Wellington and a new beginning
So I spent many days in the Victoria University library tracking down literature related to my Ph.D. which was focused on serine proteases from ovine pancreas and comparison to the equivalent porcine enzymes. After about six months of literature analysis, I had a few ideas and had gotten up to speed to start conducting a few experiments in the laboratory in order to try to develop a viable economic solution for the purification of elastase I. So I was trying to target only one of the many proteins present in the pancreas. I had identified several affinity matrices that could be used to isolate elastase I from the other pancreatic proteases present in the pancreatic extracts. The activation of the pro-enzymes required active trypsin which could auto activate slowly in the presence of calcium ions. Calcium was also important in the stabilization of the enzymes. It is also involved in the catalytic reaction as an atomic clock through neutron capture and beta minus radioactive decay can generate a proton which may be involved in the charge relay system present at the active site and responsible for catalytic hydrolysis of peptide bonds C-terminal to Ala, and Val in the case of elastase I, aromatic amino acids as the case for chymotrypsin and positively charged amino acids as was the case for trypsin. There were also other enzymes present including carboxypeptidase A and B, aminopeptidase and DNase, RNase, amylase, and lipase. So in all, there is a sweet of enzymes, a cocktail of opportunities that could be isolated from the pancreas.
My initial affinity matrices were pretty poor at binding elastase I as I was trying only simple amino acids and had used pre-activated CNBr Sepharose matrices. Most of them failed. I learned about steric hindrance and spacer arms and various coupling chemistries that would enable me to develop my organic chemistry expertise. The more I developed the affinity matrices for elastase the more I realized that isolating just one enzyme from the cocktail was going to be an issue due to wastage and the autohydrolysis issues present when dealing with pancreatic proteases. I decided to change tact rather than develop just one expensive poly alanine affinity ligand for the isolation of elastase I. I decided to look at multiple enzymes and use a staged approach to enzyme capture and ended up developing a number of affinity matrices targeting all of the various pancreatic proteases present. I read about the bifunctional coupling reagents to increase substitution levels on the matrix and applied this knowledge to create a trypsin affinity matrix that had such a high binding capacity that when I was eluting the enzyme off the matrix a crystalline precipitate formed in the elution stream which turned out to be trypsin. I also made a resin for chymotrypsin which was functional and this lead to a solution containing predominately elastase I. Therefore I achieved my goal of obtaining a highly purified elastase I by performing ion exchange after having removed trypsin and chymotrypsin using affinity chromatography. I had worked in reverse to achieve my goal and ended up creating multiple products in the process.
Half my time during my Ph.D. was spent up in Taranaki living in Stratford and working in Eltham at Renco applying these processes to produce highly purified enzymes. I managed to do a number of moderate scale enzyme purifications using affinity chromatography at several liter scales. However, a commercial outcome did not result from this work as it was not seen to be economically feasible. My time in Eltham and Stratford was quite lonely and I started developing music and videos for Vjing at dance parties and ended up buying a DV video camera and used my computer to make videos for music that I was making and that was a fun time-consuming thing to do to keep myself occupied. I would often drive down to Wellington to go dancing on Friday and Saturday nights with friends and crash on a couch. These were fun times. I also did a fair bit of fishing and played golf which balanced my time spent indoors doing the academic and Vjing. I look back fondly at this time I spent alone and the opportunity I took to grow and to think about what I wanted in my future.
When I returned to Wellington after spending a year up in Taranaki I was fully into the Wellington clubbing scene and I flatted in Haining Street. I would walk everywhere and enjoyed Wellington slowly discovering who I was. I found my home away from home and as I connected with like-minded people I grew and started to learn more about myself. Hours on the dance floor improving my moves have allowed me to both lose myself and find myself. I completed the three years of the Scholarship but still had not finished my Ph.D. So I started doing laboratory demonstrating and teaching up at Victoria University of second and third-year biochemistry classes so I could have some income. I also decided to get a student loan and ended up getting in debt but also enjoyed myself and headed overseas for one of Australia's big dance parties. The friends I had then appear to have disappeared. Where they all are now I do not know, the creatures of the night who looked like angles. The people I loved and partied with. They appeared out of the woodwork like beetles taking-flight to the call of trance and house music. We had many a communion with nature at the Gathering on Takaka hill near Howard's hole. It was a pleasure being part of something bigger than myself and to connect to others via movement. I was no saint but I think I did what I did for one reason only and that was to find and explore my mind. It's an inner space that we all travel in order to see the world from an alternative perspective so that humanity can grasp existence. I hooked up with people that put on parties and got into doing Vjing at these parties rather than dancing and then felt like I was becoming a spy looking through a camera trying to explore the big questions of life. I eventually grew tired of all the parties I felt like I needed to do something else. In 1999, when I was still completing my Ph.D. I met my wife on Guy Fawkes night on Roxbough Street, Wellington.
It's funny, I felt like I had seen her before but could not place where and when. We started talking and exchanged numbers and I finally plucked the courage to call and we had our first date as the fishbowl. We walked to the Matterhorn in Cuba street and I shook her hand to say farewell on Dixson Street. She had braces and was living at home in Kelburn with her parents at the time but there was something about her that was interesting. We talked about our past and what we wanted in life. Our paths had crossed previously as we had lived in similar areas in New Zealand both in Christchurch and Taranaki. Interestingly, she used to walk down Adams terrace between her sister's place at the bottom of the hill and her parents place up at Kelburn Parade. So I think now I recollect where I had seen her previously, it was out of my window on Adams Terrace that day when I saw that woman walking by.
So we ended up having that conversation. The one you have when you want to spend the rest of your life together. Talking about how many children the rules of engagement. I still didn't know who I was, still exploring, trying figure things out. I was given an ultimatum, her way or the highway. In other words, ditch my dance party friends, the children will be baptized Catholic and I will have to stop my mind exploration and do a Catholic marriage course and get on the straight and narrow. I decided to go all in and walked away from my old life and started a new life. I became more focused and completed my Ph.D. with a pass. Nothing outstanding but being the first of my family to obtain a Ph.D. allows me to feel proud. I was lucky. I feel like I am in the twilight zone. It was like star wars where my Ph.D. supervisor Dr. Sue Marshall had also been a Ph.D. student at Victoria University of Wellington under the supervision of Dr. Alan Clark. Now Sue had been previously at Industrial Research Limited and had moved to Taranaki and worked at Renco in Eltham. She had organized the Ph.D. position along with Alan which had resulted in me doing my Ph.D. So there was a relationship already established with Industrial Research Limited and I was given the opportunity to go and talk to some people out at Industrial Research Ltd in 2001 after completing my Ph.D. to see if I could help them with a project they had, which involved developing an RNase sequencing kit for the NIH and RNA Society. It was a six-month contract. I had to isolate six different RNase enzymes from different organisms in order to make the kit. I would have to work radioactive P32 which I have had previous experience with during my Masters in Christchurch. So I was comfortable in doing both molecular biology research using RNA and DNA as well as purifying enzymes from various sources including bacteria and fungi which was also a combination of skills I developed during my Ph.D. and Masters. So, fortunately, the project went well and I successfully produced the kits and it went worldwide. Based on that success I was given a full-time position at Industrial Research Limited. I also spent some time in Hong Kong, my first venture north and into Asian society. I spent time working at the Chinese University of Hong Kong and using an enzyme that I had isolated for introducing novel nucleoside bases into DNA structure using a pure nucleoside phosphorylase PNP in a solvent-based system.
When returning back to IRL the work I was going to focus on was related to wound healing and extracellular matrix scaffold from the red meat Industry. Having already worked for a New Zealand company as part of my Ph.D. that involved the use of co-products from the meat industry I was comfortable with that industry and would prefer that the products could be used for human health applications. Before embarking on this I had got married on the 1st of September 2001. My wife and I traveled around Europe for four months. It was eye-opening seeing another part of the world and the ancient landscape compared to the youthful nature of the New Zealand landscape. It was like a spiritual homecoming going to Menorca, the place of my father's ancestry. I remember one special occasion in Assisi, the home of Saint Francis and my own future Baptismal name, I can relate to his approach to life being a biologist and biochemist. To get up the hill, to his tomb, you have to hop into a one person cage that went up to the hill a primitive Gondola. It was a misty morning and I could not see the top of the hill. I hopped in and as I traveled slowly up I went through the mist and could not see a thing. Breaking through the mist resulted in the revelation of parting the land from the sky. It was a peaceful scene in his tomb, quiet, reflective, moving. To think about it now takes me back to that experience. It was very moving. I was alone with my thoughts and felt the peace of the location on the top of the hill in his presence. When I came down I did not realize it at the time how that moment had moved me and how
Back in New Zealand, the work on ECM crypteins resulted in the discovery of GAP537. I developed a processing method for the production of the peptide from the eye lens. The extraction method used pH precipitation of the alpha crystallins followed by enzymatic treatment with elastase I. The enzyme I had targeted during my Ph.D. My initial approach of using elastase I, resulted in the loss of the peptide as I had added calcium to the hydrolysis solution. The number of peptides generated was numerous and the one I wanted had disappeared. I did not know why but saw a precipitate and thought that this may have been responsible for the disappearance of the peptide. So I tried again this time without adding calcium. This time the peptide that I was targeting was the only one that was produced. Very strange selectivity obtained, so I took advantage of this observation so it appears that the peptide has some useful properties one of which is its ability to bind divalent cations such as calcium. I ended up using this property of the peptide to help purify it by precipitating it with calcium and then formulating it with alginate in order to develop a slow release wound healing combination wound dressing. This was tested and shown to accelerate wound healing, reduce scarring, decrease inflammation, and its property of modulating gap junction communication was clearly observed through delayed stratification of the epithelial layer. Once the peptide was removed the tissue fully differentiated. However, the peptide was also tested much later for its ability to do the photoreduction of the mineral coordinated in the C-terminus of the peptide. What I found was the peptide produced superoxides and other high energy radicals when exposed to blue and UV light. This suggested that the eye lens can also perform photoreduction. I now consider this to be part of the mechanism of action that is responsible for the wound healing potential of GAP573. The ability of high energy radicals sending out photons of light and the quantum entanglement that occurs due to radical generation. A scientist once thought that the human eye also produced light that was sent out into the environment. The ability of UV and blue light to trigger electron capture in GAP573 to generate various isotopes of calcium. The ability of the calcium radioactive isotope to release a photon of light out into the environment may be what was previously was considered many years ago. There are other aspects of light captured by the eye regarding tryptophan and kynurenine which is a cleavage product of tryptophan by indoleamine 2,3 dioxygenase so certain frequencies of light entering the eye are captured by various molecules causing photo Fenton like reactions and photoreduction events. Biological material seems to have this evolutionary time stamp within it that allows both upfield shifts and downfield shifts with respect to visual site wavelengths of electromagnetism. This is responsible for the UV catastrophe, where the amount of observed energy in a system is way more than the predicted energy levels and this aligns with photo reduction events, photo Fenton chemistry, radical generation, quantum entanglement, quantum tunneling of the hydride into the nucleus to generate the neutron. The imbalance of the number of neutrons and protons and the rebalancing of the nucleus with respect to isolates and the growth of atoms with respect to the periodic table and its energy levels related to the number of photons and its wavelength and the frequency of energy in the environment that the atom finds itself in. So a lot is going on triggered by high energy light at the UV frequency. So the quantum nature of biological systems means that the weird and wonderful world of physics is at play in biology.