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An alarming increase of antibiotic-resistant bacterial strains has made the demand for novel antibacterial agents, for example, more effective antibiotics, highly crucial. One of the oldest antimicrobial agents is elementary silver which has been used for thousands of years. Even in our days, elementary silver is used for medical purposes, such as for burns, wounds, and microbial infections. We have taken the effectiveness of elementary silver into consideration to generate novel antibacterial and antifouling agents. Our innovative antibacterial agents are hybrid peptide silver nanoparticles (CH-01-AgNPs) that are created de novo and in situ from a silver nitrate solution (AgNO3) in the presence of ultrashort self-assembling peptides compounds. The nucleation of CH-01-AgNPs is initiated by irradiating the peptide solution mixed with the AgNO3 solution using ultraviolet (UV) light at a wavelength of 254 nm, in the absence of any reducing or capping agents. Obviously, the peptide itself serves as the reducing agent. The ultrashort peptides are four amino acids in length with an innate ability to self-assemble into nanofibrous scaffolds. Using these ultrashort peptides CH-01 we were able to create hybrid peptide silver nanoparticles CH-01-AgNPs with a diameter of 4-6 nm. The synthesized CH-01-AgNPs were further characterized using ultraviolet-visible spectroscopy, transmission electron microscopy, dynamic light scattering, and X-ray photoelectron spectroscopy. The antibacterial and antifouling activity of CH-01-AgNPs were then investigated using either gram-negative bacteria, such as antibiotic-resistant Top10 Escherichia coli and Pseudomonas aeruginosa PDO300, or gram-positive bacteria, such as Staphylococcus aureus CECT 976. The hybrid nanoparticles demonstrated very promising antibacterial and antifouling activity with higher antibacterial and antifouling activity as commercial silver nanoparticles. Quantitative Polymerase Chain Reaction (qPCR) results showed upregulation of stress-related genes, e.g. osmB and bdm. Biocompatibility studies of CH-01-AgNPs, using concentrations of 0.06 mM and 0.125 mM, testing for the viability of human dermal fibroblast neonatal (HDFn) cells, showed no significant influence on cell viability. In summary, we consider hybrid peptide silver nanoparticles CH-01-AgNPs as promising biomaterials that can be utilized in various biomedical applications, in particular for wound healing and biofilm inhibition, but also for other applications, such as tissue engineering, drug delivery, regenerative medicine, and biosensing.

The in vivo 3D extracellular matrix provides a temporal regulatory environment of chemical cues. Understanding this dynamic environment will be crucial for efficient drug screening, diseases mechanism elucidation, and tissue engineering. Therefore, in vitro 3D cell culture systems with reversible chemical environments are required. To this end, we developed a non-cytotoxic agarose-desthiobiotin hydrogel to sequester streptavidin biomolecule conjugates (KD 10-11 M), which can then be displaced by the addition of biotin (KD 10-15 M). Streptavidin biomolecule conjugates were simultaneously and sequentially immobilized by changing media components. The time required for biochemical environment exchange was minimized by increasing the surface area to volume ratios and pore size of the hydrogels. We temporally controlled the cell adhesive properties of hydrogels with RGD modified streptavidin to influence endothelial cell tube formation. / Thesis / Master of Science (MSc)

Increasing interest has emerged in the field of nutrition and its role in promoting skeletal muscle growth. Recently, studies using both in vitro and in vivo models have suggested that cannabidiol – a constituent of Cannabis Sativa – can increase the growth and regenerative capacity of skeletal muscle stem cells. Other isolated compounds, such as cannabinol, have demonstrated anti-inflammatory effects in vivo. Due to the potential benefits of both compounds, our primary objective was to further elucidate the effects of cannabidiol and cannabinol on murine C2C12 myoblast proliferation and differentiation. We hypothesized that supplementation of cannabidiol and cannabinol would augment gene expression of myogenin, leading to enhanced myotube formation; as well as, induce greater gene expression of Myf5 and MyoD, accompanied by increased cell proliferation. In relation to skeletal muscle growth, myostatin and follistatin can substantially impact the regulation of hypertrophy; with down-regulation of myostatin being a potent stimulus for muscle growth, and follistatin being the antagonist to myostatin, we therefore examined if cannabidiol or cannabinol influenced these two proteins, as a possible rationale for increased myogenesis. In this study, cells were treated with either: (1) cannabidiol, (2) cannabinol, (3) or vehicle control (methanol). Cells were grown for 48 hrs in their respective media, the MTT assay was used to assess proliferation. Muscle differentiation experiments required cells to grow for seven days with media supplemented with the respective compound. The media was changed every 48 hrs. The extent of muscle differentiation was assessed via immunocytochemical and qPCR analysis. In preliminary experiments, cell proliferation was influenced by the duration of which cells were exposed to the compound and concentration of the compound within the media. It was noted that changing growth media and compound every 24 hrs augmented the proliferative response compared to leaving it on for 48 hrs for both cannabidiol and cannabinol (p<0.05). Furthermore, supplementing cells with cannabidiol at a 1 or 5 uM concentration resulted in considerable cell growth compared to vehicle control (p<0.0001). Cannabinol at 5 uM showed the same effect (p<0.0001). We also quantified the mRNA expression of genes involved in the myogenic regulatory pathway in proliferating and differentiating cells. Herein we report that using a 5 uM concentration of cannabidiol or cannabinol did not increase the expression of any of these genes in proliferating or differentiating cells. These findings help further characterize the effects of cannabidiol and cannabinol on the myogenic response. / Thesis / Master of Science (MSc) / Nutrition impacts the regulation of skeletal muscle mass, with many individuals turning to supplements as a means to improve overall health. Cannabidiol – a constituent of the cannabis plant – has been used over the past several decades for its anti-inflammatory, neuroprotective, and anxiolytic properties; however, recent evidence has revealed its potential effectiveness in promoting muscle growth. If true, there is a possibility that it can be used to target the age-related loss of muscle mass, sarcopenia, or even improve athletic performance. Other derivatives, such as cannabinol, have seldom been studied but also demonstrate anti-inflammatory effects. Therefore, this thesis further elucidates the effects of cannabidiol and cannabinol on the myogenic signaling pathway. As a model, we used the murine C2C12 cell line that recapitulates the behaviour of human myoblasts. Interestingly, the data presented herein supports the notion that cannabidiol and cannabinol only promote cell growth and have no effect on myoblast maturation and myotube formation. These findings provide a better understanding of the potential for cannabidiol and cannabinol as a nutritional supplement targeting skeletal muscle.

The human placenta facilitates many key functions during pregnancy, including uterine invasion, vascular remodeling, hormone secretion, immune regulation, and maternal-fetal exchange. Placental research, however, has been limited in part by the unrepresentative nature of traditional models. The objective of this doctoral thesis was to build and characterize novel, in vitro models that reintegrated important anatomical and environmental elements of the human placenta, thus enabling more physiologically-accurate assessments of placental function. In our first model, we manipulated the thickness of the extracellular matrix surface to promote the self-assembly of trophoblast cells into three-dimensional (3D) aggregates that exhibited increased genetic and functional markers of syncytial fusion. In our second model, we established a high-throughput platform to generate 3D trophoblast spheroids that underwent dynamic invasion and migration, expressed transcriptomic profiles redolent of the extravillous trophoblast phenotype, and responded to various drugs relevant to pregnancy. In our third model, we developed a trophoblast-endothelial co-culture model of the placental barrier that underwent syncytial fusion, exhibited size-specific barrier permeability, and functioned under physiologically-relevant oxygen tensions. In conclusion, our models may each serve as valuable tools for researchers, contribute to investigations of different aspects of placental biology, and aid in the screening of drugs and toxins for pregnancy. / Thesis / Doctor of Philosophy (PhD) / The human placenta is an important organ that helps regulate the health of both the mother and fetus during pregnancy. Researchers have traditionally studied the placenta through the use of animals or isolated cells, but these have been criticized for not being similar enough to the human placenta. Our objective was to build models that better resembled the structure and environment experienced by the human placenta within the body, such that we could better study its function. During the course of my doctoral work, I built and analyzed three models of the human placenta using human cells that were grown in three dimensions, in multiple layers, and/or in a specific environment. Our first model demonstrated that placental cell behaviour and function can be controlled by altering the thickness of the surface we grew them on. Our second model grew placental cells in three-dimensions and mimicked the invasion process into the mother’s uterus during early pregnancy. Our third model grew placental cells with blood vessel cells to form the barrier that regulates the passage of all substances between the mother and fetus during pregnancy. We also tested the impact of low oxygen on the placental barrier’s formation and function. Overall, we discovered that placental cells could indeed function more similarly to how we expect them to in the body when we design platforms that better resemble their structure and environment. Our model development work provides new information about placental biology and may serve as valuable tools in research and drug development.

No / Growing three dimensional (3D) cells is an emerging research in tissue engineering. Biophysical properties of the 3D cells regulate the cells growth, drug diffusion dynamics and gene expressions. Scaffold based or scaffoldless techniques for 3D cell cultures are rarely being compared in terms of the physical features of the microtissues produced. The biophysical properties of the microtissues cultured using scaffold based microencapsulation by flicking and scaffoldless liquid crystal (LC) based techniques were characterized. Flicking technique produced high yield and highly reproducible microtissues of keratinocyte cell lines in alginate microcapsules at approximately 350 ± 12 pieces per culture. However, microtissues grown on the LC substrates yielded at lower quantity of 58 ± 21 pieces per culture. The sizes of the microtissues produced using alginate microcapsules and LC substrates were 250 ± 25 μm and 141 ± 70 μm, respectively. In both techniques, cells remodeled into microtissues via different growth phases and showed good integrity of cells in field-emission scanning microscopy (FE-SEM). Microencapsulation packed the cells in alginate scaffolds of polysaccharides with limited spaces for motility. Whereas, LC substrates allowed the cells to migrate and self-stacking into multilayered structures as revealed by the nuclei stainings. The cells cultured using both techniques were found viable based on the live and dead cell stainings. Stained histological sections showed that both techniques produced cell models that closely replicate the intrinsic physiological conditions. Alginate microcapsulation and LC based techniques produced microtissues containing similar bio-macromolecules but they did not alter the main absorption bands of microtissues as revealed by the Fourier transform infrared spectroscopy. Cell growth, structural organization, morphology and surface structures for 3D microtissues cultured using both techniques appeared to be different and might be suitable for different applications. / (Science Fund Vot No.: 0201-01-13-SF0104 or S024) awarded by Malaysia Ministry of Science and Technology (MOSTI) and IGSP Grant Vot No. U679 awarded by Universiti Tun Hussein Onn Malaysia.

Cultivated seafood is a potential alternative protein source that can address the rising global food demand with exponentially rising human population growth. Cultivated seafood is made by growing animal cells in vitro using stem cells for edible food, eliminating the need to raise the entire animal. A crucial first step in developing cultivated seafood is creating a well-characterized cell line that can continuously grow and differentiate into desired cell types. Due to difficulties in determining optimal primary cell culture conditions, no continuous cell lines of food-relevant mollusks have been established so far. This study used the adult Eastern Oyster, Crassostrea virginica, as a model bivalve to study the decontamination, cell dissociation, and culture conditions suited for mollusk adductor muscle cells. Oyster adductor (OAD) cells were obtained via tissue explant, mechanical and enzymatic digestion. The cells were routinely monitored using an inverted microscope for phase-contrast and fluorescence imaging. Culture vessels were coated with surface proteins such as fibronectin, laminin, matrigel, and poly-d-lysine to promote cell attachment. The tissue decontamination with Penicillin-Streptomycin (100 µg/mL), Amphotericin B (0.25 µg/ml), and algaecide solution (0.03%) was effective in controlling microbial growth. OAD cells grew best at lower nutrient levels in a one-to-one ratio of Lebovitz L-15 media and artificial seawater. Lower fetal bovine serum levels, 1-5%, provided a high number of cell attachments and consistent growth in combination with 1% adult oyster whole-body or larvae extract. The tissue explant method resulted in the optimal cell dissociation from the three methods, and proceeding cultures had attached cells surviving for up to 10 days. All the plate coatings promoted cell attachment, but fibronectin provided optimal cell attachment of OAD cells. Fibroblast-like, neuron-like, epithelial-like, and rounded cells were observed. Fluorescence cell staining confirmed the presence of cytoskeleton and nuclei in the OAD cell cultures. These advances in primary cell culture methods of OAD cells may be beneficial for establishing mollusk cell lines for cultivated seafood production. / Master of Science in Life Sciences / For sustainable seafood production, alternative sources of seafood proteins are essential in ensuring food security in the future. Cultivated seafood is an alternative protein source to address this rising food demand without the need to raise, farm, or slaughter animals. In developing cultivated seafood, self-renewing stem cells of the animal of interest are grown and made into edible products. A crucial first step in making cultivated seafood is understanding the growth conditions of the primary cells taken from animal tissue. Marine mollusk composes a significant part of seafood consumption, and developing cultured mollusks can address the growing food demand as a seafood alternative. However, there are many gaps in understanding the biological and physiological requirements of mollusk cells. No continuous, self-renewing mollusk cells of food-relevant species have yet been established. This study used the adult Eastern Oyster, Crassostrea virginica, as a model bivalve to study the tissue decontamination, cell dissociation, and culture conditions suited for oyster adductor muscle (OAD) cells. OAD cells were obtained via three cell dissociation methods. Cell growth was routinely monitored using an inverted microscope. Cell-surface proteins such as fibronectin, laminin, matrigel, and poly-d-lysine were used to promote cell attachment. The tissue decontamination was effective with Penicillin-Streptomycin, Amphotericin B, and algaecide. OAD cells grew best at lower nutrient levels in the one-to-one ratio of Lebovitz L-15 media and artificial seawater. Lower fetal bovine serum levels, 1-5%, provided a high number of cell attachments and consistent growth in combination with 1% adult oyster whole-body or larvae extract. Various cell morphologies were observed in the OAD cell cultures. Fluorescence cell staining confirmed the presence of cytoskeleton and nuclei in the OAD cell cultures. These advances in cell culture methods of OAD cells may be beneficial for establishing mollusk cell lines for cultivated seafood production.

Irreversible electroporation (IRE) is a non-thermal ablation technique that allows for eradication of unresectable tumors in a minimally invasive procedure. While IRE will preferentially kill larger cells over smaller ones, it does not discriminate between cells with larger and small nuclei. Given that one of the hallmarks of cancer cell morphology is larger, more abundant nuclei, our team set out to explore the possibility of preferentially targeting this physical and geometrical characteristic. / Master of Science