<p>Human actions and the resultant global warming are leading to considerable environmental changes that are negatively impacting marine ecosystems and their biodiversity. Luidia clathrata, a starfish species, is essential to the marine ecosystem, and understanding its sensitivity to stressors can help predict its future adaptations and role in the reef ecosystem. The study involved subjecting L. clathrata to thermal stress by incrementally raising the temperature by 1°C each day for a period of seven days. Physiological responses were evaluated on two separate occasions: day 1, which corresponded to the acute stress response, and day 7, which corresponded to the chronic stress response. The results showed a minor increase in phagocytic activity during acute thermal stress, but a significant decrease during chronic exposure. Although there was a slight decrease in total coelomic plasma protein during acute thermal stress, it significantly increased during post-chronic exposure. The amputated starfish avoided using the injured arm when righting themselves, indicating the development of neurosensory potential. Total cell count increased slightly in all stressed groups during acute stress but decreased after prolonged exposure to stressors. The mortality rate of the temperature-stressed groups was 33%, indicating that prolonged exposure to temperatures exceeding expected future temperatures could be harmful to L. clathrata. To support the hypothesis at the molecular level, RNA/DNA ratios and Heat shock protein gene 90, a molecular marker for cellular stress, were studied. Although no significant differences were observed in transcriptomic level, the temperature-stressed group showed slightly upregulated hsp90 gene expression. The findings indicate that L. clathrata responds to stress similarly to vertebrates, highlighting the potential impact of climate change on marine ecosystems. This study provides a baseline for comprehending the stress response of starfish, and further research is recommended with a larger sample size and over a more extended period. It is interesting to note that the gonad and body wall extracts of starfish exhibit significant inhibitory activity against various tested pathogens. The findings suggest that starfish extracts may have potential medicinal uses as antimicrobial agents. However further research is needed to understand the mechanisms of action behind these inhibitory activities and to identify the specific compounds responsible for them.</p>

<p>  </p> <p>Metastases are responsible for approximately 90% of all cancer-related deaths, with metastatic breast cancer (BC) holding a 5-year survival rate of only 27%. Recent research has highlighted a complex dynamic between cancer cells and the tumor microenvironment as essential for the formation of macrometastases. Within this field, tissue stiffening through matrix accumulation and altered matrix organization at the primary tumor site were recently linked with sustained proliferation and increased migration of tumor cells. Separately, elevated levels of the glycoprotein, fibronectin, were correlated to poor patient survival in BC and were linked to enhanced seeding of disseminated tumor cells at metastatic sites. Through my doctoral work, we have identified several mechanisms through which accumulated fibronectin impacts the metastatic potential of BC cells. First, we identified a transient increase in extracellular fibronectin in the lungs, which peaked before overt metastasis, coupled with a non-transient increase in total lung volume. To better recapitulate physiological conditions, we then developed a novel magnetically-actuated platform with the ability to apply tensile strain on cells at various amplitudes and frequencies in a high-throughput multi-well culture plate using suspended fibrillar fibronectin for 3D cell culture that is not reliant on a synthetic substrate. Using this as a biomimetic lung model, we found that cyclic mechanical force acted as a suppressor of cancer cell growth in a biomimetic lung model, implicating the accumulation and reorganization of extracellular matrix as an attempt by the cancer cells to alter the mechanical properties of the lung tissue and resist entering dormancy. However, our results showed that BC cells could not organize extracellular fibronectin independently. Instead, BC cells altered the accumulation and architecture of fibronectin by conditioning fibroblasts through soluble factors and extracellular vesicles. We observed that the fibronectin produced by conditioned fibroblasts varied as an effect of both the method of conditioning and the phenotype of the BC cell as the conditioning source. Taken together, these results have increased our knowledge of the relationship between disseminated breast cancer cells, fibroblasts, and fibronectin architecture in the early metastatic lung niche that paves the way for further investigation on targeting disseminated BC cells during early disease intervention in order to inhibit later overt metastatic outgrowth.</p>

The wide scale use of ZnO nanoparticles (NPs) in the world consumer market has resulted in likelihood of exposure to human beings. The present study was aimed to assess the in vitro and in vivo interactions of ZnO NPs in the mammalian system and to elucidate the possible mechanism of their toxicity. Our in vitro results using human epidermal cells (A431), primary human epidermal keratinocytes and human liver cells (HepG2) demonstrated that cells exposed to ZnO NPs exhibit a decrease in cell viability which was independent of NP dissolution. ZnO NPs also induced oxidative DNA damage as evidenced by an increase in the Fpg sensitive sites. The reactive oxygen species triggered a decrease in mitochondrial membrane potential and an increase in the ratio of Bax/Bcl2 leading to apoptosis through the intrinsic pathway. In addition, ZnO NPs induced phosphorylation of JNK, P38 and P53ser15. The results from our in vivo studies using a mouse model showed that ZnO NPs induce lipid peroxidation, oxidative DNA damage and apoptosis in liver which further confirmed our in vitro findings. The data from the present study provide valuable insights into the cellular interactions of ZnO NPs and the underlying molecular mechanism of their toxicity. The results also stress the need for a comprehensive environmental health and safety assessment of engineered nanomaterials to ensure safer nanotechnology based products.

<p dir="ltr">Obesity is a worldwide epidemic that is partially linked to changing lifestyles within the modern world, including increased access to calorically dense foods and decreased energy output due to more sedentary jobs. Obesity can lead to many different health complications, such as cardiovascular diseases or Type 2 Diabetes (T2D). Obesity-induced T2D is caused by dysfunction of the insulin-producing beta cells of the pancreas. However, mechanisms that promote obesity and the mechanisms by which obesity leads to beta cell dysfunction are not fully known.</p><p dir="ltr">Spinophilin is a filamentous (F)-actin binding, protein scaffolding, and protein phosphatase 1 (PP1)-targeting protein that can regulate protein. Spinophilin has multiple actions. Spinophilin can bundle filamentous actin to modulate the cellular cytoskeleton. Spinophilin also mediates substrate phosphorylation by targeting and modulating PP1 activity. In addition, spinophilin interacts with multiple proteins, including certain G-protein coupled receptors and can scaffold them with F-actin and/or PP1. Previous studies established that spinophilin KO mice have decreased fat mass, increased lean mass, and improved glucose tolerance. Yet, how spinophilin modulates the above metabolic parameters is unclear. We found that spinophilin is expressed in hypothalamic tissue and appears to also be expressed in the feeding center of the hypothalamus, as well as in other glucose-sensing cells known as tanycytes that neighbor the arcuate nucleus and the third ventricle. We found that loss of spinophilin limited weight gain observed in both a leptin receptor db/db mouse line (Leprdb/db<i>)</i> and mice fed a high-fat diet. Moreover, we found that the decreased fat mass seen in global spinophilin KO mice, at least in the Leprdb/db mice, was not due to major differences in feeding behaviors, consistent with what was observed by other groups using high-fat diet-fed mice. </p><p dir="ltr">As spinophilin was not associated with alterations in feeding, we posited that its ability to modulate glucose homeostasis may be linked to non-neuronal actions of the protein. Previous studies have found that spinophilin may regulate adipose tissue function and <i>in vitro</i> pancreatic beta cell function; however, its role in the pancreas and beta cells <i>in vivo</i> is not well characterized. We found that spinophilin is expressed in mouse pancreas. Using proteomics-based approaches we identified multiple putative spinophilin interacting proteins isolated from intact pancreas, including: PP1, the spinophilin homolog neurabin, and myosin-9. KEGG pathway analysis of proteomic proteins identified multiple pathways regulating ER stress, such as the unfolded protein response, and cytoskeletal arrangement. We observed decreased associations of spinophilin with PP1 and neurabin and increased association with myosin-9 in obese, Leprdb/db mice as early as 6 weeks, as well as significant decreases in body weight when spinophilin was knocked out in Leprdb/db mice. Moreover, we confirmed a robust and specific increased interaction of spinophilin with myosin-9, and other cytoskeletal proteins. Additionally, we found specific spinophilin interactions with ribosomal proteins, and exocrine and digestion proteins in high-fat diet-fed mice. Using our recently generated pancreatic beta cell-specific spinophilin KO mice, we found that loss of spinophilin in mice on a high-fat diet significantly reduces weight gain and improves whole- body glucose tolerance, and loss of spinophilin specifically within the beta cells also improves whole-body glucose tolerance, with no effect on body weight, further suggesting cell type-specific and independent roles for spinophilin on body weight and glucose homeostasis.</p>

<p>  </p> <p>The role of the endoplasmic reticulum (ER) Ca2+ release channels ryanodine receptor 2 (RyR2) and inositol 1,4,5-triphosphate receptor (IP3R) in pancreatic b-cell function are emerging, but are not well defined. It has been demonstrated that ER stress brought about by RyR2 dysfunction leads to impaired insulin secretion and contributes to the etiology of type 2 diabetes (T2D). Our work contributes to the understanding of the role of RyR2 in physiological pancreatic b-cell function and how loss of RyR2 contributes to the pathophysiology of T2D.</p> <p>To investigate the role of RyR2 in pancreatic b-cell function, we utilized CRISPR-Cas9 to delete RyR2 from the rat insulinoma INS-1 cell line (RyR2KO). We found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) and were sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. We discovered that total cellular insulin content and secretion were reduced in RyR2KO cells, but more modestly reduced in IRBITKO cells. We found that <em>INS2</em> mRNA levels were reduced in both RyR2KO and IRBITKO cells, but <em>INS1</em> mRNA levels were specifically decreased in RyR2KO cells. Additionally, nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in both RyR2KO and IRBITKO cells. DNA methylation of exon 2 of the <em>INS1</em> and <em>INS2</em> genes was more extensively methylated in RyR2KO and IRBITKO cells compared to controls. Proteomics analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 regulates IRBIT levels and activity, and together maintain insulin content and secretion, and regulate the INS-1 cell proteome, perhaps via DNA methylation.</p> <p>The role of interplay between RyR2 and IP3R in Ca2+ signaling and homeostasis in pancreatic b-cell function remains understudied. Stimulation with the sulfonylurea tolbutamide resulted in markedly delayed Ca2+ transients in both RyR2KO and IRBITKO cells. Xestospongin C significantly reduced the AUC of Ca2+ in RyR2KO and IRBITKO cells. Muscarinic receptor stimulation revealed a markedly increased AUC of Ca2+ in IRBITKO cells compared to both RyR2KO and control INS-1 cells. Assessment of PLC activity revealed that basal and stimulated PLC activity were reduced in the absence of RyR2 or IRBIT. Store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion revealed a decreased SOCE amplitude only in RyR2KO cells. Given evidence that phosphatidylinositol-4,5-bisphosphate (PIP2) depletion from the plasma membrane can regulate voltage-gated Ca2+ channel inhibition, we explored electrophysiological properties of all three cell lines. The frequency of glucose-stimulated action potentials was doubled in RyR2KO cells. Additionally, whole-cell voltage-gated Ca2+ current density was doubled in RyR2KO cells, and this current was more sensitive to hydrolysis of PIP2. These results evidence crosstalk between RyR2 and IP3R, and that RyR2 plays a critical role in maintaining proper Ca2+ homeostasis, PLC activity, and electrophysiological properties in pancreatic b-cells.</p>

No description available.

<p dir="ltr">The actin cytoskeleton is a dynamic structure that regulates various important cellular processes, such as cell protrusion, migration, transport, and cell shape changes. Cells employ different actin architectures best suited for each of these functions. We have employed an agent-based model to illuminate how the actin cytoskeleton plays such functions in animal and plant cells, via dynamic interactions between molecular players.</p><p dir="ltr">Lamellipodia found in animal cells are two-dimensional actin protrusion formed on the leading edge of cells, playing an important role in sensing surrounding mechanical environments via focal adhesions. Various molecular players, architecture, and dynamics of the lamellipodia have been investigated extensively during recent decades. Nevertheless, it still remains elusive how each component in the lamellipodia mechanically interacts with each other to attain a stable, dynamic steady state characterized by a retrograde flow emerging in the branched actin network. Using the agent-based model, we investigated how the balance between different subcellular processes is achieved for the dynamic steady state. We simulated a branched network found in the lamellipodia, consisting of actin filament (F-actin), myosin motor, Arp2/3 complex, and actin crosslinking protein. We found the importance of a balance between F-actin assembly at the leading edge of cells and F-actin disassembly at the rear end of the lamellipodia. We also found that F-actin severing is crucial to allow for the proper disassembly of an actin bundle formed via network contraction induced by motor activity. In addition, it was found that various dynamic steady states can exist.</p><p dir="ltr">The actin cytoskeleton in plant cells plays a crucial role in intracellular transport and cytoplasmic streaming, and its structure is very different from the actin cytoskeleton in animal cells. The plant actin cytoskeleton is known to show distinct dynamic behaviors with homeostasis. We used the agent-based model to simulate the plant actin cytoskeleton with the consideration of the key governing mechanisms, including F-actin polymerization/depolymerization, different types of F-actin nucleation events, severing, and capping. We succeeded in reproducing experimental observations in terms of F-actin density, length, nucleation frequency, and rates of severing, polymerization, and depolymerization. We found that the removal of nucleators results in lower F-actin density in the network, which supports recent experimental findings.</p>

<p dir="ltr">CaaX proteins, comprising approximately 300 members in the human protein database, represent a diverse group implicated in fundamental cellular processes, including proliferation, differentiation, trafficking, and gene expression. To carry out such vital cellular functions, CaaX proteins need to undergo three sequential post-translational modifications (PTM) through the CaaX pathway, which consists of isoprenylation (farnesylated or geranylgeranylated), endoproteolysis, and methylation. Among the CaaX family of protein, the Ras superfamily, plays a pivotal role in cell growth and survival. Mutations in <i>Ras proteins</i> are associated with a spectrum of cancers, presenting a significant challenge for therapeutic intervention. This thesis explores the intricate landscape of PTMs of CaaX proteins, with a focus on methylation, which is carried out by membrane protein isoprenylcysteine carboxyl methyltransferase (Icmt), and its potential as a therapeutic target, particularly for Ras-driven cancers.</p><p><br></p><p dir="ltr">Icmt is unique as it is the sole methyltransferase which carries out the third PTM of methyl esterification of CaaX proteins with the aid of co-substrate SAM, which serves as the methyl donor. Additionally, how Icmt, a membrane protein localized in the endoplasmic reticulum (ER), brings these two chemically diverse molecules in close enough proximity to promote catalysis, is very intriguing and is not yet fully understood. This thesis focuses on studying the structural and functional properties of Ste14, the yeast homolog of Icmt, in order to better understand the Icmt family of proteins. Ste14 is a functional homolog of human Icmt, sharing 41% sequence identity and 62% sequence similarity. Furthermore, Ste14 can be functionally purified unlike human Icmt. Together, these attributes make Ste14 an ideal system to study.</p><p dir="ltr"><br>The first project explores Ste14 and substrate binding, focusing on residues that determine farnesylated vs geranylgeranylated substrate specificity. It is essential to note that wild-type Ste14 recognizes farnesylated and geranylgeranylated substrate equally, with no preference to one over the other. Conserved residues found in Loop 2 and Transmembrane 3 of Ste14 were mutated to alanine and assessed for their activity with AGGC, the minimal geranylgeranylated CaaX substrate. Mutants which showed nearly zero percent activity with AGGC in comparison to wild type were further analyzed to understand if this loss of mutant activity with AGGC was potentially due to the mutant's inability to bind with AGGC. A photoreactive AGGC analog was used to carry out the photolabeling experiments and residues were analyzed for their binding ability with geranylgeranylated substrate. Mutants were further analyzed to understand the effect of mutation on structural integrity, to gauge which residues are essential for catalysis and for maintaining structural integrity of Ste14. Results demonstrated that residues F80 and E98 are essential for structural stability while L81 and L82 are essential for catalysis. This project would overall help better understand the lesser studied Ste14-substrate binding.</p><p><br></p><p dir="ltr">In the second project, the focus shifts to study Ste14 and co-substrate SAM binding by using electron paramagnetic resonance spectroscopy (EPR) and site directed spin labeling (SDSL). The biophysical technique of EPR requires much less protein and serves as great tool to study conformational change Ste14 undergoes on SAM binding, 3 non conserved residues found in the SAM binding region of Ste14, were individually mutated to cysteine, and had a spin label MTSL attached to their purified active mutant forms. Through EPR the conformational changes of Ste14 during methylation specifically during SAM binding was analyzed by visualizing the movement of MTSL attached residue. Results showed of the three non-conserved residues, A223 and E227 were immobile during SAM binding while T164 residue displayed flexibility during SAM binding during SAM binding and release process. This study would help understand the protein dynamics that Icmt undergoes upon SAM binding.</p><p><br></p><p dir="ltr">The final section centers on inhibiting the third step of the CaaX pathway, which is methyl esterification, by targeting Icmt. The project involved testing a library of Icmt inhibitors and evaluating their ability to inhibit Icmt activity. Of this library of bi-substrate analog inhibitors, compounds YD 1-66, YD 1-67 and YD 1-77 emerge as promising inhibitors against human Icmt, laying the foundation for further studies to develop more potent inhibitors. This section accentuates the strategies employed to target Icmt and the potential of these inhibitors in combating Ras-driven cancers.</p><p><br></p><p dir="ltr">This thesis provides an extensive analysis of the structure and function of Ste14. The varied studies and their insights contribute to a comprehensive understanding of Icmt and pave the way for the rational design of potent chemotherapeutic inhibitors for Ras-driven cancers. The multifaceted research presented in this thesis reveals several new possibilities for targeted therapies in the field of oncology.</p>

<p dir="ltr">The Ebola virus (EBOV) is a filamentous RNA virus which causes severe hemorrhagic fever. It is one of the most dangerous known pathogens with a high fatality rate. Multiple outbreaks of EBOV have occurred since the 1970s with the most widespread outbreak starting in December 2013. This outbreak continued through May of 2016 and had a fatality rate of approximately 50%. EBOV outbreaks are recurrent because the virus is still present in animal reservoirs. Despite multiple EBOV outbreaks we still lack a clear understanding of how new viral particles are formed and spread through virus assembly and release. Given the widespread global travel, EBOV now poses a threat to the entire world. EBOV encodes for the matrix protein, VP40, which is one of the most conserved viral proteins. VP40 can form different structures leading to different functions of the protein in different stages of the EBOV life cycle. The VP40 dimer traffics to the inner leaflet of the plasma membrane to facilitate assembly and budding. The VP40 octameric ring has been implicated in transcriptional regulation. This thesis focuses on understanding in further detail the determinates of VP40 plasma membrane assembly and exit from an infected cell.</p><p dir="ltr">The assembly and trafficking of VP40 to the plasma membrane requires a network of protein-protein and lipid-protein interactions (PPIs and LPIs). Studying these interfaces is important for understanding how VP40 structure and function regulates trafficking and assembly and can shed light on therapeutic strategies to target EBOV. The alteration of host cell Ca²⁺ levels is one of the strategies that viruses use to perturb the host cell signaling transduction mechanism in their favor. Evidence has emerged demonstrating that Ca²⁺ is important for the assembly and budding of EBOV in a VP40-dependent manner. The relationship between intracellular Ca²⁺ levels and EBOV matrix protein VP40 function is still unknown. In this work we utilize biophysical techniques to study the role of LPIs and intracellular Ca²⁺ on VP40 dynamics at the plasma membrane and key residues for assembly and budding. This work highlights the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding and a critical interaction between Ca²⁺ and the VP40 dimer that are important for lipid binding at the plasma membrane.</p>

<p>Metastasis of primary mammary tumors to vital secondary organs is the primary cause of breast cancer-associated death, with no effective treatment. Metastasis is a highly selective process that requires cancer cells to overcome multiple barriers to escape the primary tumor, survive in circulation, and eventually colonize distant secondary organs. One of the important aspects of metastatic cancers is the ability to undergo epithelial-mesenchymal transition (EMT) and the reverse process mesenchymal-epithelial transition (MET) process. Constant interconversion of tumor cells between these phenotypes creates epithelial-mesenchymal heterogeneity (EMH) and interaction between these tumor cell types and the stromal cell compartment is clearly important to metastasis. In healthy tissues, stromal cells maintain the composition and structure of the tissue through the production of extracellular matrix (ECM) proteins and paracrine signaling with epithelial cells. However, little is known about how EMH promotes changes in the ECM to promote breast cancer progression and metastasis. Cancer cells also secret exosomes, nano-size extracellular vesicles, to establish intercellular communication with distant organs in order to induce metastasis. These exosomes contain a plethora of different proteins including extracellular matrix proteins and matrix crosslinking enzymes. Fibronectin, an important ECM protein, plays an active role in tumor progression and is often crosslinked by tissue transglutaminase 2 (TGM2) to promote fibrosis in cancer. Both FN and TGM2 exist in exosomes and are expressed by heterogenous breast tumors. Although FN and TGM2 have been reported to play essential roles in cancer, their involvement in metastasis remains unclear. This work utilizes a variety of approaches to investigate the role of tumor heterogeneity and ECM proteins in promoting breast cancer metastasis. In this dissertation, we establish that mesenchymal cells expressing intracellular FN are held in a stable non-metastatic mesenchymal phenotype and produce cellular fibrils containing functionalized FN capable of supporting the growth of metastatic competent epithelial cells. We introduce a novel 3D culture system consisting of a tessellated scaffold which is capable of recapitulating cellular and matrix phenotypes <i>in vivo. </i>Further, we also demonstrate breast tumor cells secrete exosomes containing TGM2 crosslinked FN fibrils to promote premetastatic niche formation and induction of metastasis.<i> </i>Using genetic approaches, we establish TGM2 is essential and sufficient to drive metastasis. Finally, we demonstrate pharmacological inhibition of TGM2 offers a potential therapeutic strategy to treat metastatic breast cancer. Altogether, our research provides insights into the mechanism through which TGM2 promotes metastatic breast cancer. This work will help in developing new drugs to target TGM2 aimed at reducing breast cancer metastasis.<br></p>