A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A₂ (PLA₂). Lipid thermal phase transition was found to have a dramatic effect on PLA₂ activity. It was also established that although membrane binding and insertion was essential for of PLA₂ activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA₂ secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA₂. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.

Up-conversion (UC) in rare-earth co-doped fluorides to convert diode laser light in the near infrared to red, green and blue visible light is applied to make possible high performance emissive displays. The infrared-to-visible UC in the materials we study is a sequential form of non-linear two photon absorption in which a strong absorbing constituent absorbs two low energy photons and transfers this energy to another constituent which emits visible light. Some of the UC emitters' most appealing characteristics for displays are: a wide color gamut with very saturated colors, very high brightness operation without damage to the emitters, long lifetimes and efficiencies comparable to those of existing technologies. Other advantages include simplicity of fabrication, versatility of operating modes, and the potential for greatly reduced display weight and depth. Thanks to recent advances in material science and diode laser technology at the excitation wavelength, UC selected materials can be very efficient visible emitters. However, optimal UC efficiencies strongly depend on chosing proper operating conditions. In this thesis, we studied the conditions required for optimization. We demonstrated that high efficiency UC depends on high pump irradiance, low temperature and low scattering. With this understanding we can predict how to optimally use UC emitters in a wide range of applications. In particular, we showed how our very efficient UC emitters can be applied to make full color displays and very efficient white light sources.

Despite not being a tangible substance, light is becoming an increasingly valuable tool in numerous areas of science and technology: the use of laser excitation of a fluorescent probe can generate incredibly detailed images of cellular structures without the need for large amounts of dissection; new types of solar cells are being produced using organic dyes to harvest light; computer data can be stored by inducing a chemical change in a compound through irradiation with light. However, before any of these materials can be applied in such a way, their properties must first be analyzed for them to be deemed viable. The focus of this dissertation is the photophysical characterization, linear and nonlinear, of a several novel organic compounds, and a europium complex, as well as using quantum chemical calculation techniques to understand some of the phenomena that are witnessed and begin to develop predictive capability. The nonlinear characterization of compounds utilizes wavelengths outside of their linear absorption range, where a focused beam can achieve the same excitation as one at half the wavelength, though this effect has a quadratic dependence on power. The potential for nonlinear excitation, or two-photon absorption (2PA), is becoming of increasing interest and importance for organic chromophores. Exciting only a small volume of material at a focal point makes it possible to nondestructively image samples in 3-dimensions, record data in multiple layers, and fabricate intricate structures through photopolymerization reactions. Lanthanides such as europium are known to exhibit sharp emission bands when excited, typically through an antenna effect due to the low probability of achieving direct excitation. This emission is long-lived, and through gating systems can readily be separated from background noise and autofluorescence (often observed in biological samples) that have much shorter lifetimes. Thus, one of the foci of this dissertation is the photophysical investigation of a series of novel lanthanide complexes, with particular attention to a europium complex.

Imaging instruments are required for accurate tumor resection during neurosurgery, especially in the case of glioblastoma multiforme (GBM) - the most common and aggressive malignant glioma. However, current intraoperative imaging techniques for detection of glioma either suffer low sensitivity and low specificity or require a significant capital cost. Advances in diffuse reflectance spectroscopy and fluorescence spectroscopy have offered high sensitivity and high specificity in differentiating tumors from normal tissues with much lower capital cost. Whereas diffuse reflectance spectroscopy alone and fluorescence spectroscopy alone has been used in limited studies to differentiate normal brain tissues from brain tumors with moderate sensitivity and specificity, low specificity and sensitivity were usually observed when studying high grade glioma (HGG) such as GBM. Furthermore, optical properties and diffuse reflectance signal of HGG and low grade glioma (LGG) have not been observed separately, and thus a relation between optical properties and glioma progression has not been established. Intraoperative differentiation of GBM and LGG can be helpful in making treatment plan at the first surgery. This thesis focuses on characterizing a previous integrated system of diffuse reflectance spectroscopy and fluorescence spectroscopy to extract optical properties and fluorescence properties of LGG and GBM. First, tissue-simulating phantom models were developed to calibrate the integrated system. The direct method and Mie theory were used to calculate optical scattering of the phantoms while Beer-Lambert’s law was used to calculate optical absorption. Second, an experimental method was introduced to recover intrinsic fluorescence because the measured fluorescence signal is likely distorted by the presence of scatterers and absorbers in tissue (i.e. hemoglobin). Third, an experimental method was developed to recover optical properties of both GBM and LGG. In addition, the sensitivity and specificity of the integrated system was optimized. / Thesis / Doctor of Philosophy (PhD)

Glioblastoma (GBM) is the most common and aggressive primary brain tumor and is comprised of transcriptionally heterogeneous cells and a complex microenvironment. Despite decades of research effort, few treatments significantly benefit clinical outcomes. This may be, in part, due to the lack of tools to directly measure functional responses of these heterogeneous cell types under therapy. This thesis aims to advance the understanding of cell type-specific therapeutic response by the development and application of integrated single cell imaging and RNA sequencing technology. Chapter 1 provides an overview of GBM and its heterogeneity, how investigation of cell type-specific phenotypes would benefit the development of GBM treatments, and current sequencing and imaging technologies to examine cell phenotypes with single-cell resolution. Chapter 2 presents a new microfluidic technology for joint single cell imaging and RNA sequencing that can link imaging-based phenotypes and transcriptional identity of the same individual cells with high throughput, molecular capture efficiency, linking accuracy, and user-friendliness. Chapters 3 and 4 present applications of this technology in investigating cell type-specificities of GBM treatments. Chapter 3 focuses on the specificities of 5-aminolevulinic acid (5-ALA), an FDA approved fluorogenic agent, used in fluorescence guided surgery and reveals 5-ALA labeling is not specific to transformed glioma cells, which encourages further studies to systematically compare its performance with potential alternatives. Chapter 4 focuses on the specificities of drug responses by presenting a functional drug screening approach that directly links cell states measured by apoptosis indicators with transcriptional states, which greatly enhances the interpretability of single cell-resolved drug perturbation assays.

Synthetic dyes that are used as color additives in foodstuffs are regulated by the Food and Drug Administration (FDA) under the Food, Drug, and Cosmetic Act (FD&C). The use of synthetic dyes not approved by the FDA, or the addition of dyes approved by the FDA above their maximum concentration limits in foodstuffs necessarily constitutes illegal food adulteration. Recently, rhodamine B (RhB), a bright-pink synthetic dye not approved for use in foodstuffs, has become an adulterant of interest due to its discovery in a large variety of food samples, and its identity as a potential carcinogen. Numerous chromatographic and spectroscopic methods have been developed for the analysis of RhB in food samples, as well as standard methods available for the analysis of synthetic dyes. However, due to the complexity of real food samples and the chemical diversity of synthetic dye concomitants, comprehensive chromatographic methods tend to be time consuming and expensive. Herein, we report a method for the determination of RhB in foodstuffs for the screening of real samples prior to subsequent full-blown chromatographic analysis, saving valuable time and resources. This screening method employs thin-layer chromatography (TLC) as a separation method, and, due to the strong yellow-orange fluorescence exhibited by RhB when excited with ultraviolet or green light, direct fluorescent measurement of RhB on the TLC plate using a fiber optic probe coupled to a commercial spectrofluorometer or to an instrumental set up for laser-induced fluorescence measurements. Qualitative analysis of RhB is based on its retardation factor, excitation and fluorescence spectra, and fluorescence lifetime. Quantitative measurements are made directly from the TLC plate to provide analytical figures of merit comparable to traditional fluorometric methods in liquid solution. The ability of the new method to determine RhB in food samples is then demonstrated with the analysis of chili powder.

Protein disulfide isomerase (PDI) is an endoplasmic reticulum (ER)-resident chaperone with oxidoreductase and isomerase activity. Unique to its normal function, PDI also appears to disassemble the A1 subunits of cholera toxin (CT) and heat-labile enterotoxin (LT). It does so using an unfolding mechanism that knocks the catalytic A1 subunit away from the rest of the holotoxin. Release of the A1 subunit is linked to the diarrheal diseases caused by V. cholerae and enterotoxicogenic E. coli (ETEC). Due to the previously established difference in disease potency between CT and LT, we investigated and established a distinction between the two toxins in their efficacy of disassembly by PDI. We further identified four amino acid differences between the CTA2 and LTA2 linkers, which connect the A1 and cell-binding B subunits of both toxins, as the basis for this difference. We believe these four amino acids result in changes to holotoxin architecture that lead to antiparallel binding of PDI to LT as opposed to CT, which translates to a loss of momentum for the physical disassembly of LT. We have shown this through algorithmic simulations of the binding event between PDI and either CT or LT. We hypothesized the unfolding mechanism of PDI, which dislodges the A1 subunit of both CT and LT, can also break down neurotoxic aggregates of β-Amyloid (AB) and α-Synuclein (AS). PDI is known to inhibit the aggregation of the amyloid proteins. We demonstrated here that PDI could also reverse oligomeric and post-oligomeric aggregates of AB and AS, respectively. Our work sheds light on the specifics of PDI's novel physical mechanism as well as introduce it as a possible therapeutic for both Alzheimer's and Parkinson's disease due to its unique ability to disaggregate early fibrillar structures of AS and AB proteins.

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