There is an urgency to make agriculture more environmentally sustainable and resilient to the changing climate that is exacerbating food insecurity and biodiversity loss. One approach to address this problem is improving crops through biotechnological genome modification, e.g., with CRISPR/Cas9. However, avoiding the regulation associated with genetically modified organism (GMO) labels is necessary for rapid, economical crop development. An alternate approach to transgenic methods of gene editing is the use of protoplasts (cells whose cell wall has been removed) for transient expression and subsequent regeneration of non-GMO, edited plants. However, efficient regeneration of plants from protoplasts is a bottleneck in the implementation of this technique. To create a universal method for protoplast regeneration, there first needs to be a baseline level of regeneration efficiency established in a model organism that is not only easy to work with but can also help us uncover the basic principles governing regeneration. To accomplish this, we are working with Arabidopsis which will allow us to demonstrate enhanced efficiency through culture conditions or ectopic gene expression (e.g., morphogenic transcription factors). Morphogenic transcription factors (MTFs) are a category of genes that coordinate the expression of multiple other genes, guiding the step-by-step formation of organs and embryos. We identified several MTFs that enhance root explant regeneration efficiency through a two-step root-to-shoot regeneration assay, and additionally distinguished the optimal timing of inducing expression of each MTF, either induced early during the callus induction step, late during the shoot induction step, or constantly induced during both steps. Characterizing the optimal induction timing for each MTF that enhances regeneration is crucial for their effective application. For example, when using transient expression in protoplasts for enhanced regeneration together with genetic modification, employing an MTF that boosts regeneration during early induction is likely to be advantageous, given that the MTF is only temporarily present alongside gene editing tools. We additionally investigated the links between these MTFs and their directly and indirectly regulated genetic targets to better understand the mechanistic control each of the MTFs have on regeneration. During these studies, we developed a baseline Arabidopsis protoplast regeneration method. Additionally, we identified five MTFs that enhance root-to-shoot regeneration and analyzed the target genes of the MTF that gave the highest regeneration efficiency. The future aim is to enhance protoplast regeneration using these MTFs. The overall goal of this research is to enhance plant regeneration to make biotechnology for crop trait improvement more broadly applicable. / Doctor of Philosophy / There is an urgency to make agriculture more environmentally sustainable and resilient to the changing climate that is exacerbating food insecurity and biodiversity loss. One approach to address this problem is improving crops through changing the genome in very specific ways. However, avoiding the regulation associated with genetically modified organism (GMO) labels is necessary for rapid, economical crop development. GMO labeling is required for plants that contain genetic characteristics that are not naturally present. To avoid integration of foreign genes while attaining a naturally occurring, beneficial trait, we can use gene editing tools that are temporarily present in a cell, which modify the genome before being degraded. This temporary expression can easily be accomplished using protoplasts, which are individual plant cells that have their cell wall removed. A protoplast with an edited genome must then be regenerated into a non-GMO, edited plant. However, efficient regeneration of plants from protoplasts is a bottleneck in the implementation of this technique. To create a universal method for protoplast regeneration, there first needs to be a baseline level of regeneration efficiency established in an organism that is not only easy to work with but can also help us uncover the basic principles governing regeneration. We accomplished this using Arabidopsis, which allowed us to demonstrate enhanced regeneration efficiency through optimizing protoplast culture conditions. Another approach for enhancing the regeneration efficiency of protoplasts is to alter which genes are present and govern the role of the cell. Overabundance of a gene responsible for plant growth and development could steer the protoplasts towards division and regeneration, when they are naturally unwilling to do so. Morphogenic transcription factors (MTFs) are a category of genes that orchestrate the levels of many other genes, guiding the step-by-step formation of organs and embryos. We identified several MTFs that enhance regeneration efficiency through a two-step root-to-shoot regeneration method, and additionally distinguished the optimal timing of inducing expression of each MTF, either induced early during the first step, late during the second step, or constantly induced during both steps. The induction timing is significant for the application of these MTFs; for instance, to enhance protoplast regeneration when the gene is only temporarily expressed alongside gene editing tools, using an MTF that enhances regeneration during early induction would be advantageous. We additionally investigated the links between these MTFs and their genetic targets that they directly or indirectly regulate to better understand the mechanistic control each of the MTFs have on regeneration. During these studies, we developed a baseline Arabidopsis protoplast regeneration method. Additionally, we identified five MTFs that enhance root-to-shoot regeneration and analyzed the target genes of the MTF that gave the highest regeneration efficiency. The future aim is to enhance protoplast regeneration using these MTFs. The overall goal of this research is to enhance plant regeneration to make biotechnology for crop trait improvement more broadly applicable.

Bone defects caused by trauma, tumor resection or disease present a significant clinical problem. Failures in 'high risk' fractures and large bone defects have been reported to be as high as 30-50%. The drawbacks associated with current bone grafting procedures have stimulated the search for improved techniques for bone repair. Tissue engineering/regenerative medicine approaches promote tissue repair by providing a combination of physical and biological cues through structural scaffolds and bioactive agents. Though they have demonstrated significant promise for bone regeneration, very little has been translated to clinical practice. The goal of this thesis was to investigate the potential of electrospun nanofiber mesh scaffolds for bone regeneration. Nanofiber meshes were utilized in a three-pronged approach. First, we validated their ability to robustly support osteogenic cell functions, including proliferation and matrix mineralization. We also demonstrated their efficacy as a cell delivery vehicle. Second, we investigated the effects of modulating nanofiber bioactivity and orientation on stem cell programming. Our results indicate that functionalization of nanofiber meshes with a collagen-mimetic peptide enhanced the migration, proliferation and osteogenic differentiation of cells. Fiber alignment improved cell migration along the direction of fiber orientation. Finally, a nanofiber mesh based hybrid system for growth factor delivery was developed for bone repair and tested in a challenging animal model. The delivery of bone morphogenetic protein (BMP) via this system resulted in the functional restoration of limb function, and in fact proved more efficacious than the current clinical standard for BMP delivery. The studies performed in this thesis have suggested novel techniques for improving the repair of clinically challenging bone defects. They indicate that the delivery of BMP via the hybrid system may reduce the dose and side effects of BMP, thereby broadening the use of BMP based bone augmentation procedures. Therefore, this nanofiber mesh based system has the potential to become the standard of care for clinically challenging bone defects, including large bone defects, open tibial fractures, and nonunions.

Regeneration of large bone defects presents a critical challenge to orthopaedic clinicians as the current treatment strategies are severely limited. Tissue engineering has therefore emerged as a promising alternative to bone grafting techniques. This approach features the delivery of bioactive agents such as stem cells, genes, or proteins using biomaterial delivery systems which together stimulate endogenous repair mechanisms to regenerate the tissue. Because bone is a highly mechanosensitive tissue which responds and adapts dynamically to its mechanical environment, application of mechanical stimuli may enhance endogenous tissue repair. While mechanical loading has been shown to stimulate bone fracture healing, the ability of loading to enhance large bone defect regeneration has not been evaluated. The goal of this thesis was to evaluate the ability of sustained osteogenic growth factor delivery and functional biomechanical loading to stimulate vascularized repair of large bone defects in a rat segmental defect model. First, we evaluated the hypothesis that the relationship between protein dose and regenerative efficacy depends on delivery system. We determined the dose-response relationship between dose of recombinant human bone morphogenetic protein-2 (rhBMP-2) and bone regeneration in a hybrid alginate-based protein delivery system and compared with the current clinically-used collagen sponge. The hybrid delivery system improved bone formation and reduced the effective dose due to its sustained delivery properties in vivo. Next, we tested the hypothesis that transfer of compressive ambulatory loads during segmental defect repair enhances bone formation and subsequent limb regeneration. We found that delayed application of axial loads enhanced bone regeneration by altering bone formation, tissue differentiation and remodeling, and local strain distribution. Finally, we evaluated the hypothesis that in vivo mechanical loading can enhance neovascular growth to influence bone formation. We found that early mechanical loading disrupted neovascular growth, resulting in impaired bone healing, while delayed loading induced vascular remodeling and enhanced bone formation. Together, this thesis presents the effects of dose and delivery system on BMP-mediated bone regeneration and demonstrates for the first time the effects of in vivo mechanical loading on vascularized regeneration of large bone defects.

The mining industry is a significant contributor to the Canadian economy. However, the mining activities can be detrimental to the environment due to the release of pollutants. Ammonia is one of the noxious and toxic contaminants associated with mining, ammonia contamination is created by the oxidizing agent in explosives. The explosives impacted mining wastewater (EIMWW) usually contains ammonia and other metal ions. The ammonia in EIMWW could harm the aquatic environment by the depletion of oxygen and its lethal toxicity to aquatic organisms. Before release to environment, EIMWW needs to be treated with an easy-to-operate method for ammonia removal at the remote mining sites. Ion-exchange (IE) with zeolite is an effective method for ammonia removal that is easy-to-operate, is not significantly impacted by cold temperature or toxicity effects. However, the traditional IE regeneration approach of using high concentration NaCl solutions creates a secondary polluting stream. Chlorine regeneration of ammonia-loaded zeolite appears to be a promising option, an evaluation of this option is the main topic of this thesis. This thesis includes three initiatives. The first is a set of multi-cycle batch loading-regeneration tests to assess the viability of ammonia removal with a commercial zeolite (SIR-600) for the treatment of a synthetic EIMWW (containing total ammonia nitrogen (TAN), K, and Ca) and to examine the performance of different ion-exchange regeneration solutions. The long-term TAN uptake of SIR-600 regenerated using a NaOCl (100 mg free Cl2/L) solution was 0.24 meq/g, which was approximately 20% lower than that after a NaCl regeneration. However, chlorine regeneration is promising because the selectivity of SIR-600 for TAN over Ca and K increased after the chlorine regeneration. To simulate recycling of the NaOCl regenerants, K and Ca were added to the NaOCl solution, it did not substantially affect the subsequent SIR-600’s ion uptake. This initiative represents a significant contribution since the earlier studies into chlorine regeneration did not investigate the impact of competing ions. The second initiative addressed concerns regarding the long-term integrity of SIR-600 arising from its exposure to high chlorine concentrations during the regeneration. The five-week long chlorine batch exposure tests with solutions of up to 1000 mg free Cl2/L showed that chlorine exposure did not significantly affect the SIR-600’s characteristics in terms of particle size distribution, surface area, FTIR spectra and ion uptake. Thus, SIR-600 has the potential for long-term use in field applications. The final initiative evaluated the feasibility of chlorine regeneration for continuous flow IE column systems used for ammonia removal from a synthetic EIMWW. Continuous flow column systems are important because these are the standard IE units used in full-scale applications. Multi-cycle column loading-regeneration tests were performed to compare the zeolite performance using a NaOCl (1000 ppm as free Cl2) solution with that using a 5% NaCl regeneration. The influence of loading duration was also assessed. The use of 6-hr loading cycles were shown to be preferable to 23-hr loading cycles because it had lower effluent concentrations and they could achieve higher overall TAN mass removals per unit time. After three operational cycles, the SIR-600 had similar TAN uptake performances (0.21 meq/g Vs. 0.21 meq/g) after NaOCl regeneration and after salt (NaCl) regeneration. This is in contrast to the lower TAN uptakes for the NaOCl regeneration in the batch tests, this indicates that batch tests are not always representative of full-scale applications. Compared to NaCl regenerated SIR-600, SIR-600 after NaOCl regeneration had a higher preference for TAN over Ca and K, which makes this type of regeneration very promising. Its only apparent limitation is that the NaOCl regeneration required a longer duration. During the NaOCl regeneration, the main mechanism appears to be the oxidation of ammonia to nitrogen gas and hydrogen ions, however the Na in the NaOCl solution also seems to have a role in the regeneration.

The Canadian mining industry is one of Canada’s largest industrial sectors, creating jobs and a significant contributor to the economy. However, the mining activities can be detrimental to the environment due to the release of pollutants. Mining extensively uses nitrogen-based explosives, creating explosive impacted mining wastewaters (EIMWW) that contains substantial quantities of ammonia which is toxic to fish and thus, it has serious environmental repercussions. Ion-exchange (IE) with zeolite is an effective method for ammonia removal as it is easily automated, has a rapid start-up, is not significantly impacted by cold temperature or toxicity effects. Thus, it is particularly suited for Canadian mines. However, the traditional IE regeneration approach of using high concentration NaCl solutions creates a secondary polluting stream. Chlorine regeneration of ammonia-loaded zeolite appears to be a promising option to avoid such a secondary source of contamination. An evaluation of this option and other alternative regeneration are the main focus of this thesis. This thesis includes two initiatives. The first is a set of multi-cycle column loading and regeneration tests to investigate the feasibility of a zeolite (SIR-600) column for ammonia removal from a synthetic EIMWW, containing K and Ca as competing ions, coupled with regeneration using different concentration of chlorine solutions and combined salt+chlorine regeneration solutions. NaOCl regeneration was quite effective, but it was slower than salt regeneration. During the NaOCl regeneration, the main mechanism appears to be the oxidation of ammonia to nitrogen gas and hydrogen ions, however the Na in the NaOCl solution also seems to have a role in the regeneration. This results in pH levels around 3 for approximately half the regeneration cycles. In the combined salt+chlorine regeneration, the incorporation of the salt leads to more rapid elusion of the three ions presumbaly because of the higher sodium concentration (205 meq/L Na versus 14 meq/L Na). The long-term total ammonia nitrogen (TAN) uptake of SIR-600 regenerated with a NaOCl and NaOCl-NaCl were fairly similar, they varied within a relatively small range (0.185meq/g - 0.202meq/g). Thus, the various regeneration schemes did not impact the TAN uptake. The only apparent limitation of NaOCl regeneration is that it required a longer duration. However, the NaOCl is very promising because it resulted in very similar TAN uptakes, the SIR-600 showed a higher preference for TAN over K and avoided creating an additional process waste stream. The second initiative addressed concerns regarding the long-term integrity of SIR-600 arising from its exposure to low pH solutions during the regeneration. Long-term batch tests were performed to expose SIR-600 to low pH conditions (pH=2, pH=3, pH=4) and the characteristics of this IE material were evaluated. The 3-month low batch exposure experiment showed that pH below 4 decreased the TAN uptake capacity by up to 58%. There was no considerable impact on the surface gravimetric analysis (TGA) and Powder x-ray diffraction (PXRD). The exposure to pH=2 and pH=3 led to breakdown of the outer surface of SIR-600 and the creation of fine particles. It also led to decreases in the BET surface area and a decrease in the TAN uptake proportional to the decrease in the BET surface area. Thus, the exposure to pHs below 4 impacts the durability of SIR-600, so SIR-600 may have to be replaced more frequently. However, regeneration with NaOCl solutions still seems very promising as it avoids the creation of a secondary waste stream.

HA (Hyaluronan, hyaluronic acid) is a predominant component of the extracellular matrix and plays diverse roles in the process of wound healing. The major significance of the biological functions of HA in wound healing arise from early studies showing that rich and prolonged existence of HA is associated with the fetal scarless wound healing. It has been hypothesized that adult wound healing can be modulated by the application of exogenous HA to make the process of wound repair more like regeneration. / In this thesis, the effects of molecular weight and concentration of HA on the biological behavior of human fibroblasts and keratinocytes have been investigated in both monolayer and 3D culture models. In addition, the mechanism for the HA-induced biological effects has also been investigated. Previous laboratory studies have primarily focused on individual cell type, but in wound healing in vivo there is considerable interaction between the keratinocytes and fibroblasts. The role of HA in keratinocyte-fibroblast interactions is not clear, so an organotypical culture model was used to investigate the keratinocyte differentiation and stratification and basement membrane formation with and without fibroblasts to explore the effect of HA on keratinocyte-fibroblast interactions. / The experimental data suggest that exogenous application of HA modulates the process of wound healing so that it becomes more like regeneration by regulating the biological functions of human keratinocytes and fibroblasts, including their interaction. By further understanding the HA-induced cell biological phenomena and involved mechanisms new approaches for wound care may be revealed. / The skin is the largest organ in the body. It consists of an outer layer, the epidermis supported by a connective tissue matrix, the dermis. The keratinocyte and fibroblast are the major cell types in the epidermis and dermis respectively. When the skin is damaged, the body will active a cascade of mechanisms to restore the tissue integrity. If the damaged tissue is limited to the epidermis, it will be restored by the process of regeneration. Regeneration involves the replacement of injured tissue by new tissue which has both form and function identical to the original tissue. If the defect involves the dermis, healing is achieved by repair which results in permanent scar formation. Wound healing is an extremely complex biological process which involves several overlapping steps; inflammation, granulation tissue formation and remodeling which are coordinated by various cell types, growth factors, cytokines and extracellular matrix components including hyaluronan. / Whilst there have been many studies looking at the effect of HA on different cell types using varying molecular parameters, concentrations and sources of HA, the result are often contradictory. Even the endogenous or exogenous application of HA have been reported to have opposite effects on cell behavior. / Gu, Hua. / Adviser: Andrew Bard. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0216. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 116-135). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

High risks imposed by natural hazards, and changes in economic, socio-cultural and technological conditions compel Turkey to transform its cities rather than promote growth. In this respect, urban regeneration became a significant concern demanding a comprehensive and integrated vision and action. Turkey will have to focus on regenerating the built environment in the near feature, rather than follow conventional trends of city-extension development. As scope and methods of urban regeneration planning differ from that of development-planning, a special legal framework and process for urban regeneration is required. The development of comprehensive urban regeneration policies to coordinate physical, social and environmental issues, together with relevant procedural steps, all accommodated within a legal framework are today the most challenging problem in urban planning. To identify the needs in detail, procedural and legal aspects of Zeytinburnu Urban Regeneration Project one of the current urban regeneration projects, related laws and draft laws, and experience abroad are comparatively reviewed here. The main finding is that the procedural steps and legal arrangements of recent regulations represent single-minded understandings of the scope of urban regeneration. There are tendencies of centralizing the powers of implementation, relying only on physical regeneration as a linear process, discouraging all forms of participation. The recent regulatory attempts in special laws concerning regeneration, as well as in laws of &amp / #8216 / development&amp / #8217 / , &amp / #8216 / local administrations&amp / #8217 / and &amp / #8216 / municipalities&amp / #8217 / need to be modified in their procedural and legal provisions. Local authorities can be charged to designate regeneration areas at 1/5000 city master plans which could be coordinated by changing an article within Development Law (3194). Tasks and responsibilities for comprehensive regeneration could be provided with the Law of Municipalities (5272), and the Law of Provincial Administration (5302). These laws could also equip local authorities with prerogatives of implementing regeneration projects. A second procedural requirement concerns the preparation of plans. This demands steps for participatory interventions as well as a versatile structure to allow feedback and returns to former stages of planning. This could be introduced by means of a regulation regarding preparation of regeneration projects. This resembles the regulation on technical specifications for the preparation of development plans of Law 3194. This can enforce the participation of local citizens and stakeholders to the process. Changing the article 13 of the Municipality Law would suffice for the realization of participation processes. The article (24n) of the Greatercity Municipalities Law that enable partnerships between public, private bodies and NGOs can be further introduced to the Municipality Law for the very necessary synergies in regeneration projects.

All-optical signal processing techniques for phase-shift keyed (PSK) systems were developed theoretically and demonstrated experimentally. Nonlinear optical effects in fibers, in particular four-wave mixing (FWM) that occurs via the ultra-fast Kerr nonlinearity, offer a flexible framework within which numerous signal processing functions can be accomplished. This research has focused on the regenerative capabilities of various FWM configurations in the context of processing PSK signals. Phase-preserving amplitude regeneration, phase regeneration, and phase-regenerative wavelength conversion are analyzed and demonstrated experimentally. The single-pump phase-conjugation process was used to regenerate RZ-DPSK pulse amplitudes with different input noise distributions, and the impact on output phase characteristics was studied. Experiments revealed a limited range over which amplitude noise could effectively be suppressed without introduction of phase noise, particularly for signals with intensity pattern effects. Phase regeneration requires use of phase-sensitive amplification (PSA), which occurs in nonlinear interferometers when the pump and signal frequencies are degenerate (NI-PSA), or in fiber directly through single-stage (degenerate) or cascaded (non-degenerate) FWM processes. A PSA based on a Sagnac interferometer provided the first experimental demonstration of DPSK phase and amplitude regeneration. The phase-regenerative capabilities of the NI-PSA are limited in practice by intrinsic noise conversion (amplitude to phase noise) and to a lesser extent by the requirement to modulate the pump wave to suppress stimulated Brillouin scattering (SBS). These limitations are relaxed in novel materials with higher SBS thresholds and nonlinearities. Degenerate FWM provides PSA in a traveling-wave configuration that intrinsically suppresses the noise conversion affecting the NI-PSA, while providing stronger phase-matched gain. Experiments confirmed superior phase-regenerative behavior to the NI-PSA with simultaneous reduction of amplitude noise for NRZ-DPSK signals. Phase-regenerative wavelength conversion (PR-WC) provides the regenerative properties of PSA at a new wavelength, and was proposed and demonstrated for the first time in this research. The parallel implementation of two FWM processes, phase-conjugation and frequency conversion, provides two idlers which exhibit interesting and useful regenerative properties. These were investigated theoretically and experimentally. Ideal phase-regenerative behavior is predicted when the contributing FWM processes are equally phase-matched, which can be maintained over any interaction length or wavelength shift provided the pump powers are properly adjusted. Depleted-pump regime PR-WC provides simultaneous phase and amplitude regeneration. Experiments confirmed regenerative behavior for wavelength shifts of the idlers up to 5 nm. Two techniques for phase regeneration of 4-level PSK signals were developed and evaluated. The first is based on parallel operation of PSAs suitable for processing 2-level PSK signals, where phase projection and regeneration are combined to recover the input data. Analysis of this scheme outlined the conditions required for effective phase regeneration and for practical implementation using known PSAs. A novel process based on FWM (parallel phase-conjugation followed by PSA) was developed and analyzed, and demonstrated using numerical simulations. These studies provide a basis for further work in this area.

<p>Zebrafish regenerate amputated caudal fins, restoring the size and shape of the original appendage. Regeneration requires generation of diverse cell types comprising the adult fin tissue. Knowledge of the cellular source of new cells and the molecules involved is fundamental to our understanding of regenerative responses. In this dissertation, the contribution made by the bone cells towards fin regeneration is investigated. Fate mapping of osteoblasts revealed that spared osteoblasts contribute only to regenerating osteoblasts and not to other cell types, thereby suggesting lineage restriction during fin regeneration. The functional significance of osteoblast contribution to fin regeneration is tested by developing an osteoblast ablation tool capable of drug induced loss of bone cells. Normal fin regeneration in the absence of resident osteoblast population suggests that the osteoblast contribution is dispensable and provides evidence for cellular plasticity during fin regeneration. To uncover the genes involved in proliferation of osteoblasts within the fin regenerate, a candidate in-situ screen was carried out and revealed bone specific expression of fgfr4 and twist3. Transgenic tools for visualization of gene expression confirmed the screen results. Knockdown of twist3 by morpholino antisense technology impedes fin regeneration. Mutant heterozygotes for twist3 were generated using genome editing reagents, which will enable loss-of-function study in future.</p> / Dissertation

Using the right planting spots when planting can provide a lot of advantages forthe plant more nutrients, less competition and redused risk of damage from pineweevil. In this study comparisons have been made between quality follow upsfor not approved and approved plantings made by SCA in the distrikt ofÅngermanland. Few significant differences were found but not approvedplantings had more opportunitis for improvement than approved plantings. Moreplantings vere not approved on moist sites.