A high degreee of structural and sequence homology exists between the EGF -1 domains of the vitamin K-dependent coagulation factors, as well as between the EGF-1 domains of individual vitamin K-dependent coagulation factors from various species. Through studies of protein evolution it has been determined the conserved amino acid residues observed are essential for protein structure while the variable residues have been implicated in specific protein-protein interactions. In the case of FVII, this interaction is the high affinity binding of its cofactor, TF, which initiates the extrinsic pathway of coagulation. 43% of the contact area of the FVII molecule in the FVIIa•TF complex is located within the FVII EGF-1 domain. A series of human FVII variants have been constructed in which the EGF-1 domain has been exchanged, either in its entirety or as single amino acid substitutions, with that of the mouse or rabbit. These species were chosen as it had previously been shown that plasma from mouse or rabbit, when combined with human TF, was able to clot at a significantly greater rate than homologous human plasma. We hypothesized that through these exchanges it might be possible to generate a human FVII variant with increased TF binding. Of the FVII variants generated, 2 human FVII point mutants have shown an increased affinity for human TF after transient expression. A75D and T83K exhibited TF binding at 200% and 150%, respectively, of that seen with wild-type human FVII. Both A75D and T83K exhibited clotting and amidolytic activity that was proportionally increased, with respect to TF binding. Computer-generated structures of these variants predict an additional hydrogen bond between the FVII and TF molecules likely to be responsible for the increased TF affinity in the T83K mutant. Intramolecular forces within the FVII molecule are predicted to have caused a conformational change with the A75D mutation which has lead to its increased TF binding. The greater affinity for TF exhibited by the human FVII point mutants A75D and T83K, as compared to wild-type human FVII, is an important step toward the creation of new class of competitive inhibitors of coagulation which are specifically directed towards its initial stage, rather than later in the cascade. However, more study is needed to determine the ability of either of these mutants to compete against each other for TF both 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 and 𝘪𝘯 𝘷𝘪𝘷𝘰. If either mutant is successful, it can then be active site inhibited by site-directed mutagenesis or through the use of chloromethyl ketones, in order to decrease its clotting and amidolytic activity. / Thesis / Master of Science (MSc)

Identifying forces that affect population dynamics can allow us to better understand the distribution and abundance of animals. Both top-down and bottom-up factors can significantly influence animal populations. Mason bees (members of the genus Osmia; Hymenoptera: Megachilidae) are important pollinators for agricultural systems and are vulnerable to exploitation by brood parasites, such as kleptoparasitic wasps. High levels of nesting density have the potential to increase rates of brood parasitism by attracting larger numbers of parasites to areas with aggregations of nests. I conducted a field study in subalpine meadows at the Rocky Mountain Biological Laboratory in Colorado, USA, to assess whether mason bees suffer increased brood parasitism as the size of nesting aggregations increases. Mason bees were allowed to nest in artificial nest boxes and establish natural variations in numbers of nesting individuals within nest boxes. Nest cells constructed by bees were then checked for the presence of kleptoparasite larvae shortly after they were completed. Overall, nest cells constructed in blocks containing multiple active bees were significantly more likely to be oviposited in by brood parasites compared to cells constructed in blocks with fewer active nesting bees. This suggests that gathering in large aggregations for nesting can negatively affect populations of mason bees, given the high levels of brood parasitism observed in areas of high nesting density. In addition, the last nest cell in mason bee nests was significantly more likely to be parasitized than inner cells, suggesting bees may be abandoning nests that are parasitized, representing a potential defensive response of bees to brood parasitism. These results have implications for the management of mason bees as agricultural pollinators, as cultivating them in large groups could reduce their survival.

abstract: Prior to the first successful allogeneic organ transplantation in 1954, virtually every attempt at transplanting organs in humans had resulted in death, and understanding the role of the immune mechanisms that induced graft rejection served as one of the biggest obstacles impeding its success. While the eventual achievement of organ transplantation is touted as one of the most important success stories in modern medicine, there still remains a physiological need for immunosuppression in order to make organ transplantation work. One such solution in the field of experimental regenerative medicine is interspecies blastocyst complementation, a means of growing patient-specific human organs within animals. To address the progression of immune-related constraints on organ transplantation, the first part of this thesis contains a historical analysis tracing early transplant motivations and the events that led to the discoveries broadly related to tolerance, rejection, and compatibility. Despite the advancement of those concepts over time, this early history shows that immunosuppression was one of the earliest limiting barriers to successful organ transplantation, and remains one of the most significant technical challenges. Then, the second part of this thesis determines the extent at which interspecies blastocyst complementation could satisfy modern technical limitations of organ transplantation. Demonstrated in 2010, this process involves using human progenitor cells derived from induced pluripotent stem cells (iPSCs) to manipulate an animal blastocyst genetically modified to lack one or more functional genes responsible for the development of the intended organ. Instead of directly modulating the immune response, the use of iPSCs with interspecies blastocyst complementation could theoretically eliminate the need for immunosuppression entirely based on the establishment of tolerance and elimination of rejection, while also satisfying the logistical demands imposed by the national organ shortage. Although the technology will require some further refinement, it remains a promising solution to eliminate the requirement of immunosuppression after an organ transplant. / Dissertation/Thesis / Masters Thesis Biology 2020

abstract: This dissertation explores discourses in the contemporary United States surrounding the creation, coding, sterilization, and general keeping of canines in order to interrogate how sex, gender, race, class, sexuality, and species together serve biopolitical formations of social control, patriarchal white supremacy, and heteronormativity. Interrogating these socially constructed and oftentimes stereotypical narratives through an interspecies lens demonstrates how taxonomies of power and systems of oppression and privilege become situated across species. This project utilizes interviews and ethnography, as well as analysis of popular culture, legislation and news media. Interspeciesism is informed by feminist influences, functioning as a framing paradigm that engages with a politicized question of the animal that explicitly acknowledges human-animal entanglements across sites that are shaped by imperialism and colonialism. This interspecies project considers the political nature of relationships between humans and canines. It suggests that people situate their own identities and power not only in relation to other humans but also to other species. Simultaneously, the interspeciesm I engage with extends analyses of biopolitics, or the regulations of living bodies, beyond humans to all species. It interrogates how contemporary U.S. society has organized and identified itself in part through the ways in which it controls and monitors canines, often in relationship to the multiple ways dogs in the U.S. are racialized, classed and gendered by specific breeds. This coding of canine bodies with various taxonomies of power is not about dog breeds’ in-and-of themselves, but instead indicates that dominant U.S. society seeks to assert control over certain populations that are constructed as undesirable and unproductive. Canines exist in a unique space in the U.S. cultural imaginary where they have multiple and oftentimes contradictory meanings that are influenced by a variety of power relations that transcend species. At stake is a critical concern regarding how interspecies bodies are made, controlled, formed, and refigured together under heteropatriarchal white supremacist modes of power. It draws attention to what these corporeal un(makings) imply for an ethics of being with, and thinking of, the other—human and animal. / Dissertation/Thesis / Doctoral Dissertation Gender Studies 2017

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Using Monte Carlo simulations and analytic methods, we examine the dynamics of inter-species competition using the Penna bit-string model. We begin with a study of the steady state with a single species, then proceed to the dynamics of competition between two species. When the species are not evenly matched in fitness, a simple differential equation provides a satisfactory model of the behavior of the system. However, when the species are equally fit, we show that a model, originally proposed to describe population genetics [Fisher,Wright], is required. When mutations are allowed between the competing species, the dynamics becomes more interesting. The mutation rate becomes a parameter that dictates the steady state behavior. If the two species are not equally fit, the value of the mutation rate determines whether the longer-lived or faster reproducing species is favored. With two species that are equally fit, the steady state varies with mutation rate from a single peaked to a double peaked distribution. This behavior is shown to be well described by an extension to the Fisher-Wright model mentioned above. Finally, we describe the preliminary results of a few new lines of investigation, and suggest ideas for further study of the dynamics of this model. / Ph. D.

Antibiotic treatment failure might be due to bacterial resistance or suboptimal exposure at target site and there is a lack of knowledge on the interaction between antimicrobial pharmacodynamics (PD) and the immune response to bacterial infections. Therefore, it is crucial to develop tools to increase the understanding of drug disposition to better evaluate antibiotic candidates in drug development and to elucidate the role of the immune system in bacterial infections. Colistin is used as salvage therapy against multidrug resistant Gram-negative infections. In this work, a whole-body physiologically based pharmacokinetic model (WBPBPK) was developed to characterize the pharmacokinetics (PK) of colistin and its prodrug colistin methanesulfonate (CMS) in animal and human. The scalability of the model from animal to human was assessed with satisfactory predictive performance for CMS and demonstrating the need for a mechanistic understanding of colistin elimination. The WBPBPK model was applied to investigate the impact of pathophysiological changes commonly observed in critically ill patients on tissue distribution of colistin and to evaluate different dosing strategies. Model predicted concentrations in tissue were used in combination with a semi-mechanistic PKPD model to predict bacterial killing in tissue for two strains of Pseudomonas aeruginosa. Finally, a toxicokinetic (TK) model was constructed to describe the time course of E. coli endotoxin concentrations in plasma and the effect on pro-inflammatory cytokine release. The model adequately described the concentration-time profiles of endotoxin and its stimulation of IL-6 and TNF-α production using an indirect response model combined with a transit compartment chain with a tolerance component to endotoxemia. The WBPBPK model developed in this work increased the knowledge on colistin tissue exposure under various conditions and could be used in drug development process to assess antibiotic efficacy or to test new drug combinations. The model describing endotoxin TK and its effect on cytokines is a new tool to be further applied in longitudinal studies to explore the immune response cascade induced by bacterial infections. The methodology applied in this thesis contributes to the development of an integrated modeling framework including physiology, drug distribution, bacterial growth and killing as well as the immune response to infection.

<p>Between October 2001 and May 2012 approximately 70% of U.S. military personnel killed in action and 75% wounded in action were the direct result of exposure to an explosion. As of 2008, it was estimated that close to 20% of all Operation Iraqi Freedom and Operation Enduring Freedom (OIF/OEF) veterans had sustained some form of traumatic brain injury (TBI). Further, blast exposure is also a civilian problem due to the increased usage of explosives in terrorist attacks. Blast injury research has historically focused on the pulmonary system and the other air-containing organs which have been shown through extensive experimentation to be susceptible to blast overpressure injury. A shift in injury pattern during recent conflicts is characterized by decreased incidence of pulmonary injuries with an increase in TBI thought to be associated with blast exposure. This increase in observation of blast TBI has resulted in a large research effort to understand mechanisms and thresholds. However, due to the relatively sudden shift, much of this research is being conducted without a proper understanding and consideration of blast mechanics and interspecies scaling effects.</p><p>This dissertation used experimental and computational finite element (FE) analysis to investigate some large questions surrounding blast TBI research. An experimental investigation was conducted to determine the effects of modern thoracic body armor usage on blast pressure exposure seen by the body. To improve FE modeling capabilities, brain tissue mechanics in common blast TBI animal model species were investigated experimentally and computationally to determine viscoelastic constitutive behavior and measure interspecies variation. Meta-analysis of blast pulmonary literature was conducted to update interspecies scaling and injury risk models. To derive interspecies scaling and injury risk models for blast neurotrauma endpoints a meta-analysis of existing experimental data was used.</p><p>This dissertation makes major contributions to the field of injury biomechanics and blast injury research. Research presented in this dissertation showed that modern thoracic body armor has the ability to lower the risk of pulmonary injury from blast exposure by attenuating and altering blast overpressure. The study shows that the use of soft body armor results in the pulmonary injury threshold being similar to that for neurotrauma. The use of hard body armor results in the threshold for pulmonary injury occurring at higher levels than that of neurotrauma. This finding is important, as it helps to explain the recent shift in injury types observed and highlights the importance of continued widespread usage of body armor not only for ballistic protection but for protection from blast as well.</p><p>This dissertation also shows the importance of interspecies scaling for investigation of blast neurotrauma. This work looks at existing in vivo animal model data to derive appropriate scaling across a wide range of brain size. Appropriate scaling for apnea occurrence and fatality for blast isolated to the head was found to be approximately equal to a characteristic length scaling of brain size, assuming similar brain geometry. By combining the interspecies scaling developed and existing tests data, injury risk models were derived for short duration blast exposures.</p><p>The contributions and conclusions of this dissertation serve to inform the injury biomechanics field and to improve future research efforts. The consideration by researchers of the recommendations presented in this dissertation for in vivo animal model testing will serve to maximize the value gained from experimentation and improve our understanding of blast injury mechanisms and thresholds. The injury risk models presented in this work help to improve our ability to prevent, diagnose, and treat blast neurotrauma.</p> / Dissertation

We classify animals into a few distinct groups: companion animals (pets and strays like dogs and cats), utility animals (animals that serve a purpose like cows farmed for dairy or meat or foxes farmed for fur), and wild animals (nondomestic animals in their natural habitat). This study will investigate empathic relationships between people and animals of these four types in two abuse scenarios (active abuse and neglect). These factors were manipulated in a 2x4 experimental design. I expect a closer relationship with humans will correlate with a higher degree of empathy for the victim such that homed companion animals will generate the most empathy, followed by stray, utility, and finally wild. Results supported this hypothesis to an extent. This research is intended to bridge gaps between empathy towards humans and empathy towards other animals. If we can manipulate the degree to which people tend to relate to animals by varying our descriptions of these animals, we can also manipulate people’s desire to protect them.