Structural study of immunophilins from wheat (Triticum aestivum) by X-ray diffraction

SIMRANJEET SINGH SEKHON University of Science and Technology 2013 국내박사

Cyclophilins belong to a family of proteins that bind to immunosuppressive drug cyclosporine A (CsA). Several membes of this protein family catalyze the cis-trans isomerization of peptide bonds preceding prolyl residues. The present study describes the structural characteristics of a cytosolic cyclophilin (TaCypA-1) cloned from wheat (Triticum aestivum L.). The purified TaCypA-1, expressed in E. coli, showed peptidyl prolyl cis trans isomerase activity, which was inhibited by CsA, with an inhibition constant of 78.3nM. The structure TaCypA-1 (native) and TaCypA-1-CsA complex were determined at 1.25 Å and 1.20 Å resolutions respectively by X-ray diffraction method. Binding of CsA to the active site of TaCypA-1 did not result in any significant conformational change in TaCypA-1 (native) structure. This is consistent with the crystal structure of human cyclophilinD-CsA complex reported at 0.96 Å resolution. The TaCypA-1 structure revealed the presence of a divergent loop of seven amino acids 48KSGKPLH54, which is a characteristic feature of plant cyclophilins. This study is the first to elucidate the structure of an enzymatically active plant cyclophilin, which shows peptidyl-prolyl cis-trans isomerase activity and also the presence of a divergent loop. FK506 binding proteins are cellular target for immunosuppressive drugs FK506 and rapamycin. Characteristic feature of FK506 binding protein is its ability to catalyse rotation of the peptide bond preceding a prolyl residue between cis-trans configurations. Cytosolic FK506 binding protein TaFKBP was cloned from wheat (Triticum aestivum). The protein was insoluble and disordered and so could not be crystallized. The TaFKBP structure was predicted by homology modeling and the predicted structure is a typical FKBP structure with five  sheets wrapping around a short  helix with an overall conical shape. The 12 amino acid residues Tyr61, Phe72, Asp73, Arg78, Phe83, Gln90, Val91, Ile92, Trp95, Tyr117, Ile127, and Phe134 involved in Peptidyl Prolyl Isomerase activity and binding of FK506 or rapamycin are conserved in TaFKBP.

Exposing Sin City: Southern California sense of place and the Los Angeles anti-myth

Sekhon, Sharon Elaine University of Southern California 2002 해외박사(DDOD)

This interdisciplinary study explores the relationship between mass media sources and national and local attitudes towards specific regions, specifically sense of place, demonstrated through the example of Southern California. Throughout the 20th century, Los Angeles developed an at once conflicted image; known as a place where impossible dreams could be achieved within the confines an agreeable climate, simultaneously, the Southern California region became known as a haven for conspicuous consumption, anti-intellectualism, lax sexuality, and violence. The origins of this imagery and how these portrayals influenced and continue to affect regional attachment to place are the central concerns of this study. Powerful arms of the mass media disseminated Los Angeles' primarily negative stereotype to the American public and to the residents of the region. Reacting in part to booster claims of the late 19<super>th</super> century, regional fiction from the 1930s and 1940s voiced criticism of the Southern California mythos and the film industry, a place many writers knew well. This literary trope was visually translated to film noir of the 1940s and 1950s during one of the highest periods of film-going attendance. Additionally, regardless of a search for journalistic objectivity, non-fictional sources of the same period used language and subject matter evocative of the regions anti-myth. This triangulation of sources created a potent vacuum in which a vision of Southern California became reified and normalized in American consciousness, but more importantly in the vision of its residents. Re-invented in various fictional forms to the present, this stereotype goes largely unquestioned in the popular culture, ultimately fostering a diminished sense of place among Los Angelenos. Involving extensive research in local and national archives, this dissertation is based on historical texts ranging from promotional literature, personal writings, mainstream fiction, poetry, and mainstream news sources to tabloid journalism, film production notes, and finally, the films themselves. This dissertation builds on extant writing on Los Angeles and the historiography of popular culture by taking seriously the political repercussions of media-made places.

Effect of drying temperature, size reduction, propane extraction, and storage temperature on the quality and shelf life of cilantro (Coriandrum sativum L.)

Sekhon, Jasreen Kaur Oklahoma State University 2013 해외박사(DDOD)

Cilantro dried at 40°C and 60°C was ground and separated into large flakes (LF), small flakes (SF), and coarse powder (CP) and subjected to continuous flow liquid propane extraction at 21-25°C. Extracted and unextracted samples were packaged in aluminum foil laminate packages and stored in a freezer (-20°C), a refrigerator (4°C), at room temperature (18-30°C) and at elevated temperature (40°C) for a period of twelve months. The effect of drying temperature, particle size, propane extraction, and storage conditions on color (L*, a*, b*, chroma, hue angle, and browning index), volatile composition, and fatty acid composition was evaluated. Major volatiles present in dried cilantro were E-2-tetradecenal, dodecanal, E-2-dodecenal, and tetradecanal. Linoleic acid and linolenic acid were the major fatty acids found in cilantro. While percent oil (%) in samples dried at 60°C was lower than those dried at 40°C, bulk density, volatile concentration, and color values were higher. No significant effect (p > 0.05) of drying temperature was observed on fatty acid composition. Volatile composition was greater in SF or LF as compared to CP. However, fatty acid concentration was higher in CP followed by LF and SF. Solvent extraction with propane lead to a positive change in color values and a decrease in volatile composition, oil content (%), and fatty acid composition. There was a significant (p < 0.05) decrease in all volatile compounds, except nonane, with increase in storage time. During storage, color quality and retention of volatile compounds in dried samples was dependent on the particle size of the sample and the storage temperature. During storage the concentration of linoleic acid and linolenic acid decreased in the first three to four months and then became stable. The stability of fatty acids in storage was dependent on particle size, solvent extraction, and storage temperature.

Bio-Inspired Enzyme Responsive Nanotechnologies for the Treatment of Bleeding Dysfunctions

Sekhon, Ujjal Didar Singh Case Western Reserve University ProQuest Dissertat 2021 해외박사(DDOD)

Transfusion of donor-derived platelets is a clinical standard-of-care to treat bleeding risks and hemorrhagic complications in hematology/oncology, surgery, trauma and congenital platelet disorders. However, these platelet products face severe logistical challenges due to their limited availability and portability, high risk of bacterial contamination and extremely short (5-7 days) shelf-life. This can be potentially addressed by designing `synthetic platelet surrogates’ that functionally and modularly mimic platelet-mediated hemostatic mechanisms and allow in vitro manufacture and scale-up for on-demand use. To this end, a novel liposome-templated procoagulant synthetic platelet (P-SP) nanoparticle system was engineered that: (i) Achieves platelet-mimetic ability of injury selective adhesion and aggregation via specific activity of vWF-binding, collagen-binding and active platelet integrin αIIbβ3-binding peptides decorated on the nanoparticle, and (ii) Mimics platelets’ procoagulant mechanism of phosphatidylserine (PS) exposure for thrombin amplification in a unique plasmin-responsive manner using a plasmin-cleavable cholesterol-tethered polyethylene glycol (PEG) mask on the nanoparticle surface. This design exploits the local plasmin upregulation at a vascular injury site to expose the procoagulant PS, amplify thrombin, and augment fibrin generation site-specifically for augmenting clot quality and stability. In vitro evaluation in human plasma confirmed that P-SP enables plasmin-responsive PS exposure, coagulation factor assembly and enhanced thrombin generation even when native platelets were depleted. In vitro analysis in human plasma and whole blood showed that P-SP significantly improves clot robustness and stability against lysis by in situ fibrin generation. Additional in vitro studies revealed P-SP’s ability to rescue hemostatic function in antiplatelet drug induced platelet dysfunction scenarios. In vivo in a mouse thrombocytopenia (TCP) model, the P-SP vesicles significantly reduced tail-bleeding time and blood loss, comparable to syngeneic platelet dose. Furthermore, in a rat liver hemorrhage model, the P-SP vesicles significantly reduced bleeding and improved survival. No sign of systemic thrombotic risks was found in either animal model. These studies demonstrate the potential of a bioinspired enzyme responsive nanotechnology platform as a viable platelet surrogate for transfusion management of bleeding when natural platelets are of limited availability or have diminished function.