Hybridization is thought to have played an important role in human evolution, with hybridizing groups having significant differences in soft tissue trait variation. Ectodermal trait variation is of interest because primate hybrids show increased atypical non-metric dental and cranial trait variation thought to be the result of interactions between parental genomes which have diverged for ectodermal trait development (including hair and tooth development). There were also differences between hybridizing hominin groups for limb measurements which have changed significantly throughout human evolution. Here a mouse model is used to look at the effect of hybridization on coat morphology and long bone length. Using standardized photographs, the differences in mean RGB values for the dorsal and ventral coat were used to determine whether the hybrids were different from their parents for pelage colour of different regions of the body, dorsal ventral colour contrast, and levels of variation in coat colour. The sample is composed of parents from one specific and three sub-specific crosses, as well as F1, F2 and first generation backcrossed (B1) hybrids. Long bone measurements of the forelimbs and hind-limbs were collected from micro-CT scans of the sub-specific F1 hybrids and their parents. Previous data have shown that hybridization can have variable morphological outcomes: hybrids can look like one of the parents, they can be intermediate, or they can have extreme traits outside of the range of variation of the parents. Our results indicate that morphological outcomes for coat colour in F1 hybrids depends on factors such as genetic distance. However, the genetic background of one of the strains used for this experiment might contribute the transgressive phenotype of some of the F1 hybrids. Hybrid morphology also changes in subsequent generations (F2 and B1) as new recombinants formed, with transgressive coat colour phenotypes sometimes appearing even if they are not present in the F1 hybrid groups. Phenotypes produced in F1 hybrids are also seen in subsequent generations of hybrids. All sub-specific F1 hybrids were transgressive for long bone length. Compared to parental groups hybrids have a different relationship between the long bones of the forelimb (ratio of humerus to ulna). This is in line with previous data from primate hybrids, that shows that changes in the relationships between different regions of the body occurs in hybrids producing novel phenotypes. The inter-membral indexes are not significantly different from one of the parents for two of the crosses. This data shows that hybridization can produce novel pelage phenotypes over multiple generations. There were many transitions in hair/skin morphology during human evolution and these tissue groups were and are under a great deal of selective pressure due to their direct interaction with the environment. Thus, understanding how these traits are impacted by hybridization will be important for disentangling how hybridization affected our evolutionary trajectory and ability to occupy new regions of the world. Post cranial data, indicates that F1 hominin hybrids might have longer limbs in relation to parental populations, more work needs to be done on the post cranial remains of posited hominin hybrids as well as pedigreed mammalian hybrids to determine if this is a pattern which can be used to identify hybrids in the fossil record.

Human serpins belong to a superfamily of serine protease inhibitors involved in the regulation of essential physiological processes, including coagulation via thrombin inhibition by AT. Inhibitory serpins undergo a remarkable folding mechanism to a thermodynamically unstable (i.e. metastable) conformation, highlighted by a long and flexible RCL, prior to secretion as soluble proteins into circulation. The serpin alpha-1-proteinase inhibitor (API) normally protects tissues from proteases released from inflammatory cells (e.g., neutrophil elastase). Importantly, a variant of API (i.e. API-Pittsburgh or API-M358R) was reported to be the cause of a fatal bleeding disorder in a young patient in the late 1970`s; the point mutation M358R at P1 of the RCL resulted in a dramatic shift in function toward thrombin inhibition. This dissertation summarizes the results from experiments performed with serpins expressed as membrane proteins tethered to the surface of mammalian cells. Serpins API-M358R, AT, HCII, and the non-inhibitory double mutant API-M358R/T345R were anchored to 293 and COS cell plasma membranes with N-terminal non-cleavable protein sequences derived from either the human asialoglycoprotein (AR) or transferrin (TF) receptors. Sub-cellular fractionation (with or without monolayer exposure to thrombin) immunoblots confirmed serpin localization to the integral membrane fraction using either anchoring approach or cell type. The blots also revealed that tethered API-Pittsburgh in particular, and AT to a lesser extent formed serpin-enzyme complex (SEC) with thrombin, while HCII and API-M358R/T345R (as expected) did not. While tethered API-M358R maintained inhibitory function, kinetic studies revealed that the rate of SEC formation was less rapid compared to its soluble counterpart. Additional testing by immunofluorescence microscopy, and flow cytometry confirmed the status of tethered API-M358R as a robust inhibitor of thrombin. That tethered serpins maintained the ability to inhibit thrombin provided the underlying rationale for the thesis hypotheses: surface displayed serpins can be used in gene therapy to temper thrombogenicity associated with certain diseased tissues (i.e. cancer), and tethered serpins can be used as a platform for screening RCL mutant libraries to identify “better” protease (i.e. thrombin) inhibitors. The potential gene therapy scheme was tested by expressing tethered API-Pittsburgh on the surface of T24/83 cancer cells constitutively co-expressing tissue factor (TF), and then measuring endogenous thrombin generation in the presence of re-calcified, and defibrinated human plasma by either discontinuous or continuous fluorescence-based thrombin generation assays (TGA). Unexpectedly, the displayed API-Pittsburgh did not appear to reduce discontinuous TGA thrombin suggesting that the difference may have been too low for accurate measurement by this method. Moreover, the results were identical when the same reaction was continuously monitored by fluorescence-based TGA, indicating that the levels of API-Pittsburgh expression were simply insufficient to effectively counter thrombin generation. The high levels were confirmed when up to 1 µM of hirudin variant 3, or soluble API-M358R, were required to completely abolish the thrombin profile. With this in mind, a measureable reduction in TGA was achieved when 293 cells were co-transfected with DNA ratios of API-M358R: TF adjusted to 9:1. Mammalian cell display, in combination with FACS/flow cytometry, has previously been employed to successfully develop improved monoclonal antibodies. However, there was never any certainty that the technique was applicable to the screening of displayed serpin mutant RCL libraries. The method was tested with a modest library degenerate only at P1; the rationale was that successful sorting would generate the expected wild-type (WT) P1 Arg (i.e. API-Pittsburgh). Unfortunately, repeated attempts did not result in enrichment, and flow cytometry was abandoned. An alternate protocol based on bacterial expression, and previously developed in our lab, was implemented in order to perform the library screens. This technique involved incubating lysates, containing soluble serpin RCL mutant candidates, with immobilized thrombin. Encouragingly, the P1 library screen identified the WT candidate at the expected frequency (5 in 150 lysates) as well as the more rare P1 mutant Lys (1 in 150 lysates). A kinetic comparison between mutant proteins containing the three basic residues revealed that P1 Arg (k2 ~ 105 M-1sec-1) was approximately two orders of magnitude more efficient than either Lys or His (both with k2 ~ 103 M-1sec-1) at inhibiting thrombin. The bacterial expression technique was then enhanced through kinetic optimization in order to facilitate the screening of more complex libraries. Analysis of the P7 to P1 (always Arg) library returned a selection of anticipated non-polar residues (5 in 100 lysates; 2 x Pro, 2 x Leu and Met) at P2. Extensive screening (~ 1300 colonies) of a second expanded library with the repeated VNN nucleotide sequence (i.e. no stop codons to eliminate truncated proteins) at P7 to P2 and P1 Arg, identified 7 x Pro mutants at P2 further confirming the results from the original library screen. Importantly, the assay also identified the novel mutant TLSATPR which registered the largest kinetic response (k2 ~ 5 x 105 M-1sec-1), and even exceeding API- M358R by a factor of ~ 3 (k2 ~ 1.5 x 105 M-1 sec-1). / Thesis / Doctor of Philosophy (Medical Science)

Human serpins belong to a superfamily of serine protease inhibitors involved in the regulation of essential physiological processes, including coagulation via thrombin inhibition by AT. Inhibitory serpins undergo a remarkable folding mechanism to a thermodynamically unstable (i.e. metastable) conformation, highlighted by a long and flexible RCL, prior to secretion as soluble proteins into circulation. The serpin alpha-1-proteinase inhibitor (API) normally protects tissues from proteases released from inflammatory cells (e.g., neutrophil elastase). Importantly, a variant of API (i.e. API-Pittsburgh or API-M358R) was reported to be the cause of a fatal bleeding disorder in a young patient in the late 1970`s; the point mutation M358R at P1 of the RCL resulted in a dramatic shift in function toward thrombin inhibition. This dissertation summarizes the results from experiments performed with serpins expressed as membrane proteins tethered to the surface of mammalian cells. Serpins API-M358R, AT, HCII, and the non-inhibitory double mutant API-M358R/T345R were anchored to 293 and COS cell plasma membranes with N-terminal non-cleavable protein sequences derived from either the human asialoglycoprotein (AR) or transferrin (TF) receptors. Sub-cellular fractionation (with or without monolayer exposure to thrombin) immunoblots confirmed serpin localization to the integral membrane fraction using either anchoring approach or cell type. The blots also revealed that tethered API-Pittsburgh in particular, and AT to a lesser extent formed serpin-enzyme complex (SEC) with thrombin, while HCII and API-M358R/T345R (as expected) did not. While tethered API-M358R maintained inhibitory function, kinetic studies revealed that the rate of SEC formation was less rapid compared to its soluble counterpart. Additional testing by immunofluorescence microscopy, and flow cytometry confirmed the status of tethered API-M358R as a robust inhibitor of thrombin. That tethered serpins maintained the ability to inhibit thrombin provided the underlying rationale for the thesis hypotheses: surface displayed serpins can be used in gene therapy to temper thrombogenicity associated with certain diseased tissues (i.e. cancer), and tethered serpins can be used as a platform for screening RCL mutant libraries to identify “better” protease (i.e. thrombin) inhibitors. The potential gene therapy scheme was tested by expressing tethered API-Pittsburgh on the surface of T24/83 cancer cells constitutively co-expressing tissue factor (TF), and then measuring endogenous thrombin generation in the presence of re-calcified, and defibrinated human plasma by either discontinuous or continuous fluorescence-based thrombin generation assays (TGA). Unexpectedly, the displayed API-Pittsburgh did not appear to reduce discontinuous TGA thrombin suggesting that the difference may have been too low for accurate measurement by this method. Moreover, the results were identical when the same reaction was continuously monitored by fluorescence-based TGA, indicating that the levels of API-Pittsburgh expression were simply insufficient to effectively counter thrombin generation. The high levels were confirmed when up to 1 µM of hirudin variant 3, or soluble API-M358R, were required to completely abolish the thrombin profile. With this in mind, a measureable reduction in TGA was achieved when 293 cells were co-transfected with DNA ratios of API-M358R: TF adjusted to 9:1. Mammalian cell display, in combination with FACS/flow cytometry, has previously been employed to successfully develop improved monoclonal antibodies. However, there was never any certainty that the technique was applicable to the screening of displayed serpin mutant RCL libraries. The method was tested with a modest library degenerate only at P1; the rationale was that successful sorting would generate the expected wild-type (WT) P1 Arg (i.e. API-Pittsburgh). Unfortunately, repeated attempts did not result in enrichment, and flow cytometry was abandoned. An alternate protocol based on bacterial expression, and previously developed in our lab, was implemented in order to perform the library screens. This technique involved incubating lysates, containing soluble serpin RCL mutant candidates, with immobilized thrombin. Encouragingly, the P1 library screen identified the WT candidate at the expected frequency (5 in 150 lysates) as well as the more rare P1 mutant Lys (1 in 150 lysates). A kinetic comparison between mutant proteins containing the three basic residues revealed that P1 Arg (k2 ~ 105 M-1sec-1) was approximately two orders of magnitude more efficient than either Lys or His (both with k2 ~ 103 M-1sec-1) at inhibiting thrombin. The bacterial expression technique was then enhanced through kinetic optimization in order to facilitate the screening of more complex libraries. Analysis of the P7 to P1 (always Arg) library returned a selection of anticipated non-polar residues (5 in 100 lysates; 2 x Pro, 2 x Leu and Met) at P2. Extensive screening (~ 1300 colonies) of a second expanded library with the repeated VNN nucleotide sequence (i.e. no stop codons to eliminate truncated proteins) at P7 to P2 and P1 Arg, identified 7 x Pro mutants at P2 further confirming the results from the original library screen. Importantly, the assay also identified the novel mutant TLSATPR which registered the largest kinetic response (k2 ~ 5 x 105 M-1sec-1), and even exceeding API- M358R by a factor of ~ 3 (k2 ~ 1.5 x 105 M-1 sec-1). / Dissertation / Doctor of Philosophy (PhD)

Adenosine kinase (AK) is a purine salvage enzyme which catalyses the phosphorylation of the 5'-hydroxyl of adenosine via ATP. AK is a key enzyme which controls the intra and extracellular concentration of adenosine (Ado). Agents which inhibit the activity of AK have been found to attenuate cellular damage, demonstrating therapeutic utility in a variety of disease processes. In order to design inhibitors of AK with increased efficacy, a better understanding of enzyme activity is required. Previously, a number of novel characteristics of mammalian AK had been discovered. It was shown that the activity of AK is influenced by the presence of pentavalent ions (PVD such as inorganic phosphate (Pi), arsenate and vanadate. A detailed study of the influence of Pi on the kinetic parameters of Chinese hamster (CH) and beef liver AK was performed. These studies suggested that the Km (Ado) decreases and the Ki (Ado) increases asymptotically in the presence of increasing concentrations of Pi. Under the same conditions, the Vmax for activity increases hyperbolically. The effect of phosphate is not limited to the mammalian form of AK. Pi, arsenate and vanadate were all found to have similar effects on AK from yeast, spinach and Leishmania donovani AK. PVI as well as the metabolite phosphoenol pyruvate were also found to stimulate the activity of the enzyme ribokinase (RK) from E. coli, which similar to AK, is a member of the PfkB family of carbohydrate kinases. Although AK and RK show little sequence similarity, the residues at the active site and the 3D structues of these two proteins are very similar. Based on sequence alignment of PfkB family members, we have identified a conserved sequence motif, NXXE, which based upon the available structural information appears to be involved in the binding of phosphate. To confirm and understand the role of this motif in Pi binding, the residues at the NXXE site were altered by site-directed mutagenesis and their effect on activity of the recombinant CH AK was examined. Though the residues at the NXXE site do not directly interact with substrate, nor the putative catalytic base, the resulting proteins were found to have greatly altered phosphate requirement, substrate inhibition characteristics and different magnesium requirements. In the AK structure, aspartic acid at position 316 is presumed to act as the catalytic base. This residue was changed to asparagine and glutamic acid by mutagenesis. The resulting proteins were found to be nearly completely devoid of activity, confirming its critical role in AK activity. The amino acid sequence at the extreme N-terminus of AK has been found to exhibit the greatest variability within and among species, though the rest of the protein remains greatly conserved. To delineate the residues that are involved in the structural stability and activity of AK, systematic deletions of the residues from both the N-and C-terminus were performed, and the structure-activity relationships were examined. It was determined that the first 16 residues of CH AK can be removed without affecting activity. Removal of the next 11 residues resulted in sequential decreases in enzyme stability and activity. These 11 residues are involved in the first B-structure of the protein and are required for the stability of the tertiary structure. All residues at the C terminus were required for activity, and involved in a hydrogen-bonding network necessary for the stability of the ATP binding site. These studies provide novel insight to the structure-activity relationship of mammalian AK as well as the PfkB family of enzymes. Our work has identified a site distal to the catalytic site, that is implicated in the PVI binding and catalytic effect. Further studies should be aimed at understanding how binding of PVI at this site influences the catalytic activity of AK. Development of inhibitors which bind to this site and modulate the activity of AK should prove very useful in this regard. / Thesis / Doctor of Philosophy (PhD)

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This research is concerned with the application of neural network techniques to the problems of classifying images in a manner that is invariant to changes in position and scale. In addition to the goal of invariant classification, the network has to classify the objects in a hierarchical manner, in which complex features are constructed from simpler features, and use unsupervised learning. The resultant hierarchical structure should be able to classify the image by having an internal representation that models the structure of the image. After finding existing neural network techniques unsuitable, a new type of neural network was developed that differed from the conventional multi-layer perceptron type of architecture. This network was constructed from neurons that were grouped into feature detectors. These neurons were taught in an unsupervised manner that used a technique based on Kohonen learning. A number of novel techniques were developed to improve the learning and classification performance of the network. The network was able to retain the spatial relationship of the classified features; this inherent property resulted in the capability for position and scale invariant classification. As a consequence, an additional invariance filter was not required. In addition to achieving the invariance property, the developed techniques enabled multiple objects in an image to be classified. When the network had learned the spatial relationships between the lower level features, names could be assigned to the identified features. As part of the classification process, the system was able to identify the positions of the classified features in all layers of the network. A software model of an artificial retina was used to test the grey scale classification performance of the network and to assess the response of the retina to changes in brightness. Like the Neocognitron, the resulting network was developed solely for image classification. Although the Neocognitron is not designed for scale or position invariance, it was chosen for comparison purposes because it has structural similarities and the ability to accommodates light changes in the image. This type of network could be used as the basis for a 2D-scene analysis neural network, in which the inherent parallelism of the neural network would provide simultaneous classification of the objects in the image.

The current project is to develop a transient expression system for Chinese Hamster Ovary (CHO) cells based on autonomous replication and retention of plasmid DNA. The expression system, named Epi-CHO comprises (1) a recombinant CHO-K1 cell line encoding the Polyoma (Py) virus large T-Antigen (PyLT-Ag), and (2) a DNA expression vector, pPy/EBV encoding the Py Origin (PyOri) for autonomous replication and encoding the Epstein-Barr virus (EBV), Nuclear Antigen-1 (EBNA-1) and EBV Origin of replication (OriP) for plasmid retention. The CHO-K1 cell line expressing PyLT-Ag, named CHO-T was adapted to suspension growth in serum-free media (EXCELL-302) to facilitate large scale transient transfection and recombinant (r) protein production. PyLT-Ag-expressed in CHO-T supported replication of PyOri-containing plasmids and enhanced growth and r- protein production. A scalable cationic lipid based transfection was optimised for CHO-T cells using LipofectAMINE-2000??. Destabilised Enhanced Green Fluorescence Protein (D2EGFP) and Human Growth Hormone (HGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of CHO-T cells with the vector pPy/EBV encoding D2EGFP showed prolonged and enhanced EGFP expression, and transfection with pPy/EBV encoding HGH resulted in a final concentration of 75 mg/L of HGH in culture supernatant 11 days following transfection.