Tight regulation of caspase-9, a key initiator of apoptosis, is required to uphold cellular homeostasis. Although it is controlled on a multifactorial level, misregulation of this process does occur, which is a characteristic of a variety of diseases from ischemic injury to cancer. Therefore it remains important to gain a detailed understanding of the mechanisms behind native caspase-9 regulatory pathways and harness these mechanisms for therapeutic purposes. Based on known mechanisms, such as the unique inhibitory complex of caspase-9 and XIAP-BIR3, development of synthetic regulators can be envisioned, while other mechanisms such as zinc-mediated inhibition and CARD activation of caspse-9 remain undefined. Intrigued by the multiple ways to control caspase-9’s activity, we sought after designing synthetic caspase-9 inhibitors in addition to defining the mechanistic details metal regulation and CARD domain activation. We report the first stabilized α-helical peptides that harness the native regulatory mechanism of caspase-9 and the BIR3 domain which lead to the understanding of the importance of exosites in inhibitory complexes. Our studies also revealed that there are two distinct zinc binding sites, one at the active site and another at a novel zinc binding site of yet unknown function in caspase-9 however this site may have the potential to control caspase-6 based on its regulatory mechanism. Furthermore, an interaction was discovered between CARD and the catalytic core of caspase-9 in the presence of a properly formed substrate binding groove, a potential mechanism utilized by the apoptosome for activation of the enzyme. All in all, the regulation of caspase-9 occurs on a variety of levels that requires almost every surface of the enzyme. Through exploring these underlying molecular details behind the various mechanisms, not only has the field of caspase-9 regulation mechanisms been extended, essential information was gained for further pursuit in an advancement towards the design of caspase-9 activators and inhibitors.

<p> The variation in sensitivity of L60T cells to gamma rays has been studied as a function of position in the cell division cycle. For a dose range of 0-12,000 rads, no significant variation was found for mitotic delay. Such was not the case for sensitivity to cell killing, which was found to increase as the cells passed from G1 through S to G2 of the division cycle. The results of mitotic delay are in disagreement with results published by other workers although the survival data agree with previous reports for a similar cell line. Results reported in connection with cell cycle determinations and mitotic delay suggest that the existence of a repair cycle operating concurrently with the normal cell cycle may be postulated. The theoretical treatment of mitotic delay given by Lea is examined and is not found to describe adequately the present results. Finally, the evidence reported here suggests that mitotic delay and radiation lethality are not separate manifestations of the same phenomenon. Experimental materials for further investigation into the repair processes involved are suggested.</p> / Thesis / Master of Science (MSc)

This thesis proposal shows the efficiency of different methods of Myostatin inhibition by using CRISPR/CAS9. With the data cataloged by this thesis, researchers will have a better understanding of what methods are better to achieve their goals. The data was collected by reading multiple scientific articles involving the usage of CRISPR/CAS9 to inhibit the Myostatin protein. The data collected from all the different studies were analyzed in the same categories. The experiment that used CRIPSR/CAS9 on in vitro specimens had a superior Myostatin inhibition overall, therefore presenting higher muscle mass. The method using CRISPR/CAS9 to inhibit the Myostatin in vivo and in vitro depends on what the researcher is trying to accomplish. By reading this thesis researchers can understand that if they choose to use an in vitro treatment the results would be way more severe than using an in vivo application treatment.

Afferent Inhibition is the reduction in motor output when Transcranial Magnetic Stimulation (TMS) of the motor cortex is preceded by peripheral nerve stimulation. Afferent inhibition can be subdivided into two circuits of Short- (SAI) and Long-Afferent Inhibition (LAI). Reliability reflects the repeatability of a measure and can be measured in terms of both absolute and relative reliability. Relative reliability refers to the ability of a measure to identify individuals on repeated testing, measured through the Intraclass Correlation Coefficient (ICC); absolute reliability is the repeatability of scores through repeated testing, measured through Standard Error of Measurement (SEM) and Smallest Detectable Change (SDC). Current literature has highlighted only the intersession reliability of SAI and LAI, but measures of the intrasession reliability are also needed. This study aims to quantify the intrasession reliability of SAI and LAI, alongside identifying the minimum number of trials needed to obtain a reliable measure. 30 healthy individuals (21.17 ± 2.84 years) took part in one session, with SAI and LAI obtained three times at 30-minute intervals. To identify the minimum number of trials required to reliably elicit SAI and LAI, relative reliability was assessed at running intervals of every 5 trials. Results indicate that SAI had moderate–high, and LAI had high-excellent relative reliability. Both SAI and LAI had high amounts of measurement error. LAI was seen to have high relative reliability when only 5 frames of data were included, whereas for SAI, ~20-30 frames of data resulted in high relative reliability. For LAI, a minimal sample size of 19 is needed to have an SDCGroup < 10, whereas for SAI, a sample size of 22 is needed to achieve the same. These results can be used to inform future work regarding the clinical utility of these measures, particularly in terms of their diagnostic ability. / Thesis / Master of Science (MSc)

This thesis encompasses five chapters. Chapter 1 details the background literature for how the inhibition of return effect manifests in 3D environments. Additional summary is provided about how positioning cues and targets within the boundary of the same objects affects the spread of IOR when compared to when attention is cued in empty space. Finally, the literature review also provides a background for whether the IOR effect is affected by a viewer-centered or world-centered frame of reference. Chapters 2-4 are empirical chapters. Chapter 2 observes a depth-specific IOR effect in a 3D composed of pictorial depth cues. Chapter 3 suggests that this depth-specificity can only occur when cues and targets are positioned in different objects rather than when positioned within a single object. Chapter 4 investigates how the distances between viewer and cue, viewer and target, and cue and target affect the magnitude of IOR, suggesting that the world-centered reference frame influences IOR. Chapter 5 serves as a general discussion and conclusion chapter, discussing the findings and implications of each empirical chapter. / The distribution of human attention in space can be modulated by spatial and temporal factors. This dissertation studied inhibition of return (IOR), a robust behavioural effect obtained through a spatial cueing paradigm where observers exhibit slower detection times to a target appearing over 300 ms after a cue in a previously cued location. Most research has studied the IOR effect in two-dimensional space; thus, it remains unclear whether, in three-dimensional space (3D) space, slower reaction times occur due to a target appearing in the same world location (defined in 3D coordinates) or in the same retinal location as the cue (i.e., anywhere along an observer’s line of sight to the cue). My thesis examines IOR in a computer-simulated 3D environment, with the location of the cue and target residing in the same versus different depth/distance position either within the same or different object and either relative to the observer or to the world environment. Following a general literature review (Chapter 1), the first empirical chapter (Chapter 2) demonstrates that IOR is depth-specific when the direction of depth switch between cue to target occurs from far-to-near space, suggesting a behavioural advantage for near space in the human attention system. Chapter 3 shows that this depth-specificity and depth-asymmetry of IOR is maintained only when cues and targets are not part of the same object; object membership can therefore override the depth-specific property of IOR in 3D scenes. Chapter 4 introduces motion of the viewpoint, showing that IOR is depth-specific when the cue and target appear in different depth locations in the world environment even when located at the same relative distance from the observer’s viewpoint. Thus, IOR could be the result of an inhibitory tag placed at a location relative to the environment rather than at a location relative to the viewpoint. / Dissertation / Doctor of Science (PhD) / Human attention can be distributed over space and affected by external events. Prior research using 2D environments has shown that some time after the first stimulus (a cue), the reaction time to a subsequent stimulus (a target) appearing in the same location is typically slower compared to when this target appears elsewhere. Thus, attention likely moves away from a previously observed to more novel location of interest. I examined, in a 3D environment, whether this “location” of reduced attention resides in the same 3D location or retinal location as that of the cue. I also assessed the impact on reaction time for when the cue and target belong to the same or different object and when their locations differ in reference to the observer or world environment. My research suggests that humans maintain a higher level of attention for nearer space when the cue previously appears at a farther location.

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Aniline oligomers have become a very interesting topic for research because of their potential application not only in organic electronics but also in smart coatings for corrosion treatment of iron and steel. A majority of the studies in the literature are focussed on the bulk or direct interaction between the organic molecules with metal substrates, without considering the native oxide film. In order to develop smart coatings (has redox activity and self-healing ability) for iron and steel, one must first understand how these oligomers interact with the native iron oxide film. In this thesis, we develop new knowledge from our fundamental understanding of the interactions of redox-active aniline oligomers with the iron oxide surface. Phenyl capped aniline dimer with two oxidation states [fully reduced (DPPD) and fully oxidized (B2Q1)] and phenyl-capped aniline tetramer (PCAT) with three oxidation states [fully reduced (B5), half-oxidized (B4Q1), fully oxidized (B3Q2)] were chosen for investigation. The former is the smallest redox active aniline oligomer but with one fewer oxidation states than polyaniline whereas the latter mimics the redox system as well as corrosion inhibition properties of polyaniline. Moreover, the phenyl-caps help both of these molecules to resist polymerization on the surface. Raman spectroscopy, mid-IR spectroscopy, atomic force microscopy (AFM), temperature programmed desorption (TPD) and electrochemical impedance spectroscopy (EIS) were used to study interactions. We demonstrate that charge transfer and interconversion to different oxidation states take place during interactions between each of these molecules with iron (III) oxides surfaces. During interaction with the surface, all three tetramer molecules and DPPD prefer standing on their edge orientation, whereas B2Q1 molecules tend to orient in lying down direction on the same surface. Having amino groups in the chain helps reduced and half oxidized molecules to strongly hydrogen bond with the surface and make them static on the surface. On the other hand, a lack of amino groups makes oxidized molecules mobile and loosely bound to the surface. Interactions and change of oxidation states impact the corrosion inhibition properties of PCAT. Strong ability of sticking to the surface and not fully oxidizing (B3Q2) during interactions makes B5 molecules superior corrosion inhibitors than B4Q1 and B3Q2 molecules. Transformation into B3Q2 form at the beginning of interaction allows B4Q1 to moderately inhibit corrosion but as it transforms back to its original form with time it becomes the 2nd best corrosion protector of iron oxide surface after B5. The study of all oxidation states and their surface interactions with iron oxide surface will open up pathways towards of designing smart coatings using aniline oligomers and other redox-active molecules. / Thesis / Doctor of Philosophy (PhD) / Aniline oligomers have become a very interesting topic for research because of their potential application not only in organic electronics but also in smart coatings for corrosion treatment of iron and steel. A majority of the studies in the literature are focussed on the bulk or direct interaction between the organic molecules with metal substrates, without considering the native oxide film. In order to develop smart coatings (has redox activity and self-healing ability) for iron and steel, one must first understand how these oligomers interact with the native iron oxide film. In this thesis, we develop new knowledge from our fundamental understanding of the interactions of redox-active aniline oligomers with the iron oxide surface. Phenyl capped aniline dimer with two oxidation states [fully reduced (DPPD) and fully oxidized (B2Q1)] and phenyl-capped aniline tetramer (PCAT) with three oxidation states [fully reduced (B5), half-oxidized (B4Q1), fully oxidized (B3Q2)] were chosen for investigation. The former is the smallest redox active aniline oligomer but with one fewer oxidation states than polyaniline whereas the latter mimics the redox system as well as corrosion inhibition properties of polyaniline. Moreover, the phenyl-caps help both of these molecules to resist polymerization on the surface. Raman spectroscopy, mid-IR spectroscopy, atomic force microscopy (AFM), temperature programmed desorption (TPD) and electrochemical impedance spectroscopy (EIS) were used to study interactions. We demonstrate that charge transfer and interconversion to different oxidation states take place during interactions between each of these molecules with iron (III) oxides surfaces. During interaction with the surface, all three tetramer molecules and DPPD prefer standing on their edge orientation, whereas B2Q1 molecules tend to orient in lying down direction on the same surface. Having amino groups in the chain helps reduced and half oxidized molecules to strongly hydrogen bond with the surface and make them static on the surface. On the other hand, a lack of amino groups makes oxidized molecules mobile and loosely bound to the surface. Interactions and change of oxidation states impact the corrosion inhibition properties of PCAT. Strong ability of sticking to the surface and not fully oxidizing (B3Q2) during interactions makes B5 molecules superior corrosion inhibitors than B4Q1 and B3Q2 molecules. Transformation into B3Q2 form at the beginning of interaction allows B4Q1 to moderately inhibit corrosion but as it transforms back to its original form with time it becomes the 2nd best corrosion protector of iron oxide surface after B5. The study of all oxidation states and their surface interactions with iron oxide surface will open up pathways towards of designing smart coatings using aniline oligomers and other redox-active molecules.

<p> Poly aniline (PANI) is capable of inhibiting corrosion on iron by inducing the formation of a passive oxide film. The mechanism by which PANI does this is unknown to the scientific community. We have used photoemission spectroscopy of thin films of a model PANI oligomer to investigate the PCAT -iron interaction. </p> <p> The oligomer chosen was a phenyl-capped aniline tetramer (PC AT). Thin films of PCAT were prepared by in-vacuum physical vapor deposition to obtain extremely thin films of thickness ranging from -5A to over 1 Onm. </p> <p> Films were investigated with a photoemission electron microscope (PEEM) using synchrotron radiation to obtain spatially resolved valence band photoemission spectra. Analysis of PEEM results suggest that PCAT is capable of migrating several microns along the substrate surface, and causes a decrease in substrate work function wherever present. </p> <p> High-resolution core level and valence band photoemission spectroscopy using a laboratory-based photon source was used to characterize the substrate and PCAT properties near the PCAT-substrate interface. Characterization of an in-situ thin film deposition reveals that the iron substrate exhibits band bending in it oxide as well as a decrease in work function by 0.7eV upon adsorption of PCAT. </p> / Thesis / Master of Science (MSc)

The process of reversible growth arrest, otherwise known as cellular quiescence or the G₀ phase denoted by withdrawal from the cell cycle, is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to/dependent on the limiting factor or circumstance inducing growth arrest. Here I characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition and oxygen limitation (hypoxia). Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients is the limiting factor inducing growth arrest. Contact inhibition and hypoxia induced FABP4 expression, whereas cycling control CEF and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. The GAS gene p20k lipocalin has been shown to exhibit similar expression patterns to FABP4, with its regulation determined by the presence of the transcription factor C/EBP-β. CEF overexpressing C/EBP-β also showed strong FABP4 induction. Furthermore, chromatin immunoprecipitation (ChIP) assays revealed that C/EBP-β bound directly to the FABP4 promoter in both normoxic and hypoxic cells, although only the latter condition induced FABP4 protein expression. In summary, these results suggest that FABP4 is induced during growth arrest specifically when oxygen is the limiting factor, as induction was not seen during growth arrest mediated by starvation-induced endoplasmic reticulum (ER) stress, where nutrients was the limiting factor. The induction of these hypoxia-responsive genes suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes. Additionally, FABP4 was shown to be associated with growth arrest and the promotion of cell survival and proliferation, as depicted by proliferation assays. Lastly, C/EBP-β not only strongly induced FABP4 expression, but directly bound to the FABP4 promoter. This suggests that C/EBP-β is a regulator of FABP4, although there may be other interacting factors acting as activators or repressors as this FABP4-C/EBP-β interaction was observed in conditions permissive and non-permissive to FABP4 expression. / Thesis / Master of Science (MSc) / The process of reversible growth arrest is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to the limiting factor or circumstance inducing growth arrest. Here we characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition (CI) and hypoxia. Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients availability is the limiting factor. CI and hypoxia induced FABP4 expression, whereas control and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. When overexpressing C/EBP-β, CEF showed strong FABP4 induction. Additionally, a direct interaction with the FABP4 promoter was observed in both normoxic and hypoxic cells, although only the latter condition induced expression. In summary, the induction of this hypoxia-responsive gene suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes and that this induction may be regulated by C/EBP-β.