Reform and Democratization in Ukraine: My Service as a Peace Corps Volunteer with an Ukrainian Local Government Organization

This paper provides a micro-analysis of democratization and reform in an Ukrainian local government institution that was the site placement for the author\u27s Peace Corps service. Through using a culturalist lens and applying open systems theory while working as a Peace Corps Volunteer, the researcher observed how one organization coped with implementing public administration reform that is the result of the Revolution of Dignity in 2013-2014. This paper contributes to existing research regarding reform efficacy in other post-Soviet spaces, and provides a foundation for furthering similar research in Ukraine

Characterizing the evolution and mechanisms of bacterial epitope perception and evasion of the plant immune systems

Both plants and animals are impacted by diverse biotic threats. To limit disease, plants use protein receptors to recognize and respond to pathogen protein epitopes or effectors. Pathogens have evolved strategies to circumvent recognition to proliferate and cause disease. Pathogens can also persist on non-hosts, leading to reservoir populations and subsequent costly outbreaks. Despite considerable resources focused on understanding the interactions between pathogens and model organisms, we lack considerable knowledge in how the natural diversity of bacterial pathogens, particularly Gram-positive actinobacteria, impact plant immune perception, colonization, and disease susceptibility. Using a combination of comparative genomics, genetics, and biochemistry, I leveraged natural genetic variation to understand the evolution of pathogen epitopes and elucidate a driver of pathogen evasion in a Gram-positive actinobacteria. Pathogen recognition and receptor signaling is crucial in host-pathogen interactions, but most studies use a single pathogen epitope and thus, the impact of multi-copy epitopes on pathogen outcomes is unknown. Through comparative genomics of thousands of plant-associated bacterial genomes, I characterized the naturally-evolved bacterial epitope landscape and their impact on pathogen outcomes. I revealed that natural variation was constrained yet experimentally testable and both epitope sequence and copy number variation altered pathogen-immune outcomes. Through genetic and biochemical analyses, I uncovered a mechanism for pathogen immune evasion, intrabacterial antagonism, where a non-immunogenic epitope blocks perception of immunogenic forms encoded in a single genome. One such intrabacterial antagonist, cold shock protein CspB, was conserved in actinobacteria including Clavibacter, a genus comprised of several crop pathogens including tomato, potato, wheat, and corn. As a non-model system, I developed a genetic toolkit to manipulate Clavibacter and test the role of CspB in blocking immune perception of one host species, tomato. While I was able to build and validate the genetic tools through deletion of several critical virulence genes, I was unable to generate a null mutant of the cspB gene in C. michiganensis, likely due to its high GC-content between 73-78%. Instead, I validated our intrabacterial antagonism model though a combination of biochemical assays and genetic transfer of cspB to another foliar pathogen of tomato, Pseudomonas syringae pathovar tomato DC3000. I show via bacterial titers that expression of antagonist cspB blocked perception of other native encoded immunogenic cold shock proteins in a receptor-dependent manner. Collectively, I revealed a mechanism for immune evasion and showcased the importance of analyzing all epitope copies within a genome. I also provided evidence that Gram-positive actinobacteria interface with the plant immune system, a paradigm previously put into question due to insufficient evidence. Finally, I developed a genetic toolkit which may aid in characterizing other genotypic-phenotypic outcomes in the non-model bacterium. While my research has shown that we can leverage natural genetic variation to generate hypotheses and understand their impact on phenotypic outcomes, major questions remain in the evolution, functional biology, and signaling in plant-microbe interactions, which is addressed in the final chapter. Findings from the research questions posed may provide critical insights for subsequent advancements in bioengineering for disease resistance

Parentification and Separation-Individuation in Siblings of Individuals with a Chronic Illness or Disability

Well siblings of children with an illness or disability constitute a population of growing interest in psychological research. Past research suggests that these individuals may be likely to adopt increased caretaking responsibilities, or a parentified role, within the family. However, to date, few studies have examined the experiences of well siblings as they relate to late adolescent development. This study extends the well sibling research to the period of emerging adulthood and examines the degree to which 18 to 25 year-old well sibling and control groups report different levels of parentification and endorse different patterns of adolescent separation-individuation. In addition, this study explores the relationship between two constructs -- parentification and separation-individuation -- that have been linked in theory but rarely, if ever, been explored in research. T-tests identified that well siblings endorsed increased emotional and overall parentification relative to controls but did not indicate that they differed significantly with regard to patterns of separation-individuation. Hierarchical regression analyses determined that perceived unfairness of parentification predicted problematic separation individuation, and that instrumental parentification predicted more adaptive, but potentially conflicted, negotiation of the separation-individuation process. These results provide evidence for the emotional parentification of well siblings beyond the childhood and early adolescent periods. In addition, this study indicates that parentification is meaningfully related to the developmental tasks of separation and individuation that characterize emerging adulthood

Impacts of Tertiary Ligands in Catalysis in Thermostable Human Carbonic Anhydrase II by Site-directed Mutagenesis and Genetic Code Expansion

Carbonic anhydrases are ubiquitous enzymes that catalyze the reverse hydration and dehydration of carbon dioxide and bicarbonate respectively. A variety of tumors in humans show overexpression of human carbonic anhydrase II (HCA II) leading to an acidic breakdown of carbon dioxide into bicarbonate. While a variety of drug inhibitors exist, many come with undesirable side effects. Using non-conical amino acids (ncAAs), we probe an adjacent residue (Phe 93) to one of three key histidine residues involved in the active site. This shifting of a histidine residue creates a non-optimal proton transfer and thus, decreasing catalytic efficiency. The goal of this study was to better understand the catalytic flexibility and efficiency of HCA II without compromising protein stability. What we found was unexpected: exchanging phenylalanine with p-bromophenylalanine (pBrF) has a slight decrease in enzymatic activity while the exchange with p-nitrophenylalanine (pNO2F) significantly increased enzymatic activity in both an optimal pH of 7.4 and a more acidic pH of 6.4 compared to WT HCA II. In addition, stability of pNO2F mutant protein is significantly increased in pH conditions ranging from 7.4 to 9.4 compared to wild-type HCA II. Finally, the stability of pBrF mutant is comparable or even slightly higher than wild-type HCA II in pH conditions ranging from 7.4 to 9.4. This data suggests that Phe 93 plays a role in influencing the coordination of His 94, one of three key residues that coordinate a zinc ion involved in protein transfer; additionally, further studies of this residue may lead to potential cancer treatments based on the pBrF mutant and industrial applications based on the pNO2F mutant

Improving patient involvement in the lifecycle of medicines : insights from the EUPATI BE survey

EUPATI Belgium (EUPATI.be) is an informal gathering of local partners who are interested in improving patient involvement in healthcare innovation and medicines research and development. EUPATI.be brings together various stakeholders from different areas related to healthcare including patients, academia and industry. In doing so, we create an innovative collaborative approach where actors from different backgrounds work toward improving patient involvement in medical research, and putting the patient at the center of the Belgian healthcare system. Previously, we performed in-depth interviews with a small group of stakeholders on patient involvement. Here, we elaborate on our previous findings by using a nation-wide survey to inquire into Belgian stakeholders' perception on patient involvement. To this end, an electronic survey was available in French, Dutch and English, and accessible for 11 months. Twelve questions were asked, including 11 multiple choice questions and 1 open question. The latter was thematically analyzed according to the framework method. A total of 117 responses were registered and descriptive statistics were performed. The majority of respondents could be categorized into patient, academia and industry, whereas policy makers, payers, and healthcare professionals were underrepresented. We identified several barriers that hamper patient involvement, which were sometimes more reported by specific stakeholder groups. Next, we found that various stakeholders still consider patient involvement as a passive role, i.e., medical subject in a clinical trial. Respondents also reported that the role of the various stakeholders needed more clarification; this was also confirmed by the level of trust amongst the various stakeholders. Existing and the wish for more collaboration with the various stakeholders was reported by almost all respondents. Based on this survey, we can define the potential of involving patients in the medical research and development in the Belgian landscape. Our results will help to understand and tackle the various barriers that currently hamper patient involvement, whilst highlighting the need for a collaborative landscape from the multi-stakeholder perspective

Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae

Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) – a residue methylated by Set2 during transcription elongation – exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast

Hydraulic transmissivity inferred from ice-sheet relaxation following Greenland supraglacial lake drainages

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Lai, C.-Y., Stevens, L. A., Chase, D. L., Creyts, T. T., Behn, M. D., Das, S. B., & Stone, H. A. Hydraulic transmissivity inferred from ice-sheet relaxation following Greenland supraglacial lake drainages. Nature Communications, 12(1), (2021): 3955, https://doi.org/10.1038/s41467-021-24186-6.Surface meltwater reaching the base of the Greenland Ice Sheet transits through drainage networks, modulating the flow of the ice sheet. Dye and gas-tracing studies conducted in the western margin sector of the ice sheet have directly observed drainage efficiency to evolve seasonally along the drainage pathway. However, the local evolution of drainage systems further inland, where ice thicknesses exceed 1000 m, remains largely unknown. Here, we infer drainage system transmissivity based on surface uplift relaxation following rapid lake drainage events. Combining field observations of five lake drainage events with a mathematical model and laboratory experiments, we show that the surface uplift decreases exponentially with time, as the water in the blister formed beneath the drained lake permeates through the subglacial drainage system. This deflation obeys a universal relaxation law with a timescale that reveals hydraulic transmissivity and indicates a two-order-of-magnitude increase in subglacial transmissivity (from 0.8 ± 0.3 mm3 to 215 ± 90.2 mm3) as the melt season progresses, suggesting significant changes in basal hydrology beneath the lakes driven by seasonal meltwater input.C.-Y.L. and L.A.S thank Lamont-Doherty Earth Observatory for funding through the Lamont Postdoctoral Fellowships. D.L.C acknowledges support from the National Science Foundation (NSF) Graduate Research Fellowship. T.T.C. was supported by NSF’s Office of Polar Programs (NSF-OPP) through OPP-1643970, the National Aeronautics and Space Administration (NASA) through NNX16AJ95G, and a grant from the Vetlesen Foundation. S.B.D. and M.D.B. acknowledge funding from NSF-OPP and NASA’s Cryospheric Sciences Program through OPP-1838410, ARC-1023364, ARC-0520077, and NNX10AI30G. H.A.S. thanks the High Meadows Environmental Institute and the Carbon Mitigation Initiative at Princeton University. This publication was supported by the Princeton University Library Open Access Fund