Biomimetic calcium phosphate (CaP) coatings imitate the trabecular bones surface structure and have shown to promote osteogenic differentiation in multipotent cells. The work of this thesis focused on the problem of former CaP coatings cracking and flaking off when being put on a bendable core structure like a 3D-printed poly (ε-caprolactone) (PCL) scaffold. The aim was to provide a chemical linkage between PCL and CaP using a star-shaped polymer (sPEG) and a phosphonate, 2-aminoethylphosphonic acid (2-AEP). First, a published CaP coating protocol was revised and investigated in terms of etching parameters for the PCL scaffold. Results presented reproducible thick coatings for all groups. The protocol was then broadened to include subsequent scaffold incubation in sPEG and 2-AEP solutions. Homogenous CaP coatings of decreased thickness presented themselves, proving feasibility. However, as is often found with physical CaP coating depositions, there were some irregular outcomes even during the same experimental group. A lower consumption of the chemical 2-AEP, for economic reasons, meant that the protocol was altered to simultaneously incubate scaffolds with sPEG and 2-AEP including preceding calculations for molar ratios. For ratios 1:1, 1:2 and 1:3, again a homogenous CaP coating was produced on most of the samples, although reproducibility issues maintained. However, the mechanical bending to induce surface cracking showed that the CaP did strongly bond to the sPEG/2-AEP, while the control CaP coating flaked off the surface in large pieces. This research demonstrates that chemically-bound CaP coatings resist flaking off the fiber surface. Future investigations should focus on the mechanisms of CaP crystallization, to improve reproducibility. / Biomimetische Calciumphosphat (CaP) - Beschichtungen imitieren die oberflächliche Struktur des spongiösen Knochens und wirkten sich bereits begünstigend auf die osteogene Differenzierung von multipotenten Zellen aus. Diese Dissertation konzentriert sich auf das Problem des Reißens und Abplatzens bisheriger CaP-Beschichtungen, wenn diese sich auf einem biegsamen Kern-Gerüst, wie einem 3D-gedruckten Polycaprolacton (PCL)-Konstrukt befanden. Das Ziel war, durch den Gebrauch eines sternförmigen Polymers (sPEG) und eines Phosphonates, 2-Aminoethylphosphonsäure (2-AEP), eine chemische Verknüpfung zwischen PCL und CaP herzustellen. Zuerst wurde ein bereits publiziertes CaP-Beschichtungs-Protokoll nachgestellt und verschiedene Ätzungsparameter untersucht. Die Ergebnisse zeigten reproduzierbare, dicke Beschichtungen in allen Gruppen. Danach wurde dieses Protokoll erweitert, indem es nun nacheinander gestellte Inkubationen in sPEG- und 2-AEP-Lösungen mit einbezog. Dünnere, homogene Beschichtungen waren das Ergebnis, was beweist, dass die Hypothese realisierbar ist. Jedoch zeigten die Ergebnisse nicht reproduzierbare Resultate. Desweiteren, war der 2-AEP Verbrauch nicht wirtschaftlich. Daher wurde das Protokoll weiterentwickelt, indem die Proben, nach vorherigen Berechnungen zu den molaren Verhältnissen, simultan mit sPEG und 2-AEP inkubiert wurden. Für die Verhältnisse 1:1, 1:2 und 1:3 wurden wiederum homogene CaP-Beschichtungen produziert. Mit der Absicht Reproduzierbarkeit zu erzielen, wurden weitere Parameter untersucht. Dies blieb jedoch erfolglos. Zuletzt wurde ein mechanischer Test durchgeführt, welcher eine verbesserte CaP-Adhäsion zu den PCL-Fasern nahelegt, wenn diese zuvor mit sPEG und 2-AEP inkubiert wurden. Zukünftige Untersuchungen werden jedoch von Nöten sein, um Daten zur Oberflächenanalyse und von weiteren mechanischen Tests bereitzustellen und um das Protokoll in Bezug auf die Reproduzierbarkeit zu verbessern.

The increasing number of nucleic acid-based therapeutics demonstrates the potential to treat diseases at the genetic level. Although oligonucleotides show clinical potential, challenges remain including nuclease degradation, rapid clearance when administered systemically, low cell permeability, and limited distribution to tissues of interest. This is largely imparted by the polyanionic phosphate backbone, which produces unfavourable electrostatic interactions at cell membranes. As a result, their clinical translation is dependent on delivery technologies that improve stability, facilitate cell entry, and increase target affinity. Spherical nucleic acids (SNAs) consist of radially orienting linear nucleic acids onto a nanoparticle core, conferring them a three-dimensional, spherical architecture. These structures enter cells readily and display distinct properties that are independent of their nanoparticle core. Accordingly, we decided to replace the intrinsically anionic phosphodiester linkage of DNA with a phosphoramidate linkage (P-N), allowing us to incorporate new functionality at the phosphate backbone. With this handle, we inserted cationic and hydrophobically modified functional groups that were compatible with nanoscale architectures, giving rise to new properties relevant in biological contexts. Specifically, amine and guanidinium derivatized functional groups provided SNAs with a ~10-fold increase in cell uptake at early incubation times compared with unmodified SNAs. This demonstrates that we can tune the behaviour of SNAs with phosphate backbone modifications in a highly controlled manner. We hypothesize that the stringent control over location and placement of functional groups within the SNA framework will afford them favourable interactions at cell membranes, not only increasing their cell uptake, but also access to alternative uptake mechanisms and potency as therapeutics. / Thesis / Master of Science (MSc) / Oligonucleotides are short synthetic sequences of DNA or RNA that have the capacity to treat diseases at the genetic level. However, they face challenges such as degradation, low cell uptake, and poor tissue distribution. To overcome this issue, we plan to incorporate chemical modifications at the phosphate backbone of oligonucleotides to make them more stable and facilitate more favourable interactions at cell membranes. Conferring oligonucleotides into a 3D arrangement further enhances their stability and cell uptake relative to linear oligonucleotides. By densely functionalizing them onto a nanoparticle core, we can create spherical nucleic acids (SNAs). We hypothesize that the modifications imparted onto the phosphate backbone of linear oligonucleotides will translate their properties into SNAs. The new properties afforded to the SNAs will provide increased cell uptake, alternative uptake mechanisms, and access to cytosolic and nuclear targets, highlighting their potency and therapeutic potential.

The HydroFloat technology was specifically developed to upgrade phosphate sands that are too coarse to be efficiently recovered by conventional flotation methods. In this novel process, classified feed is suspended in a fluidized-bed and then aerated. The reagentized phosphate particles become buoyant and report to the product launder after encountering and attaching to the rising air bubbles. Simultaneously, the hydrophilic particles are rejected as a high solids content (65-70%) underflow. The fluidized bed acts as a "resistant" layer through which no bubble/particle aggregates can penetrate. As a result, the HydroFloat also acts as a density separator that is capable of treating much coarser particles as compared to traditional flotation processes. In addition, the high solids content of the teeter bed promotes bubble-particle attachment and reduces the cell volume required to achieve a given capacity. To fully evaluate the potential advantages of the HydroFloat technology, a 5-tph test circuit was installed and evaluated in an industrial phosphate beneficiation plant. Feed to the test circuit was continuously classified, conditioned and upgraded using the HydroFloat technology. The test results indicated that the HydroFloat could produce a high-grade phosphate product in a single stage of separation. Product quality ranged between 70-72% BPL (bone phosphate of lime = 2.185 x %P2O5) and 5-10% insols (acid insoluble solids). BPL recoveries exceeded 98% at feed rates greater than 2.0 tph per ft^2 of separator cross-sectional area. These results were superior to traditional column flotation, which recovered less than 90% of the valuable product at a capacity of less than 1 tph per ft^2. / Master of Science

This thesis explores phosphate as an indicator of occupational intensity (i.e. as a function of scale and length of occupation) at shell midden sites on the central coast of British Columbia. Despite the prevalence of shell middens in coastal environments world-wide and the long history of elemental analysis in archaeology, shell middens are not routinely investigated for their chemical content. Ongoing research on the British Columbia central coast has shown clear associations between fish bone densities (NISP/L) and site area (m2), which have proven useful for characterizing variability among settlements in the region. This provided the opportunity and essential basis for investigating phosphate. Due to its general abundance, persistence, and established association with human activity, phosphate was expected to reflect previously inferred patterns in occupational intensity, which were based on fish bone density and site area data. Results show clear relationships between phosphate values, fish bone densities, and site area, which speaks to the utility of phosphate as an independent indicator of the relative intensity of residential activity among sites. / Thesis / Master of Arts (MA)

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<p> The two carboxyvinyl transferases MurA and AroA are essential for bacterial survival, and are proven or potential antibiotic targets. The reactions they catalyze are chemically challenging, involving protonation of an ethylene group in the first step, and deprotonation of a methyl in the second step. In order to probe how the enzymes promote these reactions, the reverse reactions from enolpyruvyl compounds (EP-OR) plus phosphate to phosphoenolpyruvate (PEP) plus R-OH were investigated, and compared with EP-OR hydrolysis reactions catalyzed by phosphate analogues. </p> <p> Thirteen phosphate analogues were used to study EP-OR hydrolysis. Among these phosphate analogues, many could bind to the free enzymes, but only three could promote hydrolysis. The products were pyruvate and the corresponding alcohol (S3P in AroA/EPSP reaction and UDP-GicNAc in MurA/EP-UDP-GicNAc reaction). The most effective analogue was arsenate. The mechanism of the arsenate-promoted reaction was examined in detail. The hydrolysis reaction proceeded though an arseno-tetrahedral intermediate with AroA, a similar reaction pathway to the natural reaction. This arseno-tetrahedral intermediate was converted to arsenoenolpyruvate and hydrolyzed spontaneously. MurA also likely catalyzed arseno-tetrahedral intermediate formation, and appeared to catalyze arsenoenolpyruvate breakdown, though it is possible that it was a bystander in the reaction, with the tetrahedral intermediate being formed by water attack on C2 of EP-UDP-GicNAc. There was a fast solvent exchange step before EP OR was converted to arseno-THI by AroA or MurA. This strongly indicated an oxacarbenium ion like intermediate before the arseno-tetrahedral intermediate. </p> <p> The catalytic machinery for stabilizing such an unstable oxacarbenium ion like intermediate was investigated by studying ligand binding. Based on information from all the phosphate analogues, there were evidence that the enzyme undergoes an conformational change upon binding with phosphate, by which EPSP was distorted into an oxacarbenium ion like intermediate. </p> / Thesis / Master of Science (MSc)