Modulatory effects of bag cell peptides in {\it Aplysia}: Behavioral and electrophysiological studies

Reproductive hormones have effects on the nervous system not directly related to reproductive function. In the rat, for example, luteinizing hormone releasing hormone has dramatic effects on learning and memory. The present work attempts to examine the effects of reproductive hormones on non-reproductive behaviors and the neural loci and mechanisms underlying these effects in Aplysia, an animal whose behaviors, reproductive hormones and neural circuitry have been well characterized.^ In Aplysia, the neurosecretory bag cells release several peptides that are responsible for eliciting egg laying. The effects of these peptides on the defensive tail-siphon withdrawal reflex, as well as sensitization of this reflex, were examined. Sensitization, a simple form of nonassociative learning, refers to the behavioral enhancement of a response to a test stimulus after the presentation of a strong stimulus, that may last minutes (short-term) or days (long-term). An extract of the bag cells (BCE) inhibited the baseline siphon component of the tail-siphon withdrawal reflex and suppressed long-term, but not short-term, sensitization of the reflex. Preliminary experiments suggest that BCE also affects the tail component of the tail-siphon withdrawal reflex.^ To determine the neural mechanisms underlying the inhibition of the baseline reflex, electrophysiological studies were performed using an in vitro analogue of the tail-siphon withdrawal reflex to examine the ability of BCE, as well as the individual bag cell peptides (BCPs), to modulate the circuitry of the reflex. Bag cell extract attenuated the synaptic strength of the monosynaptic connections between tail sensory neurons and tail motor neurons. When individually applied only $\beta$-BCP produced a similar attenuation. This effect of $\beta$-BCP was not dependent on changes in duration of the presynaptic action potential.^ An in vitro analogue of long-term sensitization training was developed to examine the mechanisms by which the BCPs may affect long-term sensitization of the tail-siphon withdrawal reflex. This analogue exhibited both short- and long-term facilitation of the connections between the tail sensory and motor neurons.^ The results of these behavioral and electrophysiological experiments suggest that the BCPs inhibit the tail-siphon withdrawal reflex, at least in part, by modulating the synaptic strength of the connections between the sensory neurons and motor neurons underlying the reflex. One candidate for this effect is $\beta$-BCP. Thus, the peptides which elicit egg laying may also serve other functions such as the inhibition of defensive reflexes. In addition, these experiments raise the possibility that BCPs may exert a long lasting effect ($>$24 hr), suppressing long-term sensitization of the tail-siphon withdrawal reflex. ^

Effect of parental weight status on the food packed in preschool children's lunches

Problem. Recent statistics show that over a fifth of children aged 2-5 years in 2006-2008 were overweight, with 7% above the 97 th percentile of the BMI-for-age growth charts (extreme obesity). Because poor diet is an important environmental determinant of obesity and the preschool years are crucial developmentally, examination of factors related to diet in the pre-school years is important for obesity prevention efforts. ^ Objective. The goals of this study were to determine the association between BMI of the parents and the number of servings of fruits, vegetables, and whole grains (FVWG) packed; the nutrient content of preschool children’s lunches; and norms and expectations about FVWG intake.^ Methods. This study was a cross sectional analysis of parents enrolled in the Lunch is in the Bag program at baseline. The independent measure was weight status of the parents/caregivers, which was determined using body mass index (BMI) calculated from self-reported height and weight. BMI was classified as healthy weight (BMI <25) or overweight/obese (BMI ≥25). Outcomes for the study included the number of servings of fruits, vegetables and whole grains (FVWG) in sack lunches, as well as the nutrient content of the lunches, and psychosocial constructs related to FVWG consumption. Linear regression analysis was conducted and adjusted for confounders to examine the associations of these outcomes with parental weight status, the main predictor. ^ Results. A total of 132 parent/child dyads were enrolled in the study; 59.09% (n=78) of the parents/caregivers were healthy weight and 39.01% (n=54) of the parents/caregivers were overweight/obese. Parents/caregivers in the study were predominantly white (68%, n=87) and had at least some college education (98%, n=128). No significant associations were found between the weight status of the parents and the servings of fruits, vegetables and whole grain packed in preschool children’s lunchboxes. The results were similar for the association of parental weight status and the nutrient contents of the packed lunches. Both healthy weight and overweight/obese parents packed less than the recommended amounts of vegetables (mean servings = 0.49 and 0.534, respectively) and whole grains (mean servings = 0.58 and 0.511, respectively). However, the intentions of the obese/overweight parents were higher compare to the healthy for vegetables and whole grains.^ Conclusion. Results from this study indicate that there are few differences in the servings of fruits, vegetables and whole grains packed by healthy weight parents/caregivers compared to overweight/obese parents/caregivers in a high income, well-educated population, although neither group met the recommended number of servings of vegetables or whole grains. Thus, results indicate the need for behaviorally-based health promotion programs for parents, regardless of their weight status; however, this study should be replicated with larger and more diverse populations to determine if these results are similar with less homogenous populations.^

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.