VictorJuiz/thesis huggingface dataset:
Molecular Evolution and Functional Characterization of the Visual Pigment Proteins of the Great Bowerbird (Chlamydera nuchalis) and Other Vertebrates

Visual pigments are light sensitive receptors in the eye that form the basis of sensory visual transduction. This thesis presents three studies that explore visual pigment proteins in vertebrates using a number of computational and experimental methods in an evolutionary framework. The objective is not only to identify, but also to experimentally investigate the functional consequences of genetic variation in vertebrate visual pigments. The focus is on great bowerbirds (Chlamydera nuchalis), which are a model system in visual ecology due to their spectacular behaviour of building and decorating courtship bowers. There are 4 chapters: Chapter 1 introduces background information on visual pigments and vision in birds. Among visual pigment types, the short-wavelength-sensitive (SWS1) pigments have garnered particular interest due to the broad spectral range among vertebrates and the importance of UV signals in communication. Chapter 2 investigates the evolutionary history of SWS1 in vertebrates with a view toward its utility as a phylogenetic marker. Chapter 3 investigates SWS1 evolution and short-wavelength vision in birds, with particular focus on C. nuchalis and its SWS1. The evolution of spectral tuning mechanisms mediating UV/violet vision in passerines and parrots is elucidated in this chapter using site-directed mutagenesis, protein expression, and phylogenetic recreation of ancestral opsins. While cone opsins mediate colour vision in bright light, the rhodopsin visual pigment contained in rod photoreceptors is critical for dim light vision. Detailed characterization of rhodopsin function has only been conducted on a few model systems. Chapter 4 examines C. nuchalis RH1 using a number of functional assays in addition to absorbance spectra, including hydroxylamine sensitivity and the rate of retinal release. This chapter includes an investigation into the role of amino acid mutations typical of dim-light adapted vertebrates, D83N and A292S, in regulating functional properties of bovine and avian RH1s using site-directed mutagenesis. Together these chapters describe naturally occurring mutations in visual pigments and explore the way they can influence visual perception. These represent one of the few investigations of visual pigments from a species that is not a model lab organism and form a significant contribution to the field of visual pigment biochemistry and evolution.

Keywords: bowerbird
visual pigments
opsin
vertebrate phylogenetics
molecular marker
avian vision
visual behaviour
adaptation
mutagenesis
protein expression
short wavelength sensitive 1
SWS1
spectral tuning
evolution
ancestral sequence reconstruction
maximum likelihood
rhodopsin
rod opsin
RH1
retinal release
photoactivation cycle
dim light adaptation
molecular evolution
PAML
Advisor: Chang, Belinda
Trace Bases and Acids in the Troposphere: Importance in New Particle Formation and Atmospheric Oxidation Capacity

Accurate measurements of the trace nitrogenous atmospheric species amines (NR3) and nitrous acid (HONO) are essential to understanding their chemistry and potential influence on new particle formation and oxidation capacity in the atmospheric boundary-layer, respectively. Ambient Ion Monitor – Ion Chromatography (AIM-IC) methods developed in this work have provided quantitative online observations of atmospheric amines in the gaseous and condensed phases with detection limits of pptv and ng m-3 at hourly time resolution. Size-resolved particle observations demonstrated maximum amine mass loadings in 320 – 560 nm particles, and an increase in importance relative to ammonium for the smallest particles measured (56 – 180 nm). In particular, the size-resolved samples analysed in this work indicate that bulk aerosol measurements may not be appropriate for modelling the atmospheric processes that govern the incorporation of amines and ammonia in to atmospheric particles. Measurements of HONO made during the two intensive field campaigns (NACHTT, CalNex) and a lab study provided a new perspective on the interactions of this trace compound with ground surfaces. Integrated atmospheric column measurements of HONO and NO2 during NACHTT provided clear evidence that the ground surface dominates HONO production and loss at night. Simultaneous measurements of the gas and particle phases made by the AIM-IC system during CalNex demonstrated the potential for reactive uptake of HONO on mineral dust/soil as a nocturnal sink. Similarly, the potential for nitrite salts to react with strong acids, displacing HONO during the day was suggested by this dataset. Lab study results showed that HONO is taken up irreversibly on carbonate salts and real soil extracts. Relative humidity-dependent reactive uptake coefficients were derived. Subsequent release of HONO by displacement reactions with HNO3 and HCl was also confirmed. Together, these field and lab studies have produced a new picture of HONO surface interactions by providing i) a more explicit description of a nocturnal sink of HONO that could act as a surface reservoir and ii) a new mechanism for daytime HONO formation that does not require NO2.

Keywords: Atmospheric Chemistry
Environmental Chemistry
Advisor: Murphy, Jennifer
LIMK1 Regulation of Long-term Memory and Synaptic Plasticity

The LIM-Kinase family of proteins (LIMK) plays an important role in actin dynamics through its regulation of ADF/cofilin. A subtype of LIMK, LIMK1, is mostly expressed in neuronal tissues with high levels in the mature synapse. Previous studies from the Zhen Ping Jia laboratory have shown that LIMK1-/- mice exhibit abnormal spine morphology as well as altered hippocampal synaptic plasticity. LIMK1 has been shown to interact with CREB during neuronal development (Yang et al., 2004). We propose that LIMK1 is able to phosphorylate CREB in response to a synaptic activity. We hypothesize that if LIMK1 activates CREB in mature neurons, then LIMK1 knockout mice will have decreased L-LTP and deficits in long-term memory. My results show that LIMK1 and CREB exist in a complex and are bound to each other in mature neurons. LIMK1-/- mice exhibit deficits in the late phase of long-term potentiation and specific deficits in long-term memory while short-term memory remains unaltered. Pharmacological activation of CREB attenuates the observed deficits in synaptic plasticity and long-term memory. These results show a potentially novel mechanism of CREB activation in response to synaptic activity. Moreover, using peptides to manipulate actin dynamics in LIMK1 lacking animals only has effects on early LTP and is not able to rescue the late phase LTP deficits found in LIMK1 -/- mice. These results indicate a specific role of LIMK1 long-term memory and synaptic plasticity through regulation of CREB and not through regulation of the actin cytoskeleton.

Keywords: synaptic plasticity
learning and memory
LTP
LIMK1
CREB
Advisor: Zhengping, Jia
Characterization of PilP from the Type IV Pilus System of Pseudomonas aeruginosa

Pathogenic bacteria employ a number of mechanisms to induce infection and survive in host tissues, including toxin secretion and the formation of protective multicellular structures called biofilms. Type IV Pili (T4P) are highly conserved organelles essential for both the establishment of infection and biofilm maturation. The goal of this research is to gain a molecular level understanding of the function of the highly dynamic T4P of Pseudomonas aeruginosa. The pilMNOPQ operon encodes 5 members of a transmembrane complex that facilitates pilus function. While PilQ is the putative outer membrane secretin through which the pilus exits the cell, the roles of the PilM/N/O/P proteins are less well defined. Using both in vivo and in vitro techniques our characterization of PilP has provided significant insight into organization of the apparatus. PilP is an inner membrane lipoprotein essential for T4P function, but lipidation is dispensable, suggesting that its interactions with other T4P components are sufficient for PilP function. We showed that PilN/O/P form a stable heterotrimer when expressed in E. coli, and we suggest that they form a similar subcomplex in P. aeruginosa. Additionally we were able to show that PilP is also able to interact with a periplasmic fragment of the outer membrane pore protein PilQ. Structural and bioinformatics studies suggest that the organization of PilN/O/P/Q complex is similar to that of the transenvelope complex of another important Gram-negative virulence factor – the Type II Secretion System (T2SS). Our structural and functional characterization of PilP, the PilN/O/P complex and the striking similarities between the T4P and T2S systems, as well as important differences that make each molecular machine unique, will be presented.

Keywords: P. aeruginosa
virulence factors
protein-protein interactions
Type IV Pili
Advisor: Howell, P. Lynne
Customizing the Composition of Web Services and Beyond

Web services provide a standardized means of publishing diverse, distributed applications. Increasingly, corporations are providing services or programs within and between organizations either on corporate intranets or on the cloud. Many of these services can be composed together, ideally automatically, to provide value-added service. Automated Web service composition is an example of such automation where given a specification of an objective to be realized and some knowledge of the state of the world, the problem is to automatically select, integrate, and invoke multiple services to achieve the specified objective. A popular approach to the Web service composition problem is to conceive it as an Artificial Intelligence planning task. This enables us to bring to bear many of the theoretical and computational advances in reasoning about actions to the task of Web service composition. However, Web service composition goes far beyond the reaches of classical planning, presenting a number of interesting challenges relevant to a large body of problems related to the composition of actions, programs, and services. Among these, an important challenge is generating not only a composition, but a high-quality composition tailored to user preferences. In this thesis, we present an approach to the Web service composition problem with a particular focus on the customization of compositions. We claim that there is a correspondence between generating a customized composition of Web services and non-classical Artificial Intelligence planning where the objective of the planning problem is specified as a form of control knowledge, such as a workflow or template, together with a set of constraints to be optimized or enforced. We further claim that techniques in (preference-based) planning can provide a computational basis for the development of effective, state-of-the-art techniques for generating customized compositions of Web services. To evaluate our claim, we characterize the Web service composition problem with customization as a non-classical planning problem, exploit and advance preference specification languages and preference-based planning, develop algorithms tailored to the Web service composition problem, prove formal properties of these algorithms, implement proof-of-concept systems, and evaluate these systems experimentally. While our research has been motivated by Web services, the theory and techniques we have developed are amenable to analogous problems in such diverse sectors as multi-agent systems, business process modeling, component software composition, and social and computational behaviour modeling and verification.

Keywords: Automated Composition
Preferences
Optimization
Hierarchical Task Network Planning
Planning
Planning Algorithms
Software Composition
Software Customization
Component-based Software
Web Services
Advisor: McIlraith, Sheila Ann
Mechanical and Histological Characterization of Porcine Aortic Valves under Normal and Hypercholesterolemic Conditions

Calcific aortic valve disease (CAVD) is associated with significant cardiovascular morbidity. While late-stage valve disease is well-described, there remains an unmet scientific need to elucidate early pathobiological processes. In CAVD, pathological differentiation of valvular interstitial cells (VICs) and lesion formation occur focally in the fibrosa layer. This VIC pathological differentiation has been shown to be influenced by matrix stiffness in vitro. However, little is known about the focal layer specific mechanical properties of the aortic valve in health and disease and how these changes in matrix moduli may influence VIC pathological differentiation in vivo. In this thesis, micropipette aspiration (MA) was shown to be capable of measuring the mechanical properties of a single layer in multilayered biomaterial or tissue such as the aortic valve, if the pipette inner diameter was less than the top layer thickness. With MA, the fibrosa of normal porcine aortic valves was significantly stiffer than the ventricularis; stiffer locations found only within the fibrosa were comparable to stiffnesses shown in vitro to be permissive to VIC pathological differentiation. Early CAVD was induced in a porcine model, which developed human-like early CAVD lesion onlays. Extracellular matrix remodeling occurred in the absence of lipid deposition, macrophages, osteoblasts, or myofibroblasts, but with significant proteoglycan-rich onlays and chondrogenic cell presence. These early onlays were softer than the collagen-rich normal fibrosa, and their proteoglycan content was positively correlated with Sox9 chondrogenic expression, suggesting that soft proteoglycan-rich matrix may be permissive to chondrogenic VIC differentiation. The findings from this thesis shed new light on early disease pathogenesis and improve the fundamental understanding of aortic valve mechanics in health and disease.

Keywords: heart valve
aortic valve disease
micromechanical testing
micropipette aspiration
multilayered biomaterial
histology
porcine
animal model
hypercholesterolemia
proteoglycan
lipid
Sox9
Advisor: Simmons, Craig Alexander
CREB-mediated Enhancement of Hippocampus-dependent Memory Consolidation and Reconsolidation

Memory stabilization following encoding (synaptic consolidation) or memory reactivation (reconsolidation) requires gene expression and protein synthesis. Although consolidation and reconsolidation may be mediated by distinct molecular mechanisms, disrupting the function of the transcription factor CREB (cAMP responsive element binding protein) impairs both processes. We use a gain-of-function approach to show that CREB (and CREB-coactivator CRTC1) can facilitate both synaptic and systems consolidation and reconsolidation. We first examine whether acutely increasing CREB levels in the dorsal hippocampus is sufficient to enhance spatial memory formation in the watermaze. Locally and acutely increasing CREB in the dorsal hippocampus using viral vectors is sufficient to induce robust spatial memory in two conditions which do not normally support consolidation, weakly-trained wild-type (WT) mice and strongly-trained mutant mice with brain-wide disrupted CREB function. CRTCs (CREB regulated transcription co-activators) are a powerful co-activator of CREB, but their role in memory is virtually unexplored. We show, for the first time, that the novel CREB co-activator CRTC1 enhances memory consolidation. Locally increasing CRTC1 (or CREB) in the dorsal hippocampus of WT mice prior to weak context fear conditioning facilitates consolidation of precise context memory. Last, we show that CREB or CRTC1 facilitates precise and enduring memory consolidation and reconsolidation. Acute enhancement of hippocampal CREB or CRTC1 during initial synaptic consolidation can maintain precision of remote context memory, while increasing CREB or CRTC1 just prior to reactivation of a weak remote context memory enhances context memory reconsolidation. These gain-of-function manipulations indicate that increasing CRTC1 or CREB function is sufficient to enhance the strength of new, as well as reactivated established, memories without compromising memory specificity. Together with previous results, these findings indicate that CREB is both necessary and sufficient for hippocampal-dependent memory formation, and underline its pivotal role in the hippocampal molecular machinery underlying long-term memory consolidation and reconsolidation.

Keywords: memory
CREB
hippocampus
animal model
reconsolidation
context fear conditioning
spatial memory
viral vectors
CRTC1
transcription factor
Advisor: Josselyn, Sheena
Influence of Crystalline Microstructure on Optical Response of Single ZnSe Nanowires

Semiconductor nanowires (NWs) are anticipated to play a crucial role in future electronic and optoelectronic devices. Their practical applications remain hindered by an urging need for feasible strategies to tailor their optical and electronic properties. Strategies based on strain and alloying are limited by issues such as defects, interface broadening and alloy scattering. In this thesis, a novel method to engineer the optoelectronic properties based on strain-free periodic structural modulations in chemically homogeneous Nanowire Twinning Superlattices (NTSLs) is experimentally demonstrated. NTSLs are an emerging new class of nanoscale material, composed of periodically arranged rotation twin-planes along the length of NWs. The main objective of this thesis is to establish the relationship between the electronic energy band gap (Eg) and the twin-plane spacing (d) in NTSLs, quantified using a periodicity parameter, based on ZnSe. ZnSe was chosen because of its excellent luminescence properties, and potential in fabrication of optoelectronic devices in the near-UV and blue region of the spectrum. A prerequisite to establishing this correspondence is a prior knowledge of the photoluminescence (PL) response and the nature of fundamental optical transitions in defect-free single crystal ZnSe NWs with zinc-blende (ZB) and wurtzite (WZ) crystal structures. There has been no systematic work done yet on understanding these fundamental optical processes, particularly on single NWs and in relation to their crystalline microstructure. Therefore, the secondary objective of this thesis is to study the influence of native point defects on the optical response of single ZnSe NWs in direct relation to their crystalline microstructure. The PL response from single ZB and WZ NWs was determined unambiguously, and excitonic emission linewidths close to 1 meV were observed, which are the narrowest reported linewidths thus far on ZnSe NWs. Based on this and extensive optical and structural characterization on individual NTSLs, a linear variation in Eg is shown through a monotonic shift in PL peak position from ZnSe NTSLs as a function of d, with Eg's that lie between those of ZB and WZ crystal structures. This linear variation in Eg was also validated by ab Initio electronic structure calculations. This establishes NTSLs as new nanoscale polytypes advantageous for applications requiring tunable band gaps.

Keywords: semiconductor nanowires
optical characterization
Advisor: Ruda, Harry E.
Molecular Characterization Reveals Novel Genes Implicated in Aetiology and Progression of Osteosarcoma

Osteosarcoma is the most common bone malignancy in children and adolescents with poorly understood aetiology. Recently, disease susceptibility and aetiology in several cancers have been associated with genomic copy-number (CN) change. We therefore studied the contribution of CN change in osteosarcoma. We report that individuals with osteosarcoma have increased germline structural variation compared to controls. These CN variants (CNVs) preferentially localize to genes implicated in control of osteoblast differentiation, bone mineralization and ossification. We propose that germline CNVs contribute to osteosarcoma susceptibility through deregulation of developmental processes controlled by genes contained within CNVs. Further supporting the notion that germline CNVs in individuals with osteosarcoma are pathogenic, we demonstrate that CNVs are associated with poor patient survival. Finally, we characterize two germline CNVs, at chromosome 1q43 and 2p11.2, which are overrepresented in osteosarcoma patients and propose that they contribute to osteosarcoma susceptibility through effect on neighbouring genes, which could be involved in control of microtubule dynamics and tumour suppression. We further characterize two regions in the tumour genome of osteosarcoma patients that harbour recurrent CN alterations (CNAs). These include deletions at chromosome 3q13.31 and vi ii amplifications at chromosome 7p14.1, which are the most altered regions in osteosarcoma and contest the view that CNAs in osteosarcoma are non-recurrent. Both chromosome 3q13.31 and 7p14.1 CNAs involve genes implicated in carcinogenesis, including LASMP at 3q13.31 and TARP at 7p14.1, while 3q13.31 CNAs also involve two non-coding RNAs. We further show that expression of 3q13.31 genes correlates with the presence of 3q13.31 CNAs. We report that chromosome 3q13.31 and 7p14.1 CNAs are also common in other cancers, identifying these loci as candidates with a global role in carcinogenesis. Supporting the notion that 3q13.31 deletions play a role in osteosarcomagenesis, we find that depletion of 3q13.31 genes promotes proliferation of osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1 and that genetic deletions of 3q13.31 are associated with poor survival of osteosarcoma patients. In summary, our study implicates germline and somatic CN changes in osteosarcoma and represents a model approach for elucidation of elements contributing to disease susceptibility and aetiology in human cancer.

Keywords: osteosarcoma
copy-number change
oncogene
tumour-suppressor gene
Advisor: Malkin, David
Characterizing the Roles of PilF and PilQ in Pseudomonas aeruginosa Type IV Pilus Biogenesis

Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.

Keywords: Pseudomonas aeruginosa
PilF
PilQ
protein-protein interactions
pilotin
secretin
bacterial virulence
pilus biogenesis
X-ray crystallography
lipoprotein
type IV pilus
PilF-PilQ interactions
Advisor: Howell, P. Lynne