This research examined the effects of acclimation to lowered salinity, elevated temperature, and hypoxia on aerobic metabolism of the Gulf killifish, Fundulus grandis, a common estuarine resident of the Gulf of Mexico. Standard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS), and critical oxygen tension (Pcrit) were each influenced by one or more acclimation treatments. Assessing the consistency of these traits measured in the same individuals over time, all were found to be significantly repeatable with no indication that the repeatability of any traits was affected by acclimation conditions. Significant correlations were found between SMR and Pcrit (positively correlated), between SMR and AAS (negatively correlated), between MMR and AAS (positive), and between AAS and Pcrit (negative). This study, therefore, documents the effects of acclimation on these traits, their repeatability, and correlations among them. It further suggests that repeatability of these traits is not context dependent.

INTER-SEGMENT COORDINATION VARIABILITY POST ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION SEPTEMBER 2015 DEVIN K. KELLY, B.S., UNIVERSITY OF MASSACHUSETTS AMHERST M.S., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Dr. Joseph Hamill There is an increased risk for ipsilateral graft rupture and contralateral ACL rupture following ACL reconstruction surgery (ACLR) despite return to sport clearance. The reason for this increased risk is not well understood. Previous literature has shown that decreased coordination variability is indicative of an injured system regardless of the absence of pain. PURPOSE: To quantify inter-segment coordination variability during three portions of the stance phase of gait in athletes at three progressive time points post-surgery compared to the contralateral limb (NI) and healthy controls. METHODS: Three-dimensional kinematic and kinetic data were collected for 10 ACLR and 10 healthy athletes matched for age, gender, and activity level. The ACLR group was measured at 4 weeks, 12 weeks, and when cleared to run post-surgery. Kinematic data were used in a modified vector coding technique to determine inter-segment coordination variability of lower extremity couples of interest. Statistical significance was determined using two factor multivariate ANOVAs (limb x visit) for early (1-33%), mid (34-66%), and late (67-100%) stance with alpha level set at .05. Tukey post-hoc tests were performed where appropriate. RESULTS: ACLR athletes have decreased inter-segment coordination variability of the involved lower extremity during the late stance phase of gait compared to both the contralateral limb and healthy controls at 4 weeks post-surgery. By 12 weeks post-surgery there were improvements in joint function as exemplified by inter-segment coordination variability of the ACLR involved limb becoming similar to the healthy control limb. CONCLUSION: Inter-segment coordination variability during late stance in the present study is not an indication for the increased risk for ipsilateral graft rupture and contralateral ACL rupture in ACLR athletes.

Polymer conjugates offer a way to introduce materials into the body that would normally be rejected or cause toxicity. Two polymers are investigated in vivo for uses in chemotherapeutic delivery, protein therapeutics, and DNA transfection. A novel polymer, polyMPC, has the ability to increase doxorubicin loading and its solubility, and is conjugated in a way to release its payload in a low pH environment. Through its conjugation, blood clearance time of doxorubicin is increased, and thus tumor exposure to the drug is increased with a single administration. It can be administered at ten times the concentration of free doxorubicin, and three times the concentration of Doxil®, while decreasing the cardio-toxicity normally associated with doxorubicin administration. These results show that polyMPC has the potential to increase treatment efficacy of doxorubicin. With increased circulation time, MPC polymers have additional potential for protein delivery and variations of its design were tested in linear, branched and grafted states, which show limited affect on tissue weight. An additional polymer for use in DNA transfection, NLS2, demonstrated its lack of tissue toxicity when injected intramuscularly. While continued investigation into these polymers is required, this initial data indicates their promising uses as therapeutics.

Human replication protein A (RPA) is an eukaryotic single-stranded DNA binding protein directly involved in a variety of DNA metabolic pathways including replication, recombination, DNA damage checkpoints and signaling, as well as all DNA repair pathways. This project presents 2 novel roles of RPA in the cellular response to DNA damage. The first elucidates the regulation of RPA and p53 interaction by DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) in homologous recombination (HR). HR and nonhomologous end joining (NHEJ) are 2 distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-PK, the core component of NHEJ, partners with DNA-damage checkpoint kinases ATM, and ATR to synergistically regulate HR repair of DSBs. The regulation was accomplished through modulation of the p53-RPA interaction. We show that upon DNA damage p53 and RPA are freed from the p53–RPA complex. This is done through simultaneous phosphorylation of RPA by DNA-PK, and p53 by ATR and ATM. Neither the phosphorylation of RPA nor that of p53 alone could dissociate the p53-RPA complex; furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR and NHEJ repair through the coregulation of p53–RPA interaction by DNA-PK, ATM and ATR. The second part of this project reveals a novel role of RPA32 phosphorylation in suppressing the signaling of programmed cell death, also known as apoptosis. Our results show that deficiency in RPA32 phosphorylation leads to increased apoptosis after genotoxic stress. Specifically, PARP-1 cleavage, Caspase-3 activation, sub-G1 cell population, annexin V staining and the loss of mitochondrial membrane potential were significantly increased in the phospho-deficient RPA32 cells (PD-RPA32). The lack of RPA phosphorylation also promoted activation of initiator Caspase-9 and effector Caspase-3 and -7. This regulation is dependent on the kinase activity of DNA-PK and is mediated by PUMA through the ATM-p53 pathway. Our results suggest a novel role of RPA phosphorylation in apoptosis that illuminates a new target that lies on the crossroads of DNA repair and cell death, a pivotal point that could be of importance for sensitizing cancer cells to chemotherapy.

This dissertation aimed to explore the usefulness of using force derivatives for onset detection in the isometric mid-thigh pull test. First, we examined applications of three differential calculus principles, first and second derivative, and curvature using visual detection as a reference under different baseline conditions. Second, we compared the best derivative method to a threshold-based method using visual detection as a reference. Results of our first investigation showed trivial differences between many differential calculus methods and visual detection. However, statistical differences exceeding a trivial effect was observed when instantaneous force and rate of force develop were examined. Through the first investigation, first and second derivative emerged as possible viable methods for baseline with a countermovement and for all other baseline conditions, respectively. Results of the second investigation showed similarities to the first investigation with respect to onset time. However, examination of instantaneous force and rate of force development indicated that a threshold-based method tended to overestimate compared to visual detection and a first and second derivative combined method. In fact, the difference between visual detection and the first and second derivative combined method ranged from trivial to moderate under all baseline conditions while the threshold-based method often reached a large difference. Overestimation by the threshold-method was more pronounced for rate of force development. In conclusion, while not perfect, the first and second derivative 3 combined method appears to hold possible practical potential and may be used as an assistant method for entry-level sport scientist plus using visual detection for obvious erroneous values.

There are long-standing hypotheses that endogenous ion currents act to control cell dynamics in development, wound healing and regeneration. However, the mechanisms employed by cells to detect the electric field (EF) and translate it into a discernable message to drive specific cell behaviors, such as migration, proliferation and differentiation, are not well understood. A better understanding of how cells are able to sense EFs and react to them is vital to understanding physiological mechanisms are involved in regeneration. Ion channel signaling provides a reasonable suspect for mediating these effects based on their documented involvement in proliferation, migration and differentiation. To investigate mechanisms underlying ionic regulation of critical cellular processes in non-excitable cells, a novel, in vivo assay was developed to screen multiple pharmacological inhibitors of ion channels during larval A. mexicanum tail regeneration. This assay was used to identify individual channels that were then targeted for further analysis regarding their involvement in the regenerative process. Chapter 2 presents data from a study that indicates that a wound-like response can be generated in an invertebrate model by application of exogenous, low-amplitude sine-wave electrical stimulation. This was characterized by recruitment of hemocytes at the stimulation site which was dependent on voltage-gated potassium channels. Chapter 3 presents data from a comprehensive and systematic screen of pharmacological compounds against larval salamander tail regeneration that indicates 8 specific target ion channels. This chapter also describes results indicating specific mechanisms by which these channels may be perturbing regeneration. Chapter 4 presents data that indicate that the Anoctamin 1 channel identified in the aforementioned screen is a regulator of cellular proliferation. This is shown to be accomplished via amplification of intracellular calcium surges and a subsequent increase in the activity of the p44/42 MAPK signaling cascade.

Hypoxia inducible factors (HIFs) are transcription factors and the master regulators of oxygen-dependent gene expression in animals. The focus of this thesis is the distribution of HIF protein in tissues of the fish Fundulus heteroclitus and F. grandis, two widespread species that occur in naturally hypoxic waters. Polyclonal antibodies against HIF-1α, HIF-2α, and HIF-3α were tested on proteins made in vitro and on extracts made from several tissues of normoxic and hypoxic fish. Antibodies against HIF-1α and 3α bound specifically to full length protein made in vitro, and produced bands on western blots of nuclear extracts of near the expected molecular weights for these proteins. Hypoxic exposure did not markedly increase the intensity of these bands, and mass spectrometry failed to identify HIF-1α and 3α peptides in excised gel bands. Thus, further tests of antibody specificity are needed before the tissue distribution of HIF in these fish can be confidently assessed.

Ovarian cancer is the fifth leading cause of cancer death in women between the ages of 35 and 74. With 22 thousand new cases and 15 thousand deaths annually ovarian cancer is among the most deadly cancers with a death to incidence ratio of 68%. With 70% of cases High Grade Serous Ovarian Carcinoma (HGSOC) is the most common type of ovarian cancer and causes 90% of ovarian cancer deaths. 80% of patients have reoccurrence within five years and only 15-30% of patients with recurrent metastatic ovarian cancer respond to current therapies, chemotherapy and surgery. One reason for the high reoccurrence rate is thought to be linked to the heterogeneity of tumors: there is evidence that, among tumor cells, a subpopulation is cancer stem cells (CSCs). Since CSCs are frequently drug resistant, when the patient undergoes chemotherapy many of the cells may die but the CSCs are left behind and the tumors can therefore regrow. CSCs are also more likely to undergo epithelial-mesenchymal transition which gives these cells the ability to more readily migrate and invade through the extracellular matrix, leaving the primary tumor to form metastases. One key inducer of EMT and therefore possibly of metastasis of particular interest in this project is SNAI1 (Snail). It is therefore the goal of this project to understand the growth, makeup and metastatic ability of HGSOC cell lines to test possible strategies to decrease growth of cancer and prevent metastasis. In this thesis project the phenotype, CSC population make up, and functionality of various HGSOC cell lines was examined. The cell lines assessed were A2780, Kuramochi, OVSAHO, COV318, SKOV3 and OVCAR8. A Snail knockdown OVCAR8 cell line was also assessed as described above and in a xenograft model. It was determined that the cell lines show varying phenotype from epithelial like to mesenchymal like morphology and the cell lines have varying concentrations of cancer stem cells. It was also determined that the CSC population of the HGSOC cell lines were positive for both epithelial and mesenchymal markers in the same cells. OVCAR8 stood out as a hybrid line with both epithelial and mesenchymal characteristics and was therefore chosen for the Snail knockdown model. In the Snail knockdown we observed that CSC markers were reduced, however no change between control and knockdown was seen in the in vitro functional experiments. There was a difference seen between Snail knockdown and control in the in vivo mouse xenograft model. Snail knockdown showed a trend for decreasing tumor burden in both primary and metastatic tumors and showed a significant decrease in growth of metastatic tumor at day 43. Based on these results Snail may be an important target for cancer therapy.

Atherosclerotic cardiovascular disease (CVD) is hallmarked by inflammatory immune activation, particularly by the induction of a response by monocytes. Classical (CD14++CD16-) are anti-inflammatory mediators under homeostatic conditions, while intermediate (CD14++CD16+) and non-classical (CD14LowCD16++) monocytes promote inflammation following activation. Monocyte activation and functionality is dependent upon receptor expression and ligand production by a variety of cells, including monocytes. Alterations in the expression of surface receptors often have a direct impact upon monocyte function, such as the increased pro-inflammatory cytokine production in response to activation that accompanies elevated CD14 expression or increased chemotaxis that is elicited by increased CCR2 expression. Ligand-receptor interactions also play a significant role in cell fate, including survival, proliferation, and differentiation. Monocytes are capable of differentiating into phagocytic cells known as macrophages in response to specific ligand-receptor interactions. Macrophages play a significant role in the pathogenesis and progression of CVD. Imbalance between pro-inflammatory M1 and anti-inflammatory M2 macrophages can to lead disease development and progression, such as the skewing toward the M1 phenotype that occurs in CVD. Elucidation of these mechanisms will allow for the development of targeted interventions, including pharmacological and non-pharmacological physical interventions, such as physical exercise. Therefore, this dissertation investigates the role of CD14 and CCR2 monocyte subset receptors that impact immune-mediated inflammation following ST segment elevation myocardial infarction (STEMI) as well as physical activity and cardiorespiratory endurance related differences in the acute exercise response of monocyte signaling, recruitment, and macrophage polarization and their potential role in CVD prevention.

Research in the multidisciplinary science of ecotoxicology is crucial to assess injuries to ecosystem resources from chemical spills or other stressors used to support environmental decision-making. Established guidelines recommend the use of non-standard native species in toxicity investigations. This work focused on the use of native species for aquatic toxicity assessment to make more relevant conclusions on the potential for adverse biological effects to occur as a result to single chemical exposures or exposures to a complex mixture like oil. We apply these studies to investigate petroleum product impacts from the Deepwater Horizon incident and concerns for metal toxicity in estuarine environments using a new model organism. Data generated from comprehensive toxicity testing programs were used in the first probabilistic risk assessment of Deepwater Horizon oil toxicity highlighting a lack of appropriate data and representative phyla. Novel toxicity study methods and a stress-response index were developed and demonstrated sensitivity and success in using the starlet anemone in ecotoxicology studies. Swim performance was used as new method to investigate sublethal indicators of stress resulting in varied responses from sheepshead minnows and Florida pompano. These studies further our ability for better laboratory-to-field extrapolation and for decision-making. The use of native species and complex mixtures like oil presented novel challenges in conducting aquatic toxicity studies. Special emphasis is placed on the necessity to understand the appropriate laboratory conditions for native species not typically held in the laboratory and maintaining study parameters to obtain quality data for more accurate interpretation and replication.