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With an expanding life-science sector and growing production of recombinant proteins, the need for efficient downstream processing is increasing. Certain proteins are sensitive to the harsh conditions often used in protein purification, such as low pH, which can result in aggregation and denaturation. ZCa is a domain derived from Protein A that can be used for calcium-dependent purification of antibodies without the need for acidic pH. Based on this domain, the CaRA library has been constructed, which targets other therapeutic proteins than human antibodies. Four of the proteins isolated from the CaRA library, namely CaRA_scFv_1, CaRA_scFv_2, CaRA_G-CSF_1 and CaRA_G-CSF_3, are presented here for the purification of single chain variable fragment and granulate colony stimulating factor. The four proteins were produced as monomers, trimers and hexamers in an attempt to increase the binding capacity and attached to a matrix for purification using site-specific coupling. The successful binders CaRA_scFv_1 and CaRA_scFv_2 showed high affinity for their target protein scFv and were able to selectively capture an increased number of molecules through multimerization. Calcium-dependent binding was demonstrated by elution at neutral pH using the calcium chelator citrate, thus concluding that these multimerized CaRA variants can be used to considerably increase the efficiency in scFv purification while providing excellent purity and significantly reducing the risk of aggregation.

Traumatic brain injury (TBI) is the most common injury in childhood, and it is the leading cause of disability. Early childhood is an area of specific interest because it is a period of rather significant vulnerability to longer-standing problems. Better health and behavior-related outcomes generally improve when diagnosis occurs early enough to inform evidence-based interventions adequately. However, there continues to be relatively weak identification of concussions in early childhood, and misdiagnoses often lead children to receive the incorrect intervention if they receive intervention at all. Clinicians need to identify symptoms of a concussion immediately following injury. To this end, the present study examines the literature to determine domains, and any narrow abilities impacted following a concussion. Assessments items were generated based on a review of published norm references tests and task demands analyses (n = 18). Testing items were cross-referenced using developmental literature to ensure they were appropriate for assessment for children age 3 years. Via the Delphi survey method, a heterogeneous panel of experts (Round 1 n = 17; Round 2 n = 13), including physicians, psychologists, school nurses, speech and language pathologists, and athletic trainers, offered their opinion regarding what areas are impacted following a concussion. The panel provided clarification on the operational definitions and agreed that the testing items, indeed, were developmentally appropriate. The group also decided that a paraprofessional could administer the items with minimal training, which is an essential consideration because children in early childhood are often cared for by professionals untrained in assessment, working in daycare or preschool settings. The present study concludes that, indeed, an evaluation of concussions symptoms that are like traditional sideline assessments is possible. However, the results of this assessment are only preliminary, and there was no evidence for validity based on response processes or relations to other variables; likewise, reliability data are unavailable at this time. Recommendations for future research are included, and ideas to move toward standardization are presented. Recommendations for the training of paraprofessionals in these assessment procedures, too, are outlined. / School Psychology

Objective: Grit is a noncognitive trait related to perseverance and consistent pursuit of long-term goals. Research on grit and aging provides evidence that grit increases with age and may be protective of cognitive and everyday functioning. However, no studies to date have examined relations between concurrently measured grit, cognitive abilities, and everyday functioning. This study tested two hypotheses: 1) that grit would predict cognitive performance and that this relation would be moderated by clinical diagnosis of cognitive status (i.e., healthy vs. mild cognitive impairment; MCI), and 2) that grit would predict everyday functioning and that this effect would be mediated by compensatory strategy use. Methods: Sixty-one older adults were recruited from the Penn Memory Center’s National Alzheimer’s Coordinating Center (NACC) cohort, including forty healthy controls with normal cognition and twenty-one individuals with mild cognitive impairment (MCI). Participants completed tests of verbal episodic memory, executive functioning, grit, compensatory strategy use, and everyday functioning. Results: Grit was not associated with cognitive functioning in either domain. Instead, memory performance was predicted only by clinical status (healthy vs. MCI), and executive functioning was predicted by clinical status, depressive symptoms, and years of education. Grit was negatively associated with everyday functional difficulties; however, there was no indirect effect of compensatory strategy use. Additionally, grit was moderately correlated with depression symptoms (r = -0.41). Conclusions: Grit is predictive of preserved everyday functioning, but not cognitive functioning, in a sample of healthy older adults and individuals with MCI. Mechanisms explaining the role of grit on everyday function remain elusive, though secondary analyses support that grit also influences affective well-being and may have a weaker role in the context of cognitive impairment. / Psychology

A novel belt alternator starter (BAS) is proposed to replace the starter and alternator in a hybrid electric vehicle. The BAS designed utilizes an 18 rotor, 12 stator pole switched reluctance machine (SRM) configuration, with concentrated bar windings wound in parallel. Through iteration of various machine geometry parameters, the SRM can meet the torque and speeds demands over standardized drive cycles described by the US Environmental Protection Agency. / With the depletion of oil wells and changing global climate, a large emphasis is placed on the research, development and adoption of electric vehicles (EVs) to replace vehicles driven by internal combustion engines (ICEs). However the global supply chain is still not ready for such a large demand in EVs; therefore hybrid electric vehicles (HEVs) aim to ease the transition between ICEs and EVs. The research outlined in this thesis investigates the design of a 18 stator, 12 rotor pole (18/12) configuration switched reluctance machine (SRM) utilizing novel technologies for use as a belt alternator starter (BAS) motor in an HEV. Background research on current trends and technologies for electric motors and vehicles is performed before evaluating initial geometry for the motor core to be designed. Initial geometry is brought into JMAG to develop an electromagnetic model and begin the geometry optimization. The 18/12 design process highlights how changes to motor parameters from a geometry and winding standpoint will affect motor performance. After the motor core geometry yields suitable performance, a mechanical design is proposed encompassing the rotary assembly, cooling as well as solutions for mounting. / Thesis / Master of Applied Science (MASc) / Hybrid electric vehicles are becoming more prevalent as stricter restrictions are placed on fuel economy and emissions targets. Full electric vehicles on the other hand have not yet become the standard form of transportation due to the limits on range and infrastructure. Because of this, automotive manufacturers are researching and developing new methods in which they can meet these restrictions and limitations. Switched reluctance motors aim to be a solution to meet these demands while forging a new path by alleviating the demand on rare earth metals for the motor core. In this thesis, a design is proposed to fill an existing role in vehicle electrification best suited for a belted alternator starter.

Mild blast-induced traumatic brain injury (bTBI) is a modality of injury that has been of major concern considering a large number of military personnel exposed to the blast wave from explosives. bTBI results from the propagation of high-pressure static blast forces and their subsequent energy transmission within brain tissue. Current literature presents a neuro-centric approach to the role of mitochondria dynamics dysfunction in bTBI; however, changes in astrocyte-specific mitochondrial dynamics have not been characterized. As a result of fission and fusion, the mitochondrial structure is constantly altering shape to respond to physiological stimuli or stress insults by adapting structure and function, which are intimately connected. Dysregulation of the protein regulator of mitochondrial fission, DRP1, and upregulation in the phosphorylation of DRP1 at the serine 616 site is reported to play a crucial role in astrocytic mitochondrial dysfunction, favoring fission over fusion post-TBI. Astrocytic mitochondria are starting to be recognized to play an essential role in overall brain metabolism, synaptic transmission, and neuron protection. Mitochondria are vulnerable to injury insults leading to the worsening of mitochondrial fission and increased mitochondrial fragmentation. In this study, a combination of in vitro and in vivo bTBI models were used to examine the effect of blast on astrocytic mitochondrial dynamics. Acute differential remodeling of the astrocytic mitochondrial network was observed, accompanied by an acute (4hr) and sub-acute (7 days) activation of the GTP-protein DRP1. Further, results showed a time-dependent reactive astrocyte phenotype transition in the rat hippocampus. This discovery can lead to innovative therapeutics targets to help prevent secondary injury cascades that involve mitochondria dysfunction. / Doctor of Philosophy / Blast-induced traumatic brain injury (bTBI) is a modality of injury that has become prominent considering a large number of military personnel exposed to a blast wave caused by explosives. Blast injury results from the energy transmission of the blast wave to the brain. Within the brain, there are specialized cells, called astrocytes, that help maintain a healthy environment. This work investigates the role that astrocytes play during the injury recovery process. Within the astrocytes, there are organelles called mitochondria, that help maintain the energy for the cell. The number and function of mitochondria can change in response to the brain injury. They can increase in number by a process called fission and they can decrease in number by a process called fusion. These events effect the function of the mitochondria. Researchers have methods that can identify changes in the number and function of the mitochondria. In this work, astrocyte mitochondrial dynamics were examined and compared using models of bTBI. We found significant changes in the mitochondria of astrocytes, which could lead to an unhealthy environment in the brain. This discovery can lead to new treatments for patients that may improve their quality of life following bTBI.