Conventional wastewater treatment in the U.S. is an energy dependent and carbon dioxide emitting process. Typical mechanical systems consume copious amounts of energy, which is most commonly produced from fossil fuel combustion that results in the production of CO2. The associated organic load is also metabolized by microorganisms into CO2 and H2O. As the desire to reduce CO2 output becomes more prominent, it is logical to assess the costs of conventional treatment methods and to compare them to alternative, more sustainable technology. Vegetated Sand Bed (VSB) and Reed Bed (RB) systems are green technologies that provide environmentally superior treatment to conventional systems at a fraction of the cost both environmentally and economically. Using mass balance equations the net CO2 produced from wastewater treatment at 3 conventional facilities, (Amherst, MA, Ithaca, NY and Shelburne-Buckland, MA) and 3 VSBs, (Lloyd, NY, Shushufindi Slaughterhouse, Ecuador and Shushufindi Municipal Facility, Ecuador), will be estimated. Carbon dioxide sources considered are BOD5 microbial respiration, power demand, and sludge treatment. Using the BOD5 reduction and the average daily flow from each of the conventional facilities, hypothetical VSB and RB systems will be sized for the 3 conventional systems. The land area for each hypothetical VSB and RB and the CO2 reduction for equal treatment are estimated for each conventional facility. Estimates of annual CO2 production for Amherst, Ithaca, and Shelburne-Buckland, are 3,021 metric tons, 5,575 metric tons, and 158 metric tons of, respectively. The annual CO2 reduction potential for the conventional facilities Amherst, Ithaca, and Shelburne-Buckland, when compared to VSB and RB technology is estimated to be 74.0%, 83.2%, and 86.3% respectively. VSB and RB technology also provide promising results for sustainable wastewater treatment and reuse. Ammonium and nitrate reduction at the Joseph Troll Turf Plot VSBs were 72% and 88% respectively. The mean ammonium microbial growth rate constant was – 0.14 d-1 and the mean nitrate microbial growth rate constant was – 0.23 d-1. The implications are ammonium and nitrate reduction is possible with VSB and RB technology. Further investigation to understand the processes and fate of nitrogen including separate testing of ammonium and nitrate reduction are recommended.

ABSTRACT Oceano Community Health Plan Phillip Brennan Recent, mounting research shows that chronic disease, the leading causes of death and primary driver of health care costs, cannot be effectively addressed through education or preventative health alone. A physical environment that promotes health—through access to healthy food, opportunities for physical activity, quality housing, transportation options, and safe schools—is an integral part of making our communities healthier. This research and accompanying Healthy Community Plans will serve as a way for the County to begin looking in-depth at the ways the built environment (our streets, parks, and neighborhoods) contribute or detract from the health of the community. Though the creation of a healthy general plan may be unattainable for the County in the short term, a focus on a small yet cohesive part of the county presents an opportunity to affect these changes. Under the direction of the SLO County Health Agency and the Health Commission, we have written Healthy Community Plans for the unincorporated communities of Cayucos and Oceano, California. Both of these plans were greatly informed by their respective communities through input garnered through outreach, interviews, surveys and personal interactions with community members. This project examines the relationship between the built environment and public health, and explores ways planning professionals are beginning to address health issues through infrastructure, land use, creative zoning, and planning strategies that promote health and active living in policy. The planning documents, modeled after health elements currently being included in general plans throughout California, have integrated the fields of planning and public health to provide Cayucos and Oceano an assessment of its residents’ health, a description of the current built environment conditions that may be helping or hindering physical activity and access to nutritious food sources, as well as establish goals, policies and implementation strategies that will set a course of action toward healthier communities. Key Words: planning, public health, physical activity, built environment, community, active transportation

The best known correlate of protection provided by vaccines is the presence of pathogen specific antibodies after immunization. However, against the Human Immunodeficiency Virus-1 (HIV-1) the mere presence of antibodies specific for the viral Envelope (Env) protein is not sufficient to provide protection. This necessitates in depth study of the humoral responses elicited during infection and by vaccination. While a significant amount of effort has been invested in studying the evolution of antibody responses to viral infection, only limited progress in understanding antibody responses elicited through vaccination has been made. In the studies described here, I attempt to rectify this deficiency by investigating how the quality of a humoral response is altered with the use of different immunization regimens, in particular a DNA prime-protein boost regimen, or with the use of different model HIV-1 Env gp120 immunogens. In a New Zealand White (NZW) rabbit model, we demonstrate that the broader neutralizing activity elicited with the DNA prime-protein boost regimen may be the result of the elicitation of a higher avidity antibody response and a unique profile of antibody specificities. Specifically, use of a DNA prime-protein boost regimen elicits antibodies targeted to the CD4 binding domain of the HIV-1 Env, a specificity that was not frequently observed when only protein based immunizations were administered. We extended this analysis to sera from healthy human volunteers who participated in early phase HIV vaccine trials utilizing either a protein alone immunization regimen, a canarypox prime-protein boost immunization regimen, or a DNA prime-protein boost immunization regimen. Evaluation of sera from these trials demonstrated that the use of a DNA prime-protein boost regimen results in an antibody response with greater neutralization breadth characterized by an increased frequency and titer of antibodies targeted toward the CD4 binding site (CD4bs). In addition to this, the antibody response elicited by the DNA prime-protein boost regimen also exhibited the capability to mediate antibody dependent cell-mediated cytotoxicity (ADCC) activity as well as activation of the complement system. Additionally, in an attempt to better understand the capabilities of antibodies elicited by a DNA prime-protein boost regimen, we generated gp120 specific monoclonal antibodies (mAbs) from a single DNA primed-protein boosted NZW rabbit. Analysis of mAbs produced from this animal revealed that use of this immunization regimen elicits an antibody repertoire with diverse epitope specificity and cross reactivity. Furthermore, these select mAbs are capable of neutralizing heterologous HIV isolates. Further application of mAb generation in rabbits may provide a valuable tool to study immunogenicity of different vaccines and immunization regimens. Concurrently, while demonstrating that a DNA prime-protein boost regimen elicits a higher quality antibody response than that observed with other leading techniques, we also demonstrated that immunogen selection can play a vital role in the quality of the resulting antibody response. By immunizing with two closely related but phenotypically distinct model gp120 immunogens, known as B33 and LN40, we demonstrated that disparate gp120s have different intrinsic abilities to raise a heterologous neutralizing antibody response. Additionally, we showed that residues found within and flanking the b12 and CD4 binding sites play critical roles in modulating neutralizing activity of sera from animals immunized with LN40 gp120, indicating that the broader neutralizing activity seen with this immunogen may be due to differential elicitation of antibodies to this domain.

In this dissertation, the roles of endocytosis and phagocytosis pathways in a variety of clinically relevant scenarios were examined. These scenarios include antibody-mediated internalization of cell surface proteins, titanium wear-particle uptake in failed joint replacements, and polymeric microparticle uptake and immune responses for drug delivery or adjuvant use. The use of antibodies specific for cell surface proteins has become a popular method to deliver therapeutics to target cells. As such, it is imperative to fully understand the ability of antibodies to mediate internalization and endosomal trafficking of the surface protein that it recognizes, so that drug delivery can be optimized. By comparing the internalization and endosomal localization of two different antibody-bound proteins, the transferrin receptor (TfR) and rabies G, we have found that there is a specific antibody-mediated internalization pathway that occurs when an antibody binds to a cell surface protein. Interestingly, the internalization pathway induced by antibody binding is different than that seen with recycling receptor internalization after ligand binding. This may have broad implications for the future development of antibody-based therapeutics. Joint replacement failure is a major clinical problem. Studies have indicated that a large amount of metal and polyethylene wear debris is found in the synovial membrane and tissue surrounding failed replacements. Through examination of the immune response following uptake of titanium particles, our results suggest that titanium wear-particle induced inflammation and subsequent joint replacement failure may be due to activation of the NLRP3 inflammasome, leading to increased IL-1ß secretion and IL-1 associated signaling. These findings introduce IL-1 as a target for potential therapeutics for patients exhibiting significant inflammation. Polymeric microparticles have been widely used in a variety of therapeutic applications, including drug delivery and vaccine adjuvants. It is essential to understand the ability of such particles to either activate or inhibit an immune response following uptake. Through comparison of particles with varying surface morphology, we have determined that particles with regions of high surface curvature (budding) are more immunogenic than particles with low surface curvature (spherical). Budding particles were more rapidly phagocytosed and induced higher levels of the inflammasome-associated cytokine, IL-1ß, when exposed to mouse macrophages. Additionally, budding particles induced a more rapid neutrophil response in vivo, when compared to spherical particles. These findings have broad implications for the development of future targeting vehicles for delivery of vaccines, drugs, proteins, and siRNA therapeutics.

Virus infection is sensed by the innate immune system through germline encoded pattern recognition receptors (PRRs). Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) serve as PRRs that recognize different viral components. Microbial nucleic acids such as Ribonucleic acid (RNA) are important virus-derived pathogen-associated molecular patterns (PAMPs) to be recognized by PRRs. Virus recognition may occur at multiple stages of the viral life cycle. Replication intermediates such as single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) are detected by the RNA-sensing PRRs that initiate innate and adaptive immune responses. Triggering of the innate immune system is a critical event that can shape the adaptive immune response to virus infection. Better vaccination strategies that lead to improved T-cell and antibody responses are needed for protection against pathogens. We sought to delineate the RNA-sensing PRR pathways that are activated during infection with an RNA virus, the signaling mediators involved and the influence on subsequent virus-specific adaptive immune responses. To analyze the role of RNA-sensing PRRs in T-cell immune responses in vitro, we performed direct co-stimulation experiments on CD4+ T-cells of high purity. We utilized synthetic RNA-like immune response modifiers (IRMs) R-848 (MyD88-dependent) and poly I:C (MyD88-independent) as RNA PAMPs to determine the direct effects of RNA-sensing PRR activation on CD4+ T-cells. RNA PAMPs can act directly on CD4+ T-cells and modulate their function and phenotype. Maximal direct co-stimulatory effects were observed in CD4+ T-cells cultured with poly I:C compared to R-848. The cytoplasmic dsRNA-dependent protein kinase R (PKR) was also involved in poly I:C-mediated signaling in CD4+ T-cells. We found differences in the RNA-sensing PRRs activated by R-848 between mouse and human CD4+ T-cells. We observed minimal direct co-stimulatory effects by R-848 in mouse CD4+ T-cells. In contrast, augmentation of Th1 responses by R-848 was observed in human CD4+ T-cells. TLR8 activation in human CD4+ T-cells may explain the observed differences. We next explored the signaling pathways activated by RNA PAMPs in conventional dendritic cells (cDCs) and CD4+ T-cells that drive Th1 CD4 T-cell responses in isolated cDC/CD4 T-cell interactions. Allogeneic cDCs and CD4+ T-cells of high purity were cultured together with R-848 and poly I:C in MHC congenic mixed leukocyte reactions (MLRs). R-848 and poly I:C stimulation of type I IFN production and signaling was essential but not sufficient for driving CD4+ Th1 responses. The early production of IL-1α and IL-1β was equally critical. To analyze the role of RNA-sensing PRRs in T-cell immune responses in vivo, we utilized a mouse model of heterosubtypic influenza A virus (IAV) infections. Using MyD88-/-, TLR7-/- and IL-1-deficient mice, we explored the role of MyD88-signaling in the generation of heterosubtypic memory CD4+ T-cell, CD8+ T-cell and antibody responses. We found that MyD88 signaling played an important role in anti-IAV spleen and lung CD4+ T-cell, spleen CD8+ T-cell and Th1 antibody immune responses. Anti-IAV lung heterosubtypic CD8+ T-cell responses were not dependent on MyD88 signaling. Our in vitro and in vivo results show the pivotal role of RNA-sensing PRR pathway activation in T-cell immune responses. Understanding the complexity of the PRR pathways involved during viral infections and defining the subsequent immune response would have important implications for the generation of more effective vaccine strategies.

N. meningitidis is a common commensal of the human upper respiratory tract and a leading cause of bacterial meningitis and septicemia worldwide. The classical pathway of complement (C) is essential for both naturally acquired and vaccine induced immunity against N. meningitidis. Qualitative and/or quantitative differences in anti-meningococcal antibodies (Abs) in serum is one reason for variations in C-dependent bactericidal Ab activity among individuals. I showed that IgG isolated from select individuals could block killing of group B meningococci by Abs that were otherwise bactericidal. Ligand overlay immunoblots revealed that these blocking IgG Abs were directed against a meningococcal antigen called H.8, Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when blocking Ab binding to meningococci was inhibited (or competed for) using either synthetic peptides corresponding to H.8 or a non-blocking mAb against H.8. Further, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab)2 fragments alone generated by pepsin treatment were ineffective. Blocking required IgG glycosylation; deglycosylation of blocking IgG with peptide:N-glycanase (PNGase) eliminated blocking. C4 deposition mediated by a bactericidal mAb directed against a meningococcal vaccine candidate, called factor H-binding protein (fHbp), was reduced by blocking Ab. Anti-fHbp-mediated C4 deposition was unaffected, however, by deglycosylated blocking IgG. Although preliminary, our data suggests blocking of serum bactericidal activity by human anti-H.8 blocking antibody may require mannan-binding lectin (MBL), which itself is a complement activator. Also, whether MBL recruits a complement inhibitor(s) that facilitates blocking remains to be determined. In conclusion, we have identified H.8 as a meningococcal target for novel blocking antibodies that are commonly found in human serum. Blocking Ab may reduce the efficacy of meningococcal vaccines. We propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.

Human papillomavirus infection is the cause of more than 99% of cervical cancer cases. The current vaccine is ineffective therapeutically; highlighting the need for continued papillomavirus research. One avenue that could be explored in this regard is the function of the papillomavirus E2 regulatory proteins. HPV E2 represses expression of the viral E6 and E7 oncoproteins. Reintroduction of E2 into cervical carcinoma cells results in growth arrest and cellular senescence. Understanding the mechanism of how E2 regulates the early promoter may be key to developing new therapeutic and prophylactic vaccines. Here, we describe regulation of E2 through acetylation and possibly through direct interaction with a novel cellular interacting protein, RINT1. Histone acetyltransferase (HAT) proteins have been demonstrated to interact with Bovine Papillomavirus (BPV) and Human Papillomavirus (HPV) E2 proteins as well as enhance E2 dependant transcription luciferase reporter plasmid containing E2 binding sites. We demonstrate that HATs p300, CBP, and pCAF are limiting for E2 dependant transcriptional activation and that each protein functions independently. We have also identified that BPV-1 E2 is a substrate for acetylation by p300. Mutants of E2 that cannot be acetylated on lysines 111 or 112, display abnormal transcriptional phenotypes. Cells deficient in p300 display similar transcriptional defects that are intensified by CBP depletion. We propose that acetylation of BPV-1 E2 is necessary for transcriptional activation. Acetylation generates a binding site through which a co-factor may interact via a bromodomain. Regulation of E2 dependent transcriptional activation through a post-transcriptional modification represents a novel method through which BPV-1 controls gene expression. We also present evidence for a direct interaction between BPV-1 E2 and the cellular factor RINT1. This interaction does not appear to be critical for transcriptional regulation; however, several other functional pathways are indicated by the cellular complexes in which RINT1 functions. Some of these, such as ER/Golgi vesicular transport and hTERT independent telomere maintenance, are pathways in which E2 has no known role. Further investigation into regulation and consequences of E2 acetylation and the biological significance of the interaction between E2 and RINT1 could prove important in understanding the complex role of E2 in papillomavirus infection.

This thesis explores three state-run green building programs (California, Minnesota and New York) through comparative case-study analysis. Main topics discussed include cost benefits, health benefits, certification (i.e. LEED or other), and lessons learned from direct interviews with program staff.

The need for access to clean, healthy water is a subject that surrounds the world. The United States is fortunate to have the latest technology for access to water and treatment of water; however, one subject that hits at the heart of the nation is water security. A breach in the water systems in the U.S. would cause direct public harm because the population relies on water to sustain life. Protecting potable water systems is necessary because of natural disasters, but it has gained importance since the September 11, 2001 attacks on the U.S. Threats on the water systems from intentional attacks cause high risk in affecting public health. Water Security is an area of defense that has recently been evolving and research in this subject has increased. Detection methods are of high importance in protecting from contamination. This thesis determines the most detrimental contaminants; discusses the importance of detection methods, sensor networks, and Contamination Warning Systems (CWS); provides prevention and detection devices currently available; and explains in depth the threats, prevention, detection, response, and rehabilitation associated with water contamination events. NOTE: Both archival copies of the manuscript were missing pages 208-210.

Cities within California are beginning to incorporate urban agriculture into their land use designations. Prompted by residents and local organizations, cities are hoping to capture the benefits that urban agriculture provides. Research has shown that urban agriculture renews and beautifies neighborhoods, provides healthy food choices, increases public health, has the potential to help with stormwater runoff, creates jobs, and fosters community. In the last few years, several California cities have made headlines as they have adopted new zoning codes that include urban agriculture. In reviewing these new zoning codes and exploring the topic of urban agriculture, it became evident that just because an urban farm was small, organic and provided certain benefits that it was not free from impacting its surroundings. As more urban agricultural ventures are established within cities, planners have to carefully consider their effect. One such impact could be stormwater pollution. There is insufficient research to determine whether there is a relationship between urban agriculture and stormwater, however, studies on conventional agriculture and urban landscaping (mainly urban lawns) show that each of these areas pollute the local water bodies with sediment, chemicals, and nutrients. Is urban agriculture different? This thesis utilizes two case studies within California, the City of Oakland and the City of San Diego, to examine the similarities and differences between each city’s urban agriculture ordinances and evaluate whether or not the cities have adjusted stormwater requirements in parallel with these ordinances. Interview responses and site visits in each city were analyzed and compared to expound upon the approaches each city engaged. Using the collected data and analysis as a base, a set of guidelines was created for managing stormwater runoff from urban agriculture.