Biotechnologyhttp://hdl.handle.net/10211.3/10211.8_142024-03-29T13:40:29Z2024-03-29T13:40:29ZCharacterizing Curli Accessory Proteins CsgE/CsgFSunder Rajkumar, Shrutihttp://hdl.handle.net/10211.3/2171882020-08-12T20:58:55Z2020-08-12T00:00:00ZCharacterizing Curli Accessory Proteins CsgE/CsgF
Sunder Rajkumar, Shruti
The term curli is used to define cell surface organelles that are present on Gram-negative bacteria such as E. coli and Salmonella. These organelles are hair-like filaments comprised of protein. The curli facilitate functions such as bacterial motility, cell aggregation, biofilm formation and host colonization. The assembly of curli involves six proteins CsgA, CsgB, CsgC, CsgE, CsgF and CsgG. CsgA and CsgB are the curli structure proteins while CsgC, CsgE, CsgF and CsgG help in proper curli assembly. It is thought that CsgE and CsgF are periplasmic chaperone proteins that help export CsgA/CsgB through the periplasm to the outer membrane. The main goal of this project was to characterize regions of CsgE/CsgF that maybe involved in the structure and/or function of curli fibers. A series of single cysteine mutants of CsgF (T23C, S44C, S66C, T97C T123C) were expressed in E.coli, purified and labeled with environment-sensitive fluorophore IAEDANS. The protein structure of
the unlabeled and labeled mutants were investigated using Circular dichroism (CD) and compared with the CsgF wild type (WT). The CD spectra of CsgF WT contained a broad negative peak based around 218 nm while the unlabeled mutants S44C, S66C and T23C had negative peaks at 210, 216 and 208 nm respectively. The labeled mutants S44C, S66C and T23C contained negative peaks centered at approximately 218, 210 and 206 nm respectively. Structural differences were observed in the unlabeled T23C,S44C and labeled S66C, T23C compared to the WT. This could be due to protein denaturation or because these sites influenced the structure of the protein. Protein bioinformatics analysis was conducted to identify significant conserved regions that maybe important structure and/or function for CsgE. The residues that were absolutely conserved were found to be located at the positions 33,37,44,47,48,51,71,76,78,109 and 134 which may be used to elucidate the mechanistic details of curli assembly. Future direction - mutation of the residues at positions 33,51,71 and 76 to cysteines. Cysteine is the selected residue as it is known to have role in stabilizing protein structure.
2020-08-12T00:00:00ZMetabolic characterization of induced pluripotent stem cells from the critically endangered Northern White RhinocerosGao, Chenyuanhttp://hdl.handle.net/10211.3/2171722020-08-06T19:42:09Z2020-08-06T00:00:00ZMetabolic characterization of induced pluripotent stem cells from the critically endangered Northern White Rhinoceros
Gao, Chenyuan
The Semester-in-Residence project was conducted in San Diego Zoo Institute for Conservation Research, conservation genetics department. The purpose of this study is to discover the metabolic properties of induced Pluripotent Stem Cell (iPSC) lines from the critically endangered Northern White Rhinoceros (NWR). We perform a series of experiments to characterize the metabolic properties of NWR iPSCs, including mitochondria detection, ATP quantification, glycolysis capacity and oxygen consumption level detection. Our results show (1) The NWR iPSCs contain high levels of viable mitochondria. (2) The intracellular ATP level in NWR iPSC is 9.55±1.53 fmol, which is less than human induced Pluripotent Stem Cells (hiPSCs). (3) NWR iPSCs and hiPSCs both primarily rely on glycolysis instead of oxidative phosphorylation for energy generation. This glycolysis and oxygen consumption data also suggest that NWR iPSCs are at primed pluripotent states. This knowledge is firstly proposed for NWR iPSCs and support a long-term international collaborative conservational project to save NWRs. There are some recommendations to enhance this study, which include: introducing more hiPSC and NWR iPSC lines; introducing positive and negative controls for the glycolysis study; including beta-oxidation study; collaborating with epigenomic study to fortify our conclusion that NWR iPSCs are at primed pluripotent states.
2020-08-06T00:00:00ZCharacterization, Standard Operating Procedure and Validation Document Generation of the Flexicon PF7 for Reagent Fill ManufacturingCastaneda, Fernandohttp://hdl.handle.net/10211.3/2171592020-08-06T18:02:38Z2020-08-06T00:00:00ZCharacterization, Standard Operating Procedure and Validation Document Generation of the Flexicon PF7 for Reagent Fill Manufacturing
Castaneda, Fernando
This Semester-in-Residence project was conducted at lllumina, Inc., HQ in San Diego, CA between the manufacturing and pilot plant departments. The intent of this Semester-in-Residence project was to complete characterization activities related to the Flexicon PF7 Filler, and generate User Requirement Specifications (URS), Standard Operating Procedures (SOP), and Installation Operational Qualification (IOQ) documentation for the validation of the Flexicon PF7 Filler. One of the requirements under current good manufacturing practices (cGMP) is to validate manufacturing equipment to ensure the equipment operates and is appropriate for the intended use. A URS, SOP, and IOQ documentation were generated in order to validate the Flexicon PF7 Filler. The purpose of the URS is to layout the minimum requirements that the Flexicon PF7 Filler must have in order to meet the intended use in Reagent Manufacturing. The SOP was created to provide the specific details needed to properly operate the equipment so that it operates as intended. The IOQ protocol is required in order to verify that the minimum installation and operational requirements of the Flexicon PF7 are met with regards to the URS. All validation related documents, the URS, SOP, and IOQ were successfully created and approved. The characterization study was completed and is going under review. The characterization study outputs will serve as inputs to the performance qualification (PQ) that will be executed after the IOQ is successfully executed. Future recommendations are to characterize the effects of liquid surface tension with relation to dispense volume.
2020-08-06T00:00:00ZEvaluation of Psychomotor Skill AssessmentDages, Christinahttp://hdl.handle.net/10211.3/2171552020-08-06T17:23:10Z2020-08-06T00:00:00ZEvaluation of Psychomotor Skill Assessment
Dages, Christina
This Semester-In-Residence Project was conducted at Pima Medical Institute (PMI) in San Marcos California for use within the Veterinary Technician Program. The goal of this project was to establish the need to implement standardized criteria for evaluation of instructor assessment of psychomotor skills. In order to do so, four psychomotor skills were chosen to be independently assessed by five instructors within the Veterinary Program at PMI’s San Marcos, CA campus. The skills assessed were 1) aseptic closed gloving technique, 2) intravenous (IV) catheter placement, 3) K9 intravenous injection, and 4) K9 intramuscular injection. Instructors were asked to virtually assess student performance from a prescreened list of five videos for each psychomotor skill, providing a pass or fail grade. Instructors were also asked to provide comments as to their reasoning for a particular grade. The pass or fail score and comments from each evaluator were then compared to assess continuity across instructors within the same institution.
Results from the study demonstrated a lack of continuity between instructors across multiple videos. Statistical analysis using a chi square (χ2) test for each of the psychomotor skills determined a significant difference between the instructor’s assessment. This ultimately means some students might earn a passing mark on a psychomotor skill per one instructor and fail based on the score of another evaluator. However, there was similarity within instructor comments as to a particular reason for either passing or failing a student. For example, a student’s ability to maintain sterility or use of proper technique, etc. Students in the program must successfully complete all required psychomotor skills to use them in a real- world clinical setting as an entry level veterinary technician. Discrepancies between individual instructor evaluation of psychomotor skills must be resolved at the level of the evaluator.
The importance of this data relates to students entering clinical settings with insufficient technical skills and therefore compromising patient safety. All medical professionals, regardless of profession, are expected to uphold the highest standards of patient care and safety. Inconsistencies across instructor assessment of required program psychomotor skills ultimately lowers this threshold, striking a need to standardize assessment methods.
It is recommended that PMI San Marcos’s veterinary program rewrite all rubrics to include specific procedural steps to improve instructor assessment continuity. The rubrics should also convert the current pass or fail grading system to a more streamline system that includes categories in which instructors can score a student based upon the comprehension and success of attempt. Another recommendation of this study is to ensure that the newly formatted rubrics are written by subject matter experts that have a minimum of five years’ experience in a hospital setting and licensure as a Registered Veterinary Technician (RVT) or Doctor of Veterinary Medicine (DVM). Further studies should be conducted across other PMI locations and programs to compare evaluator continuity.
2020-08-06T00:00:00Z