147 research outputs found
SHAPE - A UNIFYING CONCEPT IN DOCUMENT LAYOUT
No full textText objects have traditionally been constrained to be rectangular,
a constraint inherited by modern computer document preparation systems.
However, a variety of tasks in document formatting benefit from a more
general notion of shape. This paper describes such a representation,
suitable for graphic composition at both low and high levels of
document layout. Intermediate in generality and complexity between
rectangles and arbitrary polygons, it comprises separate left and right
margin functions composed piecewise of linear segments. Such shapes
can be compared, conbined, modified and generated using simple, economical
algorithms. This notion of shape appears to provide the correct level
of abstraction for elegant solutions to several knotty problems in
existing systems. Software methods making use of shape have been
implemented in JOT [Bonham 1985], an interactive documentation system
under development as part of the University of Calgary JADE project
[Witten et al 1983].We are currently acquiring citations for the work deposited into this collection. We recognize the distribution rights of this item may have been assigned to another entity, other than the author(s) of the work.If you can provide the citation for this work or you think you own the distribution rights to this work please contact the Institutional Repository Administrator at [email protected]
Katamari Kart: A serious and hilarious sub/urban game for more serendipitous, playful and friendly public art
No full textThe article presents the sub/urban game-method Katamari Kart, where people roam industrial and suburban areas collecting waste materials and progressively building a large and mobile public sculpture. This game-method departs from established concepts and practice in neighbourhood improvement as it tries to evade capital expenditure and embraces uncertainty and friction in bringing together various stakeholders, does not aim to look stylish or even be useful, decelerates daily life, promotes self-sufficiency in cultural life, and creates long-term wellbeing through play. That is, it is a degrowth approach to neighbourhood improvement. Rumen Rachev positions the Katamari Kart game-method as an expression of the ‘Kiwi way’ and discusses the role it can play in making our future neighbourhoods more serendipitous, playful and friendly, and a ref lection of the diverse people and things that reside there – all without capital expenditure or the support of the authorities. Finally, Alex Bonham, author of the book Play and the City: How to Create Places and Spaces to Help Us Thrive, discusses the Katamari Kart game-method in the context of adult play, our settler history, keeping up appearances, the Be a Tidy Kiwi campaign, play and wellbeing, and conflicting responses to frugality
Class of 1951, Indiana University School of Law
No full textIndiana Law School Class of 1951 in front of Maxwell Hall. Front Row: James Fredrick Risk, Lyman Keith Kern, Edwin Ragon Smith Jr., Mary Emily Williamson, John A. Kesler, William Fasig Downey, Mary Elizabeth Parish, Robert Howard Brown Second Row: Robert James Wilson, Harold Ralph Johnson, William Hill Turner, Warren Adams Jackman, Thomas Adelbert Nutting, Edward Thurston, James Leslie Puckett, Robert Oral Aders Third Row: Keith Kern, Robert William Bonham Jr., Waldo F. Beebe, James David Nafe, Frank Nathaniel Howard Jr., Charles Robert Parr, William Snyder Hall Fourth Row: Donald Gordon Speyer, Edward Meyers, Chester Robert Hobbs, Robert Edgar Horn, Robert Monroe Newkerk Fifth Row: George Peter Pappas, William Franklin Radcliffe, Thomas Dale Salwasser, Jack T. Beaverstine, Samuel Earl Bressner Sixth Row: Brooks Charles Pinnick, Jack Bowers Joel, Edward Jefferson Peck, Max Cohen, James Borter Sparks, Richard Hilmer Stozek, Harrison Jacob Mellman, Art Daronatsy, Gerald Keith Hodson, Earl Sheets, Joseph Douglas Schmidthttps://www.repository.law.indiana.edu/composite/1007/thumbnail.jp
Class of 1951, Indiana University School of Law
No full textIndiana Law School Class of 1951 in front of Maxwell Hall. Front Row: James Fredrick Risk, Lyman Keith Kern, Edwin Ragon Smith Jr., Mary Emily Williamson, John A. Kesler, William Fasig Downey, Mary Elizabeth Parish, Robert Howard Brown Second Row: Robert James Wilson, Harold Ralph Johnson, William Hill Turner, Warren Adams Jackman, Thomas Adelbert Nutting, Edward Thurston, James Leslie Puckett, Robert Oral Aders Third Row: Keith Kern, Robert William Bonham Jr., Waldo F. Beebe, James David Nafe, Frank Nathaniel Howard Jr., Charles Robert Parr, William Snyder Hall Fourth Row: Donald Gordon Speyer, Edward Meyers, Chester Robert Hobbs, Robert Edgar Horn, Robert Monroe Newkerk Fifth Row: George Peter Pappas, William Franklin Radcliffe, Thomas Dale Salwasser, Jack T. Beaverstine, Samuel Earl Bressner Sixth Row: Brooks Charles Pinnick, Jack Bowers Joel, Edward Jefferson Peck, Max Cohen, James Borter Sparks, Richard Hilmer Stozek, Harrison Jacob Mellman, Art Daronatsy, Gerald Keith Hodson, Earl Sheets, Joseph Douglas Schmidthttps://www.repository.law.indiana.edu/composite/1007/thumbnail.jp
Effects of Fyn-related kinase activity on breast cancer cell proliferation, migration, invasion and colony formation
No full textThe human Fyn-related kinase (FRK) is a member of subfamily of Src-related kinases family. FRK is 54 kDa non-receptor tyrosine kinase protein composed of 505 amino acids. FRK consists of three functional domains: Src homology 3 (SH3), SH2 and kinase domain, as well as a putative tyrosine kinase regulator at the C-terminus. FRK has a conserved auto-regulatory tyrosine residue within its kinase domain. It has been reported that FRK is repressed in about 30 % of human breast cancer samples. Over-expression of FRK in breast cancer cells of the mammary gland was shown to suppress cell growth by interacting, phosphorylating and stabilizing the tumor suppressor PTEN, thus inhibiting AKT/PI3K signaling. Although it has been suggested that FRK is a tumor suppressor gene, the effects of activated FRK on cell proliferation, migration and invasion are unclear. Likewise, the signaling pathways regulated by the activation of FRK have not been yet fully characterized. We hypothesize that the activation of FRK is essential for the regulation of its cellular functions. Mutation of the C-terminal auto-regulatory tyrosine 497 to phenylalanine (FRK-Y497F) resulted in the constitutive activation of FRK. We generated stable cell lines expressing either the FRK wild type (FRK-WT) or FRK-Y497F from triple negative breast cancer MDA-MB-231 cells. The introduction of FRK-Y497F in MDA-MB-231 cells significantly suppressed their proliferation, migration, invasiveness and colony formation as compared to cells that expressed the FRK-WT gene. Over-expression of either FRK-WT or FRK-Y497F in MDA-MB-231 cells inhibited the phosphorylation of AKT, STAT3, JNK and P38 MAPK as compared to either the MDA-MB-231 parental cells or those that were transfected with the empty vector. Our results suggested that FRK represses cell proliferation, migration, invasiveness and colony formation at least in part by the inhibiting the activation of AKT/PI3K, JAK-STAT and MAPK signaling pathways
The role of Chfr and Ubc13 in mitosis.
No full textThe Chfr checkpoint is a point at which a cell checks whether it is safe to enter mitosis. Chfr is a protein that functions at this particular checkpoint to ensure safe entry into mitosis, but the molecular mechanism by which this protein functions is not entirely clear. The hypothesis in this thesis is that Ubc13, Chfr, and Uev1/Mms2 function together in mitosis. The results were observed using immunocytochemistry, the mitotic shake off procedure, Western blot analysis, and coimmunoprecipitation. High Ubc13, Mms2, and Chfr-Ub levels at the interphase-early prophase transition, indicate that these proteins function together at the Chfr checkpoint. Localization of Chfr to decondensed chromatin in interphase cells and to decondensing chromatin in telophase cells indicates a decondensing function for Chfr. Interaction between Chfr and Ubc13, Chfr and phosphorylated histone H3, as well as Ubc13 and phosphorylated histone H3, further indicates that these proteins may function together at the Chfr checkpoint, because phosphorylated histone H3 is a mitotic protein at that particular point in mitosis. Localization of Chfr, Ubc13, and Mms2 to the centrosomes, indicates that they function together at these sites to target substrates important in centrosome maturation, separation, and spindle formation. Furthermore, there are two molecular states of Chfr: Chfr and Chfr-Ub. Chfr is predominant at late prophase, whereas, Chfr-Ub is predominant at interphase-early prophase. Chfr increases in level upon nocodazole exposure at late prophase to counteract the mitotic stress; and it also looses its ubiquitin signal upon passage into mitosis. High Ubc13 and Mms2 levels coincide with high Chfr-Ub levels at the interphase-early prophase transition, indicating that they function together at the Chfr checkpoint. The ubiquitin signal could be either K-48-linked or K-63-linked in nature. The Chfr, Ubc13, and Mms2 protein complex could function through a self ubiquitination-decondensation-Chfr destruction-recondensation mechanism. Chfr could bind to pH3 and its auto-ubiquitin signal to serve as a bulky modification that hinders chromosome condensation
Zhangfei suppresses the growth of Medulloblastoma cells and commits them to programmed cell death
No full textMedulloblastoma cells do not contain detectable amounts of the bZIP protein Zhangfei. However, previous work has shown that expression of this protein in cells of the ONS-76 line, derived from a human medulloblastoma, causes the cells to stop growing and develop processes that resemble neuritis (a characteristic of differentiated neurons). Zhangfei-expressing cells eventually die. My objective was to determine the molecular mechanisms by which Zhangfei influences ONS-76 cells. My strategy was to infect ONS-76 cells with adenovirus vectors expressing either Zhangfei or the control E. coli protein â-galactosidase (LacZ) and then to compare the following parameters in Zhangfei and LacZ-expressing cells: a) markers of apoptosis, autophagy and macropinocytosis (the three main pathways of cell death); b) transcripts for genes involved in neurogenesis and apoptosis; c) phosphorylation of peptide targets of selected cellular protein kinases; and d) active transcription factors. Zhangfei-expressing cells appeared to succumb to apoptosis as determined by the expression of phosphatidylserine on the cell surface and intensity of nuclear staining with the DNA dye Hoechst. Increased staining for autophagic vesicles and upregulated expression of autophagy response genes in these cells indicated that they were undergoing autophagy, possibly associated with apoptosis. My analysis of steady-state transcripts for genes involved in apoptosis and neurogenesis and functional protein kinases in Zhangfei-expressing cells indicated that the mitogen-activated protein kinase (MAPK) pathway was active in these cells. In addition, I found that the transcription factor Brn3a as well as factors implicated in differentiation were also active. These observations led me to hypothesize that Zhangfei enhances the expression of Brn3a, a known inducer of TrkA, the high-affinity receptor for nerve growth factor (NGF). TrkA then binds in an autocrine manner to NGF, triggering the MAPK pathway and leading to differentiation of ONS-76 cells into neuron and glia-like cells, eventually bringing about cell death by apoptosis and autophagy. I tested this hypothesis by showing that Zhangfei could enhance transcription from the isolated Brn3a promoter, that ONS-76 cells produce NGF as detected in a bioassay, and that antibodies against NGF and inhibitors of TrkA and selected components of the MAPK pathway could partially restore the growth of Zhangfei-expressing ONS-76 cells. My work supports previous work highlighting the importance of NGF-TrkA signaling in the outcome of medulloblastomas and shows how Zhangfei is able to trigger this pathway
Development of the PHEARLESS system and its application for directed protein mutagenesis and characterisation of putative enzyme activity
No full textBacteriophages (phage) are considered a primary resource for developing new therapeutic treatments for bacterial infections. Phage will likely be used either as an alternatives to antibiotics or in cooperative treatment with antibiotics. However, a mixed blessing of phage and phage proteins is that they are highly selective for their target bacteria. The advantage of this is that the natural bacterial fauna of the patient is undisturbed. Future therapeutic phage and phage-derived protein treatment for bacterial infections will likely be administered as a cocktail, of several phage or phage-derived proteins. More phage and phage-derived proteins with high antimicrobial activity and suitable pharmacokinetics must be identified to develop these cocktails. Current methods for identifying phage-derived proteins include their purification and testing against target bacteria. However, many phage-derived proteins have properties that make them difficult to purify, including limited solubility. The inability to efficiently and cost-effectively purify these proteins means that many potentially effective antimicrobial proteins are overlooked. The PHEARLESS (Phage-based Expression, Amplification and Release of Lytic Enzyme Species) assay system developed here consists of a bacterial expression strain where both the expression of the protein of interest and cell lysis to release that protein are controlled via a simple chemical induction. What makes this system unique is the coordination of two modules, one controlling expression of the protein of interest and the second a biological switch modified for cell lysis. The system can test any protein expressed in E. coli against any target bacterial species that can be co-plated with E. coli, without a requirement for protein purification. The system provides for a simple and rapid method to test for antimicrobial activity. The E. coli expression strain carries a bacteriophage 186 prophage that has been genetically engineered to take control of its lytic cycle via a separate chemically-inducible module integrated elsewhere in the bacterial genome. Cumate (p-isopropyl benzoate) was used as the chemical inducer, and allowed tight control of the activation of the prophage lytic cycle. The first version of the PHEARLESS system was developed using a multicopy plasmid to express the antimicrobial protein of interest. Expression of the protein of interest was placed under control of a bacteriophage 186 late promoter, thus linking gene expression and cell lysis. This plasmid-based system allows rapid assessment of antimicrobial activity of any gene cloned into the expression plasmid. A second variant of the system was developed to facilitate recovery of the gene of interest following cell lysis. In this case, the plasmid carrying the gene of interest has been site-specifically integrated into the phage 186 prophage genome, such that cumate-induced cell lysis also leads to phage production, where every phage particle carries a copy of the gene. Continued progeny phage propagation through the inclusion of a sacrificial propagation strain ensures continued protein expression. This version of the PHEARLESS assay system it is also suitable for mutant library screening. A proof of principle test confirmed that the PHEARLESS assay system is capable of high throughput screening of a library of mutants, using a mutated endolysin protein, and screening for revertants with restored activity against S. aureus. Bioinformatic searches of S. aureus genomic sequences for putative phage-associated endolysins provided a list of possible anti-microbial enzymes, and a small sample of these were tested for activity using the PHEARLESS system. Similarly, the system can be used for testing engineered variants of known endolysins and the impact of protein linker sequences was examined. In summary, the tools developed here will help discover new proteins for anti-microbial therapeutics and can be employed to optimize and investigate their chemical properties.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202
Biochemical and structural studies of dosage compensation members : MSL1, MSL3, and MOF from Drosophila melanogaster
No full textDosage compensation is the key regulatory process employed in Drosophila melanogaster to equalize the level of gene transcripts between the single X chromosome in males (XY) and the two X chromosomes in females (XX). Dimorphic sex chromosomes evolved by the severe degeneration of the Y chromosome, giving rise to an imbalance between the heterogametic sex and the homogametic sex. Vital to the viability of male Drosophila is the dosage compensation complex (DCC), a ribonucleoprotein complex that mediates the precise two-fold transcription of the single male X chromosome. The DCC is comprised of five proteins: male-specific-lethal proteins (MSL) 1, 2, and 3, male absent-on-the-first (MOF), maleless (MLE), and two non-coding RNAs. The complex specifically co-localizes along the male X chromosome in a reproducible manner, resulting in acetylation of lysine 16 of the N-terminal tail of histone H4. The exact mechanism of recruitment and spreading of the DCC along the male X chromosome remains unclear; recent studies propose a multi-step mechanism involving DNA sequence elements, epigenetic marks, and transcription. Understanding how dosage compensation functions provides insight into the interplay between gene regulation and chromatin remodelling. The goal of this project was to better understand how Drosophila MSL1, MSL3, and MOF interact and how their interaction modulates MOF’s acetyltransferase activity. Recombinant protein constructs were cloned and over-expressed in a bacterial expression system permitting future structure determination by X-ray crystallography. The dMSL1820-1039 construct consisted of the C-terminal domain, reported to be able to interact with both dMSL3 and dMOF. dMSL3186-512 contained the domain required for the interaction with dMSL1 and dMOF. dMOF371-827 was comprised of the catalytic domain, the CCHC zinc finger, and the chromodomain, as the N-terminal region does not encode any known domains. All three recombinant proteins were successfully cloned, over-expressed, and purified to homogeneity. Recombinant dMOF371-827 was determined to acetylate histones. Interaction studies using GST pull-down assays and size exclusion chromatography determined that dMSL1820-1039 and dMOF371-827 did not interact above background levels. Moreover, size exclusion chromatography revealed dMSL3186-512 and dMOF371-827 did not interact nor did the three recombinant proteins form a stable complex
Regulation of constitutive platelet-derived growth factor receptor degradation by the 105 kilodalton isoform of ankyrin3
No full textDeregulation of platelet-derived growth factor receptor (PDGFR) signaling is a driving event in glioblastoma, promotes tumor progression epithelial to mesenchymal transition (EMT) in multiple cancers, modulates the tumor stroma to facilitate tumorigenesis and reduces tumor uptake of chemotherapeutics. Previous studies identified the 105 kDa isoform of ankyrin3 (Ank105) as a binding partner of the PDGFR signaling machinery and demonstrated that expression of Ank105 promoted PDGFR degradation (Ignatiuk et al., 2006)(Ignatiuk et al., 2006)(Ignatiuk et al., 2006). Receptor tyrosine kinases are targeted for degradation via endocytosis and ubiquitin-dependent trafficking to the lysosome. It was hypothesized that Ank105 promoted the constitutive degradation of the PDGFR and attenuation of PDGFR signaling by facilitating endocytosis of the PDGFR and targeting the PDGFR for lysosomal degradation via an ubiquitin-dependent mechanism. The studies in this thesis characterized the effects of Ank105 expression on PDGFR signaling and protein expression levels, determined the endocytic pathways involved in Ank105-mediated PDGFR degradation and studied the role of ubiquitin binding in Ank105 function. The most robust effect of Ank105 expression on the PDGFR was constitutive degradation as PDGFR protein expression levels in Ank105-expressing cells were significantly reduced compared to NIH 3T3 cells in the absence of PDGF ligand. Low constitutive PDGFR levels resulted in attenuated pro-proliferative AKT and mitogen-activated protein kinase (MAPK) signaling in response to ligand stimulation. To determine the endocytic requirements for Ank105-mediated constitutive PDGFR degradation, a constitutive PDGFR degradation assay was developed and the effects of several small molecule endocytosis inhibitors were evaluated. Additionally, the small molecule endocytosis inhibitors were validated by determining the effects of these inhibitors on low density lipoprotein (LDL) uptake and ligand-induced PDGFR degradation in Ank105-expressing cells. Both LDL uptake and ligand induced PDGFR degradation are known to proceed by a clathrin and dynamin dependent mechanism of endocytosis. In Ank105-expressing cells, both LDL uptake and ligand incuded PDGFR degradation were dependent upon clathrin and dynamin function. Interestingly, constitutive PDGFR degradation in Ank105-expressing cells was not dependent upon CME, but required dynamin activity. Expression of Ank105 may promote clathrin-independent, dynamin-dependent, constitutive endocytosis of the PDGFR. Additionally, acute inhibition of either lysosomal or proteasomal degradation strongly impaired constitutive PDGFR degradation, whereas ligand-induced PDGFR degradation was less sensitive to protein degradation inhibitors, while LDL uptake was unaffected. It was unclear if PDGFR was degraded in the proteasome or if the proteasome was involved in sorting of PDGFR to the lysosome for degradation. Ubiquitination of receptors is required to target them for degradation. Ank105 was assayed for the ability to interact with ubiquitin and ubiquitinated proteins. Interestingly, Ank105 bound ubiquitin in vitro via the spectrin binding domain and co-immunoprecipitated with several ubiquitinated proteins, suggesting a role for Ank105 in the sorting of ubiquitinated proteins for degradation. Furthermore, Ank105 co-immunoprecipitated with a number of high and low molecular weight proteins in the absence of PDGF stimulation. Identification of Ank105 binding partners would provide further insight in the mechanism of Ank105-mediated constitutive PDGFR degradation. In summary, Ank105 promoted the attenuation of PDGFR signaling via alteration of constitutive PDGFR endocytosis and targeting of constitutive PDGFR for degradation, potentially through interaction with ubiquitin and ubiquitinated proteins. Reduction of constitutive PDGFR levels in cancers with PDGFR driver mutations, acquired PDGF responsiveness and stromal expression of PDGFR, could significantly reduce tumor proliferation, tumorigenesis and increase effectiveness of chemotherapeutics
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