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Historically, the role of immune cells in the nervous system was predominantly examined throughthe lens of disease. In recent years, studies have shown that the complex, orchestrated events of immune activity throughout embryonic and postnatal critical periods are crucial for proper nervous system development. While previous studies have suggested limited immune heterogeneity in the adult brain, the diverse roles of the hippocampus in cognition and pathological development would suggest variation of immune cells in this region. Specifically, the hippocampus is known to be a site of adult neurogenesis. However, fundamental traits of immune cells in this region have not been well characterized. In chapter one, I present a summary of literature that discusses what was previously known of immune regulation of adult neurogenesis during health and disease. In chapter two, I compare different reporter lines and marker genes to evaluate responses in various cell types in the neurogenic niche and in other regions of the brain in the context of injury and pharmacological modulation. I discuss preliminary evidence suggesting microglial depletion may result in phenotypic changes in astrocytes throughout the hippocampus. In chapter three, I provide evidence of heterogeneity in myeloid-lineage cells in the hippocampus. I leveraged the highthroughput nature of cell suspension based single cell RNA-sequencing to collect transcriptomes of over 20,000 myeloid lineage cells from murine hippocampi. Using a series of bioinformatic techniques, I was able to computationally dissect different populations within this system and found spatial mapping of one distinct subset specifically localized to the neurogenic niche of the hippocampus. The transcriptomic signature of these cells alongside immunoreactivity to candidate genes, and morphological properties of this population resemble those of reactive microglia associated with the restriction of neurodegenerative diseases. In chapter four, I discuss how the immune landscape of the hippocampus responds to perturbation using a model of Focused Ultrasound mediated Blood-Brain Barrier opening. Subtypes of myeloid lineage cells change in composition and in transcriptomic response. We find distinct, temporally defined transcriptional responses in microglial and macrophage populations, indicating discrete roles for microglia and macrophages in immune activity during the transition from acute to chronic response. Together, these findings point towards diverse properties of microglia in the adult hippocampus.

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Many signaling pathways which are classically understood for their roles in early development are also known to be involved in tissue maintenance and adult energy homeostasis. Furthermore, dysfunction of these signaling pathways results in human diseases such as cancer. An in depth understanding of how developmentally important signaling pathways function in the adult will provide mechanistic insights into disease and potential new therapeutic targets. Herein Chapter 1, the Wnt, fibroblast growth factor (FGF), and Hedgehog (Hh) signaling pathways are discussed and examples of their relevance in development, adult homeostasis, and disease are provided. Wnt signaling provides an example of this concept as it has well described roles during both development and adult metabolism.<div><br></div><div> Work included in Chapter 2, investigates the regulation of adult energy homeostasis by a member of the endocrine FGF family, FGF19. The three endocrine FGFs, FGF19 (FGF15 in mice), FGF21, and FGF23 have well described roles in the regulation of metabolic processes in adults. While FGF23 is primarily involved in the regulation of phosphate and vitamin D homeostasis,FGF19 and FGF21 have shown similar pharmacological effects on whole body metabolism. Here, the importance of adaptive thermogenesis for the pharmacological action of FGF19 is explored. UsingUCP1KO animals we show that whole-body thermogenesis is dispensable for body weight loss following FGF19 treatment.<br></div><div><br></div><div>Finally, the potential involvement of Hh signaling in mediating the hyperphagia driven obesity observed in certain ciliopathies is explored in Chapter 3. Emerging evidence suggests cilia play an important role in the regulation of feeding behavior. In mammals, the hedgehog pathway is dependent on the primary cilium as an organizing center and defects in hedgehog signaling share some clinical symptoms of ciliopathies. Here, we characterized the expression of core pathway components in the adult hypothalamus. We show that neurons within specific nuclei important for regulation of feeding behavior express Hh ligand and members of its signaling pathway. We also demonstrate that the Hh pathway is transcriptionally upregulated in response to an overnight fast. This work provides an important foundation for understanding the functional role of Hh signaling in regulation of energy homeostasis. In its entirety, this work highlights the emerging clinical relevance of developmentally critical pathways in diseases associated with dysfunction of adult tissue homeostasis, such as obesity.<br></div>

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Traumatic brain injury, peripheral neuropathies, and other neurodegenerative diseases exhibit diverse clinical manifestations but are connected by their underlying trigger, axonal degeneration. These diseases cause extensive morbidity and mortality worldwide, as treatments are palliative and no curative treatments exist. SARM1 has recently emerged as a therapeutic target for these diseases as knockdown prevents axonal degeneration and ameliorates disease prognosis. Later, it was shown that SARM1 hydrolyzes NAD+ in response to degenerative stressors. Given that NAD+ supplementation delays axonal degeneration, we expect therapeutically targeting SARM1 will be efficacious for neurodegenerative diseases. However, the design of SARM1 therapeutics is limited by the dearth of knowledge surrounding its NAD+ hydrolase activity and active structural state. Illuminating this black box has been hindered by technical difficulties in obtaining pure active protein. To circumvent these issues, I began by studying SARM1 in lysates. I synthesized truncated constructs and developed three different assays, which enabled me to characterize the kinetic activity. I also established a high–throughput screening pipeline to identify inhibitors and screened >4,000 compounds. Recently, I identified additives (i.e., PEG and citrate) that activate SARM1 by ~2,000–fold, making it feasible to study the purified protein. I found that the additives enhance activity by inducing SARM1 to form a multimeric precipitate. To further interrogate the role multimerization plays in activity, I performed detailed mutagenesis and cell culture studies. The insights from this thesis have aided in our understanding of this elusive enzyme and provided strategic direction for future SARM1 investigation and drug development.

A major challenge in developing disease altering therapeutics for the treatment of Parkinson’s disease (PD) has been the delivery of compounds across the blood-brain barrier (BBB) to the central nervous system (CNS). While direct surgical infusion has been utilized to deliver compounds to the brain that don’t cross the BBB, issues of poor biodistribution in the CNS due in part to properties of the molecules being delivered and/or infusion device protocols have limited the widespread success of this invasive approach. To avoid the issues of surgically delivering compounds to the CNS, numerous studies have examined the use of intranasal administration as a non-invasive delivery method. The data presented in this dissertation examines intranasal administration of dopamine neuron stimulating peptide-11 (DNSP-11), a small, amidated peptide with neuroprotective and restorative properties, and its effects on the nigrostriatal system in animal models of PD. Here we demonstrate that severely lesioned 6-hydroxydopamine (6-OHDA) F344 rats repeatedly administered DNSP-11 intranasally exhibited a decrease in damphetamine- induced rotation, dopamine (DA) turnover, and an increase in tyrosine hydroxylase positive neuronal sparing. Additionally, tracer studies indicated rapid distributed throughout the CNS and CSF following a one-time bilateral intranasal dose of 125I-labeled DNSP-11. These results demonstrate that DNSP-11 can be delivered to the CNS intranasally, and maintains its neuroactive properties on the nigrostriatal system in a rat model of PD. In a dose escalation study of DNSP-11, we evaluated the efficacy of repeated intranasal administration in awake, vertically chaired trained, 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) hemiparkinsonian rhesus macaques using an atomizer system over a 10-week period. Here we report that animals did not exhibit observable adverse effects at the DNSP-11 concentrations examined, bilateral increases in fine motor performance of the upper limbs, and changes in tissue levels of DA and its metabolites. Finally, tracer studies indicated signal present throughout the CNS and CSF following a one-time bilateral intranasal dose of 125I-labeled DNSP-11. These studies support the efficacy of the repeated intranasal administration of DNSP-11 in awake Rhesus macaques over 10-weeks, while also enhancing motor performance and striatal neurochemistry in a non-human primate model of PD.

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Plant physiologist Bohumil Němec is one of the most important people in the history of Czech science. In this thesis, crucial concepts and theories that B. Němec developed in his scientific and popularizing publications are described and put in context. In addition, materials from the personal fond of B. Němec located in the Archive of the CAS were used. Particular questions tackled in this thesis are those of stimuli transmission in plants, the starch-statolith theory of gravitropism, morphaesthesia, organogenes, Němec's approach to evolutionary theory, and genetics, and the division of organisms in two groups. Moreover, Němec's role in the contemporary debates about vitalism, mechanism, and the effect of these two philosophies on the approach towards scientific research is indicated. By that, Němec's transition from the mechanism to the so-called physiology of stimuli (Rheizphysiologie) is illustrated. The outcome of the thesis is broadening the knowledge in the history of plant physiology, especially the history of the natural sciences in the Czech context.