Team:Calgary/Human PracticesImposter

Human Pratices

Human Practices

Andrew Symes

Member

DRY LAB

Prabhu Dhatt

Member

DRY LAB

Sarah Walker

Member

DRY LAB

Rylan Marianchuk

Member

DRY LAB

Jake Liu

Member

DRY LAB

Jean-David Reasseau

Member

DRY LAB

Sebastian Alvarez

Member

DRY LAB

Nimaya De Silva

Member

WET LAB

Michaella Atienza

Member

WET LAB

Cassie Sillner

Member

WET LAB

Christian Emond

Member

WET LAB

Mike Wahba

Member

WET LAB

Sravya Kakumanu

Member

WET LAB

Jacob Grainger

Teaching Assistant

TEACHING ASSISTANT

Dr. Marija Drikic

Instructor

INSTRUCTOR

Dr. Elke Lohmeier-Vogel

Instructor

INSTRUCTOR

Dr. Mayi Arcellana-Panlilio

Instructor

INSTRUCTOR

Pheophorbide

Creating value from waste

pheophorbie hp is important because... our reasoning.

things thingsthingsthings.

Human Practices Timeline

Our early background research identified pheophorbide as a candidate for use as a therapeutic agent for photodynamic therapy. Photodynamic therapy (PDT) is a medical treatment which utilizes photosensitizing compounds, such as Pheophorbide, in the presence of specific wavelengths of light in order to produce a directed cytotoxic effect. PDT is currently being investigated for applications in cancer treatment and there already exist several commercial photosensitizers such as Photofirin. We were interested in Pheophorbide’s potential as a PDT photosensitizing agent, but we had to talk to someone to learn more about it.

Dr. Ronald Moore is a professor of Surgery and Oncology at the University of Alberta and currently serves as the Mr. Lube Chair in Uro-Oncology Research. He has over 25 years of experience in researching novel therapies for genitourinary malignancies.

Our discussion with Dr. Moore gave us an excellent overview of PDT’s current usage as well as what considerations doctors make when choosing a photosensitizing agent for PDT. Generally, the world of experimental cancer research is constantly in flux, changing with new developments and discoveries. While the current consensus is positive, tumorous cells substantially retain photosensitizers, the future's uncertain and the demand for photosensitizing agents in the future is shrouded in darkness.

Following our conversation with Dr. Moore, it was immediately apparent that we could not put all our eggs into one PDT-basket. We needed to explore other ways. During our literature review, we identified PDT as a potential method to treat fungal infestations. After our meeting with Dr. Moore we realized that pheophorbide has more potential as an anti-fungal agent than as an experimental cancer treatment drug.

After the meeting with Dr. Moore, we immediately had to learn more about working with fungi and anti-fungal assays. Through background research, we had the idea to use the “drop method”, applying drops of pheophorbide to different locations on an agar plate, with varying concentrations to see if it inhibited fungal growth. However, we needed to validate our experimental design. So we met with Dr. Heather Addy.

Dr. Heather Addy is a mycologist and plant biologist who specializes in plant-fungal interactions at the University of Calgary. Her expertise in the field meant we absolutely had to talk to her to inform the design of our anti-fungal assays.

Dr. Addy gave us the idea to use the “disc method”, immersing paper discs in solubilized pheophorbide, placing them around a fungal culture placed in the centre of a potato dextrose agar plate. The fungal colony would grow and eventually come into contact with the pheophorbide discs. Over the course of the experiment, we would measure the distance from the centre of the plate to the edge of the fungal colony’s growth. If pheophorbide does in fact have an inhibitory effect on fungal growth, then there would be a decreased rate of growth for the portions of the colony interacting with pheophorbide.

Dr. Addy generously put us in touch with Fran Cusack, a Biological Sciences Technician who prepares fungal samples for classes. Fran was kind enough to provide us with samples of Pestalotiopsis microspora and Sclerotinia sclerotinium, the same fungus which commonly afflicts canola crops.

We now had the information and tools at our disposal, to begin testing pheophorbide’s application as an anti-fungal agent. However, there was still a gap in our knowledge regarding the what the average Albertan farmer goes through when faced with fungi, like Sclerotinia.
To say John Mayko is merely an Agronomy Specialist from Mundare, Northern Alberta would vastly undersell his involvement with the Alberta Canola industry and his contribution to yOIL.

Receiving his Masters degree in Engineering Agrology from the University of Alberta, John is the president of Apex Agrology Services and currently sits on the Board of Directors for the Alberta Canola Producers Commission. His many years as a Senior Agri-Coach meant he could give us a clear indication of how fungi affect Albertan farmers.

John himself has had to deal with fungus. According to him, “Anybody growing canola in Alberta will have to deal with it”. There is no question that fungus is an issue for canola farmers, but what is being done about it?

Unfortunately, there is no fix for fungus. Once a crop has been afflicted by fungal blight, it must be discarded, there is no turning back the clock. At the early bloom stage, every farmer must make a decision whether or not to apply anti-fungal treatments to their crop. It is a costly proposition ($20-$30 per acre according to Dr. Kelly Turkington) which does not give a 100% guarantee.

From John, we learnt that our pheophorbide application would have to be preventative not prescriptive. He also gave us the indication that for our product to be viable, it would have to be cheaper to apply than current methods.

Now that we had an idea of the farmer’s perspective towards fungi, it was time for us to learn the pathologist’s perspective.
Dr. Turkington holds a Masters Degree and Ph.D in Plant Pathology, focusing on the epidemiology of sclerotinia stem rot in canola. For the last 23 years, Kelly has been working as a Research Scientist for Agriculture and Agri-Food Canada at the Lacombe Research Centre in Alberta.

We consulted him to learn more about the progression of Sclerotinia fungus, its impact on farmers and preventative and prescriptive measures to combat it.

According to Kelly, the cost to treat fungi like Sclerotinia can be around $20-$30 per acre, severely reducing a farmer’s bottom line. To assess risk of fungal growth, farmers employ a checklist by assigning point values to certain factors including the plant, the host and the environment. This is a very broad indication of risk and is not an exact science as there is ambiguity in the checklist. Some companies have started using DNA-based chips to quantitatively determine the percentage infestation of a plant.

In determining if Pheophorbide would be an ideal anti-fungal agent, he directed us to consider the full chemical profile of pheophorbide. Not just it’s effects on fungi but also on non-target organisms. Another consideration is the societal aspect of such a product. Members of the farming community and industry only care if the product is cheap and effective, but society as a whole tends to support products which are “organic” or come from the environment in a responsible manner.

Chlorophyll Repurposing

Cleaner chlorophyl removal

To assist in the dynamic characterization by other teams we looked to develop a methodology that allows for the calculation and aggregation of Brownian motion measurements for each amino acid in a sequence. The Brownian motion measurement chosen was the Root Mean Square Fluctuation(RMSF) calculated for every atom of a protein in ten picosecond intervals.

The RMSF data was calculated from a nanosecond Molecular Dynamic Simulation(MDS) completed within GROMACS, an industrial MDS software. These values were then averaged over each amino acid, this ensured that the unit of measurement was observed on a scale that was modifiable by teams. This resulted in a series of curves that quantitatively expressed the dynamics for each amino acid.

Chlorophyll Repurposing
During the initial stages of project design, we were contemplating whether to use a biofilm layer (1) containing chlorophyll-binding proteins to bind the chlorophyll molecules, and use this to filter the chlorophyll out of the canola oil. To gain an idea into the feasibility of using a biofilm, team members contacted Dr. Harrison, a microbiology professor at the University of Calgary who focuses on biofilms as a research interest. Dr. Harrison advised us to consider the amount of chlorophyll present in the oil compared to the amount of protein that can be present on a biofilm layer to capture all of the chlorophyll present. Additionally, he cautioned us that a biofilm is composed of bacteria, and thus the biofilm would have to be composed of bacteria which would not degrade the oil, as otherwise this would result in a lower yield of oil. Dr. Harrison spoke of some positive aspects to this project venture including the ability to kill the bacterial communities that comprise the biofilm using UltraViolet light, autoclaving and boiling without destroying the biofilm or denaturing the proteins in the process. Dr. Harrison also offered us the ability to use his equipment such as reactors and pumps to grow up the biofilm. He also told us to do more research into hydrophobic biofilms that would be able to withstand oil being passed through. Through this meeting, Dr. Harrison gave us valuable feedback on using biofilms as a chlorophyll filtration system, and gave us points to conduct further research in.
we also spoke with Dr. Lewis of the biochemistry department who caused us to rethink our approach. Similar to Dr. Harrison, Dr. Lewis advised us to consider the binding capacity of the protein of interest compared to the amount of chlorophyll needed to be removed. Additionally, he encouraged us to consider the industrial viability of any system we eventually decided on. For example, he suggested the use of a reusable magnetic bead that can be used in combination with a column to strip the oil of the chlorophyll molecules. The main takeaway from this meeting however, was the fact that proteins can only exist in aqueous environments-- which oil is certainly not. Even though the proteins would be existing on the biofilm surface, it would still be in the presence of oil. Thus, we reached a dilemma--we wanted to use a chlorophyll binding protein which could not exist in a hydrophobic environment like oil.