Monitoring Subcellular Calcium by Designed Calcium Sensors and the Calcium Sensing Receptor Structure and Function

Calcium (Ca2+) regulates various biological and pathological functions via calcium dynamics and interacting with key calcium binding proteins such as the calcium sensing receptor (CaSR). In this dissertation, the first X-ray structure of the extracellular domain of CaSR was determined by engineering mammalian expression systems. The revealed Ca2+/Mg2+ and Trp derivative L-1,2,3,4-tetrahydronorharman-3-carboxylic acid (TNCA) binding sites and key determinants contribute to the functional cooperativity of CaSR in cells. Magnesium (Mg2+) acts as a heterotropic cooperative co-agonist with calcium to co-activate the function of CaSR, including calcium oscillations. TNCA potentiates CaSR co-activation and recovers a loss of function caused by mutation at the dimer interface calcium binding site. Several mutations of the main Ca2+/TNCA binding site at the hinge region eliminate CaSR activity. Mutations S272A and D216N at the hinge region lead to a loss of Ca2+ binding and complete loss of cooperative binding for Tb3+ using bacterially expressed protein and Trp-sensitized FRET assay. Efforts in the development of new CaSR therapeutics using structure-based drug design were also explored. Next, we aimed to monitor endoplasmic/sarcoplasmic reticulum (ER/SR) mediated subcellular Ca2+ dynamics using our designed calcium sensors CatchER+ and CatchER+-JP45. Using highly inclined laminated optical (HILO) microscopy, we report calcium dynamics in the ER/SR with differential calcium responses to 4-cmc for release and recovery indicating differential Ca2+ signaling from Ca2+ and protein expression subcellular microdomains. We find Ca2+ dynamic differences between the localized high Ca2+ release region of the junctional SR for E-C coupling with targeted CatchER+-JP45 to ryanodine receptor over the global Ca2+ ER/SR regulation of CatchER+ sensor. To understand ER Ca2+ dynamics in neurons, we utilized our sensor CatchER+ and high-resolution HILO imaging to show that 100 µM DHPG induced mGluR1/5 activation leads to IP3R Ca2+ release as well as Ca2+ uptake throughout the soma and dendrites. The differential release and uptake for the ER Ca2+ dynamics in response to DHPG indicates subcellular microdomains throughout the neurons as well. These sensors will significantly impact Ca2+ dynamics research and molecular basis of ER Ca2+ related diseases by exposing Ca2+ dynamics, function, mobility, and trafficking in the ER/SR

The Hip in Hip Hop: Toward a Discipline of Hip Hop Studies

Editorial

Population substructuring in Schreibers' long-fingered bat (Miniopterus schreibersii) in South Africa

Schreibers' long-fingered bat, Miniopterus schreibersi migrates seasonally between winter (hibernacula) and summer (maternity) colonies in South Africa. Previous behavioural studies suggested that roost fidelity is well developed in this species, and that juvenile dispersal may be limited, possibly in both sexes. If males and/or females are strongly philopatric, this may lead to restricted gene flow among colonies, resulting in genetically distinct breeding subpopulations. The population structure of M. schreibersii in South Africa was investigated using microsatellites and mitochondrial DNA (mtDNA), with the aim of determining the degree of genetic differentiation among colonies, and the extent and direction of bat movement among the colonies. A genomic library was constructed for M. schreibersii, and was screened for (CA)0 and (GA)0 microsatellite repeats. Five novel, highly polymorphic loci were identified. These five loci, and an existing mammalian microsatellite locus, were amplified in. 301 individuals, sampled from ten colonies throughout South Africa. Significant genetic heterogeneity exists within the M. schreibersii population, such that the population can be subdivided into three partially discrete breeding subpopulations. Little genetic differentiation exists between colonies withi

Novel method to detect unlabeled inorganic nanoparticles and submicron particles in tissue by sedimentation field-flow fractionation

Journal ArticleA novel methodology to detect unlabeled inorganic nanoparticles was experimentally demonstrated using a mixture of nano-sized (70 nm) and submicron (250 nm) silicon dioxide particles added to mammalian tissue. The size and concentration of environmentally relevant inorganic particles in a tissue sample can be determined by a procedure consisting of matrix digestion, particle recovery by centrifugation, size separation by sedimentation field-flow fractionation (SdFFF), and detection by light scattering

Measuring the Food Environment: A Systematic Technique for Characterizing Food Stores Using Display Counts

Marketing research has documented the influence of in-store characteristics—such as the number and placement of display stands—on consumer purchases of a product. However, little information exists on this topic for key foods of interest to those studying the influence of environmental changes on dietary behavior. This study demonstrates a method for characterizing the food environment by measuring the number of separate displays of fruits, vegetables, and energy-dense snack foods (including chips, candies, and sodas) and their proximity to cash registers in different store types. Observations in New Orleans stores (N = 172) in 2007 and 2008 revealed significantly more displays of energy-dense snacks than of fruits and vegetables within all store types, especially supermarkets. Moreover, supermarkets had an average of 20 displays of energy-dense snacks within 1 meter of their cash registers, yet none of them had even a single display of fruits or vegetables near their cash registers. Measures of the number of separate display stands of key foods and their proximity to a cash register can be used by researchers to better characterize food stores and by policymakers to address improvements to the food environment

The Roles Elementary School Counselors Perform in the Education of Students with Autism Spectrum Disorders

This nation-wide study investigated elementary school counselors (ESC) self-reported: (a) professional background and training; (b) general knowledge of autism spectrum disorders (ASD); (c) attitudes towards ASD; and (d) roles performed with students identified with ASD. Also investigated was the predictive relationships between professional background, training, knowledge, and attitudes on roles (counseling, consultation, curriculum, and coordination) performed with students identified with ASD. Descriptive statistics were utilized to address professional background, training, knowledge, attitude and characteristics of ESC participants. These variables were also examined in relationship to the four role types. Multivariate analyses of variance (MANOVA) and Pearson’s correlation coefficients were used to test for significant relationships. A series of four multiple regression analyses predicting each of the total roles scores for counseling, consultation, curriculum, and coordination were also conducted. Results of the study suggest (a) ESC have limited training experiences of ASD, leading to self-education about this population of students, (b) ESC possess general knowledge about ASD, (c) overall, ESC have positive attitudes towards ASD, and (d) ESC perform all conceptualized roles in the education of students with ASD. Regression models revealed eight predictors found to influence roles: total knowledge, attitudes, geographic setting, U.S. region, years practiced, conference training, self-education, and ASD caseload. Significantly associated with performing roles across all four domains was the number of students with ASD on ESC caseload

STUDY OF EDUCATIONAL EQUITY IN HAWAI‘I: EXAMINING THE DISTRIBUTION OF QUALIFIED TEACHERS AND STUDENT OUTCOMES

Ph.D