Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type I (NF1) is a common genetic disease that affects over 200,000 patients in North America, Europe, and Japan. Individuals with NF1 display a wide variety of pathologies; importantly, 15-40% of NF1 patients are affected by plexiform neurofibromas. Neurofibromas are complex tumors consisting of tumorgenic Schwann cells surrounded by endothelial cells, fibroblasts, and inflammatory mast cells. These peripheral nerve sheath tumors contribute significantly to the morbidity and mortality associated with NF1. Currently, no medical therapies exist for treating neurofibromas. Recent evidence indicates that the hematopoietic tumor microenvironment carries out a crucial function in the formation of plexiform neurofibromas. Neurofibromatosis is the result of mutations at the NF1 locus, which encodes the GTPase activating protein neurofibromin. Neurofibromin is a negative regulator of the proto-oncogene Ras. Ras hyperactivation is the molecular basis of NF1 associated phenotypes, and it has been demonstrated that restoration of Ras signaling to wild type levels can correct NF1 associated phenotypes in vitro and in vivo. In keeping with the long term goal of detecting potential molecular targets for medical therapies to treat human plexiform neurofibromas, we have identified the kinase Pak1 as a possible downstream intermediary of Ras signaling in NF1 deficient cells. Studies described here utilized murine genetic models to study the effects of genetic inactivation of Pak1 on molecular signaling and cellular functions related to neurofibromas. We demonstrate that inactivation of Pak1 leads to correction of SCF mediated gain-in-function phenotypes seen in Nf1 haploinsufficient mast cells, in vivo and in vitro. However, by using a conditional Nf1 knockout mouse that is a reliable model of plexiform neurofibroma formation, we shown that loss of Pak1 alone in the hematopoeitic compartement is not sufficient to prevent neurofibroma formation. Additionally, we describe a key role for Pak1 in regulating PDGF and TGF-β mediated fibroblast functions, in vitro and in vivo. These studies provide insight into the causes of debilitating tumors related to a common genetic disease, and this research could potentially lead to the development of medical therapies for these tumors, increasing the quality of life for tens of thousands of affected individuals each year.

No description available.

Sexual reproduction is an important process amongst eukaryotic organisms, with one function being to maintain genetic variation. The idea that complex eukaryotic species can persist for millions of years in the absence of sex defies fundamental evolutionary dogma, yet a group of organisms known as ancient asexuals were thought to have evolved clonally under deep evolutionary time. Prominent among these are the arbuscular mycorrhizal fungi (AMF), which are obligate plant symbionts that colonize the root cells of plants and extend their hyphae into the soil assisting the plant in acquiring key nutrients. Unlike most eukaryotes, AMF cells are multinucleate with thousands of nuclei moving through a continuous cytoplasm. Genomic analyses have identified a putative mating-type (MAT) locus within the nuclear genomes of model AMF Rhizophagus irregularis, a region that in other fungi dictates the process of sexual reproduction. Additional findings demonstrated that AMF strains carry one of two nuclear organizations. They can be either homokaryotic (AMF homokaryons), where all nuclei within the cytoplasm are virtually identical, or heterokaryotic (AMF dikaryons), where two MAT-locus variants co-exist within the cytoplasm. Despite a lack of observable traits indicative of sex, this homo/heterokaryotic dichotomy is reminiscent of the nuclear organization of sexual fungi. My research aims to build on these findings to investigate the actual role of the MAT-locus in driving AMF reproduction. To address this, I build my thesis into three main chapters. The first chapter reviews our current understanding of AMF genetics and what drives genome evolution in these organisms. The second chapter establishes a relatively easy, inexpensive, and reproducible approach to genotype known MAT variants of R. irregularis in natural and experimental conditions. The last chapter uses experimental crossings between strains to assess cytoplasmic compatibility and nuclear exchange. I demonstrate that dikaryotic spore progenies can be formed after co-culturing two distinct AMF homokaryotic strains. Further analyses of various genomic regions also reveal possible recombination in homokaryotic spore progenies from co-cultures. Overall, this research provides new experimental insights into the origin of genetic diversity in AMF. These findings open avenues to produce genetically new AMF strains in the lab using conventional crossing procedures and provide a glimpse of the mechanisms that generate AMF genetic diversity in the field.

No description available.

Adolescent type 2 diabetes (T2D) diagnoses are on the rise. Consistent with the adult literature, preliminary evidence in adolescents suggests that T2D is associated with reduced brain volume and white matter microstructural integrity. As part of the Cognitive Performance in Adolescents with T2D (CPAT2D) study, this project aimed to test whether T2D diagnosis is associated with poorer cognitive performance in adolescents. Five adolescents with obesity and T2D (60% female; body mass index [BMI] percentile 98.2 ± 2.0; age 16.7 ± 1.1 years) were recruited and matched to two control adolescents with obesity but without T2D (50% female; BMI percentile 99.9 ± 0.2; age 15.9 ± 1.3 years) on at least three of the following characteristics: age, sex, pubertal stage and habitual sleep duration. All participants wore a wrist actigraphy device for seven consecutive nights to measure sleep at home and then completed two neuromotor cognitive tasks at a laboratory testing session assessing motor preparation (simple reaction time task) and executive functioning (affective shifting task [AST]). Control data were available through the Sleep Manipulation in Adolescents at Risk of Type 2 Diabetes (SMART2D) study. Premotor reaction time outcomes in either task and proportions of commission and omission error trials in the AST were subsequently analyzed. Based on this preliminary participant sample, there is no evidence to suggest that adolescents with compared to without T2D perform differently on the neuromotor cognitive tasks. The results should be confirmed once the intended sample size is reached. In the meantime, clinicians should monitor for changes in cognitive function in adolescents with T2D, perhaps by asking about academic achievement. The majority of our sample exhibited sub-optimal movement behaviours; to preserve overall health, adolescents with obesity and/or T2D should strive to meet sleep, physical activity and screen time recommendations for their age group.

Studies in third language acquisition (L3) add an exciting dimension to the field of language acquisition and present many interesting lines of research. One issue unique to L3 acquisition is the effect of second language (L2) experience on L3 acquisition. Because L3 learners have already experienced the process of language acquisition, it seems likely that the experiences, knowledge, and skills they may have gained while learning an L2 would transfer to, and even enhance, their ability to acquire an L3. It also seems reasonable to believe that the type of language previously studied would have an effect on learners' abilities to acquire additional languages of a similar type. Many research studies have affirmed these theories showing that L3 learners have an advantage in language learning which is absent in L2 learners. Other studies have shown L2 typology, level of proficiency, and extent of L2 experience to be contributing factors in adult L3 learners who had studied French or Spanish as an L2 for varying lengths of time, and were studying French, Spanish, Korean, Japanese, or Russian as an L3. Participants' L2 experience, including length of study and language type, was compared to their scores on an L3 speaking assessment consisting of pronunciation, grammar, vocabulary, and fluency ratings. A linear stepwise multiple regression analysis showed a modest trend in which length of L2 experience did have an effect on L3 acquisition. Although this trend did not achieve statistical significance, a hypothetical analysis showed that the trend became significant with the omission of three outliers. An analysis of variance demonstrated that type of language experience did not significantly affect L3 acquisition since participants from all five L3 groups received comparable scores on the speaking assessment. Another analysis of variance, however, showed language type to be a highly significant factor in the acquisition of L3 pronunciation. The results of the study suggest that length of L2 exposure does seem to affect L3 acquisition to some extent, although the trend found from the data was modest. The study also concluded that language typology was not a significant contributing factor in L3 acquisition, with the exception of its effect on the acquisition of L3 pronunciation.

The aspiration of oral and gastric substances is a well-known risk for ventilator associated pneumonia (VAP) in the intubated, mechanically ventilated (MV), patient of the intensive care unit (ICU) population. The gastric biomarker pepsin and the oral biomarker salivary amylase have been identified as evidence of aspiration prior to the manifestation of acute pulmonary illness. In an effort to decrease the risk for aspiration, several evidence based nursing practices are in place. Actions include 30 degree head of the bed positioning, oral care, suctioning, and circuit change interval protocols, as well as the administration of medication with the objective of reducing acid reflux. Additional recommendations concern the type of endotracheal tube (ETT) used to ventilate the intubated patient. The continuous subglottic suctioning endotracheal tube (CSS-ETT) features an additional port which continually suctions secretions that accumulate above the inflated endotracheal cuff. Patients with standard endotracheal tubes (S-ETT) receive manual, as needed suctioning of accumulated secretions in the mouth and the oropharynx per agency protocol. Research of the critical care population has demonstrated a decreased instance of VAP using CSS-ETT as compared to S-ETT utilization. This study sought to compare the incidence of the biomarkers pepsin and salivary amylase in the suctioned oral and tracheal secretions of patients with S-ETT compared to patients with CSS-ETT. Part of the protocol of a descriptive, comparative study of the clinical indicators for suctioning established the collection of the paired suctioned oral and tracheal aspirates. Those collected aspirates were analyzed for a pilot study of pepsin and amylase analysis. This study compares the incidence of aspirates in oral and tracheal secretions by endotracheal tube type.; The intention of this study was that it would assist in demonstrating beneficial aspects of the selection of the CSS-ETT. It is considered that further investigation with a larger population group could add statistical significance.; Tracheal aspirates were obtained with a closed tracheal suction device while oral secretions were obtained with a suction catheter designed to reach the oropharynx. Biomarkers assayed were the gastric marker pepsin and the oropharyngeal marker salivary amylase. Assays of pepsin and salivary amylase were performed using standard procedures in a specialty diagnostic laboratory. Specimens were obtained from 11 subjects: 8 male and 3 female. The majority were Caucasian (n=9), had a CSS-ETT (n=8), were on mechanical ventilation in the synchronized intermittent mandatory ventilation mode, and on tube feedings (n=9) located in the stomach (n=7). The mean age was 56 years. Feeding tubes were placed in 9 patients, and the majority of the tubes were Dobbhoff. Pepsin was found in the oral secretions of 62.5% (n = 5) of the CSS-ETT subjects, while 50.0% (n = 4) had pepsin in the tracheal aspirate. Pepsin was found in the oral secretions of 66.7% (n = 2) of the S-ETT subjects, and 66.7% (n = 2) had pepsin in their tracheal aspirate. All subjects of both groups (n = 11) had oral salivary amylase detected. Salivary amylase was detected in the tracheal aspirate of 100% (n = 3) of the S-ETT subjects versus 62.5% (n = 5) in CSS-ETT group. Based on the results of this study, there was a reduction in the number of subjects who had oral compared to tracheal aspirate pepsin in the CSS-ETT group (n = 5 oral versus n = 4 tracheal) tube type. The S-ETT group had equal number of subjects with oral (n = 2) and tracheal pepsin detected (n = 2). However, the results when comparing the S-ETT and the CSS-ETT groups were not statistically significant (p = 0.898 pepsin oral and 0.621 tracheal pepsin). There may be clinical significance. It appears that the CSS-ETT was beneficial in that group; two fewer subjects had pepsin in their tracheal aspirate (n = 5 oral versus n = 4 tracheal aspirate pepsin).

The passage of the 2002 Farm Bill and the removal of the peanut quota system revealed underlying fundamental problems in the Virginia peanut industry. Lower yields and high costs plague producers at the farm level, acreage levels continue to decline and it is doubtful that peanut production in Virginia will continue at levels seen in the past. The structured market due to the quota system has provided little incentive in the past for technological investment. Investment in technology such as high oleic peanuts and capturing value at the consumer level are seen as ways to improve the situation in Virginia. In particular increased coordination at all levels of the supply chain would be needed to ensure that the consumer is brought a product with characteristics they desire. The literature provides ample information regarding the formation of alliances and coordination in general. According to Cozzarin and Barry (1998), vertical integration, similar to vertical alliances are set up for the following reasons: mitigating transactions costs, taking advantage of output or input price differentials of a competitor, and reducing uncertainties in costs and/or prices. Cozzarin and Barry (1998) also note that there is an increasing move toward vertical coordination in many agriculture sectors, the reasons cited for the current trend include: a) the growing influence of consumers in controlling the agri-food agenda; b) the increasing marketing power of large food companies; and (c) technological changes that necessitate coordination. Of these three reasons, the peanut industry falls under the first two. Vertical coordination is seen to be a solution when two or more entities are able to accomplish more efficiently their objectives than they are able to on their own. For the peanut industry, the agency theory and in particular principal-agent theory is the most applicable to the peanut industry. A linear model is used to examine the effects of increased coordination along the supply chain. The linear model also provides a snapshot of how decisions made at the farm level reverberate through the entire supply chain. The linear model includes the comparison of increased profits due to premiums at the consumer level. Results of the linear model indicate that the Virginia peanut industry will have difficulty maintaining current production levels without investment in the sector, without changing the way the supply chain operates. Principal-agent theory and specifically the work done on contracts in the pork and poultry industries provide a framework within which the peanut industry could avoid asymmetric information and moral hazard. This study attempts to identify underlying problems along with possible solutions or the Virginia peanut industry. / Master of Science

Geobacter sulfurreducens possesses type IV pili that are considered to be conductive nanowires and a crucial structural element in biofilm formation, enabling electron transfer to insoluble metal oxides in anaerobic sediments and to graphite anodes in microbial fuel cells. The molecular mechanism by which electrons are transferred through the nanowires to the electron acceptor is not fully understood. Prior to the work described in this thesis, the gene (pilA) encoding the structural pilus subunit had been identified, but little was known about the functional translation start codon, the length of the mature secreted protein, or what renders the pili conductive. Using mass spectrometry, I found that a tyrosine residue (Y32) near the carboxyl terminus of the mature PilA protein is posttranslationally modified by attachment of glycerophosphate. I studied the significance of Y32 for biofilm formation on various surfaces and for growth of G. sulfurreducens with insoluble electron acceptors. A mutant in which Y32 was replaced by phenylalanine lacked the glycerophosphate; biofilm formation on graphite surfaces was severely diminished and current production in microbial fuel cells was initiated only after a long lag phase. Moreover, cells with Y32F mutation in the pilA gene exhibited growth deficiency when Fe(III) oxide was the sole electron acceptor. My data confirm the role of G. sulfurreducens pili in biofilm formation and electron transfer to Fe(III) oxide and identify an amino acid in the PilA protein that is essential for these two processes. I also confirmed the existence of two functional translation start codons for the pilA gene and identified two isoforms (short and long) of the PilA preprotein by series of genetic complementation experiments. The short PilA isoform is found predominantly in an intracellular fraction, and seems to stabilize the long isoform and influence the secretion of several outer surface c-type cytochromes. The long PilA isoform, on the other hand, is required for secretion of PilA to the outer surface of the cell, a process that requires co-expression of pilA and the nine genes on its 3’ side. The long isoform is essential for biofilm formation on various surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxide. This study provides new insight concerning the function and biogenesis of Geobacter type IV PilA, as well as a foundation for further research that will be conducted on microbial nanowires.

Aging is amongst the strongest risk factors for neurodegenerative disease and elevated Type I interferon (IFN) signaling has been associated with both normal aging and central nervous system (CNS) diseases. Type I IFN is normally produced by nucleated cells in response to the detection of viral pathogen associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs). More recently it has been appreciated that Type I IFNs are also produced in response to endogenous stimuli, in the absence of viral pathogens. While Type I IFN signaling has been shown to be elevated in human and murine brains during normal aging, the underlying cause was unknown. Here we demonstrate by flow cytometry that aging results in increased size and numbers of mitochondria in the murine brain. Despite identifying increased mitochondrial number and mitochondrial DNA content, we found no change to mitochondrially-encoded transcripts, suggesting either deficits in mitophagy or augmented biogenesis due to insufficient oxidative phosphorylation. Interestingly, mitochondrial numbers correlated with elevated Type I IFN signaling in aging, linking mitochondria to the age-dependent innate immune response in the CNS. Using genetically engineered mice, we excluded roles for two critical innate immune pathways, STING and IRAK4, in the age-dependent increase in Type I IFN signaling in the brain. Notably, we subsequently identified a mitochondrially restricted innate immune protein, mitochondrial antiviral signaling (MAVS) as an essential molecular mediator of the age-dependent Type I IFN response; MAVS deficiency in aged mice restored Type I IFN signaling in the CNS to the levels observed in adult wildtype mice. Further, using intracerebroventricular (icv) administration of antisense oligonucleotides (ASO) as an orthogonal approach, we reduced MAVS transcript and protein expression within the CNS and thereby reduced Type I IFN signaling. Our data demonstrate a specific and selective role of MAVS expression in the CNS in Type I IFN signaling in aging. To investigate the relationship between mitochondrial aging and MAVS activation, we isolated cytoplasmic and mitochondrial RNA from young and aged animals as MAVS is most studied for its response to RNA ligands. Upon transfection into reporter cells, we found that mitochondrial RNA, but not cytoplasmic RNA, from both young and aged mice was sufficient to induce Type I IFN reporter activity in a MAVS-dependent manner. Furthermore, we attempted to mimic the increase of mitochondria observed in the aging CNS by transferring mitochondria from young and aged animals to recipient cells. Mitochondrial transfer also induced MAVS-dependent Type I IFN signaling in wildtype, but not MAVS null, mouse embryonic fibroblasts (MEFs). Collectively, our findings suggest that the accumulation of mitochondria in aging serves as a robust source of MAVS pathway ligands and implicate a novel link between mitochondrial aging and MAVS-mediated innate immune signaling in the CNS.