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Application of Berry phases and Wannier functions in solids
No full textThe Berry phase is the geometrical phase acquired by a quantum state after evolving along a loop in parameter space. Wannier functions, which are Fourier transforms of Bloch functions, have centers related to the Berry phases of Bloch functions acquired along a loop winding through the Brillouin zone. The application of Berry phases and Wannier functions in solids is the main topic of this thesis, and we present two case studies to illustrate these applications. In the first case study, we demonstrate that Berry phase plays an essential role for phonons in the absence of time-reversal symmetry (TRS). The conventional approach to phonons treats them as harmonic oscillators, which does not fully account for the effects of broken TRS in magnetic systems. A recent attempt to rectify this involves considering the force induced by the velocity of atoms, which appears naturally after implementing the Born-Oppenheimer approximation, with the coefficient being the nuclear Berry curvature. We have calculated the nuclear Berry curvature under the assumption that all other degrees of freedom are fully relaxed with respect to the nuclear coordinates. The results show that the main contribution comes from spin precessions during the loop of nuclear coordinates. However, the quasiparticles associated with spin precessions, magnons, have energy scales similar to those of phonons. This contradicts the aforementioned assumption, indicating that phonons and magnons need to be treated on an equal footing. This is achieved by utilizing adiabatic theory, which will be discussed in detail in this thesis. Our case studies reveal that degenerate phonons can have different energies in CrI with atoms rotating, thus forming chiral phonons, and Raman-active even-parity phonons in CrO can acquire some infrared activity. In the second case study, we use Berry phases and Wannier functions to predict the corner charges for two-dimensional (2D) finite flakes. In the one-dimensional (1D) case, the bulk-boundary correspondence tells us that the end charge of a finite 1D chain can be predicted by the bulk polarization (modulo ). The bulk polarization is related to the Berry phase of Bloch functions calculated along a loop winding through the Brillouin zone, which is equivalent to calculating the polarization using the centers of Wannier functions. For a 2D finite flake, the corner charge can be predicted by the sum of edge polarizations for the two edges intersecting at the corner and the interior quadrupole moment. Similar to the bulk polarization, edge polarizations and quadrupole moments can also be calculated using Wannier functions. Edge polarizations can be calculated using 1D ribbon geometries, where one direction is periodic and the other is finite, containing multiple unit cells. The quadrupole moment can be calculated using a 2D bulk. We show that although these quantities are individually gauge-dependent, their gauge dependence cancels out when summed, indicating that we must work under the same Wannier gauge when calculating them. The direct calculation of corner charges for the 2D finite flake validates our theory. Besides the two cases about Berry phase applications, we will also discuss a collaborative study with experimentalists on Humble defects, a kind of planar defect observed in some semiconductors. Although there are several types of Humble defects, only the (a)-type was observed in GeSi alloy. Our density functional theory calculations show that this type has the lowest energy among the various types. We also simulate the core-level electron energy loss spectra for the Si atoms in both the defect and bulk regions, and our simulations agree well with experimental results.Ph.D.Includes bibliographical reference
Immune targeted shortwave infrared emitting nanoprobes for the prognostication of cancer
No full textAdvances in the detection and treatment of breast cancer over the previousdecades has led to remarkable improvements in patient outcomes, thanks in large
part to early detection modalities and targeted treatments. However, breast cancers
that lack the three common receptors for targeted treatment, termed triple-negative
breast cancer (TNBC), remain difficult to treat and have higher rates of metastatic
relapse. Recently, immune checkpoint inhibitors (ICIs) have gained traction as a
therapeutic option for TNBC due to their ability to reinvigorate the native immune
response to the tumor. Variable response rates to ICIs and poor stratification of
patients prior to therapy has stymied progress in this approach and spurred interest
in the development of novel technologies to better prognosticate disease progression
and therapy response in TNBC patients.
To fulfill this clinical need, researchers have been investigating biomarkers
and detection methods to identify the metastases and therapy non-responders earlier
for enhanced precision medicine. Targeting key promoters of metastatic niche
formation is one pathway to earlier detection of metastases, which can support
improved clinical decision making. Immunosuppressive populations, such as the
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myeloid derived suppressor cell (MDSC), have recently been identified as markers
of metastatic disease due to their role in preparing the pre-metastatic niche (PMN)
for tumor colonization. Accumulating evidence demonstrates that these
immunosuppressive populations, previously overlooked, may be also be a significant
source of immunotherapy evasion within the primary tumor and thus serve as
valuable biomarkers for prediction of therapy response. Although tumor infiltrating
lymphocyte index and PD-L1 expression were established as potential prognostic
indicators for immunotherapy response, their accuracy in patient stratification is
severely limited. It has become clear that when evaluating tumors for immunotherapy
responsiveness there is a need for a more robust evaluation of the tumor immune
environment, including evaluation of immunosuppressive populations in addition to
the cytotoxic lymphocytes. Designing a tool for the evaluation of the tumor associated
immune population can provide a complementary approach to assessing the
prognostic outlook of patients in regard to metastasis formation and immunotherapy
response.
This dissertation is focused on the development of molecularly targeted
optical nanoprobes for the tracking of immune dynamics in TNBC, and how this
correlates with disease progression and immunotherapy responsiveness. We use
rare earth metal-doped nanoprobes that, when excited by near infrared light (NIR, λ
= 980 nm), emit in the shortwave infrared (SWIR, λ = 1000-1700 nm). These SWIRemitting nanoprobes permit improved optical imaging at greater tissue depths, with
reduced tissue scattering and autofluorescence compared to traditional optical
imaging. We engineer these nanoprobes for the molecular targeting of MDSCs within
the tumor microenvironment and lung pre-metastatic niche. We demonstrate that the
non-invasive imaging of MDSCs within the lungs was able to identify the pre-
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metastatic niche in a model with high metastatic propensity, compared to a model
with low rates of metastasis. Further, we use MDSC targeted probes to track changes
in immune dynamics associated with response to combination therapy with an ICI
and doxorubicin in the tumor microenvironment. We present observations suggesting
that dual imaging of MDSCs and cytotoxic T cell targeted nanoprobes could provide
a better metric for stratifying response to immunotherapy. The findings of this thesis
reinforce the value of SWIR imaging to identify biomarkers for cancer prognosis, such
as immune cell dynamics, laying the groundwork for the development of a tool that
can stratify patients in terms of metastatic risk and immunotherapy responsiveness.Ph.D.Includes bibliographical reference
The tissue-specific role of 8-oxoguanine dna glycosylase in skeletal muscle and adipose tissue in energy metabolism
No full text8-oxoguanine DNA glycosylase 1(OGG1) is a DNA glycosylase enzyme localized in mitochondria and nucleus. The primary function of OGG1 is to initiate the base excision repair process to repair oxidized guanine. OGG1 function contributes to the maintenance of genomic integrity and thus protects animals from pathological conditions such as cancer, progressive neurological disorders, and metabolic syndrome. Mitochondrial DNA is prone to oxidative damage due to its proximity to proteins involved in oxidative phosphorylation. Guanine has the least oxidation potential among DNA bases, and it gets easily oxidized. The oxidized guanine product, 8-oxo-7,8-dihydrogunanine (8-oxoG) can pair with adenine. Unrepaired oxidized guanine products can cause G·C→T·A transversion. Prior studies have shown that OGG1 deletion in mice can disrupt metabolic homeostasis and decrease skeletal muscle function. Previous studies have also shown that human-OGG1(h-OGG1) overexpression in mice (Ogg1Tg) can protect them from diet-induced obesity and show improved glucose tolerance. All these metabolic studies have been done on whole-body knockout mice or on whole-body transgenic mice. We still did not know the tissue-specific role OGG1 plays in the metabolism of mice. To elucidate the OGG1 role in major metabolic organs, RNA-Seq analyses in the liver, skeletal muscle (SM), and adipose tissue (AT) were performed. The AT and SM of Ogg1Tg mice showed significant differential expression of genes. The liver of Ogg1Tg, however, showed a minimal difference. We used Ogg1Tg mice to study the role of OGG1 in skeletal muscle function. Our study shows that when OGG1 is overexpressed mice have improved skeletal muscle function. Ogg1Tg mice showed more than two times exercise endurance enabled by a higher skeletal muscle glycogen content. Ogg1Tg mice also showed increased muscle lipid and a higher mitochondrial content in the muscle. The skeletal muscle of Ogg1Tg mice showed more than a thousand-fold increase in the myokine Fgf21 expression. Together, these results show that OGG1 in the skeletal muscle of Ogg1Tg mice is better functioning due to improved mitochondrial functioning and FGF21 action. Two novel conditional knockout mice were developed and used to study the tissue-specific contribution of OGG1 in modulating whole body energy metabolism. In the adipocyte OGG1 knockout mice (AKO) mice, OGG1 in the adipocyte was deleted under adiponectin CRE promoter and was used to study the role of adipocyte OGG1 in the modulation of whole-body energy metabolism. The skeletal muscle OGG1 conditional knockout mice (MKO) was developed under human--actin promoter to study the role skeletal muscle OGG1 plays in whole-body energy metabolism. The AKO mice were prone to diet- induced obesity with a decrease in glucose clearance after 12 weeks of 60% high-fat-diet (HFD). AKO mice showed delayed glucose clearance and dyslipidemia, as shown through higher plasma triglyceride (TAG). MKO mice showed a better glucose clearance after the 12-week HFD and showed no evidence of dyslipidemia and diet-induced obesity. The exercise endurance of MKO mice was lower compared to the control mice. This thesis proves that OGG1 plays a tissue-specific role in modulating metabolism under different conditions, such as acute exercise and a hypercaloric diet in which 60% of the total calories are derived from fat.Ph.D.Includes bibliographical reference
3D dynamic blind zone modeling and analysis for high-resolution roadside LiDAR sensors
No full textThe key to any Advance Driver Assistance System (ADAS), autonomous technology, or human driving is to accurately sense the surroundings. Conventionally, Autonomous Vehicles (AV) mainly depend on the equipped sensors to sense the surroundings and make decisions based on the sensor data along with the supportive information from other Connected Vehicles (CV) and Roadside Units (RSU). Limited by the height of the vehicle, the on-vehicle sensors are significantly affected by the occlusions. Coupled with the glacially increasing CV penetration rate, AVs will have to stay in a world full of blind zones for an extended duration of time. AVs will continue to navigate a world filled with blind zones for an extended period. These blind zones are also critical considerations for human driving maneuvers. Human drivers face more severe blind zones due to a more limited field of view, although the flexibility of drivers' eyes compared to fixed cameras can slightly mitigate these blind zones.Roadside sensors have advantages over on-vehicle sensors and driver views in reducing occlusions for two main reasons. First, roadside sensors can be installed at much higher elevations than vehicles, effectively mitigating occlusions. Second, although the installation of roadside sensors is constrained by the terrain, they can be situated distantly, which grants them the ability to sense from distinct viewpoints and further reduce blind zones. The recent deployment of the roadside sensor fusion system provides opportunities to systematically analyze the occlusion-caused blind zone and detect near-misses caused by blind zones. This dissertation conducts blind zone analyses from the perspective of roadside sensors, though the model can also be adapted for on-vehicle sensors. First, Sensor Coverage Models (SCM) for different roadside sensors are calculated based on sensor characteristics. Second, a Digital Surface Model (DSM) with a world coordinate system is built from the 3D model to represent the background, including both terrain and infrastructure. The simulated sensor coverage model SCM and the sampled background model DSM are calibrated and validated with actual Light Detection And Ranging (LiDAR) data. Metrices are designed to cross compare the dense SCM and DSM with the sparse LiDAR point cloud data. For static blind zone analysis, the SCM is projected onto the DSM coordinate system as sensor placement. For dynamic blind zone analysis, vehicle trajectories are incorporated to update the dynamic DSM. Sensitivity analyses are conducted to test how different parameters affect the blind zones, and recommendations have been made based on these findings.Ph.D.Includes bibliographical reference
Development of hordein-based delivery vehicles to improve the absorption of phytochemicals
No full textGrowing awareness regarding the impacts of diet on human health has led to expanding consumer demands for incorporating of healthy ingredients and nutraceuticals into food and beverage products. Among which, phytochemicals are a promising class of nutraceutical candidates with numerous health-promoting efficacies. However, the integration of phytochemicals toward food and beverage products can often be challenged with unpleasant sensory profiles, incompatibility with food matrixes, limited absorption, etc. As a result, rational design of appropriate delivery strategies is thus crucial to ensure the proper incorporation and consumption of phytochemicals. Hordein, the major prolamin protein found within barley grains, is of great interest in this thesis as a biomaterial building block with great potential due to its appealing and unique properties: sustainable, biodegradable and biocompatible status, strong hydrophobicity, self-assembly capability, reduced susceptibility to gastrointestinal digestions, controlled release behaviors, and more. Therefore, with the aims to develop functional hordein-based biomaterials and to investigate their application potential in improving the absorption of phytochemicals, this thesis is composed with contents in 1) acquisition of hordein from barley grains and assembly of hordein-hyaluronic acid (H-HA) complexes, 2) analysis of in vitro bioaccessibility and bioactivity of dihydromyricetin-hordein-hyaluronic acid (DMY-H-HA) complex nanoparticles as delivery system for dihydromyricetin, 3) modification of hordein by lauric acid and fabrication of hordein colloidal particle based Pickering emulsions, and 4) development of hordein-based Pickering double emulsions for co-encapsulation of water-soluble and lipid-soluble nutraceuticals. Specifically, the thesis started with the extraction of hordein from barley grain flour, and the protein content as well as polypeptide composition of the extracted hordein were confirmed using the Kjeldahl method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, H-HA complexes were successfully assembled through liquid-liquid dispersion combined with electrostatic deposition. Investigations toward the physiochemical characteristics revealed that H-HA complexes exhibited reduced particle size and polydispersity and enhanced zeta-potential, when compared to single-layer hordein nanoparticles. Meanwhile, it was found that electrostatic interaction, hydrogen bonding, and hydrophobic interaction were responsible for the corresponding protein-polysaccharide complex formation. Moreover, in comparison to single-layer hordein nanoparticles, H-HA was demonstrated with improved stability profiles regarding various environmental conditions.
In addition, dihydromyricetin (DMY) is a plant flavonoid with numerous health-beneficial properties, however, its application potential could be challenged by its limited bioavailability. Therefore, H-HA was further utilized and assessed as a nanoparticle delivery system for DMY (DMY-H-HA). In this study, the physiochemical characteristics of the DMY-H-HA were evaluated, the morphology of the complex nanoparticles was observed with scanning electron microscopy (SEM), and the encapsulation and loading profiles were also determined. Through in vitro and ex vivo investigations, it was found that DMY-H-HA exhibited enhanced gastrointestinal dissolutions and intestinal permeations compared to that of the unencapsulated DMY. Additionally, the absorption pattern of the complex nanoparticles was clarified by the Caco-2 cells cellular uptake study, and anti-inflammatory analyses with RAW 264.7 macrophage cells also suggested the improved bioactivity of DMY-H-HA compared to DMY aqueous suspension. Results gathered from this study demonstrated the capability of DMY-H-HA as a delivery system for DMY, while also providing additional support for the applicability of hordein for functional food and beverage considerations.
In the following section, lauric acid-modified hordein (LAH) colloidal particles were prepared by antisolvent co-precipitation into the alkali environment. LAH colloidal particles and the influences of lauric acid were characterized, and lauric acid was found to be effective regarding hydrophobic modification of the hordein particles. Meanwhile, hordein-based oil-in-water (O/W) Pickering emulsions were fabricated with the prepared colloidal particles and evaluated systematically; It was demonstrated that the colloidal particle, particle concentration, and oil phase fraction can exert significant impacts on the stability, microstructure, and rheological property of the fabricated emulsions. More importantly, robust high internal phase Pickering emulsion was also achieved in this study, as superior stability, enhanced droplet packings, and elastic and gel-like behaviors were determined to be associated with such emulsions at elevated levels of oil phase fraction.
For instance, hordein-based water-in-oil-in-water (W1/O/W2) Pickering double emulsions (HPDE) were developed for co-encapsulation of water-soluble riboflavin and lipid-soluble carnosic acid (CA). Formulation developments were carried out for the respective primary and secondary emulsions, and the impacts of formulation parameters were investigated. At the same time, the droplet structure, interfacial morphology, and cross-section of the HPDE were visualized using cryo-scanning electron microscopy (Cryo-SEM). Moreover, HPDE exhibited satisfactory encapsulation stability and release profile under simulated gastrointestinal conditions for the W1 encapsulated riboflavin. On the other hand, HPDE was indicated with a more rapid rate of in vitro lipolysis and resulted in enhanced bioaccessibility of the oil phase loaded CA, compared to that suspended in the bulk oil. In addition, when applied to digestions using the TIM-1 dynamic gastrointestinal model, HPDE had led to 2.0- and 2.5-fold increases in the cumulative bioaccessibility of the encapsulated CA in the jejunum and ileum compartment, respectively, in comparison to that of the unencapsulated format.Ph.D.Includes bibliographical reference
Caring women: midwives and female healers in New Granada, 1700-1810
No full textIn this dissertation I investigate midwives' and other female healers' lives and medical practices in New Granada between 1700 and 1810. I do this by analyzing their participation in criminal trials. I begin in 1700 to cover the rise of the Bourbons in Spain, a dynasty that developed imperial reforms that sought greater control over social, economic, and political practices in the Americas, including botany, medicine, midwifery, and surgery. Paradoxically, reforms meant to police colonial populations more efficiently, and that also sought to professionalize medicine, brought higher participation of female experts and witnesses in criminal trials. In what type of criminal cases did these women participate and how? What do the cases reveal about them? Who were they in a sociological sense? What type of medical care did they provide to their suffering patients? How are we to understand their sometimes contradictory roles? These historical questions are integral to this dissertation. I treat women as central actors in the provision of healthcare during New Granada’s late colonial period. In doing so, I show that medical knowledge and the “ability to care” gave them social prestige and legitimacy. Midwives and female healers subverted traditional gender roles because their “calling” forced them to be mobile, independent, and economically self-reliant. They were summoned at odd hours of the night and left their houses without hesitation. They aided women who decided to terminate a pregnancy even if this contravened royal and canon law. They supported women who survived sexual and domestic violence by testifying on their behalf. Women's healing practices made use of local botanical knowledge and expertise garnered from caring for others. Caring, a gendered role in a patriarchal society, opened avenues for social mobility and respect. Yet caring played a much more ambiguous role that we may initially imagine. I understand caring as a multifaceted practice imbued with power; power over life and death, power over bodies, power over knowledge in the context of a legal process. As such, care can mean healing but also harming. Care can take the form of support but also of neglect. Care can mean assisting women through miscarriage but also policing women’s sexuality. My research builds from feminist theory to understand the centrality of care while attending to care’s problematic sides and forms. My objective is to show the complexity of midwives' and female healers' experiences and actions in the late colonial period.Ph.D.Includes bibliographical reference
Powder mixing in continuous manufacturing: understanding of mixture properties, mixing dynamics and homogeneity
No full textContinuous manufacturing of pharmaceutical oral-dose drug products has seen substantial progress over the last two decades, driven by collaborations among academic institutions, industries, and regulatory agencies. Advancements in the design of new equipment and integrated processes, along with the implementation and integration of process analytical technology with process control, and the incorporation of digitalization, have facilitated enhancements in process efficiency, improved product quality, and increased process understanding. The work in this dissertation provides deep understanding of powder mixing in continuous pharmaceutical manufacturing using multivariate analysis tools and mathematical models, which can be applied to process and formulation development with material sparing approach. It is also highlighted that the accomplishment of the work can provide new insights for manufacturing tablets without using a blender, which could significantly reduce the cost of manufacturing. In this work, mixture models were developed to predict mixture properties from ingredients. Two methods were proposed: Particle Least Squared method and mixing-rule-based method. Non-linear relationships between mixture properties and compositions were revealed, and packing mechanism with different size ratio category can provide theoretical explanation. It was found out that the Partial Least Squares (PLS) method is suitable for properties with roughly linear relations, and the mixing-rule-based method can capture non-linearities with minimal experimental effort.
The second aim focuses on the powder characterization and process development in feeding-blending system and demonstrates the suitability of a continuous manufacturing route for micronized and granular drug products. Due to scarcity and potency of the API, a surrogate that has similar bulk properties to the API’s was selected based on statistical analysis of the Rutgers material database. Importantly, for the continuous blender, process parameters such as blender speed and throughput were varied to characterize mixing behavior for six cohesive blends, that each contained different grades of excipients, and one granular blend. The residence time distribution (RTD) of the material in the continuous blender was performed using a pulse of a second surrogate, which was similar to the API and easily detectible by spectroscopic methods and characterizing the profile of the material as it exited the blender. Multivariate analysis was used to analyze the relationships between process parameters, mixture properties and mixing performances. By performing each of these steps, operators were able to identify the suitability and characteristics of blends based on the performances in the unit operations.
The third aim works on expansion of standardized methods for characterizing RTD by assessing tracer materials and quantities effect on RTD of a continuous manufacturing line. Statistical method such as analysis of variance and multivariate analysis of variance were utilized to test the statistical significance across different tracers and masses. This work facilitates broader selection criteria for tracers in RTD studies and also demonstrates the mixing ability of the feed frame to disperse the disruptions caused by the tracer. Along with the findings, the fourth aim was inspired to investigate the necessity of using a physical blender to manufacture drug products. Three mixing scenarios were evaluated and compared including batch mixing, continuous mixing, and feed frame mixing only. It was exciting to find that, for an easy-flowing high-dose formulation, blending solely within feed frame can achieve excellent blend homogeneity. Additionally, harder tablet properties can be acquired compared to the conventional manufacturing approach, which offers a lower-shear alternative for manufacturing shear-sensitive formulations. The findings from this work, a potential “no blender” manufacturing route, can reduce significant amount of time for pharmaceutical product development and costs for manufacturing.Ph.D.Includes bibliographical reference
The syntax and semantics of headless relative clauses
No full textHeadless relative clauses exhibit dual characteristics: morphologically, they resemble clauses, while distributionally, they function as nouns, adjectives, or adverbs. This dual nature complicates a comprehensive understanding of their syntactic and semantic properties. This dissertation advances our understanding of headless relative clauses by addressing four syntactic and semantic issues. First, I examine the syntactic transformation of nominal headless relatives from CPs to NPs/DPs. Through a detailed cross-linguistic investigation, I propose that this transformation requires a categorical [N]/[D] feature provided by the daughters of the headless-relative node. In [+wh] headless relatives, overt fronted wh-phrases supply the [N]/[D] feature, whereas in [-wh] headless relatives, external nominal heads can provide this feature. Next, I explore whether all arguments within a headless relative clause can be equally relativized when they are phonologically null. A preference for the object reading over the subject reading is observed in Mandarin doubly-gapped headless relatives. I argue that this asymmetry stems from a general preference for nested movements over crossing movements, as well as the unavailability of generic null objects with most Mandarin verbs. I then turn to the semantic composition of headless relative clauses. I observe that pied-pipings in English free relatives are in general degraded. Based on this observation, I propose a compositional analysis where the meaning of free relatives is sensitive to the form of their fronted strings. Finally, I extend the discussion to the readings headless relatives can have and their parallels with modified bare nouns. Drawing on the similarities between English free relatives and modified bare nouns, I argue that the distribution of kind and non-kind readings in these two constructions should be captured in a unified manner. Additionally, I discuss how their non-kind readings differ in terms of maximality.Ph.D.Includes bibliographical reference
Structure-function relationship of collagen-iii mutations in vascular Ehlers-Danlos Syndrome
No full textThe extracellular matrix (ECM) is a dynamic 3D network essential for tissue development, repair, remodeling, and homeostasis. Collagen, a major structural protein in the ECM, plays a crucial role in maintaining structural integrity and mechanical properties of tissues and organs while regulating critical biological functions. Fibrillar collagens possess a distinctive triple helix structure characterized by a (Gly-X-Y)n repeating sequence. Variations within this sequence can lead to triple helix misfolding, causing heritable connective tissue disorders. The most common variations are single-point missense mutations that replace glycine with a bulkier amino acid (Gly→X).This dissertation aims to elucidate the impact of specific Gly→X substitutions in collagen-III on the tissue structure and function associated with vEDS, a severe, life-threatening condition. Mutations in collagen-III may lead to its abnormal synthesis, secretion, and/or function. Acquiring mutated, full-length collagen with specific mutation from vEDS patients to understand structure-function relationships has proven to be difficult. The discovery of bacterial collagen-like proteins (CLPs) as a model system has enabled systematic investigation of sequence-structure-function relationship in normal and pathological collagens. CLPs self-assemble into a triple helix, similar to animal collagen, and provide a blank template to introduce specific ligand-binding sites and study protein-collagen interactions. In this study, we engineered CLPs with a common integrin-binding site found collagen-III - GFPGER - integrin-binding site (IBS) and introduced Gly→Ser/Arg substitutions either within or outside the IBS. Using a combination of biophysical techniques, enzymatic digestion assays, ELISA, and computational modeling, we demonstrated that Gly→Ser/Arg substitutions differentially impact the triple helix structure, making it susceptible to trypsin digestion, and altering its interaction with integrin receptors, with Arg having a significantly greater impact. Molecular dynamics (MD) and steered MD (SMD) simulations were utilized to explore how Gly→Ser/Arg substitutions affect the triple helix structure and mechanical properties at the molecular level. Arg substitution induced local bulging in the triple helix at the substitution site and disrupted hydrogen bonding compared to Ser substitution. SMD simulations indicated that an Arg substitution led to a substantial reduction in the Young’s modulus of the triple helix.
To isolate CLPs containing Gly→X substitutions from full-length constructs, we designed peptides to include a TEV protease cleavage site instead of the commonly used trypsin cleavage site. The triple helix renders collagen resistant to trypsin digestion. CLPs with TEV sites enabled recovery of full-length constructs for both control and Gly→X mutated sequences, whereas Gly→X mutated CLPs with trypsin cleavage sites were fragmented by the enzyme due to the impact of the mutation on the triple helix. CLPs were formed into 3D hydrogels using Michael-type thiol-maleimide addition reactions to present IBS in a native (Gly→X substitutions outside the IBS) versus distorted triple helix (Gly→X substitutions within the IBS). Rheological studies showed that bulkier amino acid substitutions, particularly Gly→Arg substitutions, influenced the shear storage modulus of hydrogels. Cellular assays demonstrated that Gly→Arg substitutions altered human smooth muscle cell behavior, including cell adhesion, spreading, proliferation, and ECM secretion. This findings emphasize the critical role of both the identity and location of the substituted amino acid in determining the loss of function either in terms of cell-matrix interactions and/or mechanical properties. impacting both tissue stiffness and cell function, including ECM production, and tissue remodeling capabilities
Using MD simulations, we also investigated the relationship between Gly→X substitutions and their surrounding amino acid sequence. We designed two sequences: a short synthetic peptide with (GPP)16 repeats and a peptide derived from the human collagen-III sequence. Both sequences included a GRPGER IBS with and without Gly→X substitutions. Our results showed that Gly→X substitutions had a more significant impact on the triple helix structure of human collagen-III sequences compared to the highly stable (GPP)16 sequences. Additionally, we demonstrated that capping Gly→X substitutions at different locations with varied imino acid content had differential effects on the triple helix structure. Our findings demonstrate that Gly→X substitutions within imino acid-rich sequences have a lower impact on the triple helix compared to those within imino-poor sequences.
Overall, the results presented in this thesis show the impact of Gly→X substitutions on cell-matrix interactions and mechanical properties. This study advances our understanding of mutation-specific effects, utilizing bacterial CLPs to investigate disease mechanisms and guide future therapeutic development for vEDS.Ph.D.Includes bibliographical reference
Behavioral and Neurophysiological Processing of Auditory Memory is Altered by Noisy Backgrounds
No full textComprehension of sound in noise is a remarkable feat of auditory systems. Associative learning about the behavioral salience of sound signals, moreover, involves physiological changes to receptive fields in the auditory cortex (ACx) (Bieszczad & Weinberger, 2010; Weinberger 2015). As such, ACx neurophysiological plasticity induced by associative learning may facilitate hearing those remembered salient signals in noisy backgrounds, i.e., relative to novel or insignificant sounds. Several ACx mechanisms have emerged as key effects of both auditory memory and background noise, including changes to receptive field properties like sound-evoked threshold and tuning bandwidth. In this regard, learning-induced changes in the receptive fields of specific sound signals that mimic ACx function in noisy backgrounds may effectively promote their detection in noise and facilitate sound-cued adaptive behavior. Recent investigations have also shown that learning-induced ACx plasticity can be facilitated by treating subjects with an HDAC-inhibitor (histone deacetylase 3, HDAC3i) while they learn an association between a signal acoustic frequency and reward6,7. However, the extent to which the effect of HDAC3i persists in novel backgrounds of noise remains unknown. Given the established effect of learning and HDAC3i, the thesis is set to explore learning-, noise level-, and HDAC3 inhibition-dependent processing of sound-specific memories. Thus, the current study utilizes a rodent (rat) model of sound-reward learning for a 5.0 kHz (60 dB) pure tone frequency cue with in vivo auditory cortical (A1) multiunit electrophysiological recordings (as in Rotondo & Bieszczad, 2020). We assessed behavioral and A1 responses to signal and non-signal frequency cues presented under different signal-to-noise ratios (SNR) (+0, +20 and +40dB SNR). Findings revealed that (i) the behavioral expression of sound-specific memory is blunted by the addition of background noise and (ii) the neural expression of sound-specific memory is protected by an HDAC3-inhibitor applied during learning. Overall, our attempt to build a comprehensive model of the epigenetic regulation of neuroplasticity in auditory cortex and sound-specific memories has a translational value for improving the ability to achieve successful hearing-related therapeutics in real world environments where sounds are seldom encountered in isolation.M.S.Includes bibliographical referencesIncludes vit