Metalloproteins have evolved to contain specific combinations of primary and secondary coordination spheres within their active sites that contribute to the high activity and selectivity of their chemical transformations. One approach to developing small molecule analogs with comparable reactivity is to design molecular constructs that exhibit similar control over aspects of the primary and secondary coordination spheres. This dissertation describes the design and reactivity of multifunctional ligands that position hydrogen-bond acceptors within the secondary coordination spheres of transition metal complexes. Two classes of ligands were studied: the first is a tetradentate, tripodal ligand ([MST]3-) that was previously demonstrated to support formation of heterobimetallic complexes containing a Mn center and a redox inactive metal ion. The extension to the Fe analog is described here, as well as the continued examination of the influence of group 2 metal ions on the properties and reactivity of both the Mn and Fe complexes. The most important finding of these studies is that Ca(II) and Sr(II) ions impart nearly identical influences on the Mn and Fe centers—a result with relevance to biological water oxidation. Reaction of [MnMST]– and [FeMST]– with oxygen-atom transfer reagents in the absence of secondary metal ions uncovered a limitation of the [MST]3- ligand system in which weak C–H bonds are susceptible to intramolecular oxidation. In order to address this limitation, ligand derivatives were designed that removed weak C–H bonds, which led to the observation of a putative Fe(IV)–oxo intermediate.

The last two chapters of this dissertation describe the development of a new type of ligand that utilizes a tridentate, redox-active framework that has been shown to provide access to multi-electron transformations at first row transition metal centers. The first derivative of this ligand was shown to form dinuclear complexes of Fe, Co, and Cu with a pre-organized binding pocket for secondary metal ions. Modification of the steric bulk of the ligand prevented formation of these dinuclear complexes and instead supported mononuclear Fe and Co centers. The structural, electronic, and electrochemical properties of both of these series of complexes are described.

Coronaviruses are enveloped RNA viruses capable of causing a spectrum of diseases including respiratory, enteric, and/or systemic diseases in a wide variety of hosts. The major structural proteins of coronaviruses include the nucleocapsid (N), spike (S), membrane (M), and envelope (E) proteins. The viral Spike protein largely determines host range and tissue/cellular tropism and is responsible for promoting fusion of the viral and host cell membranes, leading to cell entry and infection. A literature review of coronaviruses in companion animals is included in this thesis (Chapter 1), which highlights the virology, epidemiology, clinical and pathologic features of coronaviruses of the cat, ferret, dog, horse, and alpaca.Feline infectious peritonitis (FIP) is one of the most complex and interesting viral diseases of domestic cats, caused be the FIP virus (FIPV), a biotype of feline coronavirus (FCoV). FIP has an immune-mediated pathogenesis involving multiple host and virologic factors. FIP presents as a spectrum of clinical signs including cavitary effusions, anorexia, persistent fever, lymphopenia, and lesions of pyogranulomatous vasculitis and peri-vasculitis that may or may not include central nervous system or ocular involvement. Untreated, FIP is generally considered to be fatal once clinical signs appear. FCoV includes two biotypes, feline enteric coronavirus (FECV) and FIPV. FIPV spreads systemically and is thought to arise from a discrete set of mutations in the more benign FECV, which is confined to the alimentary tract. These coronaviruses are further classified into serotypes I and II based on Spike-associated antigenic differences. Serotype II FCoV initially arose as the result of a series of recombination events between feline and canine coronavirus (CCoV) and is the less prevalent serotype naturally circulating in feline populations worldwide. As a result of recombination, the serotype II FIPV Spike protein has its origin in CCoV, which serves as the viral ligand for cell entry. Although FIPV serotype I is the more prevalent viral serotype in cats with naturally occurring FIP, serotype II has been more extensively studied in vitro due to the relative ease in propagating this virus in tissue culture systems. Consequently, more is known about the biology of serotype II FIPV than the more biologically relevant serotype I. Serotype II FIPV utilizes the cell surface protein feline aminopeptidase N, the ligand of the viral Spike protein, while the receptor for serotype I remains unknown. An understanding of viral receptor biology is a key facet in decoding viral pathogenesis, informing mechanisms of disease, viral dissemination, and potential vaccine strategies. The recent development of a feline cell line that effectively propagates serotype I FIPV, FCWF-4 CU, offers an opportunity to expand our understanding of serotype I FIPV biology. FCWF-4 CU is a culture adapted feline cell line derived from FCWF-4 cells available through the ATCC. Importantly, these two FCWF cell lines are variably permissive to the propagation of serotype I and II FIPV. In Chapter 2, we determined the targeted gene expression patterns in four feline cell lines, utilized normal feline tissues to determine the immunohistochemical expression patterns of two known coronavirus receptors, ACE2 and DC-SIGN, and compared the global transcriptomes of the two closely related FCWF-4 cell lines. We identified six differentially expressed transcripts with potential to explain the differential FIPV replication kinetics. The discovery of effective, available, and affordable antiviral treatments has been a focus of veterinary research for more than 10 years and recent advances in antiviral therapies for HIV, hepatitis C virus, ebolavirus and SARS CoV-2 have paved the way for similar advances for FCoV. In Chapter 3, we screened 90 putative antiviral compounds for efficacy and cytotoxicity against FIPV serotype II (WSU-79-1146) using real-time RT-PCR based screens and identified 26 compounds with antiviral activity against FIPV representing differing drug classes and mechanisms of action. Further, based on the success of combinatorial therapy strategies in human patients with HIV or hepatitis C, we strategically combined different antiviral compounds in order to identify additive or synergistic effects (combined anticoronaviral therapy, or CACT). Although we demonstrated additive and/or synergistic effects for several antiviral combinations, ultimately a select few monotherapies demonstrated superior efficacy overall. In Chapter 4, we reapproached antiviral assessment using an improved biological colorimetric assay and compared the antiviral efficacies of GC376, nirmatrelvir, remdesivir, GS-441524, molnupiravir (EIDD-2801), and -D-N4-hydroxycytidine (EIDD-1931) against both serotype I and II FIPV, as monotherapies and CACT. We also determined the pharmacokinetic properties of three antiviral compounds, molnupiravir, GS-441524, and remdesivir in cats in vivo as a step towards establishing dose in an antiviral treatment protocol for clinical use. An understanding of viral cell entry, dissemination and persistence are concepts that are also relevant to viral-vectored gene therapy. As a parallel adjunct study to the coronavirus projects, we created a lentiviral-vectored gene therapy approach to treating cats with chronic renal disease-associated anemia. Chronic renal disease (CRD) is a common disease of aged cats, often associated with clinically significant nonregenerative anemia as a result of reduced renal production of erythropoietin (EPO). Multiple approaches to the management of CRD-associated anemia have been attempted, including the use of gene therapy. In Chapter 5, we designed a series of third-generation lentivirus-based vectors to encode and produce the native feEPO protein in tissue culture experiments and in rodent models in vivo. The vectors were designed to include a pharmacologic safety mechanism through the incorporation of the “suicide gene” HSV-TK. This gene product allows for the pharmacologic termination of the therapeutic effect in the event of supraphysiologic polycythemia. We hypothesized that cells transduced in vitro by a lentiviral vector encoding native feline erythropoietin would express feEPO mRNA and biologically active feEPO protein and that this expression could be terminated by the administration of ganciclovir (GCV, the nontoxic substrate of HSV-TK). The in vitro assays facilitated optimization of the vector for three in vivo studies in rats and genetically modified anemic mice in which we demonstrated a significant elevation of blood packed cell volume in treated animals relative to control animals.

Introduction: The purposes of this study were to assess the extent to which latent trajectories of female intimate partner violence (IPV) victimization exist; and, if so, use negative childhood experiences to predict trajectory membership.

Methods: We collected data from 1,575 women at 5 time-points regarding experiences during adolescence and their 4 years of college. We used latent class growth analysis to fit a series of personcentered, longitudinal models ranging from 1 to 5 trajectories. Once the best-fitting model was selected, we used negative childhood experience variables—sexual abuse, physical abuse, and witnessing domestic violence—to predict most-likely trajectory membership via multinomial logistic regression.

Results: A 5-trajectory model best fit the data both statistically and in terms of interpretability. The trajectories across time were interpreted as low or no IPV, low to moderate IPV, moderate to low IPV, high to moderate IPV, and high and increasing IPV, respectively. Negative childhood experiences differentiated trajectory membership, somewhat, with childhood sexual abuse as a consistent predictor of membership in elevated IPV trajectories.

Conclusion: Our analyses show how IPV risk changes over time and in different ways. These differential patterns of IPV suggest the need for prevention strategies tailored for women that consider victimization experiences in childhood and early adulthood. [West J Emerg Med. 2012;13(3):272–277.]