Bovine virus diarrhea (BVD) is a complicated disease to discuss as it can result in a wide variety of disease problems from very mild to very severe. BVD can be one of the most devastating diseases cattle encounter and one of the hardest to get rid of when it attacks a herd. The viruses that cause BVD have been grouped into two genotypes, Type I and Type II. The disease syndrome caused by the two genotypes is basically the same, however disease caused by Type II infection is often more severe. The various disease syndromes noted in cattle infected with BVD virus are mainly attributed to the age of the animal when it became infected and to certain characteristics of the virus involved.

Diseases caused by BVD infection

Fetal BVD infections (infection of the unborn calf): The result of a fetal infection with the BVD virus is usually determined by the age of the fetus at the time of infection. The virus is capable of passing from an infected cow to the unborn fetus which is particularly vulnerable to the BVD virus during the first 6 months of pregnancy. Death of the fetus is common if the infection occurs during the first 120 days of pregnancy and the cow will lose the pregnancy. However, if the fetus survives an early infection, it will be born without a detectable antibody titer and be persistently infected (PI) with the BVD virus. During the first 120 days of gestation, the fetus has an underdeveloped immune system and does not recognize the BVD virus as foreign. The fetus does not mount an immune response against the virus, remains infected, and does not have a detectable anti-BVD titer. It is not uncommon for the surviving fetus to be malformed; blindness, skeletal abnormalities and under-developed brains are common defects noted in such calves. A BVD PI calf may appear normal, be weak at birth, grow poorly, be susceptible to respiratory diseases, and die before they can be weaned. They may also grow normally, reach breeding age, and produce more persistently BVD infected calves (The virus is passed from generation to generation). PI carriers can only be created by infection with BVD virus during the first 110-120 days of pregnancy. These animals shed billions of virus particles every day in their urine, feces, and saliva, and are a source of infection for other animals in the herd. If the fetus becomes infected after 120 days of pregnancy, there may be an abortion but usually, because this aged fetus has a more developed immune system and can elicit an immune response against the BVD virus, a healthy calf is born that has a good level of BVD antibody titer.

Subclinical BVD infections: Most animals that become infected with BVD never show signs of disease caused by the virus; however infection can lower the animal’s resistance to other infections, which could result in illness. For example, in feedlot calves, BVD infection may go unnoticed, but the lungs become susceptible to infection with bacteria such as Mannheimia haemolytica (previously called Pasteurella haemolytica) and other agents that cause “shipping fever”. Some people believe that BVD is one of the most significant disease organisms involved with respiratory disease of cattle. Severe acute BVD infections: This disease syndrome is usually (but not always) associated with Type II BVD virus infection. The affected animals will exhibit high fevers (107-110 F), occasional diarrhea, respiratory disease, and they will not eat. Peracute BVD can affect cattle of all ages and often results in death of the animal within 48 hours of disease onset regardless of age.

Acute BVD infections: The classic, acute form of BVD is characterized by a fever of 104-106 F, discharge from the nose and eyes, erosions of the muzzle and in the mouth, and diarrhea that may contain mucus and blood. Diarrhea is usually present in every herd that has an outbreak of acute BVD, but diarrhea is not present in every animal that has acute BVD. The percentage of the herd exhibiting clinical disease and dying can vary extremely; however, if "secondary infections" are controlled, most animals survive the acute disease. This syndrome usually occurs in cattle 6 to 24 months of age. Acute Mucosal disease: An animal persistently infected with BVD virus is not able to mount any defense against becoming subsequently infected with a different BVD virus. When a BVD infection is superimposed on a PI animal, mucosal disease usually results. Acute mucosal disease is characterized by fever, profuse, watery diarrhea, erosions of the mouth, lack of appetite, discharge from the eyes and nose, and occasionally lameness. Secondary infections, such as pneumonia and mastitis, are common. Cattle with acute mucosal disease usually die within 3 to 10 days.

Chronic Mucosal disease: Some cattle that develop mucosal disease do not die as soon as expected but rather become chronically infected. Cattle with chronic mucosal disease are poor doers, and may have persistently loose stools or intermittent diarrhea, chronic bloat, decreased appetite, weight loss, erosions between the claws, or non-healing skin lesions. Discharge from the eyes and nose, bald spots due to loss of hair, and long-term lameness are also common. Cattle with chronic mucosal disease rarely survive beyond 18 months and ultimately die.

Treatment and Prevention of BVD infections There is no effective treatment for infection with BVD, but most cases are subclinical and self-limiting. Antibiotics, fluid and supportive therapy may be indicated to control secondary infections. Offering highly palatable feed could tempt sick animals to eat needed nutrients. Vaccination of susceptible cattle has been the principal approach to the prevention and control of BVD. However, preventing the introduction of BVD into your herd and identifying and eliminating PI animals from your herd are important steps to take to control the disease.

Vaccinate calves: Calves should be vaccinated twice with a modified live virus (MLV) vaccine before leaving the herd of origin. Ideally, BVD vaccinations should be completed in the calves at least 30 days prior to weaning, but whatever program you initiate needs to fit with your management system. Check with your veterinarian for specific recommendations for your herd.

Vaccinate the cow herd: It is difficult to provide blanket recommendations for vaccinating the cow herd, but some general guidelines can be given. Unvaccinated heifers and cows should be properly vaccinated before breeding to ensure protection for the fetus. All bulls should be properly vaccinated before putting them out with the cows or heifers and new additions should be properly vaccinated before adding them to the herd. Modified live virus vaccines can be safely used in open cows (there are new MLV vaccines safe for pregnant cows if the cows have been previously vaccinated with certain products) and provide long-lasting protection. Killed vaccines are safe for all cattle, but usually don’t provide as strong an immune response and may need more frequent booster vaccinations. Again, check with your veterinarian for specific recommendations.

Prevent introduction of BVD into your herd: BVD virus is shed from cattle in the feces and in secretions from the nose and mouth. BVD is also readily transmitted by aerosol droplets and direct contact. Avoiding contact with other cattle is therefore an important step to take to prevent infection from entering your herd. “Good fences make good neighbors”. It is especially important to keep pregnant cows less than 120 days pregnant separated from other cattle. New introductions into your herd need to be tested for PI status.

Eliminate PI animals from your herd: Until recently, testing cattle for PI infection was prohibitively expensive but now there are tools available making it feasible to test for and eliminate these “typhoid Mary” animals from the herd. There are two types of test available, one using a skin sample and one using a blood sample: Immunohistochemistry – for this test, a small notch of skin is taken from the edge of the ear, easily done using a pig ear-notching tool. The triangular piece of skin removed should be ¼ to ½ inches per side. Depending on the laboratory the sample will be sent to, the removed skin is placed either in a vial containing formalin or an individual plastic bag. All samples must be clearly labeled with the animals’ identification number. PCR – this test requires that a blood sample in a “purple top” tube be taken and submitted. Again, all samples must be clearly labeled with the individual animal ID.

Samples can be sent to a number of different laboratories; three are listed below. Be sure to contact the lab and talk to your veterinarian before taking and sending samples – if you take the wrong samples, all your work may be wasted. Be aware that it is possible to have “false positive” results – some animals may test positive when they are not truly persistently infected, and may need to be re-tested. Your veterinarian can help interpret the results of the testing. (Thank you to Dr. John Maas for the following information) 1. Tulare branch of the California Animal Health & Food Safety Laboratory (CAHFS)

CAHFS-Tulare Phone (559) 688-7543 18830 Road 112 Fax (559) 686-4231 Tulare, CA 93274

Sample description: Ear notch (triangle notch ¼ to ½ inch per side) in zip lock bag (or whirl pack bag). Refrigerated—not frozen. Ship overnight (not for Saturday arrival). Technique: Immunohistochemistry. Cost: $16.50 per 1-5 samples, i.e. $33.00 for ten (10) samples and $33.00 for 6 samples. Additional one time accession fee is also charged.

2. University of Nebraska, Lincoln, NE

Veterinary Diagnostic Center University of Nebraska Fair Street and East Campus Loop P.O. Box 82646 Lincoln, NE 68501-2646 Phone (402) 472-1434 Fax (402) 472-3094

Sample description: Ear notch (triangle notch ¼ to ½ inch per side) in neutral-buffered formalin. Leak proof tubes are mandatory for containers. Do not hold skin samples in formalin for more than 7 days prior to submission. Technique: Immunohistochemistry. Cost: Accession fee: $7.00 per each shipment (submission). First sample: $12.00. Two (2) to 6 samples: $20.00, multiples of 6: $20.00/six samples.

3. Davis branch of CAHFS.

CAHFS-Davis University of California, Davis West Health Sciences Drive Davis, CA Phone (530) 752-7578 Fax (530) 752-6253

Sample description: whole blood, refrigerated (not frozen). Ship in leak proof containers on ice bags (gel bags). Technique: PCR. Cost: $22.70 for the first sample, $5.65 per each sample after the first. Additional one time accession fee is also charged.

The ancient philosophers emphasized the virtues of balance and moderation, which resonate across cultures. This principle extends to scientific realms, notably evident in oxygen homeostasis. While insufficient or excessive oxygen levels can be detrimental, the optimal threshold remains elusive. Throughout Earth's history, oxygen levels have fluctuated significantly, prompting the questions how organisms respond to varying oxygen tensions. This thesis examines the impacts of oxygen in diverse disease settings and investigates cellular and organismal responses to aberrant oxygen levels.

First, we delve into the “optimal” oxygen level for mitochondrial disorders. Mitochondria diseases are devastating conditions without effective therapies, affecting thousands of children globally. Hypoxia emerges as a potential therapeutic avenue, previously explored in Leigh Syndrome, showcasing extended survival and mitigation of associated deficits. This study investigates hypoxia's generalizability across mitochondrial disorders, focusing on thiamine and pyruvate dehydrogenase deficiencies, highlighting its therapeutic potential beyond Leigh Syndrome. This study also reveals that for certain disease setting, the ambient oxygen level (21% oxygen) can cause toxicity and exacerbate disease, prompting a thorough investigation into the potential toxic effects of oxygen.

While excess oxygen is toxic to all known organisms, the underlying molecular mechanisms remain largely unexplored. This work delves into the effects of excess molecular oxygen, uncovering its effect on destabilizing a subset of iron-sulfur cluster (ISC)-containing proteins. This disruption hampers crucial processes such as diphthamide synthesis, purine metabolism, nucleotide excision repair, and electron transport chain (ETC) function. Our findings extend to a mouse model and primary human lung cells. In particular, we reveal that the ETC is the “Achilles' heel” in hyperoxia, leading to decreased mitochondrial oxygen consumption and subsequent tissue hyperoxia. This cyclic damage affects additional ISC-containing pathways. Notably, primary ETC dysfunction exacerbates lung tissue hyperoxia.

After observing the destabilizing effects of hyperoxia on ISC proteins, our subsequent step involves a systematic exploration of the influence of diverse oxygen tensions on protein turnover. We demonstrate that the lung proteome shows the most significant changes. Remarkably, extracellular matrix (ECM) proteins are stabilized in the lung under both hypoxia and hyperoxia, implying post-translational regulation in ECM remodeling. Furthermore, we identify MYBBP1A as a potential hyperoxia transcriptional regulator, specifically involved in rRNA homeostasis. Overall, our study underscores the intricate interplay between oxygen tensions and protein turnover, highlighting key mediators of oxygen-dependent signaling pathways. Our work has broad implications for disease including hyperoxia-related pathologies such as bronchopulmonary dysplasia, ischemia-reperfusion injury, aging, and mitochondrial disorders.

Lastly, we investigate the nexus between impaired proteostasis, varying oxygen tensions, and cardiac diseases. Leveraging human iPSC-derived cardiomyocytes, this research reveals a significant reduction in global protein turnover rates under hypoxia, particularly affecting biosynthetic and catabolic pathways. Notably, the study identifies potential targets for prolyl hydroxylases, suggesting their role in regulating responses to chronic hypoxia. This comprehensive exploration illuminates the nuanced effects of oxygen levels on proteostasis in cardiac pathology, paving the way for deeper insights into therapeutic interventions for cardiovascular diseases.

Collectively, these comprehensive investigations offer insights into the intricate interplay between oxygen tensions, cellular responses, and the pathophysiology of disease. They pave the way for interventions across diverse disease states impacted by oxygen homeostasis.

Background: G-protein signaling pathways regulate many aspects of cardiovascular development and disease. A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate these signaling events into specific physiological processes. One family member, AKAP13, integrates Gs, Gq/11, and G12/13 signals through binding of protein kinase A, C (PKC), and D (PKD) and encoding an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. AKAP13 is required for mouse development as null embryos die by E10.5 and exhibit cardiovascular defects. Additionally, the Rho-GEF and PKC-PKD binding domains mediate cardiomyocyte hypertrophy in isolated cardiomyocytes. However, the specific developmental processes regulated by AKAP13 and the requirements for the Rho-GEF and PKC-PKD binding domains during development and cardiac hypertrophy are unknown.

Methodology/Principal Findings: We used an RNAi, loss-of-function approach, to determine if AKAP13 mediates differentiation of mouse embryonic stem (ES) cells or angiogenic processes in human umbilical vein endothelial cells (HUVECs). We found that AKAP13 knockdown did not decrease ES cell differentiation into Nkx2.5-GFP-positive cardiac mesodermal cells or functionally beating cardiomyocytes. Similarly, the expression of endodermal, endothelial, or cardiac structural marker genes was not affected. We also found that AKAP13-deficient HUVECs had normal tube formation, VEGF-induced wound healing, and VEGF-induced PKD phosphorylation. We then generated AKAP13-truncation mutant mice to determine if the Rho-GEF and PKC-PKD binding domains are required for development. Surprisingly, homozygous mutant mice were born at normal Mendelian ratios and had normal survival, fertility, and cardiac structure. Finally, to determine if these domains mediate β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size increased normally in mice lacking the Rho-GEF and PKC-PKD binding domains. However, these hearts failed to increase cardiac ejection fraction or fractional shortening.

Conclusions: These results indicate that AKAP13 is not required for differentiation into cardiovascular tissue, the formation of functional cardiomyocytes, or angiogenic processes. They also indicate that the AKAP13 Rho-GEF and PKC-PKD binding domains are not required for mouse development or the cardiac structural response to β-adrenergic-induced hypertrophy. However, these domains are required for the proper cardiac functional response to β-adrenergic stimulation.

Embedded within the structure of much American environmental regulation is a distinction between the new and the existing. Existing products or sources of pollution are often grandfathered out of new regulatory requirements or receive other forms of transition relief. Such treatment reflects a recurrent political challenge facing makers of environmental policy: whether and how to mitigate regulatory burdens when policy change upsets settled expectations and investment commitments.

This dissertation first presents a survey of transition policies in various areas of environmental regulation, and then explores whether and how these policies might be explained by existing theories of regulation. The next four chapters present detailed case studies drawn from the trucking and pesticide industries, describing both the emergence of transition relief and the later efforts of policymakers to address problems arising out of this relief. These case studies demonstrate that although numerous variables affect transition policy, the degree of transition relief in a regulatory program is substantially influenced by the cost impacts of that program on incumbents, a factor which in turn is shaped by the composition and competitive dynamics of the regulated industry.

The development of an organism from conception to adulthood requires the specification of cell types to distinct fates. In an adult organism, tissues can similarly undergo dramatic transformation, altering their structure, physiology, and overall biochemical properties. In both of these paradigms, the study of gene transcription and its contribution to protein content in the cells has been the primary focus. While clearly important, more recently, alternative splicing and microRNA regulation have been recognized as significant processes that can have crucial regulatory influences on the diversity of mRNAs produced and their over-all expression in the cell.

In this dissertation, I have set out to characterize the molecular changes that occur in two distinct cellular paradigms, muscle remodeling in the uterus throughout pregnancy and the differentiation of embryonic stem cells to distinct fates using DNA microarray technology. To achieve this goal, I developed several new software applications, designed specifically to assess the relevance of coordinated gene regulatory events along biological pathways (GenMAPP and GO-Elite) and characterize sequence level functional attributes of proteins and mRNAs regulated by alternative splicing (AltAnalyze).

Studies of the mouse uterus during gestation reveal novel coordinated transcriptional networks regulating quiescence, contraction, and involution when multiple time-points are considered. Analysis of alternative splicing in mouse and human embryonic stem cell differentiation uncovered novel mechanisms for the regulation of protein domain and microRNA binding site inclusion and at least for one gene, Tcf3, the requirement of splicing to properly promote the early steps of embryonic lineage commitment down multiple paths. In summary, I have used novel computational methods and genomic resources to uncover new regulatory networks and potential biological mechanisms from both differentiating and transitioning cells, that involve regulation at the level of transcription, alternative splicing, and translational regulation by micoRNAs.

Genetic association studies have yielded a wealth of information on specific variants that lead to the development of disease, but our mechanistic understanding for most of them is lacking. Unbiased, comparative study of protein-protein interactions (PPI) for protein coding variants is a very promising approach to fill this gap. To yield data that can inform us on tissue-specific pathologies (e.g. cardiovascular, neurological disease) we need to study PPIs in the cell types affected by the disease. In a similar fashion, studying proteins expressed endogenously would allow us to overcome issues related to gene expression or dose-dependent protein function, which is a feature of many of these pathologies.

Here I present my work trying to unravel the mechanism mutations in the BAG3 chaperone gene lead to disease. To address this, I used a combination of induced pluripotent stem (iPS) cell technology (to generate cardiac myocytes), genome engineering (to induce specific disease-modifying mutations and protein fusions) and affinity purification coupled to mass spectrometry (to compare PPIs between variants). The results show that disease-related variants of BAG3 display a different profile of protein partners, some of them cardiac-specific, pointing towards interactions that potentially underlie the disease mechanism and tissue specificity. We also observed a cell culture phenotype for BAG3 deficiency and for some of the protein-coding variants, which sets the ground for the exploration of the role of BAG3 interactions in disease.

Overall, this piece of work proves the value of studying cardiac disease-related genetic variability using a disease-relevant iPS-derived model and genome engineering to explore multiple variants. In particular, we provide new clues on the role of BAG3 in the heart, and how that role is compromised by disease-related mutations. We hope that the knowledge gained on BAG3 genetics and mechanism will be useful for the development of therapeutic strategies in the future.