Rationale: The pathogenesis and progression of pressure-overload heart failure (HF) encompasses aberrations in gene regulation, leading to maladaptive cardiac hypertrophy, ventricular remodeling, and contractile dysfunction. The trigger for maladaptation and HF is signaling through the G protein, Gq, and one downstream effector for this pathway is activation of non-canonical activity of G protein-coupled receptor kinase-5 (GRK5). This kinase, following hypertrophic stimuli, can translocate and accumulate in the nucleus of cardiomyocytes. The nuclear targeting of GRK5 is mediated by an amino-terminal (NT) domain that can bind calmodulin (CaM), which is required before its nuclear translocation. Objective: This study attempted to thwart GRK5-mediated pathology in pressure-overload maladaptation and HF by cardiomyocyte expression of a peptide encoding the NT of GRK5 (GRK5nt) that includes this CaM binding domain. Methods and Results: In vitro studies in myocytes showed that Gq-coupled receptor mediated hypertrophy was abrogated with GRK5nt expression and this included attenuation of pathological gene expression and NFAT activity. We confirmed that the GRK5nt binds to Ca2+-CaM, prevents its association with endogenous GRK5, and prevents its nuclear translocation. We generated cardiac-specific GRK5nt transgenic mice and showed in vivo that expression of this peptide prevents hypertrophic nuclear translocation of GRK5 and these mice exhibit significantly less cardiac hypertrophy, ventricular dysfunction, pulmonary congestion, and cardiac fibrosis following chronic transverse aortic constriction. Conclusions: Together our data support a role for GRK5nt as an inhibitor of pathological nuclear GRK5 signaling for HF prevention. / Biomedical Sciences

In this study, we assessed the contributions of plasma membrane (PM) microdomain targeting to the functions of H-Ras and R-Ras. These paralogues have identical effector-binding regions, but variant C-terminal targeting domains (tDs) which are responsible for lateral microdomain distribution: activated H-Ras targets to lipid ordered/disordered (Lo/Ld) domain borders, and R-Ras to Lo domains (rafts). We hypothesized that PM distribution regulates Ras effector interactions and downstream signaling. We used tD swap mutants, and assessed effects on signal transduction, cell proliferation, transformation, and tumorigenesis. R-Ras harboring the H-Ras tD (R-Ras-tH) interacted with Raf, and induced Raf and ERK phosphorylation similar to H-Ras. R-Ras-tH stimulated proliferation and transformation in vitro, and these effects were blocked by both MEK and PI3K inhibition. Conversely, the R-Ras tD suppressed H-Ras-mediated Raf activation and ERK phosphorylation, proliferation, and transformation. Thus, Ras access to Raf at the PM is sufficient for MAPK activation and is a principal component of Ras mitogenesis and transformation. Fusion of the R-Ras extended N-terminal domain to H-Ras had no effect on proliferation, but inhibited transformation and tumor progression, indicating that the R-Ras N-terminus also contributes negative regulation to these Ras functions. PI3K activation was tD-independent; however, H-Ras was a stronger activator of PI3K than R-Ras, with either tD. PI3K inhibition nearly ablated transformation by R-Ras-tH, H-Ras, and H-Ras-tR, whereas MEK inhibition had a modest effect on Ras-tH-driven transformation but no effect on H-Ras-tR transformation. R-Ras-tH supported tumor initiation, but not tumor progression. Whereas H-Ras-tR-induced transformation was reduced relative to H-Ras, tumor progression was robust and similar to H-Ras. H-Ras tumor growth was moderately suppressed by MEK inhibition, which had no effect on H-Ras-tR tumor growth. In contrast, PI3K inhibition markedly suppressed tumor growth by H-Ras and H-Ras-tR, indicating that sustained PI3K signaling is a critical pathway for H-Ras-driven tumor progression, independent of microdomains. In the second phase of the study, we investigated the combinatorial use of two drugs currently either in active use as anti-cancer agents (Rapamycin) or in clinical trials (OTX008), as a novel strategy to inhibit H-Ras-driven tumor progression. H-Ras anchored to the plasma membrane shuttles from the lipid ordered (Lo) domain to the lipid ordered/lipid disordered border upon activation, and retention of H-Ras at these sites requires Galectin-1 (Gal-1). We have previously found that genetically-mediated Lo sequestration of H-Ras inhibited MAPK signaling but not PI3K activation. Here we show that inhibition of Gal-1 with OTX008 sequestered H-Ras in the Lo domain, blocked H-Ras-mediated MAPK signaling, and attenuated H-Ras-driven tumor progression in mice. H-Ras-driven tumor growth was also attenuated by treatment with mTOR inhibitor Rapamycin, and this effect was further enhanced in tumors driven by Lo-sequestered H-Ras. These drugs also revealed bidirectional cross-talk in H-Ras pathways. Moreover, dual pathway inhibition with OTX008 and Rapamycin resulted in nearly complete ablation of H-Ras-driven tumor growth. These findings indicate that membrane microdomain sequestration of H-Ras with OTX008, coupled with mTOR inhibition, may support a novel therapeutic approach to treat H-Ras mutant cancers. / Cell Biology

Osteoactivin (OA) is a novel osteogenic and repair factor. It has the ability to regulate cell proliferation, adhesion, differentiation, and synthesis and regulation of extracellular matrix proteins in various cell types under both normal and pathological conditions. Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased 3-fold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation ex vivo. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts ex vivo was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-β1 and TGF-β receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. OA expression increases during tissue degeneration and regeneration, fracture repair, and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA’s expression with repetitive overuse injuries is unknown. We sought to evaluate in an animal model of upper extremity repetitive overuse, at low force loads: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72), a protein known to increase during metabolic stress and be involved in cellular repair. We hypothesized that OA is functioning as a growth factor during periods of tissue repair. Young adult, female Sprague-Dawley rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks, and were compared to control rats. Quantitative PCR, Western blot analyses and immunohistochemistry showed increased OA mRNA and protein expression in flexor digitorum muscles of 6-week HRNF rats, compared to controls. OA protein levels increased similarly in 6-week HRNF flexor digitorum tendons. Increased OA immunostaining was localized to the myofiber sarcolemma, macrophage-like cells and tenocytes. In muscles, Western blot analyses showed progressive increases in MMP-1, -2 and -3, whereas tendons had increased MMP-1 and -3, with HRNF task performance. ELISA and immunohistochemistry showed increased HSP72 in 6-week HRNF muscles, and co-localization with OA in the myofiber sarcolemma. HSP72 increased in 6-week HRNF tenocytes, compared to controls. Inflammatory cytokines IL-1alpha or beta showed transient increases at 3 weeks in muscles and tendons, while IL-1alpha was significantly decreased in 6-week HRNF muscles. The simultaneous increases of MMPs and HSP72 with OA, factors involved in tissue repair, supports a role of OA in tissue regeneration after repetitive overuse. We extended the study above to examine the expression of OA during high repetition high force loading in our animal model of upper extremity overuse, in combination with anti-inflammatory drug, to evaluate OA’s link to inflammatory processes. Young adult female rats underwent an initial training period to learn the task (10 min/day, 5 days/wk, for 6 wks), before then performing a high repetition high force (HRHF) task for 11 weeks (2 hours/day, 3 days/week). Results were compared to age-matched control (C) rats. At the end of HRHF task week 3, two cohorts of HRHF rats received 5 intraperitoneal injections of saline (HRHF+Veh) or anti-rat TNF-a (HRHF+anti-TNF) across 4-7 weeks, as did controls (C+Veh and C+anti-TNF). Two other cohorts rested during weeks 4-7 with or without treatment (HRHF+anti-TNF/Rest and HRHF+Veh/Rest), to parallel its partner group. Motor behavior was assessed and revealed decreased grip strength in HRHF+Veh rats beginning immediately post training (HRHF task week 0), and that anti-TNF-α treatment prevented this grip strength decline. The 4-week anti-TNF-α therapy extended maintenance of grip strength near control levels through week 9, despite no further treatment after week 7. By experimental week 11, ELISA showed no significant differences in OA levels in forearm flexor digitorum muscles, and histomorphometry showed no difference in the circumference of this muscle in any HRHF group, compared to controls, matching findings of no gain in grip strength above control levels in any HRHF group. However, ELISA of distal radius and ulna homogenates showed increased OA levels in HRHF+anti-TNF rats, as well as increased IL-18 in bones of both anti-TNF treated HRHF groups (HRHF+anti-TNF and HRHF+anti-TNF/Rest rats), compared to controls. Micro-CT analysis showed that rats receiving anti-TNF-α treatment, with or without rest, had increased bone mass (detected as increased trabecular bone volume, thickness, and number and reduced trabecular separation), compared to the other groups. Histomorphometry showed increased osteoblast numbers in HRHF+anti-TNF rats, compared controls, yet decreased osteoclast numbers, compared to HRHF+Veh rats, indicative of increased bone anabolism in anti-TNF-a treated rats. Thus, these findings suggest that TNF-α blocks OA expression in bones, and that its increase when combined with prolonged repetitive loading, enhances osteoblast activity and bone formation. / Cell Biology

Extracellular ATP is now recognized as a neurotransmitter and neuromodulator (Bean, 1992) whose effects on bovine adrenal chromaffin cells include both the release of Ca$\sp{2+}$ from intracellular stores and secretion of catecholamines (Kim, 1989). Since chromaffin cells co-secrete 150 mM ATP along with catecholamines and neuropeptides, ATP has considerable opportunity to act as a modulator of secretion via its effects on cytosolic Ca$\sp{2+}$. Nucleotides and their analogues were used to characterize ATP effects, yielding an agonist efficacy order for the release of Ca$\sp{2+}$ from internal stores of ATP = UTP $>$ ADP $>$ 2-MeSATP, $\alpha,\beta$-MeATP, identifying the receptor as a P$\sb{\rm 2U}$ subtype of purinoceptor (O'Connor et al., 1991). In contrast, the efficacy order for the receptor mediating secretion, 2-MeSATP $>$ ATP $> \alpha,\beta$-MeATP, ADP, UTP places it in the P$\sb{\rm 2Y}$ receptor subtype (Gordon, 1986). Thus, two separate receptors are responsible for Ca$\sp{2+}$ release and secretion. Additionally, agonists were found to be more effective at both receptors in the absence of extracellular Mg$\sp{2+}$, suggesting that ATP uncomplexed with divalent cations may bind preferentially to the receptors. The release of Ca$\sp{2+}$ from internal stores was further investigated using ATP and bradykinin (BK), both of which release Ca$\sp{2+}\ via$ inositol (1,4,5)-trisphosphate (Ins(1,4,5)P$\sb3$). In the absence of extracellular Ca$\sp{2+}$, both ATP and BK increased cellular levels of Ins(1,4,5)P$\sb3 \sim 2$-fold within 30 s. Studies of single cells revealed that 93% released Ca$\sp{2+}$ in response to BK whereas ATP triggered release from 40% of the cells. The ATP-responsive cells were a subset of the BK-responsive cells, indicating that spatial separation between ATP and BK-sensitive Ca$\sp{2+}$ stores is intracellular. Furthermore, saturating concentrations of BK and ATP yielded an additive release of Ca$\sp{2+}$. Taken together with previous data from our lab (Kim, 1989), these results indicate that it may be possible for two agonists to activate the release of Ca$\sp{2+}$ from two spatially distinct Ca$\sp{2+}$ stores through a single second messenger, Ins(1,4,5)P$\sb3$. In addition, several parameters of chromaffin cells loaded with fura-2 were examined. The autofluorescence of chromaffin cells illuminated with light from 300-400 nm was found to alter the spectrum of fura-2-loaded cells. Also, fura-2 leakage from the cells over a period of $\sim$1 h was found to reach levels of 40-60% of the initial signal; for comparison of Ca$\sp{2+}$ signals during this time period, a method of normalization of the signal was used. Moreover, in the absence of Mg$\sp{2+}$, the removal of extracellular Ca$\sp{2+}$ from the cells occasioned a large intracellular Ca$\sp{2+}$ transient.

Revised and corrected version: the year of the author's bachelor’s degree. / Leucine Zipper-like Transcription Regulator 1 (LZTR1) is responsible for encoding a member of the BTB-Kelch superfamily, which interacts with the Cullin3 (CUL3)-based E3 ubiquitin ligase complex. Researchers have discovered mutations in LZTR1 in glioblastoma, schwannomatosis, and Noonan syndrome. However, the exact function of LZTR1 in cancer development or human growth remains unclear. A previous study in the Neel lab showed there was a cell-context dependent regulation of pan RAS (K/N/H RAS) levels by LZTR1. This thesis's main objective was to understand the underlying mechanisms for the above observation. The LZTR1 gene has gained importance in human well-being due to its essential role in cellular activities and its link to various diseases. Investigating the range of functions of LZTR1 and the underlying mechanisms will offer new perspectives on disease prevention and therapeutic approaches. These results provide hints for deciphering the mechanisms of RAS degradation and controlling the RAS/MAPK signaling pathway.

Detailed understanding of skeletal development, especially in the craniofacial space, requires close observation of growth patterns along with understanding of the genetics involved. Prior work on craniofacial development has focused on imaging data taken at specific timepoints on fixed specimens. The zebrafish offers advantages as a model organism to address the extant challenges in this field of research as many aspects of skull formation and general skeletal development are conserved from zebrafish to mammals. Zebrafish remain optically clear well into the juvenile stage, encompassing the core period of bone development in the skull. Taking advantage of many fluorescent transgenic lines marking cells that contribute to skeletal development, our lab has developed protocols to perform confocal live imaging multiple times on individual fish. I have used those imaging protocols for two main objectives: 1) to establish atlases of tissue patterning during cranial development; and 2) to characterize craniofacial and general skeletal development in mutants identified through forward genetic screens. Using serial low-magnification confocal imaging, fish carrying transgenic markers for osteoblasts (sp7:mCherry) and chondrocytes (col1a1:eGFP) were tracked for approximately 30 days, focusing on bones of the skull vault. We defined three stages of growth: initiation by osteoblasts on a cartilage scaffold; a period of rapid planar growth by the frontal and parietal bones; and a plateauing of growth and maturation of the sutures. We captured confocal Z-stacks which were converted into 3D models. To further understand the complex cell interactions of craniofacial development, we used a transgene marking osteoclasts, bone-resorbing cells which represent the innate immune component within the bone. Osteoclasts are critical in homeostasis of adult skeletal structures, but there is limited information on the role of osteoclasts in craniofacial development. We used confocal microscopy of live transgenic fish to determine the location of osteoclasts in the developing skull daily over several weeks, from 5.18 mm to 9.6 mm standard length (approximately 15 to 34 days post fertilization). The overall distribution of osteoclasts is consistent among individuals, but they are sparse in most areas of the skull and the exact locations vary among fish and across developmental time points. We observed osteoclasts concentrating at areas of remodeling associated with neuromasts and their associated nerves, the hyomandibular foramina and around the supraorbital lateral line. This suggests that they play a special role in bone remodeling around neuromasts and nerves during craniofacial development. In csfr1amh5 mutants lacking functional osteoclasts, the morphology of the cranial bone was disrupted in both areas. The hyomandibular foramen is present in the initial cartilage template, but after ossification has begun, the diameter of the canal is significantly smaller in the absence of osteoclasts. The diameter of the supraorbital lateral line canals was also reduced in the mutants, but the more significant difference was the paucity of small foramina in and around the canals, which allow passage of efferent nerves through the bone. These findings define important and previously unappreciated roles for osteoclast activity during craniofacial development. With this data on baseline developmental benchmarks and tissue patterning in wild-type individuals we then focused on two skeletal mutants, toth and jinx, identified in a genetic screen. toth was originally identified due to severely lagging growth and structural skull abnormalities, including numerous pits in the frontal and parietal bones. Mutants for toth have a dramatic accumulation of osteoclasts on the frontal and parietal bones corresponding to the pits in the bone. jinx mutants have craniofacial irregularities but have additional skeletal abnormalities, including sporadic fusion of vertebral bodies. Using the sp7:mcherry transgenic line and fluorescent vital staining of mineralized bone, I have documented uneven mineralization and vertebral fusion events in live jinx mutants during development. We have localized the genes for toth and jinx based on filtering bulked sequencing for single nucleotide polymorphisms. I have further refined the mapping of jinx using microsatellite markers to implicate the candidate gene entpd5a, which has previously been implicated in bone mineralization in zebrafish. I am performing additional experiments to verify the gene identity and mechanisms underlying the defects. My thesis presents a body of work demonstrating the power of live imaging in zebrafish to reveal developmental processes. We created an atlas of bone and cartilage morphology during normal skull development and building on that foundation demonstrated previously unknown requirements for osteoclasts in sculpting skull morphology. We have applied the same imaging tools to characterize developmental processes in two zebrafish mutants with craniofacial and skeletal defects, and in future experiments will be able to correlate those defects with specific genetic lesions.

Cellular automata (CA) are discrete dynamical systems comprised of a lattice of finite-state cells. At each time step, each cell updates its state as a function of the previous state of itself and its neighbours. Fuzzy cellular automata (FCA) are a real-valued extension of Boolean cellular automata which "fuzzifies" Boolean logic in the transition function using real values between zero and one (inclusive). To date, FCA have only been studied in disjunctive normal form (DNF). In this thesis, we study FCA in conjunctive normal form (CNF). We classify FCA in CNF both analytically and empirically. We compare these classes to their DNF counterparts. We prove that certain FCA exhibit chaos in CNF, in contrast to the periodic behaviours of DNF FCA. We also briefly explore five different forms of fuzzy logic, and suggest further study. In support of this research, we introduce novel methods of simulating and visualizing FCA.

Cellular automata (CA) are discrete dynamical systems comprised of a lattice of finite-state cells. At each time step, each cell updates its state as a function of the previous state of itself and its neighbours. Fuzzy cellular automata (FCA) are a real-valued extension of Boolean cellular automata which "fuzzifies" Boolean logic in the transition function using real values between zero and one (inclusive). To date, FCA have only been studied in disjunctive normal form (DNF). In this thesis, we study FCA in conjunctive normal form (CNF). We classify FCA in CNF both analytically and empirically. We compare these classes to their DNF counterparts. We prove that certain FCA exhibit chaos in CNF, in contrast to the periodic behaviours of DNF FCA. We also briefly explore five different forms of fuzzy logic, and suggest further study. In support of this research, we introduce novel methods of simulating and visualizing FCA.

Cellular automata (CA) are discrete dynamical systems comprised of a lattice of finite-state cells. At each time step, each cell updates its state as a function of the previous state of itself and its neighbours. Fuzzy cellular automata (FCA) are a real-valued extension of Boolean cellular automata which "fuzzifies" Boolean logic in the transition function using real values between zero and one (inclusive). To date, FCA have only been studied in disjunctive normal form (DNF). In this thesis, we study FCA in conjunctive normal form (CNF). We classify FCA in CNF both analytically and empirically. We compare these classes to their DNF counterparts. We prove that certain FCA exhibit chaos in CNF, in contrast to the periodic behaviours of DNF FCA. We also briefly explore five different forms of fuzzy logic, and suggest further study. In support of this research, we introduce novel methods of simulating and visualizing FCA.

Cellular automata (CA) are discrete dynamical systems comprised of a lattice of finite-state cells. At each time step, each cell updates its state as a function of the previous state of itself and its neighbours. Fuzzy cellular automata (FCA) are a real-valued extension of Boolean cellular automata which "fuzzifies" Boolean logic in the transition function using real values between zero and one (inclusive). To date, FCA have only been studied in disjunctive normal form (DNF). In this thesis, we study FCA in conjunctive normal form (CNF). We classify FCA in CNF both analytically and empirically. We compare these classes to their DNF counterparts. We prove that certain FCA exhibit chaos in CNF, in contrast to the periodic behaviours of DNF FCA. We also briefly explore five different forms of fuzzy logic, and suggest further study. In support of this research, we introduce novel methods of simulating and visualizing FCA.