Ascetosporea is a class of poorly studied unicellular eukaryotes that function as parasites of marine invertebrates. These parasites cause mass mortality events in aquaculture species such as oysters and mussels. The economic importance of these aquaculture species should lead to more attention on the genomics of Ascetosporea and their place on the evolutionary tree of life. With the onset of global warming and rising sea levels and temperatures, many emerging pathogens have been seen and until these are sequenced and analysed, it is difficult to make any conclusions about their relationships and evolution. As there aren’t many genomes and transcriptomes available for Ascetosporea, their position in the larger eukaryotic tree of life remains hypothetical. To attempt to remedy this lack of information, the Burki lab has recently generated sequencing data through sample collection and sequencing for these organisms (genomes and transcriptomes). A curated dataset of the various eukaryotic species was previously created and newly sampled and sequenced Ascetosporean genomes of Paramarteilia sp., Marteilia pararefringens, Paramikrocytos canceri, etc. from multiple sampling locations like Ireland, Norway, Sweden, and the UK were included. These could increase the genomic and transcriptomic data available for Ascetosporea and help to resolve the relationships within Ascetosporea. A few reasons why this group has not yet been placed on the tree of life are that the samples are from host tissue, which makes it difficult to sequence these parasites. These Ascetosporeans have also been seen to be very fast-evolving. After building phylogenetic relationships with single gene trees to allow for the identification of possible contaminants and paralogs, it was seen that there was a lot of contamination in Ascetosporea, due to the sampling being from host tissue material (hosts are open to the environment). After cleaning and filtering the possible contaminated genes, the trees were remade and a possible link between a fungal group called Microsporidia and Ascetosporea was observed in a few genes. This was hypothesized to be lateral gene transfer between the two groups resulting from their similar lifestyles and infection of invertebrates. There were complications like contamination and short blast hits that arose during analysis, and these could be caused by problems by fragmentation in the genome. This fragmentation could have negative effects on genome annotation predictions and consequently phylogenetic and phylogenomic analysis. Due to this and the challenging nature of collecting samples, the read coverage for the genomes is low but it can be used to perform phylogenetic and phylogenomic studies using currently available data and methods. Another expected result was that the sequenced data had contaminants, and a thorough and comprehensive search would have to be conducted on a dataset-wide level to remove any contaminants.

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Traditional methods for dermal wound closure such as sutures and staples are invasive and can result in soft tissue trauma, increasing the likelihood of localized inflammation and infections. Alternately, while tissue adhesive alternatives can effectively seal and adhere to the wounds, they can also present safety concerns relating to immunogenic responses and tissue toxicity. Herein, we fabricate injectable, adhesive, and cytocompatible poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)-dopamine (DA) hydrogels co-crosslinked via hydrazone and self-polymerized dopamine crosslinks that exhibit high water retention, improved tissue adhesiveness, and effective tissue regeneration properties. POEGMA-DA hydrogels exhibit independently tunable gelation properties based on their dual crosslinking mechanism, allowing for gelation as fast as 24 s (allowing for injection and rapid filling of irregularly-shaped wounds) while achieving relevant compressive moduli of up to 37 kPa and in vitro skin adhesion strengths of up to 1.2 kPa. The POEGMA-DA hydrogels induced no significant inflammation while demonstrating high interfacial adhesiveness in a stented skin excisional mouse model, enabling efficient dermal tissue regeneration by supporting collagen remodelling and enabling the regeneration of hair follicles, sebaceous glands, and blood vessels at the excision site over the 14-day study timeline. As such, injectable POEGMA-DA hydrogels represent a relevant non-toxic and adhesive alternative wound closure system for treating deep dermal wounds. / Thesis / Master of Applied Science (MASc) / Effective wound healing and subsequent tissue regeneration after a physical injury requires a moist sterile environment, the presence of oxygen, nutrients and enzymes, an efficient blood supply to the wound site, and a controlled inflammatory response to initiate the healing process. External methods of closing the wound to prevent infection aid in faster healing like sutures, staples, and liquid sealants which can result in infections and/or the stimulation of an inflammatory response that can hinder tissue restoration. Hydrogels, water-swellable polymer networks, represent an alternative solution that can both suppress infection while simultaneously promoting wound healing. Hydrogels have a similar structure to soft tissues like skin and can thus provide a supportive environment for cells to promote tissue regeneration and restore tissue structure and function. The swelling of hydrogels in water is highly beneficial for providing moisture at the wound site; however, this high degree of water retention also means they have a hard time sticking to tissues. To address this challenge, hydrogels can be modified with a component naturally derived from marine mussels that allows them to stick to their wet habitats, helping hydrogels to stick to the wound site while healing. In this thesis, mussel-inspired hydrogels are designed and can spontaneously gel and stick to a wound site to accelerate the restoration of the structure and function of skin. These biodegradable and injectable hydrogels are effective in accelerating wound closure with minimal evidence of scarring while suppressing negative inflammatory reactions and restoring the structure of skin by promoting the regeneration of hair follicles, sebaceous glands and blood vessels.

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<p>Adhesives  are  found  in  almost  every  aspect  of  the  modern  world.  They  are  found  in plywood used in buildings, electronics, shoes, plumbing and in almost every facet of your daily life. Nature also has an abundance of these adhesives that are used fora multitude of applications. Some  animals, like  the  blue  mussel, use  their  adhesive  for  protection  against  ocean  waves  and predators  while other animals, such  as  the  spider, use  it  to  trap  prey. Investigation  of  theses adhesives has led to the identification of several different proteins that allow for these animals to make  their  adhesive.  Some  of  them  are  composed of rare  amino  acids that  while  other  animals use  a combination  of  inorganic  and  organic  components.  Understanding  of  these  unique adhesives  can be  a  boon  for designof future  adhesives  that  do  not  have  the disadvantagesof current day commercialized glues.</p> <p>Increasing interest  in  the  restoration  of  natural  oyster  reefs  and  the  cement  that  holds them  together  has  resulted  in  the  identification  of  the  Shelk2  protein  that  is  found  both  in  the mantle  of  the  oyster’s  shell  as  well  as  the  cement  that  holds  the  reefs  together. Gaining  an understanding  of  how  this  protein  functions  and  its  part  in  the  oyster  reef  could  be  quite beneficial  for  projects  investing  in  reef  restorations  as  well  as  underwater  adhesion.  Gathering protein  from  the  animal  for  experimentation  and  characterization  can  be  labor  intensive  and extremely challenging. Luckily, cloning technology has become a useful tool for the expression of large quantities of proteins that can be difficult or impossible to gather from the native animal. Using <em>E. coli</em>, it  is  possible  to design  and express  this protein  in  hopes  of  gaining  a  better understanding of its impact on oyster settlement and adhesion.</p> <p>Sustainability is a major downside to current day adhesives that current technologies have not  been  able  to  solve. Most adhesives  that  are  on  the  market  today  are  primarily  derived  from petroleum. Current  research  has  begun  investigating  alternatives  to  the  large   epoxy   and formaldehyde  adhesive  market,  but  the  barrier  of  entry  is  hard  to  overcome.  To  replace  these glues  the  new  material  must  be  affordable,  non-petroleum  derived,  and  available  on  a  massive scale.  These  requirements  are  hard  to  meet  for  many  materials  and  due  to  that  the  current  bio-adhesive are generally very low strength.</p> <p>The work presented here will detail the characterization, and expression of some of these natural  adhesives that  have  been  found  in  the  Eastern  oyster. Another  aspect of  this  work includes the synthesis of a new bio-based adhesive system. Utilizing biomimetic chemistry along with  sustainably  sourced  materials  a  new  adhesive  has  been  formulated that  has  comparable adhesive strength to current day commercial adhesives.</p>

The dwarf wedgemussel (Alasmidonta heterodon) is a federally endangered freshwater mussel that once ranged from New Brunswick to North Carolina, but now only exists in isolated populations throughout its diminished range. Laboratory propagation in conjunction with augmentation or reintroduction is considered a critical component of dwarf wedgemussel restoration. My thesis aimed to I) develop in-vitro propagation techniques including methods to minimize microbial contamination for two Alasmidonta species and II) compile and assess critical fish assemblage information at existing dwarf wedgemussel presence and absence locations to inform future restoration activities. For the first objective (Chapter 2) I assessed three methods of contamination mitigation (media change frequency, concentration of the antifungal Amphotericin B, and method of antifungal replenishment). Across all experiments, higher levels of contamination severity had a negative impact on transformation success. In my experiments, dishes that had media changes every other day (vs. those changed daily and every 3 days) had the highest contamination and the lowest glochidia transformation success. Treatments with the lowest (0 µg/mL) concentration of the antifungal, Amphotericin B, and got a low-dose replenishment of Amphotericin B (vs frequent media changes) had the highest transformation success. The fungus was identified as Candida parapsilosis, a common fungus found in aquatic and human environments; future propagation efforts should use mitigation methods that are specific to the fungal contaminant. In the assessment of host fish near dwarf wedgemussel locations (Chapter 3), two fish repeatedly emerged as indicators of dwarf wedgemussel occurrence and abundance: the tessellated darter (Etheostoma olmstedi) and brown trout (Salmo trutta). Tessellated darter was positively related to dwarf wedgemussel which supports the importance of tessellated darter as a host fish in the wild. The negative relationship between dwarf wedgemussel and brown trout may indicate a difference in habitat between the species or brown trout’s predation upon wild hosts. Results from this project will be used in conjunction with habitat and genetic information to inform future restoration plans in the northeast and add to the growing body of literature on in-vitro propagation of freshwater mussels.

The studies described herein focused on the use of intermittent halogenation to control biofouling of water intake systems by the zebra mussel, Dreissena polymorpha, the comparative response of zebra mussels and the Asian clam, Corbicula fluminea, to a surfactant -based chemical control agent, the nontarget impact associated with the control agent, and the use of the Asian clam as a biomonitor of the control agent. Effects of intermittent (2-4 hr/day) treatments with chlorine or bromine at levels of 0.5 and 1.0 mg/L (total residual oxidant) upon settling of zebra mussel veligers were examined in studies conducted in a field laboratory on western Lake Erie. Veliger densities in the water column at the field site peaked at 530/L, while mussel densities on settling monitors reached 147,083/m² over the course of the study period. Zebra mussel settling in test systems treated with the halogens was reduced by as much as 91 % in comparison with controls, although mussel densities of up to 6,044/m² still occurred. Treated mussels which remained settled had growth rates similar to controls, and reached 2-4 mm length over 30 days. The intermittent halogen treatments had no significant impact on either adult zebra mussels or Asian clams. The studies indicate that while the treatment regimes may reduce zebra mussel densities within intake systems, the threat of eventual fouling due to cumulative settling remains. / Ph. D.