Reducing the amount of error and introduced data variability increases the accuracy of Western blot results. In this study, different methods of normalization for loading differences and data alignment were explored with respect to their impact on Western blot results. GAPDH was compared to the LI-COR Revert total protein stain as a loading control. The impact of normalizing data to a control condition, which is commonly done to align Western blot data distributed over several immunoblots, was also investigated. Specifically, this study addressed whether normalization to a small subset of distinct controls on each immunoblot increases pooled data variability compared to a larger set of controls. Protein expression data for NOX-2 and SOD-2 from a study investigating the protective role of the bradykinin type 1 receptor in angiotensin-II induced left ventricle remodeling were used to address these questions but are also discussed in the context of the original study. The comparison of GAPDH and Revert total protein stain as a loading control was done by assessing their correlation and comparing how they affected protein expression results. Additionally, the impact of treatment on GAPDH was investigated. To assess how normalization to different combinations of controls influences data variability, protein data were normalized to the average of 5 controls, the average of 2 controls, or an average vehicle and the results by treatment were compared. The results of this study demonstrated that GAPDH expression is not affected by angiotensin-II or bradykinin type 1 receptor antagonist R-954 and is a less sensitive loading control compared to Revert total protein stain. Normalization to the average of 5 controls tended to reduce pooled data variability compared to 2 controls. Lastly, the results of this study provided preliminary evidence that R-954 does not alter the expression of NOX-2 or SOD-2 to an expression profile that would be expected to explain the protection it confers against Ang-II induced left ventricle remodeling.

Premature babies are at risk of death from immature lung development. For this reason, pregnant mothers at risk for preterm delivery are administered dexamethasone (DEX), a synthetic glucocorticoid that promotes fetal lung development. However, exposure to DEX in utero is associated with low birth weight and cardiovascular development pathologies. Moreover, our lab found that DEX administration in-utero leads to a sex-specific increase in stress-induced tachycardia in female, but not male offspring. This project seeks to expand on this preliminary finding of the heart by examining local effectors of activity from the sympathetic system (tyrosine hydroxylase and catechol-o-methyltransferase). Tyrosine hydroxylase was measured as it catalyzes the rate limiting step of norepinephrine synthesis while catechol-O- methyltransferase was studied as it catalyzes the degradation of norepinephrine. Acetylcholinesterase was used to measure parasympathetic activity as it catalyzes the degradation of the primary neurotransmitter of the parasympathetic nervous system, acetylcholine. Analyses of sympathetic as well as parasympathetic activity were done to determine influences of in-utero DEX exposure on autonomic regulation in adulthood. Pregnant rats were administered DEX (0.4 mg/kg, i.p.) or vehicle (20% w/v 2-hydroxypropyl ß- cyclodextran) at gestation days 18-21, with euthanasia of offspring occurring at around the time the offspring reached 13-15 weeks of age. Left ventricles and right atria were pulverized, processed and subjected to western blot analysis to determine expression of proteins of interest. Males exposed to DEX in-utero saw a decrease in tyrosine hydroxylase expression in left ventricle and right atrium when compared to vehicle control, a difference not seen with females. In addition, catechol-o-methyltransferase expression was increased in right atria from male, but not female rats. Acetylcholinesterase expression was reduced in the right atria of female, but not male rats. The present findings suggest reduced norepinephrine signaling in the heart of male, but not female DEX-exposed offspring. Given that we have previously found that female, but not male rats exhibit exaggerated stress-induced tachycardia, our current findings suggest that males possess a sex-specific compensatory mechanism allowing the heart to resist increased sympathetic signaling from the brain, one that females do not possess. The underlying mechanics of this proposed mechanism are unclear, and further investigation is needed in this subject to determine the significance of the findings from our study.

Conrad Hal Waddington was an embryologist and theoretical biologist. His early experimental work investigated aspects of embryonic induction and the properties of the organizer first identified by Hans Spemann and Hilde Mangold, while his later studies focused on genetic assimilation. Waddington is probably best known for developing the concept of the epigenetic landscape, and he also held significant interest in many different areas ranging from the visual arts and poetry to philosophy. Throughout his career, Waddington maintained that the arts were integral to science, and he continued to draw inspiration from the arts for his own work.

The Cell-Theory was written by Thomas Henry Huxley in Britain and published in 1853 by The British and Foreign Medico-Chirurgical Review. The twenty-two page article reviews twelve works on cell theory, including those in Germany by Caspar Friedrich Wolff in the eighteenth century and by Karl Ernst von Baer in the nineteenth century. Huxley spends much of The Cell-Theory on a cell theory proposed in the late 1830s by Matthias Schleiden and Theodor Schwann in Germany. Schleiden and Schwann maintained that the cell was the most fundamental unit of life and that the nucleus was the most significant cellular component. Huxley, instead, promoted an epigenetic theory of the cell, for which properties of life emerge from the outer cytoplasm, cell membrane, and wall (the periplast), as opposed to the inner contents of the cell, including the nucleus (the endoplast). Huxley's arguments in The Cell-Theory influenced future scientists about the role of epigenetic processes in embryology and development.

Caspar Friedrich Wolff is most famous for his 1759 doctoral dissertation, Theoria Generationis, in which he described embryonic development in both plants and animals as a process involving layers of cells, thereby refuting the accepted theory of preformation: the idea that organisms develop as a result of the unfolding of form that is somehow present from the outset, as in a homunculus. This work generated a great deal of controversy and discussion at the time of its publication but was an integral move in the reemergence and acceptance of the theory of epigenesis.

The epigenetic landscape is a concept representing embryonic development. It was proposed by Conrad Hal Waddington to illustrate the various developmental pathways a cell might take toward differentiation. The epigenetic landscape integrates the connected concepts of competence, induction, and regulative abilities of the genes into a single model designed to explain cellular differentiation, a long standing problem in embryology.

Best known for his contributions to the field of embryology, Karl Ernst von Baer also pursued a variety of other areas of study including medicine, botany, zoology, and anthropology. Committing his life to scientific research, von Baer's work led to the advancement of the understanding of mammalian reproduction, development, and organ functioning. His embryological discoveries ultimately led him to a view of development that supported epigenesis and refuted long-held thinking about preformation. Karl Ernst von Baer was born on 28 February 1792 in Piep, Estonia, to first cousins Juliane Louise von Baer and Magnus Johann von Baer. As one of ten children, von Baer spent his childhood in Coburg with his father's brother Karl and his wife, Baroness Ernestine von Canne.

Introduced by Conrad Hal Waddington in 1942, the concept of epigenetics gave scientists a new paradigm of thought concerning embryonic development, and since then has been widely applied, for instance to inheritable diseases, molecular technologies, and indeed the human genome as a whole. A genome contains an embedded intricate coding template that provides a means of genetic expression from the initial steps of embryonic development until the death of the organism. Within the genome there are two prominent components: coding (exons) and non-coding (introns) sequences. Exons provide coding by transcribing a gene into a protein, while introns do not have this capacity. On top of these coding sequences lie mechanisms that dictate the overall capability of a gene without changing the underlying nucleotide sequence of DNA; these mechanisms are primarily known as epigenetic factors.

Alzheimer’s Disease (AD) is the most prevalent form of dementia and is the sixth leading cause of death in the elderly. Evidence suggests that forms of stress, including prenatal maternal stress (PMS), could exacerbate AD development. To better understand the mechanism linking PMS and AD, we investigated behavior and specific epigenetic markers of the 3xTg-AD mouse model compared to aged-controls in offspring of stressed mothers and non-stressed mothers.

Stress and stress-related disorders increase the risk of Alzheimer’s Disease (AD) later in life. Some evidence suggests that prenatal maternal stress (PMS) can exacerbate AD. However, the effects of PMS on AD have not been as well studied. Epigenetic changes have been shown to contribute to AD and this is a possible mechanism by which PMS could accelerate AD. Thus, the present study aimed to investigate the effects of PMS on histone modifications, which change gene expression through alterations made to chromatin structure and thereby DNA accessibility. We utilized female 3xTG-AD mice and performed spatial and learning memory assessments between 5 and 6 months of age. Tissue was analyzed for AD pathology and epigenetic markers at 6 months of age were assessed PMS was shown to influence histone modifications H3K4me3 and H3K27me3 in a manner known to promote the expression of genes associated with neurodegeneration. Further, PMS impaired spatial memory, and, interestingly, the data resembled the pattern of H3K4me3 expression across groups, suggesting that this epigenetic modification could modulate the learning and memory effects of PMS. While the presence of hallmark AD pathologies were not accelerated by PMS, PMS did increase early tau phosphorylation events. Thus, this evidence suggests that PMS impairs spatial memory through epigenetic modifications and may potentially exacerbate AD later in life.