This work is divided into two parts : (I) cell surface binding, uptake, and storage of iron in brown algae, and (II) boron uptake, localization, and speciation in brown algae. Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. Although the iron uptake and storage mechanisms of terrestrial/higher plants have been well- studied, the corresponding systems in marine algae have received far less attention. Chapter I focuses on these mechanisms in two brown seaweeds : Ectocarpus siliculosus and Macrocystis pyrifera. Through the course of experiments, it was found that a significant amount of iron was bound extracellularly. While this phenomenon is widely recognized and has prompted the development of experimental protocols to eliminate its contribution to iron uptake studies, its potential biological significance as a concentrated iron source for marine algae is only now being recognized. In this study, using an interdisciplinary array of techniques, we explore the nature of the extensive and powerful iron binding on the surface of both laboratory and environmental samples of Ectocarpus and Macrocystis. We propose that the surface binding properties of Ectocarpus and Macrocystis allow it to function as a quasi-biological metal ion "buffer" facilitating iron uptake under the widely varying external iron concentrations found in coastal marine environments. Short-term radiolabeled iron uptake studies verified that iron is taken up in a time- and concentration-dependent manner consistent with an active transport process. Using a combination of Mössbauer and XAS spectroscopies, a mineral core has been identified as the likely storage form. Genomic and microscopic analyses indicate that this iron store is more likely vacuolar-based than of protein origin. In chapter II, I describe boron uptake, speciation, localization and possible biological function in Macrocystis and Ectocarpus. In contrast to the generally boron-poor terrestrial environment, the concentration of boron in the marine environment is relatively high (0.4 mM) and while there has been extensive interest in its use as a surrogate of pH in paleoclimate studies in the context of climate change-related questions, the relatively depth independent, and the generally non- nutrient-like concentration profile of this element have led to boron being neglected as a potentially biologically relevant element in the ocean. Results herein indicate that boron is taken up by a facilitated diffusion mechanism against a considerable concentration gradient. Furthermore, in both Ectocarpus and Macrocystis some boron is most likely bound to the cell wall constituent alginate and the photoassimilate mannitol located in sieve cells. These results indicate possible biological roles for boron as an osmoprotectant and/or metabolite transporter

The following document contains the results of Ph.D. thesis research that was focused toward the complete structural characterization of molecules that were effective in the treatment and prevention of cancer. The use of marine sediment-derived actinomycetes as a source for the chemical entities led to the isolation of three suites of molecules with unprecedented carbon skeletons each of which displayed some anticancer properties. In the first project, three cancer cell cytotoxins, piperazimycins A-C (1-3), were isolated from the fermentation broth of a Streptomyces sp., cultivated from marine sediments near the island of Guam.¹ The structures of these cyclic hexadepsipeptides were assigned by a combination of spectral, chemical and crystallographic methods. The piperazimycins were found to be composed of rare amino acids, including hydroxy-acetic acid, [alpha]- methyl-serine, [gamma]-hydroxypiperazic acid and [gamma]- chloropiperazic acid. The novel amino acid residues 2- amino-8-methyl-4,6-nonadienoic acid and 2-amino-8-methyl-4, 6-decadienoic acid were found as components of piperazimycins A and C, respectively. When screened in the National Cancer Institute's 60 cancer cell line panel, piperazimycin A exhibited in vitro cytotoxicity toward multiple tumor cell lines with a mean GI₅₀ of 100 nM. In the second project, chemical evaluation of the saline fermentation broth of several strains of the obligate marine actinomycete Salinispora arenicola led to the identification of three new macrolide polyketides designated arenicolides A-C (1-3). The planar structures, elucidated via spectroscopic and chemical methods, consisted of 26-membered polyunsaturated macrolactones containing repeating vicinal hydroxyl methoxyl moieties. The stereochemistries of 1-3 were assigned by a combination of J-based configurational analyses and chemical derivatization. The arenicolides displayed moderate cytotoxicity in an in vitro colon adenocarcinoma cell-line (HCT-116) assay and also in the National Cancer Institute's 3 cancer cell line panel. Due to their structural novelty, the NCI has also recently agreed to reevaluate the cytotoxic activity of the arenicolides in the 60 cancer cell line panel. The final chapter is focused on the characterization of a novel class of likely polyketide derived molecules that exhibit inhibitory activity against the enzyme aromatase. These potent aromatase inhibitors, the pyridinopyrones, were isolated from a marine sediment-derived Streptomyces sp and their chemical structures were solved by various spectral methods

Photoreceptors are highly specialized sensory neurons with unique metabolic and physiological requirements. These requirements are partially met by Müller glia and cells of the retinal pigment epithelium (RPE), which provide essential metabolites, phagocytose waste, and control the composition of the surrounding microenvironment. A third vital supporting cell type, the retinal microglia, can provide photoreceptors with neurotrophic support or exacerbate neuroinflammation and hasten neuronal cell death. Understanding the physiological requirements for photoreceptor homeostasis and the factors that drive microglia to best promote photoreceptor survival has important implications for the treatment and prevention of blinding degenerative diseases like retinitis pigmentosa and age-related macular degeneration.

Measuring animal behavior in the context of experimental manipulation is critical for modeling, and understanding neuropsychiatric disease. Prepulse inhibition of the acoustic startle response (PPI) is a behavioral phenomenon studied extensively for this purpose, but the results of PPI studies are often inconsistent. As a result, the utility of this phenomenon remains uncertain. Here, we deconstruct the phenomenon of PPI and confirm several limitations of the methodology traditionally utilized to describe PPI, including that the underlying startle response has a non-Gaussian distribution, and that the traditional PPI metric changes with different stimuli. We then develop a novel model that reveals PPI to be a combination of the previously appreciated scaling of the startle response, as well as a scaling of sound processing. Using our model, we find no evidence for differences in PPI in a rat model of Fragile-X Syndrome (FXS) compared with wild-type controls. These results in the rat provide a reliable methodology that could be used to clarify inconsistent PPI results in mice and humans. In contrast, we find robust differences between wild-type male and female rats. Our model allows us to understand the nature of these differences, and we find that both the startle-scaling and sound-scaling components of PPI are a function of the baseline startle response. Males and females differ specifically in the startle-scaling, but not the sound-scaling, component of PPI. These findings establish a robust experimental and analytical approach that has the potential to provide a consistent biomarker of brain function.

Neuroinflammation commonly accompanies neurodegeneration, but the specific roles of resident and infiltrating immune cells during degeneration remains controversial. Much of the difficulty in assessing myeloid cell-specific functions during disease progression arises from the inability to clearly distinguish between activated microglia and bone marrow-derived monocytes and macrophages in various stages of differentiation and activation within the central nervous system. Using an inducible model of photoreceptor cell death, we investigated the prevalence of infiltrating monocytes and macrophage subpopulations after the initiation of degeneration in the mouse retina. In vivo retinal imaging revealed infiltration of CCR2⁺ leukocytes across retinal vessels and into the parenchyma within 48 hours of photoreceptor degeneration. Immunohistochemistry and flow cytometry confirmed and characterized these leukocytes as CD11b⁺CD45⁺ cells. Single-cell mRNA sequencing of the entire CD11b⁺CD45⁺ population revealed the presence of resting microglia, activated microglia, monocytes, and macrophages as well as 12 distinct subpopulations within these four major cell classes. Our results demonstrate a previously immeasurable degree of molecular heterogeneity in the innate immune response to cell-autonomous degeneration within the central nervous system and highlight the necessity of unbiased high-throughput and high-dimensional molecular techniques like scRNAseq to understand the complex and changing landscape of immune responders during disease progression.

Microglia respond to damage and microenvironmental changes within the central nervous system by morphologically transforming and migrating to the lesion, but the real-time behavior of populations of these resident immune cells and the neurons they support have seldom been observed simultaneously. Here, we have used in vivo high-resolution optical coherence tomography (OCT) and scanning laser ophthalmoscopy with and without adaptive optics to quantify the 3D distribution and dynamics of microglia in the living retina before and after local damage to photoreceptors. Following photoreceptor injury, microglia migrated both laterally and vertically through the retina over many hours, forming a tight cluster within the area of visible damage that resolved over 2 wk. In vivo OCT optophysiological assessment revealed that the photoreceptors occupying the damaged region lost all light-driven signaling during the period of microglia recruitment. Remarkably, photoreceptors recovered function to near-baseline levels after the microglia had departed the injury locus. These results demonstrate the spatiotemporal dynamics of microglia engagement and restoration of neuronal function during tissue remodeling and highlight the need for mechanistic studies that consider the temporal and structural dynamics of neuron-microglia interactions in vivo.

Background

The ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging.

Methods

We recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1-GFP line (Cx3cr1^+/GFP), naïve microglia in Cx3cr1-Dendra2 mice (Cx3cr1^+/D2) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo.

Results

Dendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration.

Conclusions

The ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1-Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation.

Men with high-grade prostate cancer (HGPC) are at greatest risk of disease progression. Clinical risk factors associated with castration-resistant prostate cancer (CRPC), metastases, and prostate cancer-specific mortality (PCSM) were identified in a contemporary HGPC cohort. Clinical data was collected from men diagnosed with Gleason sum (GS) ≥8 at the Greater Los Angeles Veterans Affairs (GLA-VA) Healthcare System between 2000 and 2013. Multivariable competing risks regression analyses assessed progression to CRPC, metastases, and PCSM within three treatment strata. The cumulative incidence of disease progression was calculated at 2, 5, and 10-year time points. Review of 2149 prostate cancer cases yielded 322 with HGPC. Median survival times for cancer-specific and overall mortality were significantly shorter in men treated with primary androgen deprivation therapy (ADT) (P = 0.0002 and P < 0.0001). Multivariable analyses revealed that clinical stage N1, GS 10, and treatment with primary ADT were significantly associated with increased risk of CRPC, metastases, and PCSM. Significant differences in these outcomes were not observed in men treated with radical prostatectomy (RP) when compared to those treated with radiation therapy combined with short-term ADT (XRT-ADT). Ten-year event rates of progression to CRPC, metastases, and PCSM, for men treated with primary ADT, were 45.5%, 25.4%, and 25.1%, respectively. In conclusion, GS 10 and lymph node involvement, as well as primary ADT treatment in men with HGPC was associated with increased risk of CRPC, metastases, and PCSM. Curative-intent treatment with RP or XRT-ADT is associated with reduced progression rates and death in men with HGPC.

Background

Depression and anxiety-related disorders are common among adolescents. Research attention to early adolescence and low-income ethnically diverse populations is limited.

Aim

To conduct screening for depression and anxiety at an early age with attention to gender and socioenvironmental context within a low-income setting.

Method

Mixed methods included the PHQ-9A and GAD-10 screening instruments and ethnographic interviews.

Results

75 ethnically diverse middle school students were included. Mean years age was 11.2 (0.74). Females had higher PHQ-9A sum scores than males (p = .002, Mann-Whitney test) and higher GAD-10 sum scores than males (p = .016, Mann-Whitney test). After controlling for multiple comparisons, girls had higher mean responses on three PHQ-9A items (p < .006, two-sided t-test) and only one GAD-10 item (p < .005, two-sided t-test). Ethnographic interviews revealed contexts associated with girls' experiences of depression and anxiety, including gender-based violence in both school and home environments. Salient for girls and boys alike were worries about consequences of COVID-19 for family with respect to illness, death, job loss, economic hardship. Fears over student perceptions of intensified discrimination and racism in school and community were prominent. These problems were experienced by students as barriers to educational engagement.

Conclusion

Specific attention to early adolescence is needed to identify emergence of subsyndromal conditions which may benefit from therapeutic attention to reduce symptom severity, identify sociocultural, structural, and gender-specific stressors, and to enhance educational engagement.