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The mortality rate of cancer establishes it as a leading global health concern, prompting significant investment into cancer research. While the effects of cancer are well known, the understanding of specific sources of cancer therapy resistance are not. In this study, our goal was to develop innovative methods to address current shortcomings in cancer treatment and understanding. To do this, we studied exosome-mimetic nanovesicles as an immunotherapeutic platform and fluorescence lifetime imaging as a means to measure cancer-associated enzyme activity at a single cell level.

Through the use of a novel method of production, we generated nanovesicles from dendritic cells in high yields and leveraged the antigen-presenting and costimulatory properties of dendritic cells for induction of a T cell immune response. We demonstrate that these nanovesicles are able to present antigens in functional immune stimulatory complexes and retain parental ability to activate CD8+ T cells. Additionally, these nanovesicles were shown to mediate activation of T cells through indirect means. Here, nanovesicles are taken up by bystander dendritic cells, thereby delivering antigen to the dendritic cell and conferring T cell stimulatory capability. Next, we investigated the application of fluorescence lifetime imaging to measure cancer-associated cytochrome P450 enzyme activity at the single-cell level. We demonstrated this approach provides detailed insights into cellular heterogeneity and localized enzyme activity. Additionally, we showed that sensitivity and dynamic range can be tuned to enzyme activity and levels by altering excitation and emission wavelengths.

These advancements offer new and promising avenues to enhance nanoparticle-based immunotherapy and understanding of the role of enzyme activity and cellular heterogeneity in cancer progression. Ultimately, the methods developed contribute to improving therapeutic strategies and personalized medicine.

The mortality rate of cancer establishes it as a leading global health concern, prompting significant investment into cancer research. While the effects of cancer are well known, the understanding of specific sources of cancer therapy resistance are not. In this study, our goal was to develop innovative methods to address current shortcomings in cancer treatment and understanding. To do this, we studied exosome-mimetic nanovesicles as an immunotherapeutic platform and fluorescence lifetime imaging as a means to measure cancer-associated enzyme activity at a single cell level.

Through the use of a novel method of production, we generated nanovesicles from dendritic cells in high yields and leveraged the antigen-presenting and costimulatory properties of dendritic cells for induction of a T cell immune response. We demonstrate that these nanovesicles are able to present antigens in functional immune stimulatory complexes and retain parental ability to activate CD8+ T cells. Additionally, these nanovesicles were shown to mediate activation of T cells through indirect means. Here, nanovesicles are taken up by bystander dendritic cells, thereby delivering antigen to the dendritic cell and conferring T cell stimulatory capability. Next, we investigated the application of fluorescence lifetime imaging to measure cancer-associated cytochrome P450 enzyme activity at the single-cell level. We demonstrated this approach provides detailed insights into cellular heterogeneity and localized enzyme activity. Additionally, we showed that sensitivity and dynamic range can be tuned to enzyme activity and levels by altering excitation and emission wavelengths.

These advancements offer new and promising avenues to enhance nanoparticle-based immunotherapy and understanding of the role of enzyme activity and cellular heterogeneity in cancer progression. Ultimately, the methods developed contribute to improving therapeutic strategies and personalized medicine.

Abstract: The energy band structure (dispersion relation between energy and wavenumber) is fundamental information essential for elucidating the charge transport properties of semiconductors. For organic semiconductors, the valence band structure, which is responsible for
hole transport, has been measured by energy-dependent and angle-resolved photoelectron spectroscopy since the 1990s. However, the conduction band structure, which is responsible for electron transport, has not yet been reported.
 

We have developed a new technique, angle-resolved low-energy inverse photoelectron spectroscopy [1], and succeeded in measuring the conduction band structure of organic semiconductors for the first time [2]. Based on the experimental results, we propose a new polaron model, "partially-dressed polaron" model. This study evidences the polaron formation in high-mobility organic semiconductors has a significant impact on electronic conduction.

[1] Y. Kashimoto, S. Ideta, H. Sato, H. Orio, K. Kawamura, H. Yoshida,
Rev. Sci. Instrum., 94, 063903 (2023). Selected as Editor’s Pick
[2] H. Sato, S. A. Abd. Rahman, Y. Yamada, H. Ishii, H. Yoshida, Nature
Mat. 21, 910 (2022).

Metal halide perovskites have gained interest in optoelectronic applications such as photovoltaics, lasers, LEDs, transistors, and photodetectors due to their excellent semiconducting properties considering their low cost. Metal halide perovskite (HP) photovoltaics have rapidly increased in power conversion efficiency (PCE), which now exceeds 25%. HPs have gained attention in these applications due to their high tolerance towards defects, long charge carrier diffusion lengths, high charge carrier mobility, high optical absorption, and bandgaps that are tunable over a large range. Even though HP photovoltaic PCEs are improved these are still not commercially available due to them showing lower stability and energy loss due to severe charge recombination at the surface and interfaces in the device . Treating the HP surface with surface ligands has become a promising approach to improve photovoltaic performance, defect passivation, and interfacial energetics. In this dissertation,  the influence of ammonium functionalized p – conjugated ligands on MAPbI3 perovskite energetics, photovoltaic performance, and interfacial charge transfer is investigated. With the thiophene ligands, a drastic PCE drop was observed for p-i-n devices, and improved PCE was obtained for n-i-p devices. With PDI surface ligands no significant change was observed for photovoltaic performance.  Two-dimensional metal halide perovskites (2D HP) have captured interest in the field due to their improved stability against air, moisture, and light relative to their 3D counterparts. 2D HPs have a layered structure, where the organic spacer cations are sandwiched between layers of inorganic octahedra. This organic layer in 2D HPs adds additional protection against moisture and oxygen ingression and other degradation pathways . These materials are used as the active layer in LEDs and solar cells and as capping layers in 3D HPs. 2D perovskites demonstrate remarkable structural variabilities, where the properties can be modified by changing the layer thickness, the halide anion, and the spacer cation. To make devices with 2D perovskites we need to understand the influence of the organic spacer cations on the optoelectronic properties of these materials . In this work, we  investigate the influence of the dipole magnitude and the direction of a series of functionalized PEAI derivatives as organic spacer cations on the ionization energy and the electron affinity of 2D tin halide perovskites. However, determining ionization energy and electron affinity in HPs could be quite difficult as several methods are being used in data interpretation for HPs . In this work, we propose a method to assign the energy levels in 2D HPs by correcting for the instrumental resolution in ultraviolet and inverse photoemission spectroscopy.

Metal halide perovskites have gained interest in optoelectronic applications such as photovoltaics, lasers, LEDs, transistors, and photodetectors due to their excellent semiconducting properties considering their low cost. Metal halide perovskite (HP) photovoltaics have rapidly increased in power conversion efficiency (PCE), which now exceeds 25%. HPs have gained attention in these applications due to their high tolerance towards defects, long charge carrier diffusion lengths, high charge carrier mobility, high optical absorption, and bandgaps that are tunable over a large range. Even though HP photovoltaic PCEs are improved these are still not commercially available due to them showing lower stability and energy loss due to severe charge recombination at the surface and interfaces in the device . Treating the HP surface with surface ligands has become a promising approach to improve photovoltaic performance, defect passivation, and interfacial energetics. In this dissertation,  the influence of ammonium functionalized p – conjugated ligands on MAPbI3 perovskite energetics, photovoltaic performance, and interfacial charge transfer is investigated. With the thiophene ligands, a drastic PCE drop was observed for p-i-n devices, and improved PCE was obtained for n-i-p devices. With PDI surface ligands no significant change was observed for photovoltaic performance.  Two-dimensional metal halide perovskites (2D HP) have captured interest in the field due to their improved stability against air, moisture, and light relative to their 3D counterparts. 2D HPs have a layered structure, where the organic spacer cations are sandwiched between layers of inorganic octahedra. This organic layer in 2D HPs adds additional protection against moisture and oxygen ingression and other degradation pathways . These materials are used as the active layer in LEDs and solar cells and as capping layers in 3D HPs. 2D perovskites demonstrate remarkable structural variabilities, where the properties can be modified by changing the layer thickness, the halide anion, and the spacer cation. To make devices with 2D perovskites we need to understand the influence of the organic spacer cations on the optoelectronic properties of these materials . In this work, we  investigate the influence of the dipole magnitude and the direction of a series of functionalized PEAI derivatives as organic spacer cations on the ionization energy and the electron affinity of 2D tin halide perovskites. However, determining ionization energy and electron affinity in HPs could be quite difficult as several methods are being used in data interpretation for HPs . In this work, we propose a method to assign the energy levels in 2D HPs by correcting for the instrumental resolution in ultraviolet and inverse photoemission spectroscopy.

The current standard of care (platinum-based drugs) for the treatment of different forms of malignancy have been very effective in the clinic, however the negative side effects associated with the administration of these platinum based-drugs remains an unsolved problem. Gold based molecules are among a few metal complexes that have been developed over the years in search for better chemotherapy drugs. While the anticancer mechanism of action of platinum-based drugs is well known to involve DNA damage, the mechanism of action of gold based small molecules remains a subject of debate. It is understood that gold-based complexes exhibit non-cisplatin like anticancer mechanism of action, hence the potential to overcome resistance seen in patients with recurrent tumors after initial remission with platinum-based drugs. Herein, we report efforts to elucidate the mechanism of action of novel gold-based anticancer agents with very potent inhibitory effect against triple negative breast cancers and ovarian cancer. A recurring observation from the mechanism of action studies is the perturbation of mitochondria physiology by these complexes. These includes; perturbation of mitochondria bioenergetics, depolarization of mitochondria membrane potential of the cells, increased mitochondria ROS production, depletion of mitochondria DNA, and disruption of mitochondria dynamics. Modified versions of the lead molecules were developed as probes to monitor in vitro localization of the complexes and facilitate elucidation of the mechanism of action. Target identification studies with a biotinylated lead complex unveiled heme oxygenase 2 (HMOX2) as a novel target in gold medicinal chemistry. Preliminary target validation studies revealed for the first time, HMOX2 as an upstream regulator of the MYC proto-oncogene. These findings uncover a new strategy for targeting tumor cells and reinforces the belief that small molecules can serve as probes to interrogate the complex cancer biology system and unveil new strategies for development of better chemotherapeutic agents.

The current standard of care (platinum-based drugs) for the treatment of different forms of malignancy have been very effective in the clinic, however the negative side effects associated with the administration of these platinum based-drugs remains an unsolved problem. Gold based molecules are among a few metal complexes that have been developed over the years in search for better chemotherapy drugs. While the anticancer mechanism of action of platinum-based drugs is well known to involve DNA damage, the mechanism of action of gold based small molecules remains a subject of debate. It is understood that gold-based complexes exhibit non-cisplatin like anticancer mechanism of action, hence the potential to overcome resistance seen in patients with recurrent tumors after initial remission with platinum-based drugs. Herein, we report efforts to elucidate the mechanism of action of novel gold-based anticancer agents with very potent inhibitory effect against triple negative breast cancers and ovarian cancer. A recurring observation from the mechanism of action studies is the perturbation of mitochondria physiology by these complexes. These includes; perturbation of mitochondria bioenergetics, depolarization of mitochondria membrane potential of the cells, increased mitochondria ROS production, depletion of mitochondria DNA, and disruption of mitochondria dynamics. Modified versions of the lead molecules were developed as probes to monitor in vitro localization of the complexes and facilitate elucidation of the mechanism of action. Target identification studies with a biotinylated lead complex unveiled heme oxygenase 2 (HMOX2) as a novel target in gold medicinal chemistry. Preliminary target validation studies revealed for the first time, HMOX2 as an upstream regulator of the MYC proto-oncogene. These findings uncover a new strategy for targeting tumor cells and reinforces the belief that small molecules can serve as probes to interrogate the complex cancer biology system and unveil new strategies for development of better chemotherapeutic agents.

Anaerobic bacteria and archaea thrive in seemingly inhospitable environments because they are extremely energy efficient. Their efficiency is based in large part on their ability to conduct electron transfer bifurcation ('bifurcation') at strongly reducing potentials, thereby producing extremely potent reducing agents able to fix nitrogen and make molecular hydrogen. This chemistry is made possible by the use of a flavin as the site of bifurcation, supported by a specialized protein environment and mechanisms that control the flow of individual electrons.

Bifurcating electron transfer flavoproteins (Bf-ETFs) are versatile protein modules that provide the bifurcating capability associated with several metabolic functions. Bf-ETFs enable use of low-energy electron reserves such as NADH to charge the carriers ferredoxin and flavodoxin with high-energy electrons. Bf-ETFs possess two flavin adenine dinucleotide (FAD) cofactors. The bifurcating FAD (Bf-FAD) receives two electrons from NADH, and distributes them through two distinct pathways. One pathway involves exothermic electron transfer to a high- potential acceptor via the second FAD, the ET-FAD (electron transfer FAD). This provides the driving force to send the second electron to a lower potential (higher-energy) acceptor.

Investigations described herein elucidated the crystal structure and internal dynamics of flavodoxin (Fld), a high-energy acceptor in the bifurcation process. 19F NMR was used to examine conformational heterogeneity and dynamics of Fld free in solution, to characterize the flexibility of a 20-residue stretch of Fld's peptide chain that is believed to mediate interaction between Fld and ETF. Temperature-dependent NMR studies, alongside paramagnetic relaxation investigations comparing Fld in both its oxidized and semi-reduced forms, detailed internal dynamics pivotal to Fld's interactions with diverse partner proteins.

Complementary research explored conformational dynamics of ETF, employing small-angle neutron scattering (SANS). This revealed notable divergence from published structures, demonstrating presence of a more extended conformation in solution. Significant reduction- triggered conformational change was also discerned via SANS by comparing the fully oxidized and reduced states of ETF. Molecular dynamics simulations-based data modeling suggests coexistence of multiple ETF conformations, ranging from extended to compact, in solution.

Finally, conformational consequences of complex formation between ETF and a partner protein were examined. We demonstrated isolation of a complex between ETF and its high- potential acceptor butyryl CoA dehydrogenase (BCD). Innovative application of segmental deuteration of BCD in combination with SANS, enabled comprehensive insights into the conformational adaptations made by ETF upon complex formation. Contrast variation SANS, utilizing 80% deuterated BCD, was used to identify the match point, paving the way for advanced analysis of the complex's structural dynamics.

This work enriches comprehension of the roles played by dynamics in bifurcation, and advances new technical approaches for future explorations of conformational changes within multidomain proteins.

Anaerobic bacteria and archaea thrive in seemingly inhospitable environments because they are extremely energy efficient. Their efficiency is based in large part on their ability to conduct electron transfer bifurcation ('bifurcation') at strongly reducing potentials, thereby producing extremely potent reducing agents able to fix nitrogen and make molecular hydrogen. This chemistry is made possible by the use of a flavin as the site of bifurcation, supported by a specialized protein environment and mechanisms that control the flow of individual electrons.

Bifurcating electron transfer flavoproteins (Bf-ETFs) are versatile protein modules that provide the bifurcating capability associated with several metabolic functions. Bf-ETFs enable use of low-energy electron reserves such as NADH to charge the carriers ferredoxin and flavodoxin with high-energy electrons. Bf-ETFs possess two flavin adenine dinucleotide (FAD) cofactors. The bifurcating FAD (Bf-FAD) receives two electrons from NADH, and distributes them through two distinct pathways. One pathway involves exothermic electron transfer to a high- potential acceptor via the second FAD, the ET-FAD (electron transfer FAD). This provides the driving force to send the second electron to a lower potential (higher-energy) acceptor.

Investigations described herein elucidated the crystal structure and internal dynamics of flavodoxin (Fld), a high-energy acceptor in the bifurcation process. 19F NMR was used to examine conformational heterogeneity and dynamics of Fld free in solution, to characterize the flexibility of a 20-residue stretch of Fld's peptide chain that is believed to mediate interaction between Fld and ETF. Temperature-dependent NMR studies, alongside paramagnetic relaxation investigations comparing Fld in both its oxidized and semi-reduced forms, detailed internal dynamics pivotal to Fld's interactions with diverse partner proteins.

Complementary research explored conformational dynamics of ETF, employing small-angle neutron scattering (SANS). This revealed notable divergence from published structures, demonstrating presence of a more extended conformation in solution. Significant reduction- triggered conformational change was also discerned via SANS by comparing the fully oxidized and reduced states of ETF. Molecular dynamics simulations-based data modeling suggests coexistence of multiple ETF conformations, ranging from extended to compact, in solution.

Finally, conformational consequences of complex formation between ETF and a partner protein were examined. We demonstrated isolation of a complex between ETF and its high- potential acceptor butyryl CoA dehydrogenase (BCD). Innovative application of segmental deuteration of BCD in combination with SANS, enabled comprehensive insights into the conformational adaptations made by ETF upon complex formation. Contrast variation SANS, utilizing 80% deuterated BCD, was used to identify the match point, paving the way for advanced analysis of the complex's structural dynamics.

This work enriches comprehension of the roles played by dynamics in bifurcation, and advances new technical approaches for future explorations of conformational changes within multidomain proteins.

Despite notable advancements in the design and synthesis of novel chemotherapeutic drugs in recent years, cancer continues to be a leading cause of mortality in the United States. Metallodrugs, especially gold complexes, have played a prominent role in the search for the next generation chemotherapeutic agents. Herein, I report on the synthetic strategies proffered towards rational design of stable gold-based agents, studied their reactivity in biological environment and proffer insights into the cytotoxic mechanism of action in diseased and normal cells. Stable gold complexes exist mostly in +I or +III oxidation states. Research into Au(I) complexes has overshadowed Au(III) complexes due to the reemergence of auranofin for treatment of other conditions including cancer. 

Despite been isoelectronic to cisplatin, Au(III) complexes exhibit different chemistry, reactivity, and molecular target compared to their platinum counterpart. My work has developed synthetic tools exploring the use of chiral bisphosphine ligands (Quinox P*) to synthesize cytotoxic Au(III) bisphosphine enantiomers that show similar response when administered to cancer cells. Further studies on chiral bisphosphine ligands using R-DuPhos ligands (where R is methyl or isopropyl) identified for the first-time products from the speciation studies of chiral Au(III) complex with L-glutathione (L-GSH), a biological reductant in cells. Furthermore, I carried out structure activity relationship studies with bisphosphine ligands. Thus, reacting different bisphosphines with di-µ-chlorido biphenyl digold(III), gold(III) bisphosphine complexes were obtained with the phosphine backbone or side chains dictating stability and reactivity. The complexes showed very high physiological stability in L-glutathione while also demonstrating serum stability for 24 h. Further mechanism studied showed that compared to cisplatin, these complexes act as mild mitochondria uncoupler comparable to other protonophores such as FCCP. This, to the best of my knowledge is the first Au(III) mitochondria uncoupling agent. Also, I studied the impact of degree of cyclometallation on two set of cyclometalated gold(III) complexes bearing either a phenylpyridine (C^N) or biphenyl (C^C) Au(III) backbone. While the neutral C^C complexes showed improved electrochemical and biological stability (in L-glutathione), they showed lower cellular responses in cancer cells when compared to the C^N counterpart. Furthermore, longer alkyl chain complexes were not soluble in biological media. To overcome physicochemical barriers encountered in long alkyl chain gold(III) dithiocarbamate, encapsulation with bovine serum albumen was carried out. This resulted in improved solubility, cytotoxicity, and cellular uptake of the drug into cancer cells