Patients with end-stage heart failure requiring heart transplantation die simply because allografts are in short supply. Cardiac xenotransplantation from genetically-modified pig source animals has been proposed to bridge the gap between supply and demand. However, there are two predominant barriers to clinical translation and are poorly understood. The first, is a type of primary graft dysfunction, termed perioperative cardiac xenograft dysfunction (PCXD), which causes failure of the xenograft within 48 hours after transplantation. The second, post-transplantation xenograft growth, which causes a life-limiting diastolic heart failure within the first month after transplantation. We demonstrate that PCXD can be overcome with cardiac preservation techniques that minimize ischemia, either with blood-based cardioplegia induction or non-ischemic continuous preservation (NICP). In a heterotopic PCXD model, we further demonstrate that PCXD is likely a phenomenon rooted in xenograft dysfunction resulting from activation of innate immunity within the xenograft. While further studies need to be done, we demonstrate evidence that PCXD is augmented by the synergistic inflammatory effects of both cardiopulmonary bypass and cross-species transplantation. We also provide evidence that polymorphisms from TLR4 of S. scrofa (swine) compared to H. sapiens, may explain a possible target for the unique inflammatory signaling, that results in xenograft dysfunction after cardiac xenotransplantation. We also demonstrate that post-transplantation xenograft growth is multifactorial, but can be limited by growth hormone receptor knockout donors, along with genetic modifications that reduce immunogenicity of the xenograft. This growth, within 6 months after transplantation, is not a result of physiologic mismatch or cardiomyocyte hypertrophy. Therefore, recipients do not need to be treated for tachycardia and hypertension as previously thought. Lastly, we demonstrate the clinical translation of cardiac xenotransplantation by applying knowledge and expertise obtained from these studies. Expanded access (“compassionate use”) FDA authorization was based on demonstration of principle from our preclinical model. An ECMO-dependent patient without other therapeutic options was transplanted a genetically-modified cardiac xenograft, with 10-gene edits, combined with non-ischemic cardiac preservation and anti-CD40 monoclonal antibody-based immunosuppression. The patient was able to successfully wean from ECMO, participate in active rehabilitation and survived 60 days after transplantation without evidence of rejection.

The Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β-galactosidase, which we are using as a model for polyextremophilic enzymes. Divergent amino acid residues in this 78 kDa protein were identified through comparative genomics and hypothesized to be important for cold activity. Six amino acid residues were previously mutated and five were shown by steady-state kinetic analysis to have altered temperature-dependent catalytic activity profiles via effects on Km and/or kcat compared to the wild-type enzyme. Double-mutated enzymes were constructed and tested for temperature effects from 0-50 ºC, including two new tandem residue pairs, two potential loop insertions, and pairwise combination of the single residue mutations. The observed temperature and salinity dependent kinetic effects were compared to root-mean-square fluctuations to determine structural mechanisms of polyextremophilic activity. All the mutated enzymes were found to be more catalytically active at higher and/or less active at colder temperatures compared to the wild-type, with both Km and kcat effects observed for the two tandem mutations. For pairwise combinations, a Km effect was seen when the surface-exposed F387L mutation located in a domain A TIM barrel α helix 19 Å from the active site was combined with two internal residues, N251D or V482L. When another surface-exposed residue, I476V, was paired with N251D or V482L, primarily a kcat effect was observed. The temperature dependent kinetic effects were continued to 25-50 ºC with a dominant Km effect observed in all mutated enzymes. By deleting the identified insertions/loops, we are able to determine the kinetic effects of these insertions. The domain B deletion resulted in a less active enzyme overall converging with the wild-type at higher temperatures while the domain C deletion resulted in reduced cold-activity and improved activity at higher temperatures. Domain B may confer cold-activity without changing the thermal stability while domain C confers cold activity, at least in part, at the expense of activity at higher temperatures. With molecular dynamics simulations, increased flexibility was observed with higher temperature and salinity and in the mutations, indicating that the enzyme’s function is related to its flexibility and there is a balance required for optimal polyextremophilic activity.

T cell activation occurs when a T cell receptor (TCR) engages with cognate agonistic peptides in the context of Major Histocompatibility Complexes (pMHCs) on antigen presenting cells (APCs). This initiates a series of intracellular signaling events proximal to the TCR and associated CD3 complexes, mediated by unique kinases together with scaffolding and lattice molecules. The downstream cascades ultimately lead to transcriptional changes that promote the cellular program of conventional T cell activation—for example, cytoskeletal rearrangement, cytokine production, cellular proliferation, and differentiation. Understanding signaling mechanisms not only allows us to decipher the regulation of T cell activation but also to manipulate immune responses pharmacologically. TCR signaling by agonistic pMHCs is well studied, but much less is known about signaling by a parallel universe of self-peptides that also engage TCRs in vivo. The focus of this thesis is to understand how T cells perceive these signals without fully acquiring effector responses as a result. The central hypothesis of my thesis is that self-peptide ligands signal uniquely through the TCR to alter the fate and function of a T cell both with and without presence of their cognate agonist. We evaluated this hypothesis using approaches that globally increased self-peptide presentation in vivo (using FLT3L to generate more DCs), deprived T cells of self-peptide (by culturing away from APCs) or stimulated a TCR with a known self-peptide in the presence or absence of the strong agonist. The significant findings from these studies are that (i) boosting self-peptide presentation transiently increases a narrow T cell effector subset; (ii) depriving T cells of self-engagement lowers basal phosphorylation in the key signaling adapter LAT; (iii) self-peptides do not trigger cellular activation on their own, but synergize to enhance activation as measured by CD69, pERK, and several other parameters (iv) self-peptides initiate TCR signaling up to the level of pMEK and (v) self-peptides elicit a unique transcriptional profile. Together, these results not only define the unique contributions of self-peptides to T cell activation but also demonstrate a distinct wiring profile in the TCR-signaling network that limits self-peptide sensing at the ERK step.

Ovarian cancer (OvCa) is the most fatal gynecological malignancy due to delayed clinical presentation, passive metastasis, frequent tumor recurrence, and lack of effective targeted therapies. OvCa tumors predominantly shed as single cells or spheroids and disseminate throughout the peritoneal cavity. Spheroids are considered critical metastatic units that contribute to disease recurrence and chemoresistance. Despite recent advances in treatment strategies, there has been little improvement in patient survival. There is an urgent need to develop a better understanding of molecular mechanisms that are fundamental for OvCa dissemination, in order to provide alternative therapeutic options for long-term remission. Matriptase, a membrane-anchored serine protease (MASP), and its substrate protease-activated receptor-2 (PAR-2) both exhibit elevated expression in OvCa compared to normal ovary tissues which worsens patient survival, suggesting a critical role for activation of this pathway in OvCa progression. Overactive matriptase, induced by an imbalanced ratio with its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) has been implicated in disruption of barrier integrity and epithelial carcinogenesis, although the mechanism is not known. This study, demonstrates a functional role for an imbalanced matriptase:HAI-1 ratio in OvCa progression, and identifies a matriptase/PAR-2/PI3K/Akt/MMP-9/E-cadherin signaling axis that disrupts cell-cell interactions and promotes formation of pro-metastatic loose spheroids in vitro, in in vivo orthotopic xenograft models, and in patient-derived tumor cells. This matriptase/PAR-2 pro-metastatic signaling is in direct contrast to another MASP family member, testisin, suggesting a novel mechanism of biased agonism that orchestrates OvCa dissemination. Since over-activity of MASPs is associated with advanced OvCa, we have developed a MASP-activated pro-drug based on a re-engineered anthrax toxin (PAS:LF) that is selectively activated by zymogen-activating proteases on the tumor cell surface to inhibit tumor cell-survival pathways. This therapeutic strategy is radically different from anti-proliferative mechanisms of standard chemotherapeutic agents. We have demonstrated anti-tumor efficacy in a range of OvCa cells and spheroids in vitro, in various in vivo orthotopic xenograft models of OvCa dissemination, in patient ascites-derived tumor cells and in patient-derived xenograft models in vivo, with no off-target adverse effects. Clinical translation of these preclinical findings could establish PAS:LF as a promising treatment strategy to improve patient outcomes.

Two isoforms of the hERG (human Ether-á-go-go-Related Gene) potassium channel, hERG1a and hERG1b, associate in the heart to form the rapid delayed rectifier current (IKr), which is primarily responsible for repolarizing cardiac action potentials. Inherited mutations in both hERG1a and hERG1b cause Long QT Syndrome type 2 (LQT2), marked by prolonged cardiac repolarization and sudden death arrhythmia. The assembly of hERG1a subunits with hERG1b subunits increases the number of hERG1b subunits at the plasma membrane, but the mechanism by which hERG1a enhances hERG1b is not completely understood. Recently, it was reported that when expressed in trans, the hERG1a N-terminal region markedly increased hERG1b currents and increased biotinlabelled hERG1b protein at the plasma membrane surface. A direct structural interaction between the hERG1a N-terminal residues 216-220 and the hERG1b N-terminal region was characterized and proposed to be necessary for the upregulation of hERG1b (Johnson et al., JBC 2022). However, hERG1b channels with a deletion of the unique N-terminal 1b domain did not have a measurable increase in current density or in biotinylated protein when co-expressed with hERG1a N-terminal region pieces. This indicates that the 1b domain is required for the increase in hERG1b by hERG1a. Interestingly, the 1b domain-deleted hERG1b channels displayed a large leftward shift in the voltage dependence of activation when co-expressed with the full hERG1a N-terminal region. This is unexpected, as the 1b domain was not previously considered as a determinant of hERG gating. Here, we will investigate the mechanism of this large leftward shift and determine the role of the 1b domain in the gating properties of hERG1a/hERG1b heterotetrameric channels.

Autism Spectrum Disorder (ASD) is a neurological disorder characterized by repetitive, restricted behaviors, interests and activities along with a range of challenges in communication skills and social interaction. Pathway and genetic analyses have indicated a strong connection between ASD and the dysregulation of Ca2+ signaling. In particular, genetic mutations within the L-type Ca2+ channel (LTCC) have been shown to be causative of ASD. In fact, mutations within the CaV1.2 LTCC have been identified as one of the few monogenic causes of ASD, resulting in a multisystem disorder known as Timothy Syndrome (TS). TS patients harbor a single de novo point mutation within CaV1.2, which causes severe cardiac arrhythmias, long-QT syndrome and ASD. TS is one of the most penetrant genetic forms of ASD, making it an ideal model system with which to gain traction on understanding the role of Ca2+ dysfunction in ASD. Interestingly, we identified marked differences in channel gating due to distinct TS mutations. Such differences may underlie the differential phenotypes of select mutations, potentially explaining why some pathogenic mutations do not lead to ASD. To probe the effects of these mutations on neuronal function, we utilized human induced pluripotent stem cell (iPSC)-derived neurons. Patch clamp recordings of these cells yield distinct LTCC currents, enabling evaluation of the mutations in the context of a human neuron. Moreover, we utilized iPSC-derived 3D neuronal organoids to gain a deeper understanding of the effect of CaV1.2 dysfunction at a network level. Overall, as Ca2+ disruption may be a recurrent feature of ASD, studying rare mutations such as TS, may provide insight into the pathogenesis ASD beyond TS.

The links between stress and psychiatric illnesses are numerous and bidirectional. Stress is highly correlated to psychiatric illness, increasing the risk of illness and often precipitating its onset. The primary class of neuroendocrine stress hormones, glucocorticoids, is often dysregulated in patients with major depression. Understanding the neurobiological changes underlying these findings is crucial for the development of improved therapeutic strategies and better patient outcomes. Prior work suggests that the hippocampus is stress-sensitive, substantially altered in major depression, and plays an important regulatory role in HPA axis function. Our understanding of the processes regulating the stress response is incomplete, particularly in regards to the various brain regions responsible for its central regulation. In this document I strive to clarify our understanding of a specific component of stress regulation, namely the hippocampal contribution, which could prove important to understanding the various links between stress and psychiatric illness. To do this, I targeted the ventral hippocampus with injections containing virus that expressed the light-sensitive ion channel channelrhodopsin (ChR-EYFP) in glutamatergic neurons. I determined the extent of glutamatergic hippocampal terminal expression in brain regions thought to regulate the paraventricular nucleus of the hypothalamus (PVN), and then stimulated those terminals while recording from corticotropin-releasing factor-positive (CRF+) neurons in the PVN in a whole-cell voltage clamp recording configuration. Using parasaggital brain sections, I was able to induce optically-evoked inhibitory post-synaptic currents in CRF+ neurons of the PVN, demonstrating the first direct functional evidence of hippocampal inhibition of the PVN. I used a similar injection scheme to record GAD+ neurons in the BNST. Finally, I utilized in vivo optical stimulation of hippocampal terminals within the BNST during acute restraint stress and observed a significant decrease in circulating levels of corticosterone. My data indicates that the hippocampus sends dense projections to the BNST and optical excitation of these hippocampal terminals produce inhibitory currents in CRF+ neurons of the PVN. Our data suggests a disynaptic hippocampus-BNST-PVN circuit of regulation by which the hippocampus inhibits the CRF+ PVN cells. The in vivo results indicate that the hippocampal inhibition of these neurons is capable of functionally inhibiting the HPA axis.