Exploring Unique Aspects of Apicomplexan Cell Biology Using Molecular Genetic and Small Molecule Approaches

The Phylum Apicomplexa contains a number of devastating pathogens responsible for tremendous human suffering and mortality. Among these are Plasmodium, which is the causative agent of malaria, Cryptosporidium, which causes diarrheal illness in children and immuncompromised people, and Toxoplasma gondii, which causes congenital defects in the developing fetus and severe disease in immunocompromised people. T. gondii also serves as a model organism for other members of this phylum due to the relative ease of parasite culture and manipulation. Although effective treatments exist for some diseases caused by these apicomplexan parasites, drug resistance for others is widespread, perhaps most notably in Plasmodium species. Development of new therapeutic agents is needed to combat this resistance and alleviate disease burden. It is important that the drugs target parasitic cell components not conserved in humans in order to minimize side effects and drug toxicity. However, in order to target unique processes, a better understanding of apicomplexan biology must be gained. One approach toward understanding the unique biological processes of apicomplexan parasites is to study proteins conserved among the Phylum Apicomplexa, but not present in other organisms. One such protein, photosensitized INA-labeled protein 1 (TgPhIL1) was identified previously. The work presented in this dissertation describes targeted disruption of this gene in T. gondii, which results in parasites with an altered shape and a fitness defect in both tissue culture and a mouse model of infection. Another approach to understanding the unique processes of apicomplexan parasites is to perturb them using small molecules. By identifying the targets of the small molecules, a more detailed understanding of the process can be gained. To this end, a small molecule screen was performed in T. gondii in order to identify small molecules that inhibit the apicomplexan-specific and essential process of host-cell invasion. In addition to identifying 24 invasion inhibitors, 6 enhancers were also identified. One of these enhancers, compound 112762, was shown to enhance invasion of other apicomplexan parasites as well. Described herein are attempts to identify the target(s) of this compound. A derivative of this compound was linked to an affinity resin, and TgProfilin was identified as a putative target that may interact covalently with 112762. Additionally, affinity chromatography was used to demonstrate non-covalent binding of a T. gondii FK506-binding protein to 112762. Finally, based on a report in the literature of a compound nearly identical to 112762 that inhibits yeast and mammalian protein arginine methyltransferase 1 (PRMT1), it was hypothesized that 112762 might target TgPRMT1 in T. gondii. Supportive of this hypothesis, 112762 was shown to inhibit TgPRMT1 in vitro, to inhibit parasite protein methylation in vivo, and to bind the 112762 affinity resin. TgPRMT1 knockout parasites are being generated in order to determine whether they show resistance to compound 112762. As a result of this work, three potential targets of 112762 in T. gondii have been identified. This work opens the door for future studies aimed at understanding and controlling invasion by apicomplexan parasites and other processes specific to the Phylum Apicomplexa

The Impact of Paid Sick Days on Public Health in an Elementary School Population

Background: The societal impact of Paid Sick Days (PSDs) has not been fully addressed in Vermont. Evidence suggests that PSDs benefit the well being of the employee in addition to saving expenses for the employer and the state. PSDs prevent the spread of diseases such as influenza and allow the ailing individual to receive proper medical attention. Inadequate PSDs not only affect the individual who needs time away from work due to illness, but extend to their entire family. Studies have documented the adverse effects from lack of PSDs on the ability for parents to care for their child. The following facts are known: • 7 states require private sector employees to provide “flexible” PSDs for family members (Vermont does not). • 66% of employers in Vermont do not provide PSDs for their employees. • Parents with PSDs or vacation are 5.2 times more likely to take time off from work to care for their sick child. We hypothesize that elementary aged children of working parents, who have an insufficient amount of PSDs, are more likely to attend school with an acute illness and are more likely to receive inadequate health care (i.e., miss well child check ups).https://scholarworks.uvm.edu/comphp_gallery/1015/thumbnail.jp

Environmental Studies Program Graduates As Leaders in Regional Environmental Nonprofits: What Sustains Them and What Influence Did Their Education Have on Them As Committed Environmental Professionals?

This phenomenological study investigates the stories of ten committed, regional environmental non-profit (ENP) professionals who were environmental studies program (ESP) undergraduates, exploring what sustains them in their work, with a focus on the role their undergraduate academic and extracurricular experiences play in supporting their commitment. Financial hardships, public valuation of environmental work, the need to perform multiple roles within the organization, complexities in ecological practice, the associated complexities in human interactions, and a slow timeline for change were the major challenges reported by the participants in their work with regional ENPs. Despite challenges, they persist in their work, often feeling called to the work and prompted by a desire for ethical accord between their personal values and professional lives. Participants describe their academic programs as helping them understand the nature and scale of the environmental challenges. Study abroad, field research, internships, and community-based class projects were all mentioned as academic experiences that allowed them to explore their identities as environmentalists and develop inter and intrapersonal skills. Involvement in student clubs and organizations also provided opportunities for identity development and inter and intrapersonal skill building. Outdoor experiences provided participants with opportunities to develop a sense of place, influencing their future decisions about where to live and work. Recommendations for ESPs, drawn from the research, include that funding for study abroad programs and field research should be made available to ensure all can access these experiences. Also recommended is having academic courses with a range of levels of activism, coupled with a range of class and club offerings on campus, which allows students to choose their level of engagement. Given attachments to place formed during the undergraduate years, and the subsequent desire for graduates to remain in those places, organizations interested in strengthening the environmental movement in particular regions could do so by increasing the strength of ESP offerings in those regions. In addition, once in the job, continued learning was vital in sustaining participants in their work; greater awareness of, and access to professional development is needed. Mentoring offers another avenue for supporting on-going learning. These regional ENP professionals are success stories, having persisted – even thrived – in a difficult profession. Despite the difficulties, they maintain generally positive personal and professional outlooks. Understanding their stories provides data about what can to be done to help sustain the existing leadership and improve the preparation of the future regional ENP leadership

Elucidating the Role of the University CEO\u27s Spouse in Development, Alumni Relations, and Fund Raising

Historically fund raising, or development, in higher education was the purview of only private four-year colleges; today, however, virtually all institutions of higher education are engaged in this endeavor. Attaining an institution‘s fundraising goals has become an integral part of a university president‘s or chancellor‘s role, consuming a high percentage of the CEO‘s time. While the president works very closely with the development office in garnering private support, there is often another player significant to the success of a university‘s development efforts. ―Hired‖ along with the CEO, the individual serves a major role, but has no job description and often works without a contract or remuneration. This is the spouse of the CEO. This study employed qualitative methodology to elucidate the role of the university CEO‘s spouse in development, alumni relations, and fund raising. The research focused on the traditional president‘s spouse, a female married to a male CEO. Seventeen interviews with spouses, development officers, and university trustees at seven different public land-grant universities were conducted to explore several questions: whether the spouse‘s role in development is formalized; whether the spouse was aware of the school‘s expectations for her in this area; the role of professional development staff in assisting the first lady; whether the spouse is recognized or compensated for her duties; how the spouse‘s role could be improved; and what could be done to make the role more satisfying, productive, or efficient. Several thematic areas were addressed regarding the first lady: (a) interview processes, (b) qualities, (c) support of the president, (d) role in development, (e) role in the university community, (f) acknowledgement, and (g) public opinion. The findings provide a multifaceted view of the role of the university president‘s wife in development. Recommendations for improving the role of the presidential spouse in university development include fostering open communication between all parties, tailoring the role to the individuals and institutions involved, and acknowledging the role of the spouse

Investigation of the Molecular Basis of Receptor Mediated Iron Release from Transferrin

Human serum transferrin (hTF) is a bilobal glycoprotein that plays a central role in iron metabolism. Each lobe of hTF (N- and C-lobe) can reversibly bind a single ferric iron. Iron binds to hTF at neutral pH in the plasma; diferric hTF binds to specific hTF receptors (TFR) on the cell surface and the complex undergoes receptor mediated endocytosis. The pH within the endosome is lowered to ~5.6 and iron is released from hTF. Apo hTF remains bound to the TFR and recycles back to the cell surface. Upon fusion with the plasma membrane, apo hTF dissociates from the TFR and is free to bind more iron and continue the cycle. The iron release process is complicated by various factors which include pH, anions, a chelator, lobe-lobe cooperativity and interaction with the TFR. All of these influence iron release in a complex manner. Because they are intricately linked, it is difficult to determine the effect of any single parameter. We have utilized stopped-flow and steady-state fluorescence and urea gel electrophoresis to dissect the iron release process as a function of lobe-lobe interactions, the presence of the TFR, and changes in pH and salt concentration. Application of recombinant protein production and site-directed mutagenesis has allowed us to generate a variety of hTF constructs in which the iron status of each lobe is completely controlled. Thus, we have created authentic monoferric hTFs unable to bind iron in one lobe, diferric hTFs with iron locked in one lobe and diferric hTF in which iron can be removed from both lobes. Importantly, we have produced the soluble portion of the TFR (sTFR) to analyze interactions between hTF and the sTFR and to monitor iron release from hTF/sTFR complexes. Together, we are able to provide a more precise picture of iron release from the two lobes of hTF in the presence and absence of the TFR. Steady-state fluorescence emission scans and urea gel electrophoresis provide a qualitative evaluation of the iron status of each construct after a predetermined incubation in iron removal buffer (i.e. an endpoint). However, these techniques do not provide information regarding the kinetic pathway to reach that endpoint. Combined with stopped-flow fluorescence time-based kinetics, a more precise assessment of the iron release process has been obtained. We have determined that changes in pH and salt affect endpoint iron release from the C-lobe, but not the N-lobe, however, the kinetics of iron release from both lobes are highly sensitive to pH and salt. Kinetic analysis in the absence and presence of the sTFR reveals the complexity of the iron release process. In the absence of the sTFR, the kinetics of iron release are insensitive to the iron status of the opposite lobe. However, in the presence of the sTFR, the kinetics of iron release from both lobes are affected by the iron status of the opposite lobe. Determination of conformational changes induced by anion binding, lobe-lobe communication and sTFR interactions have now been confidently assigned. We have created kinetic models of iron release from diferric hTF ± the sTFR and incorporated specific events pertaining to anion binding, lobe-lobe communication and conformational changes associated with sTFR interactions. We provide irrefutable evidence that a critical role of the sTFR is to accelerate the rate of iron release from the C-lobe, while decreasing the rate of iron release from the N-lobe such that the two lobes effectively release iron on a time scale relevant to one cycle of endocytosis

Promoting Physical Activity in Local Communities: Understanding Health, Nutrition, and Physical Activity Needs in Winooski, VT

Introduction: Since the Winooski YMCA opened in March 2008, enrollment has been much lower than expected, with only 200 members enrolled by September 2008. One goal of the YMCA is to promote the health of the community by increasing involvement in physical activity in Winooski. Regular exercise is associated with enhanced health and decreased risk of diabetes, cardiovascular disease, as well as many cancers. In order to promote physical activity in the Winooski community, the YMCA set a goal to increase their enrollment to 500 members by December 2008.https://scholarworks.uvm.edu/comphp_gallery/1005/thumbnail.jp

Stopping Marijuana Increases Alcohol Use: An Experimental Verification of Drug Substitution

Many, if not most, drug abuse counselors and treatment programs recommend abstinence from all psychoactive substances, in part, because of a fear that clients who decrease or stop their use of one drug will substitute another. Research to confirm this notion of substitution, however, mostly fails to show that abstinence from one drug increases use of another. A within-subjects study investigated whether consumption of alcohol and other substances changed during marijuana abstinence. Using an ABA design, 28 individuals who met Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSMIV; American Psychiatric Association [APA], 2000) criteria for either cannabis dependence or abuse and were not trying to stop their marijuana use completed an 8-day baseline period in which they used marijuana and other drugs as usual, then a 13-day marijuana abstinence period, and finally a 7-day return-to-baseline period. Marijuana abstinence was induced by a previously-validated contingent compensation schedule. Participants called a voicemail system daily to provide self-report of marijuana and alcohol use and visited the laboratory twice per week to provide self-report of caffeine, cigarette, and other illicit drug use, to complete self-report measures on psychological symptoms such as withdrawal and craving, and to submit urine samples to biochemically verify marijuana abstinence. Alcohol use significantly increased from a mean of 2.6 drinks/day (SD=1.0) during the baseline period to 3.0 drinks/day (SD=1.0) during the marijuana abstinence period (p=0.03), a 15% increase. Alcohol use then significantly decreased to 2.5 drinks/day (SD=1.3) during the return-to-baseline period (p=0.03), a 17% decrease. Although alcohol substitution occurred during marijuana abstinence, substitution of cigarettes, caffeine, and non-marijuana illicit drugs did not occur. Individuals with a diagnosis of past alcohol abuse or dependence substituted alcohol to a greater degree (52% increase) than those without this past history (3% increase). Increases in alcohol drinks/day correlated with increases in marijuana withdrawal discomfort scores and with increases in alcohol craving scores from the baseline to the marijuana abstinence period. Problems related to alcohol did not significantly increase from baseline to marijuana abstinence. This study provides empirical validation of the clinical notion of drug substitution and suggests that clinicians’ concerns about drug substitution may be valid, but this study’s results need to be replicated in individuals who seek treatment for marijuana problems. Whether substitution reduces the ability to abstain from marijuana also needs to be tested. If alcohol substitution does occur and interferes with the ability to quit marijuana, this would be important empirical support for the clinical practice of recommending abstinence from all substances

Adapting a Learning 2.0 Program

A presentation on a pilot technology training program at the Dana Medical Librar

“I Feel Like I’m Safe Again:” the Middle School Experiences of Immigrants in a Multilingual/Multicultural Setting

As the number of immigrants and refugees grows in the US, the linguistic and cultural diversity that comprises the middle grades classroom continues to increase. Given the need for resources and specific attention to linguistic and cultural strategies for these populations, this three year ethnographic study examined the schooling experiences of young adolescent immigrant and refugee students in a small town located in a rural state. Historically a homogeneous area, this community recently became a multilingual/multicultural setting. I documented the schooling experiences of my participants utilizing ethnographic methods including participant observation, interviews, and document analysis. My data describe how immigrant and refugee students internalized middle grades organizational structures such as teaming and multiage grouping. The findings suggest much variability among the students’ experiences, ranging from little or no academic, emotional, and/or social support to such high levels of support that students felt ostracized and disempowered. The implications for researchers center on expanding the current research in middle grades best practice to include a new set of voices, while practical implications focus on creating a safe environment where immigrants can express themselves and feel comfortable asking for the level of support they need

All Signs Point South: A Canadian’s Journey Through an American Student Affairs Program

The path to a career in student affairs is rarely a simple one. Aspiring professionals come into this field from many different institutional and educational backgrounds and for a myriad of reasons, but as unique as each of these individuals are, they share some similarities. Framed by literature on both general career decisions and those specific to student affairs and higher education, this article tracks the path of a Canadian student on his journey through an American graduate program. As the author approaches graduation, he reflects on the choices that led him to where he is today, the choices that will guide his next steps in the profession, and the implications those choices have for other Canadian students who wish to enter the field