a pyrethroid; N=217) and Naled (an organophosphate; N=154). A susceptible lab-reared colony was used for comparison. Microplate assays were completed to investigate elevated levels of detoxification enzymes present as well as AChE. PCR was used to amplify the VGSC and ace-1 genes. Amplicons were sequenced and aligned to determine if mutations were present. No evidence of the ace-1 mutation was found in any mosquitoes, but the kdr mutation was seen in all semi-resistant and resistant individuals exposed to Sumithrin. Microplate data revealed significant differences between certain detoxifying enzymes within mosquitoes collected from Sutter and Yolo Counties exposed to both Sumithrin and Naled. The data obtained from this study suggests that resistance to Sumithrin in both populations is carried out by both metabolic and target site insensitivity, while resistance to Naled is caused by metabolic resistance only.
39
Andersen, Ryan Otto 1979. "Dissecting Stem Cell Self-Renewal: The Roles of Mitotic Kinases in Drosophila Neuroblast Asymmetric Cell Division." Thesis, University of Oregon, 2011. http://hdl.handle.net/1794/12065.
Full textAbstract:
x, 60 p. : ill. (some col.)Regulation of stem cell self-renewal versus differentiation is critical for embryonic development and adult tissue homeostasis. Drosophila larval neuroblasts divide asymmetrically to self-renew and are a model system for studying stem cell self-renewal. Here, we identify two proteins involved in distinct steps of the cell cycle that regulate neuroblast self-renewal. We first describe three mutations showing increased brain neuroblast numbers that map to the aurora-A gene, which encodes a conserved kinase implicated in human cancer. Clonal analysis and time-lapse imaging in aurora-A mutants show single neuroblasts generate multiple neuroblasts (ectopic self-renewal). This phenotype is due to two independent neuroblast defects: abnormal atypical protein kinase C (aPKC)/Numb cortical polarity and failure to align the mitotic spindle with the cortical polarity axis. numb mutant clones have ectopic neuroblasts, and Numb overexpression partially suppresses aurora-A neuroblast overgrowth (but not spindle misalignment). We conclude that Aurora-A and Numb are novel inhibitors of neuroblast self-renewal and that spindle orientation regulates neuroblast self-renewal. We next identified an sgt1 (suppressor-of-G2-allele-of-skp1 ) mutant that had fewer neuroblasts. We found that sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of "neuroblast tumors." Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1, or AMPKα all show a similar apical cortical phenotype (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. We propose that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors. This dissertation includes published and unpublished co-authored material.
Committee in charge: Dr. Bruce Bowerman, Chair; Dr. Chris Doe, Advisor; Dr. Tory Herman, Member; Dr. Judith Eisen, Member; Dr. Kenneth Prehoda, Outside Member
40
Mendoza, J. Alexander Hoang. "Development of a codon-optimized Latrodectus hesperus MaSp1 synthetic gene for bacterial protein expression using a seamless cloning strategy." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/174.
Full textAbstract:
Spider silk has outstanding mechanical properties, displaying high tensile strength and extensibility. The unique combination of strength and great extensibility make it one of the toughest materials in the world. Of the seven different spider silks, dragline silk, the lifeline silk of the spider, represents one of the most renowned fiber types that has extraordinary properties. As a result, many labs across the globe are racing to manufacture synthetic dragline silk fibers. With the production of synthetic dragline silk fibers, there are unlimited commercial applications. In this study, we developed several codon-optimized MaSp1 minifibroin constructs for recombinant protein expression in bacteria. These recombinant MaSp1 minifibroin constructs were engineered to contain the N-terminal domain (NTD), different copies of internal block repeats (ranging from 2 to 64 copies of 35 amino acid blocks), and the C-terminal domain (CTD). The NTD and CTDs were derived from the natural cDNA sequences of black widow spiders, while the internal block repeats were generated from synthetic DNA fragments that were codon-optimized for expression in Escherichia coli . Different numbers of internal block repeats were created using a specialized seamless cloning strategy. By applying this seamless cloning strategy, we successfully multimerized MaSp1 block repeats that approach the natural fibroin size. Moreover, through the construction of a customized NTD-CTD spidroin construct, multimerized block repeats from any fibroin can be rapidly inserted to facilitate minifibroin protein expression in bacteria. Overall, this strategy as well as the created vectors, should help advance the silk community in the production of synthetic silk fibers that have properties that more closely resemble natural fibers.
41
Wang, Xiaodong. "Studies of CyP40 and β-tubulin in the Arnt-dependent signaling pathways." Scholarly Commons, 2006. https://scholarlycommons.pacific.edu/uop_etds/2634.
Full textAbstract:
Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner Ah receptor nuclear translocator (Arnt). The AhR/Arnt heterodimer binds to the enhancer element DRE to regulate target gene expression. It is known that the formation of the ligand-dependent AhR/Arnt/DRE complex requires protein factors in vitro. The first aim is to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR/Arnt/DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CYP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR-Arnt-DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex. The second aim is to determine the role of β-tubulin in Amt-dependent signaling pathways. From the insect Sf9 cytosol, β-tubulin enriched fraction (F5) was isolated which suppresses the AhR/Arnt/DRE complex formation in a gel shift assay. Tubulin enriched from pig brain had a similar inhibition of the AhR gel shift complex, suggesting that β-tubulin in F5 is likely responsible for the action. Using the TALON resin, β-tubulin was co-precipitated with the baculovirus 6His-Arnt, showing that β-tubulin interacts with Arnt. β-tubulin was examined to decide its role in the hypoxia inducible factor-1α (HIF-1α) signaling which is also Arnt-dependent. Gel shift data using HIF-1α and Arnt showed that F5 suppressed the formation of the HIF-1α/Arnt/HRE complex. Subsequently the Sf9 β-tubulin was cloned and about 95% of its full-length sequence was identified. The amino acid sequence of Sf9 β-tubulin shares high sequence identity with human β-tubulin. Upon transient transfection of a plasmid containing a human β-tubulin cDNA into MGF7 or Hep3B cells, the HRE-driven luciferase activity was clearly suppressed. In conclusion, we have evidence supporting that β-tubulin inhibits the Arnt-dependent signaling and the mechanism may involve the interaction between Arnt and β-tubulin.
42
Albertini, Niccolo'. "New approaches to scientific visualization in virtual immersive environments for science and humanities." Doctoral thesis, Scuola Normale Superiore, 2018. http://hdl.handle.net/11384/85998.
Full textAbstract:
Virtual environments and 3D visualization have introduced a new element in the process of data analysis, allowing the researcher to observe and perceive information in a more natural way.
The structure of a molecule or even only the 3D representation of a physical quantity are examples of how a graphical representation can help understanding the data itself and the subsequent and eventual communication to the public.
The use of scientific data is not only a visual act.
The fundamental element is to allow the researcher to understand and manipulate information, testing hypotheses and looking for significant results, and that’s the point where the method of interaction becomes critical; with the use of existing human-computer interface systems it all becomes intuitive, functional and simple, allowing a more natural approach.
Virtual Reality provides advanced visualization and data manipulation solutions applicable to multiple disciplines related to each other as chemistry and cultural heritage studies.
The advent of new mass technologies from smartphones to the Head Mounted Display has allowed to see and manipulate interactive graphic information even for those who practice field research, providing a valuable help to understand and use the data.
This thesis will show the fields in which visualization techniques can be applied, particularly in the field of chemistry and cultural goods, referring to case studies developed in recent years.
43
Andorf, Sandra. "A systems biological approach towards the molecular basis of heterosis in Arabidopsis thaliana." Phd thesis, Universität Potsdam, 2011. http://opus.kobv.de/ubp/volltexte/2011/5117/.
Full textAbstract:
Heterosis is defined as the superiority in performance of heterozygous genotypes compared to their corresponding genetically different homozygous parents. This phenomenon is already known since the beginning of the last century and it has been widely used in plant breeding, but the underlying genetic and molecular mechanisms are not well understood.
In this work, a systems biological approach based on molecular network structures is proposed to contribute to the understanding of heterosis.
Hybrids are likely to contain additional regulatory possibilities compared to their homozygous parents and, therefore, they may be able to correctly respond to a higher number of environmental challenges, which leads to a higher adaptability and, thus, the heterosis phenomenon.
In the network hypothesis for heterosis, presented in this work, more regulatory interactions are expected in the molecular networks of the hybrids compared to the homozygous parents. Partial correlations were used to assess this difference in the global interaction structure of regulatory networks between the hybrids and the homozygous genotypes.
This network hypothesis for heterosis was tested on metabolite profiles as well as gene expression data of the two parental Arabidopsis thaliana accessions C24 and Col-0 and their reciprocal crosses. These plants are known to show a heterosis effect in their biomass phenotype. The hypothesis was confirmed for mid-parent and best-parent heterosis for either hybrid of our experimental metabolite as well as gene expression data. It was shown that this result is influenced by the used cutoffs during the analyses. Too strict filtering resulted in sets of metabolites and genes for which the network hypothesis for heterosis does not hold true for either hybrid regarding mid-parent as well as best-parent heterosis.
In an over-representation analysis, the genes that show the largest heterosis effects according to our network hypothesis were compared to genes of heterotic quantitative trait loci (QTL) regions. Separately for either hybrid regarding mid-parent as well as best-parent heterosis, a significantly larger overlap between the resulting gene lists of the two different approaches towards biomass heterosis was detected than expected by chance. This suggests that each heterotic QTL region contains many genes influencing biomass heterosis in the early development of Arabidopsis thaliana. Furthermore, this integrative analysis led to a confinement and an increased confidence in the group of candidate genes for biomass heterosis in Arabidopsis thaliana identified by both approaches.Als Heterosis-Effekt wird die Überlegenheit in einem oder mehreren Leistungsmerkmalen (z.B. Blattgröße von Pflanzen) von heterozygoten (mischerbigen) Nachkommen über deren unterschiedlich homozygoten (reinerbigen) Eltern bezeichnet. Dieses Phänomen ist schon seit Beginn des letzten Jahrhunderts bekannt und wird weit verbreitet in der Pflanzenzucht genutzt. Trotzdem sind die genetischen und molekularen Grundlagen von Heterosis noch weitestgehend unbekannt. Es wird angenommen, dass heterozygote Individuen mehr regulatorische Möglichkeiten aufweisen als ihre homozygoten Eltern und sie somit auf eine größere Anzahl an wechselnden Umweltbedingungen richtig reagieren können. Diese erhöhte Anpassungsfähigkeit führt zum Heterosis-Effekt. In dieser Arbeit wird ein systembiologischer Ansatz, basierend auf molekularen Netzwerkstrukturen verfolgt, um zu einem besseren Verständnis von Heterosis beizutragen. Dazu wird eine Netzwerkhypothese für Heterosis vorgestellt, die vorhersagt, dass die heterozygoten Individuen, die Heterosis zeigen, mehr regulatorische Interaktionen in ihren molekularen Netzwerken aufweisen als die homozygoten Eltern. Partielle Korrelationen wurden verwendet, um diesen Unterschied in den globalen Interaktionsstrukturen zwischen den Heterozygoten und ihren homozygoten Eltern zu untersuchen. Die Netzwerkhypothese wurde anhand von Metabolit- und Genexpressionsdaten der beiden homozygoten Arabidopsis thaliana Pflanzenlinien C24 und Col-0 und deren wechselseitigen Kreuzungen getestet. Arabidopsis thaliana Pflanzen sind bekannt dafür, dass sie einen Heterosis-Effekt im Bezug auf ihre Biomasse zeigen. Die heterozygoten Pflanzen weisen bei gleichem Alter eine höhere Biomasse auf als die homozygoten Pflanzen. Die Netzwerkhypothese für Heterosis konnte sowohl im Bezug auf mid-parent Heterosis (Unterschied in der Leistung des Heterozygoten im Vergleich zum Mittelwert der Eltern) als auch auf best-parent Heterosis (Unterschied in der Leistung des Heterozygoten im Vergleich zum Besseren der Eltern) für beide Kreuzungen für die Metabolit- und Genexpressionsdaten bestätigt werden. In einer Überrepräsentations-Analyse wurden die Gene, für die die größte Veränderung in der Anzahl der regulatorischen Interaktionen, an denen sie vermutlich beteiligt sind, festgestellt wurde, mit den Genen aus einer quantitativ genetischen (QTL) Analyse von Biomasse-Heterosis in Arabidopsis thaliana verglichen. Die ermittelten Gene aus beiden Studien zeigen eine größere Überschneidung als durch Zufall erwartet. Das deutet darauf hin, dass jede identifizierte QTL-Region viele Gene, die den Biomasse-Heterosis-Effekt in Arabidopsis thaliana beeinflussen, enthält. Die Gene, die in den Ergebnislisten beider Analyseverfahren überlappen, können mit größerer Zuversicht als Kandidatengene für Biomasse-Heterosis in Arabidopsis thaliana betrachtet werden als die Ergebnisse von nur einer Studie.
44
Li, Zhiguo. "Structure, secretion, and proteolysis study of MBP-containing heterologous proteins in Pichia pastoris." Scholarly Commons, 2010. https://scholarlycommons.pacific.edu/uop_etds/2415.
Full textAbstract:
The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N -terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris , a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N -terminal partner to several C -terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C -terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C -terminal of the MBP domain, including the spacer and cargo regions, but the MEP domain could still act to enhance the secretion of certain cargo proteins. The attempt to identify the unknown protease was unsuccessful. However, in contrast to other fusion partners, MBP was secreted with the cargo when it was fused as a C -terminal peptide to an N -terminal cargo protein. These studies provide insights into the role of proteases and fusion partners in the secretory mechanism of P. pastoris , suggesting new strategies to optimize this expression system.
45
de, Moya Robert S. "Molecular Phylogenetic Analysis of Argynnis Fabricius (1807) including North American Speyeria Scudder (1872)." Scholarly Commons, 2016. https://scholarlycommons.pacific.edu/uop_etds/168.
Full textAbstract:
North American Speyeria butterflies are a group whose species hypotheses are confounded by shared wing color patterns between sympatric populations of closely related recognized species due to a putatively recent origin in evolutionary time. Previous studies of this group and the closely related Palearctic genus Argynnis , suggest that Speyeria is monophyletic but derived from within Argynnis . Sampling in these studies has either involved few basal Speyeria species, or too few Argynnis species (Simonsen 2006, Simonsen et al. 2006). Thus, no comprehensive phylogenetic analysis exists for all members that answers the question of monophyly of Speyeria , or other subgeneric taxa,and their relationship to Argynnis species. A phylogenetic analysis was completed of all North American Speyeria species and nearly all species within Argynnis , using one mitochondrial (CO1) and four nuclear genes (EF1?, WG, GAPDH, and RPS5). The results indicate that North American Speyeria is a monophyletic group, but that Palearctic Argynnis is paraphyletic. Three major lineages are identified within Argynnis sensu lato : two Palearctic and one containing both Palearctic and Nearctic species. Argynnis species representing the subgenera Argyreus , Argyronome , Childrena , Damora , Pandoriana , and Nephargynnis , belong to a well-supported lineage that split early in the evolution of the group and is comprised of species with long branches. Fabriciana and Mesoacidalia were both recovered as strongly supported lineages, except for A. clara which was recovered as sister to Speyeria . In summary, the phylogenetic analyses suggest the need for reorganization into three genera: Argynnis , Fabriciana , and Speyeria . The results have implications for the conservation of these butterflies across the temperate zone by providing a framework for understanding potential gene flow between sympatric species complexes, proper taxonomic validity, and the natural history of threatened populations of Speyeria and Argynnis butterflies.
46
Easley-Neal, Courtney Nichelle 1981. "Synapse Formation in the Zebrafish Spinal Cord." Thesis, University of Oregon, 2011. http://hdl.handle.net/1794/12028.
Full textAbstract:
xv, 102 p. : ill. (some col.)This dissertation describes research to elucidate the early steps in the process of synapse formation in the zebrafish spinal cord. One question is how presynaptic proteins are trafficked and recruited to nascent synapses. Previous work has suggested two possible models of presynaptic transport, either (1) most presynaptic proteins are transported together or (2) two types of transport packets, synaptic vesicle (SV) protein transport vesicles (STVs) and Piccolo-containing active zone precursor transport vesicles (PTVs), transport the necessary components separately. We tested these models using in vivo imaging in zebrafish spinal cord and found that the recruitment of at least three distinct transport packets during presynaptic assembly of a glutamatergic synapse occurs in an ordered sequence. First, STVs are stabilized at future synaptic sites, then PTVs, followed by a third transport packet type carrying Synapsin, a cytosolic protein that can tether SVs to actin. These results identify an order to the assembly of the presynaptic terminal in vivo, suggesting that a single synaptogenic interaction may precipitate the cascade of recruitment steps. We next examined the Cadm/SynCAM family of cell adhesion molecules, a family of proteins that has been shown to be able to induce synapse formation in vitro and was thought to play a role in recruitment of presynaptic proteins. As the role of these proteins in vivo was not well understood, we chose to examine the role of the cadms in zebrafish spinal cord. We found that zebrafish possess six cadm genes, and all are expressed throughout the nervous system both during development and in the adult. We then looked at the role of one of the Cadms, Cadm2a, in vivo in the zebrafish spinal cord. We found that knockdown of cadm2a significantly decreases the ability of zebrafish embryos to respond to touch. We also found that there is a significant reduction in the number of synapses, as shown by immunohistochemistry, formed between Rohon-Beard and CoPA neurons, the first two cell types in the touch response circuit. These data suggest that Cadm2a plays an important role in synapse formation in vivo. This dissertation contains both my previously published and unpublished co-authored material.
Committee in charge: Monte Westerfield Chairperson; Philip Washbourne, Advisor; Judith Eisen, Member; Tory Herman, Member; Mike Wehr, Outside Member
47
Luu, Tony C. "Investigation of the role of CyP40 in the aryl hydrocarbon receptor signaling pathway." Scholarly Commons, 2008. https://scholarlycommons.pacific.edu/uop_etds/2383.
Full textAbstract:
Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex containing baculovirus aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE). CyP40 was found to play a role in the AhR signaling since when the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylcholanthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidylprolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer. Coprecipitation data suggests CyP40 binds weakly to AhR, but not Arnt. We report on the progress of applying bioluminescence resonance energy transfer and chromatin immunoprecipitation techniques to further elucidate the role of CyP40 in the aryl hydrocarbon receptor signaling pathway.
48
Unis, Melod. "Peroxide reactions of environmental relevance in aqueous solution." Thesis, Northumbria University, 2010. http://nrl.northumbria.ac.uk/2284/.
Full textAbstract:
The main objective of this research programme was to determine the factors influencing the decolourisation of dyes at low pH by different peroxide species, both in the presence and absence of metal ion catalysts and, therefore, to find a set of optimal conditions for application to wastewater treatment processes. An additional study looked at whether peroxoborates were capable of acting as nucleophiles. The specific aims of the study were: to investigate the in-situ formation of peracetic acid from the equilibrium formed between hydrogen peroxide and acetic acid, and whether this can be achieved without the addition of an acid catalyst such as sulphuric acid; to study the comparative reactivity of in-situ generated peracetic acid and hydrogen peroxide towards a range of dyes used in industry; to investigate the catalytic potential of a range of metal ions towards the reaction between peroxides and dyes; to investigate the structural features of dyes that might influence reactivity (decolourisation); and to investigate the reactivities of other peracid-like peroxide species that can be generated from hydrogen peroxide (peroxoborates and peroxocarbonates).The novel aspects arising from this study were: (a) The development of a new method for the in-situ generation of peracetic acid that gives the same equilibrium yield as established methods yet does not require the addition of an acid catalyst;(the reaction was slow, but there was minimal decomposition and so it is ideal for circumstances that allow the preparation of peracetic acid well in advance of use). (b) The first comprehensive study of the bleaching potential of peracetic acid and hydrogen peroxide towards a wide structural range of dyes both in the presence and absence of metal ions (iron, manganese, silver and copper). (c) The inference that for iron-catalysed bleaching of azo dyes by peracetic acid the catalytic mechanism involves pre-complexation of iron and dye, followed by reaction of the 'activated' complex with peracetic acid rather than a free radical mechanism that might have been expected for such systems. (d) The evidence that, in contradiction to literature studies, peroxoborate species do not act as nucleophiles. As an introduction to this work the reactions of peroxyacids are described in general terms. The experimental work is divided in three parts. In Chapter two, the homogeneous preparation of peracetic acid (PAA) from acetic acid (AA) and hydrogen peroxide (H2O2) was investigated with and without the catalysis of sulphuric acid (H2SO4). The formation of PAA and total peroxide content was determined by iodimetric titration. The reaction was slow in the absence of a strong acid catalyst, and was faster with a sulphuric acid catalyst. There was no loss of total peroxide over the timescales of both reactions, whether a catalyst was used or not. The equilibrium constant for peracetic acid formation at temperature of 20 was found to be 2.04 with a catalyst, and 2.10 without catalyst. The rate constant for the hydrolysis of peracetic acid for both forward and reverse reactions increased when the sulphuric acid concentration was increased from 0.02 M to 0.32 M. Linear relationships were found between the observed rate constants and H+ concentrations at 25oC. Moreover, it was found that the preparation of peracetic acid showed a first-order dependence with respect to peroxide concentration. In Chapter three, the application of this preparation of peroxyacids to the degradation of different types of dyesstuffs was investigated. As we know, physical or other chemical methods for dye degradation are expensive and can generate secondary pollution. In this part of the study the reactions of dyes with hydrogen peroxide and peracetic acid in the absence and presence different metal ions (Fe3+, Cu2+, Mn2+ and Ag+) were investigated. The iron/peroxyacid system was found to be the most effective. Consequently, Chapter 4 evaluates the decolourization of five azo dyes under conditions of bleaching by peracetic acid in the presence of Fe3+ as a catalyst. The experiment was carried out in aqueous acidic media. Dye oxidation systems are complex because: they involve several different tautomers; there is the possibility of dye aggregation at lower dye concentrations; and the oxidant species involved can be either the undissociated peroxide acting as an electrophile, or the dissociated peroxide acting as a nucleophile. The results obtained for the reaction of azo dyes with peracetic acid without added iron, when converted to the second order rate constant for the electrophilic reaction, k2E gave a value of 4.5x10-6 dm3 mol-1 s-1 for orange II, which is very high. This may be due to trace metal ions still being present and catalysing the reaction, possibly from impurities in the dye itself. No metal ion chelators were used in the present study because the bulk of the study was designed to elucidate the effect of metal ions concentration on reaction rate. For the catalysed reactions a significantly increased rate of absorbance decrease with increasing iron concentration was observed. Saturation of iron was also demonstrated at high iron concentrations, suggesting the formation of an iron (III)-dye complex which then reacted with peracetic acid. The maximum rate of reaction was observed at an iron concentration of 0.012 M, and the results showed a reactivity order of Ponceau 4R > Amaranth > (Orange II & Carmosine) > Black PN; Orange 1 was unreactive under these conditions. Also one of the key objectives of this chapter was to determine the optimum conditions for dye degradation in terms of pH and oxidant and catalyst concentrations. The optimum conditions for maximum degradation occurred at the highest pH of 3.0 and at about 1x10-3 M iron. Evidence of the possible involvement of radicals in our studies comes from the observation of a lag phase followed by a more rapid bleaching phase in the oxidation of azo dyes by peracetic acid at the lowest iron concentrations (another possibility is that at these iron concentrations the reactive iron complex forms at a much slower rate). However, this process is slow by comparison with the rate of oxidation at higher iron concentrations that do not exhibit this lag phase; consequently, if free radical mechanisms are suggested then they are not significant compared to the proposed formation of a reactive iron-dye complex.ixThe work contained in final experimental Chapter aimed to clarify whether or not any of the peroxyborate species displayed nucleophilic characteristics and thus accelerated the rate of the reaction of hydrogen peroxide with p-nitrophenyl acetate. The pH range of 6.0 to 8.0 is critical in terms of the distribution of peroxide species for a hydrogen peroxide / boric acid system. The triganol peroxoboric acid, B(OH)2OOH, is the only significant peroxoborate species below pH 6.5. However, above this pH, increased concentrations of the monoperoxoborate anion, B(OH)3OOH, the peroxodiborate anion, (HO)3BOOB(OH)32-, and the diperoxodiborate anion (HO)2B(OO)2B(OH)22-, are formed, with diperoxoborate, B(OH)2(OOH)2- forming at higher hydrogen peroxide concentrations. Therefore this is the ideal pH range in which to elucidate any effects of borate on the reaction of hydrogen peroxide and PNPA. The observed second order rate constants (k2obs) for the reaction between p-nitrophenyl acetate and hydrogen peroxide, and the corresponding second order rate constants, k2, for the reaction of the perhydroxyl anion with p-nitrophenyl acetate was determined by equation:In borate buffer the k2 values were significantly reduced compared to other buffers; this reduction was consistent with the hydrogen peroxide complexing with borate to form a range of non-reactive (towards carbonyl groups) peroxoborate species, thus also reducing the equilibrium concentration of the perhydroxyl anion. There was no evidence for peroxoborate species that could act as nucleophiles, in contradiction of literature claims. Values of k2 in the case of phosphate buffer compared reasonably well with values in the literature of 3140 and 3520 dm3 mol-1 s-1 obtained at pH 6.8 in ionic strengths of 0.02 dm3 mol-1 and 0.1dm3 mol-1 respectively. In carbonate buffer the literature value is 3785 dm3 mol-1 s-1 at pH 10, ionic strength 0.1 M, in borate buffer.
49
Tran, Tiffany Doan. "Filarial infection in mosquitoes of Northern California." Scholarly Commons, 2016. https://scholarlycommons.pacific.edu/uop_etds/172.
Full textAbstract:
Filarial parasites are a type of nematode that requires arthropod vectors for transmission between hosts. Filarial parasites vary among species of vertebrate hosts and can cause varying symptoms in hosts, including death. The presence of filarial parasites can influence host populations and can be costly to infected areas. To evaluate the prevalence of filarial parasites in Lake County, CA, mosquitoes were collected in 2014 and analyzed for infection using polymerase chain reaction (PCR). Of 1,008 mosquito pools, six filarial parasite species were detected in 23 pools representing six mosquito species. The DNA of Dirofilaria immitis (n=6, MIR=0.26); Setaria yehi (n=9, MIR=1.44); Splendidofilaria sp. (n=4, MIR=0.20); unknown filarial parasites A (n=2, MIR=0.09), B (n=1, MIR=0.41), and C (n=1, MIR=0.05) were detected in Aedes increpitus, Aedes sierrensis, Anopheles franciscanus, Anopheles freeborni, Culex stigmatosoma, and Culex tarsalis mosquito pools. Due to the presence of D. immitis, which can lead to chronic illness and death in domestic dogs, in Lake County it is important to evaluate vector competency of D. immitis in mosquito species. Culex tarsalis was chosen due to the high abundance found in Lake County in 2014 (n=36,587). To evaluate vector competency of Cx. tarsalis in transmission of D. immitis, colony and field-caught Lake County (n=102, n=54 respectively) mosquitoes were analyzed for infectivity using decapitation. Fourteen days post feeding on infected blood, mosquitoes were decapitated to evaluate the presence of L 3 larvae; but no L 3 larvae were detected. The presence of D. immitis DNA was detected in eight colony (IR=7.8%) and fifteen field-caught (IR=23.1%) thoraces using PCR. Though no L 3 larvae were observed in decapitated mosquitoes, presence of D. immitis DNA in the thoraces of mosquitoes using PCR has previously been used as an indicator for vector competency. Thus it is probable that Cx. tarsalis is a competent vector for D. immitis.
50
Devaiah, Shivakumar P., and Cecelia A. McIntosh. "Towards Understanding of Glucosyltransferase Specifi city in Citrus Paradisi." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/350.
Full textAbstract:
Flavonoids are a broad class of low molecular weight, secondary plant phenolics characterized by the fl avan nucleus. Widely distributed in plants, food and traditional herbal medicines, more than 6000 fl avonoids have been identifi ed up to date. They are present mainly as glycosides whose phenolic hydrogen or hydrogens are substituted to sugar moiety. An increasing number of fl avonoids have attracted much attention in relation to their biological activities, including anti-viral, anti-infl ammatory, anti-bacterial, and vasodilatory activities. Present work is to understand the structure and function of a fl avonol specifi c glucosyltransferase from Citrus paradisi. The study is one of the many steps towards custom designing of the protein. We employed homology modeling, site-directed mutagenesis and yeast expression system to generate mutants of glucosyltransferase and study their substrate specifi city, regiospecifi city and kinetic properties.