Pathogen-Specific Adaptations to Conserved Signaling Pathways in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH.

Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms.

The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi.

To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.

Systematic Molecular Phenotyping: A Path Toward Precision Emergency Medicine?

Precision medicine is an emerging approach to disease treatment and prevention that considers variability in patient genes, environment, and lifestyle. However, little has been written about how such research impacts emergency care. Recent advances in analytical techniques have made it possible to characterize patients in a more comprehensive and sophisticated fashion at the molecular level, promising highly individualized diagnosis and treatment. Among these techniques are various systematic molecular phenotyping analyses (e.g., genomics, transcriptomics, proteomics, and metabolomics). Although a number of emergency physicians use such techniques in their research, widespread discussion of these approaches has been lacking in the emergency care literature and many emergency physicians may be unfamiliar with them. In this article, we briefly review the underpinnings of such studies, note how they already impact acute care, discuss areas in which they might soon be applied, and identify challenges in translation to the emergency department (ED). While such techniques hold much promise, it is unclear whether the obstacles to translating their findings to the ED will be overcome in the near future. Such obstacles include validation, cost, turnaround time, user interface, decision support, standardization, and adoption by end-users.

Resonance Energy Transfer-Based Molecular Switch Designed Using a Systematic Design Process Based on Monte Carlo Methods and Markov Chains

A RET network consists of a network of photo-active molecules called chromophores that can participate in inter-molecular energy transfer called resonance energy transfer (RET). RET networks are used in a variety of applications including cryptographic devices, storage systems, light harvesting complexes, biological sensors, and molecular rulers. In this dissertation, we focus on creating a RET device called closed-diffusive exciton valve (C-DEV) in which the input to output transfer function is controlled by an external energy source, similar to a semiconductor transistor like the MOSFET. Due to their biocompatibility, molecular devices like the C-DEVs can be used to introduce computing power in biological, organic, and aqueous environments such as living cells. Furthermore, the underlying physics in RET devices are stochastic in nature, making them suitable for stochastic computing in which true random distribution generation is critical.

In order to determine a valid configuration of chromophores for the C-DEV, we developed a systematic process based on user-guided design space pruning techniques and built-in simulation tools. We show that our C-DEV is 15x better than C-DEVs designed using ad hoc methods that rely on limited data from prior experiments. We also show ways in which the C-DEV can be improved further and how different varieties of C-DEVs can be combined to form more complex logic circuits. Moreover, the systematic design process can be used to search for valid chromophore network configurations for a variety of RET applications.

We also describe a feasibility study for a technique used to control the orientation of chromophores attached to DNA. Being able to control the orientation can expand the design space for RET networks because it provides another parameter to tune their collective behavior. While results showed limited control over orientation, the analysis required the development of a mathematical model that can be used to determine the distribution of dipoles in a given sample of chromophore constructs. The model can be used to evaluate the feasibility of other potential orientation control techniques.

The Synthesis of Novel N-Heterocyclic Scaffolds and Diazirine-Based Molecular Tags

N-Heterocycles are ubiquitous in biologically active natural products and pharmaceuticals. Yet, new syntheses and modifications of N-heterocycles are continually of interest for the purposes of expanding chemical space, finding quicker synthetic routes, better pharmaceuticals, and even new handles for molecular labeling. There are several iterations of molecular labeling; the decision of where to place the label is as important as of which visualization technique to emphasize.

Piperidine and indole are two of the most widely distributed N-heterocycles and thus were targeted for synthesis, functionalization, and labeling. The major functionalization of these scaffolds should include a nitrogen atom, while the inclusion of other groups will expand the utility of the method. Towards this goal, ease of synthesis and elimination of step-wise transformations are of the utmost concern. Here, the concept of electrophilic amination can be utilized as a way of introducing complex secondary and tertiary amines with minimal operations.

Molecular tags should be on or adjacent to an N-heterocycle as they are normally the motifs implicated at the binding site of enzymes and receptors. The labeling techniques should be useful to a chemical biologist, but should also in theory be useful to the medical community. The two types of labeling that are of interest to a chemist and a physician would be positron emission tomography (PET) and magnetic resonance imaging (MRI).

Coincidentally, the 3-positions of both piperidine and indole are historically difficult to access and modify. However, using electrophilic amination techniques, 3-functionalized piperidines can be synthesized in good yields from unsaturated amines. In the same manner, 3-labeled piperidines can be obtained; the piperidines can either be labeled with an azide for biochemical research or an 18F for PET imaging research. The novel electrophiles, N-benzenesulfonyloxyamides, can be reacted with indole in one of two ways: 3-amidation or 1-amidomethylation, depending on the exact reaction conditions. Lastly, a novel, hyperpolarizable 15N2-labeled diazirine has been developed as an exogenous and versatile tag for use in magnetic resonance imaging.