Biophysical Studies of SNARE Protein- Membrane Interactions

SNARE (Soluble NSF (N-ethylmaleimide Sensitive Fusion) Attachment Protein Receptors) proteins have been linked to the membrane fusion mechanism since 1993 as fusion proteins and have been suggested to be the minimal machinery. The complexity of the fusion process means that many questions remain unanswered as to how SNARE proteins perform their role. The most favoured model (the stalk model) does not involve proteins directly and so the influence of the SNARE proteins on lipid properties is of interest. In this thesis, work is presented which investigates how these proteins may manipulate membrane properties in order to promote fusion. Purified proteins solutions of His6-VAMP2 (Vesicle Associated Membrane Protein 2), His6-SNAP-25 (Synaptosomal-associated Protein 25) and a truncated form of Syntaxin 1A (His6-ΔN-Syx 1A, w.t aa 181-288) were obtained following bacterial over-expression. Fluorescent versions of His6-VAMP2 and His6-ΔN-Syx 1A were produced by the addition of cysteine residues to the C-terminus followed by labelling using Alexa Fluor® 488-C5-maleimide and Alexa Fluor® 555-C2-maleimide respectively. These fluorescent proteins were used to establish that the purified protein inserted into model lipid bilayers. The effect of SNARE protein incorporation on the relaxed curvature of bilayers was explored by examining giant unilamellar vesicles grown using electroformation. Bilayers containing either 1:300 His6-VAMP2: DOPC or 1:1:600 His6-SNAP-25: His6-ΔN-Syx 1A: DOPC were smaller than pure DOPC vesicles, indicating that SNARE proteins increase the relaxed curvature of the bilayer. Analysis of these vesicles by micropipette aspiration suggested that VAMP2 lowered the bending rigidity of the membrane and a reduction in the area expansion modulus relative to the pure lipid bilayer was found. The t-SNARE sample also indicated a reduction in bending rigidity but the area expansion modulus was found to increase. These latter results are thought to be due to the formation of protein aggregates. Lipid mixing assays were conducted to investigate how changes in the properties of liposome bilayers affected fusion rates. It was found that the addition of DOPE to DOPC bilayers increased the rate of hemifusion and this was also found for cholesterol addition, suggesting both components are fusogens. The rate of hemifusion rose continually upon DOPE addition but reached a plateau in the cholesterol study shortly after 10 mol%. Despite this, the fusion rates for the cholesterol study were generally higher than the same mol% DOPE added. The changes in fusion rates observed have been explained by considering the impact of the additives on the free energy and stored curvature elastic stress of membranes as well as the change in the energy of formation of intermediate structures. From the findings of this thesis it is proposed that the SNARE proteins are able to soften the membranes in which they reside. This allows the membrane to be deformed with less energy input. The strength of the SNARE complex and the force applied to the membranes during its formation increases membrane tension and reduces inter-membrane separation; promoting hemfusion. Following the action potential of the neuron it is proposed that a conformational change occurs in the synaptic SNARE complex, pulling on the hemifusion diaphragm and inducing the formation of a fusion pore

The Land\u27s Liver

Economic progress seems to consistently come with a steep price tag. The Industrial Revolution was birthed in the 1800’s, and as a result manufacturing, mining, transport, as well as other industries within our system systematically tainted viable water sources, contaminated soil, and increased greenhouse gases that include, but are not limited to, carbon dioxide and methane. By 1889 the first sewage treatment plant was installed at Washington D.C., but there were still a very small number of laws that were enacted in order to regulate the mounting amount of industrial waste. Not until 1970, almost 20 years after the fiery vision of the Cuyahoga River, did President Richard Nixon issue forth a mandate for change. In July of the same year, with Congress and the White House working collectively, the Environmental Protection Agency was formed in response to the public demand for an increase in quality standards that included the life sustaining elements; water, air, and land. On December 2nd of the same year, the Environmental Protection Agency opened its doors to fulfill its contract with America (EPA). This paper fulfilled C. Turner\u27s Honor Contract for Honors Botany. Her faculty supervisor was Professor Steven Giambrone

Relationship of Lactate Dehydrogenase & Prolactin Genotypes to Immune Function, Growth, and Composition of Replacement Heifers Grazing Tall Fescue

Objectives of this research were to determine effects of LDH enzymatic activity and circulating PRL on immune response, growth, composition, and reproduction, and to determine the effects of single nucleotide polymorphisms of LDH and PRL on immune function, growth and carcass traits, cyclicty, and fecundity of replacement heifers grazing endophyte infected tall fescue. Eighty-nine Angus based heifers had physical and ultrasound measurements along with blood samples collected at weaning, yearling, and prebreeding. Estrus detection was conducted for 10 d post each collection period, except prebreeding. Pregnancy status was verified after AI and again after bull removal. Heifers were maintained on tall fescue with corn gluten and free choice mineral supplementation. A 48 hr delayed hypersensitivy immune trial was conducted at 10 mo of age. Activity of LDH (forward and reverse) was correlated to: hormonal and enzymatic activities, growth, and carcass traits. Concentrations of PRL were associated with growth responses. Single nucleotide polymorphisms of LDH related to: hormonal and enzymatic activities, growth, carcass, cyclicity and immune responses. Polymorphisms of PRL related to carcass traits, pregnancy status, and MCV of immune response. Canonical correlations found no association between LDH activity or PRL concentration to physical set 1 traits of: body weight, hip height, and hip width, and subsequently no correlation to set 2 traits of: pelvic height, pelvic width, Longissimus dorsi area, and rib fat found in other studies. Environmental impact may have obscured anticipated results. Timing and handling appear to be of consideration to recorded measurements in regards to hormonal and metabolic connections. Growth, carcass, and other hormonal measures taken during the trial correlated amongst each other and immune responses serving as further indicators of visual manifestations recognizing the complexity of endocrine functions

Graphical properties of the bipartite graph of Spec(Z[x])\{0}

Consider $Spec(Z[x])$, the set of prime ideals of $Z[x]$ as a partially ordered set under inclusion. By removing the zero ideal, we denote G_{Z}=Spec(Z[x])\{0} and view it as an infinite bipartite graph with the prime ideals as the vertices and the inclusion relations as the edges. In this paper, we investigate fundamental graph theoretic properties of $G_{Z}$. In particular, we describe the diameter, circumference, girth, radius, eccentricity, vertex and edge connectivity, and cliques of $G_{Z}$. The complement of $G_{Z}$ is investigated as well

Associations Between Preweaning Calf Feeding Behaviors with Age at First Calving and Lactational Performance Using an Automatic Calf Feeder

The objective of this retrospective observational study was to determine whether preweaning calf behaviors and milk replacer (MR) intake from an automatic calf feeder were associated with age at first calving and first-lactation performance. Calves were housed in groups with access to an automatic calf feeder for 7 wk with a maximum milk allowance of 1,800 g/d of MR (12 L/d). Outcomes of interest included age at first calving (n = 137), first-lactation mature-equivalent 305-d (ME305) milk yield (n = 132), and first-lactation ME305 energy-corrected milk (ECM) yield (n = 132). Linear models included the fixed effects of the daily means of unrewarded visits, rewarded visits, drinking speed, and MR intake. Furthermore, breed, disease diagnosis, season of birth, and age of the calf when it was first introduced to the automatic calf feeder were included in all models. The genetic parameter for milk yield (predicted transmitting ability for milk) was included in models related to lactational performance. Feeding behaviors and milk replacer intake were not associated with age at first calving. Unrewarded visits to the automatic calf feeder were associated with ME305 milk and ECM yields. As mean daily unrewarded visits increased by 1, first-lactation ME305 milk yield and ME305 ECM yield increased by 319 kg and 224 kg, respectively. No other feeding behavior was significantly associated with first-lactation ME305 milk or ECM yields. In conclusion, unrewarded visits were positively associated with first-lactation performance, but external validation is still needed

Interlace Polynomials of Friendship Graphs

In this paper, we study the interlace polynomials of friendship graphs, that is, graphs that satisfy the Friendship Theorem given by Erdös, Rényi and Sos. Explicit formulas, special values and behavior of coefficients of these polynomials are provided. We also give the interlace polynomials of other similar graphs, such as, the butterfly graph

Athletic Trainers’ Confidence and Proficiency in Addressing Athlete Mental Health

Despite the prevalence of athletes reporting they would seek an athletic trainer’s help, athletic trainers may not feel proficient or competent in addressing mental health with their athletes (Bird, et al., 2018). Athletic trainers need proper mental health education set to a specific standard to provide an adequate continuity of care for athlete mental health. To reach this goal, a better understanding of the link between education and experiences, and athletic trainer confidence and performance is needed to determine how to achieve this outcome in a practical manner. This study attempted to investigate the relationships between education and self-efficacy and self-efficacy and subjective performance as it relates to athletic trainers performing the skills outlined in the 2020 CAATE educational standards on mental health. The ATs surveyed reported moderate-high levels of confidence and subjective performance in their abilities to perform the five competencies (identify, refer, support, develop, & implement) outlined in the 2020 CAATE standards for mental health education. The athletic trainers surveyed reported the highest levels of confidence and subjective performance in their abilities to identify, refer, and provide support to athletes with mental health conditions, rather than developing and implementing policies and procedures on athlete mental health. The statistical analysis provides evidence for self-efficacy correlations within athletic training, which provides a case for more detailed mental health education and volume of post-certification educational opportunities related to mental health for athletic trainers

Pathway Possibilities: The Lilly Endowment's Pathways for Tomorrow Initiative

Looking to the future for what supervision and mentoring will look like and be utilized

Perceived Parenting, Self-Conscious Emotions, and Forgiveness in College Students

The relations of parental warmth, self-conscious emotions, and forgiveness were investigated. University students completed self-report questionnaires online through the university-sponsored website. The questionnaires contained measures of five constructs: parental warmth, empathy, shame, self-forgiveness, and interpersonal forgiveness. From the results, the authors concluded that interpersonal forgiveness and self-forgiveness were predicted by parental warmth, empathy, and shame. Empathy mediated the relation of parental warmth with interpersonal forgiveness and self-forgiveness. Parental warmth and self-conscious emotions were important in the prediction of both interpersonal forgiveness and self-forgiveness

Functional Analysis of Cyc2 in Microbial Mat Communities of Mariana Arc and Back-arc Hydrothermal Vents

The primary production by Zetaproteobacteria at iron-rich vents of the Mariana Arc and back-arc supports high microbial diversity. In microaerophilic circumneutral pH, Zetaproteobacteria are able to produce energy by oxidizing ferrous iron (Fe2+) from the vent effluent with the presence of a key gene involved in their iron-oxidation, Cyc2, a fused cytochrome-porin. These genes are found in all Zetaproteobacteria known to date. This study combines molecular quantitative techniques with metagenomic analyses to enhance our confidence in gene quantification from environmental samples. The occurrence of cyc2 was quantified from microbial mat communities at NW Eifuku, NW Rota-1, Snail and Urashima Vents using primers designed from a composite metagenome to better understand the potential impact of Zetaproteobacteria metabolism on these iron-rich microbial communities. Based on a multiple sequence alignment and phylogenetic analysis, two distinct groups of the Zetaproteobacteria cyc2 were identified and termed Cluster 1 cyc2 and Cluster 3 cyc2 based on the nomenclature provided by Chan et al. (2018). Multidimensional scaling of Zetaproteobacteria SSU rRNA and cyc2 gene abundances showed that the presence of the two cyc2 sequence variants are strongly driven by an array of different vent fluid chemicals. We also report on the phylogenetic relationship between the two cyc2 variants and other representative FeOB, as well as the distribution of cyc2 gene abundances across the Mariana hydrothermal vent sites along the Arc and back-arc. Overall, this provides insight on the ecological and evolutionary implications of Zetaproteobacteria iron-oxidation represented at the community-level across a wide-range of geochemical parameters