RNAs are dynamic molecules that orchestrate a breadth of processes for prokaryotes and eukaryotes in both the nucleus and cytoplasm. These processes include regulation of transcription, translation, and post-translational modifications. It has been demonstrated that the spatio-temporal localization of different RNAs is an important factor in development and the correct localization of some RNAs is crucial for proper development, structural organization and function of the cell. In vivo detection of endogenous RNAs is a challenging task because of low RNA concentrations in live cells, its transient character, limited accessibility for molecular probes, and sensitivity to modification. Methods that study RNA localization utilize either hybridization techniques with pre-labeled probes in fixed cells or require modifications to target RNAs in living cells potentially altering their in vivo behavior. The goal of this project is to design and explore a new non-invasive technique that is capable of detecting unmodified endogenous RNA in living cells. This method utilizes a combination of protein complementation and split aptamer probe technology to fluorescently detect unmodified endogenous RNA in living cells. Experiments show that this approach is capable of detecting a full-length β-globin mRNA in a sequence-dependent manner in live E. coli cells. Most importantly, fluorescent detection of the endogenous inducible phosphate stress response mRNA pstC IS successfully demonstrated. This fluorescent signal Is dependent on the concentration of pstC mRNA. Furthermore, the fluorescent signal revealS a punctate localization of pstC mRNA, which dOES not overlap with nucleoid DNA. This work holds the potential for the next generation of molecular tools for basic RNA research, clinical diagnostics and genetic therapeutics.

Acute kidney injury is an abrupt loss of kidney function that develops in short time with limited effective treatments other than kidney transplantation. We have identified TMIGD1 (Transmembrane immuno- globulin domain 1) as a novel receptor expressed in various organs and tissues, mainly in cell with epithelial origin. TMIGD1 regulates cell morphology and adhesion and its extracellular domain mediates its activity. Knocking down of TMIGD1 using short hairpin RNA (shRNA) increased cell death in human kidney epithelial cells (HK2). On the other hand, HEK293 cells over expressing TMIGD1 protected cells from oxidative stress and nutrient deprivation induced injuries. Furthermore, TMIGD1 expression is reduced in vivo and in vitro kidney injury models. TMIGD1 expression was regulated by ubiquitination and degradation by proteosome 26s. Thus, we present TMIGD1 as a novel receptor that plays important roles in regulation of cell morphology, cell- cell interaction and cell survival.

Forensic analysis of a deoxyribonucleic acid (DNA) profile includes determining if DNA from a known person should be considered as a likely contributor to the biological evidence. Prior to making this determination, the number of contributors (NOC) of the DNA sample is considered. It is important to take multiple factors into account when estimating the NOC, including stutter, baseline noise, and peak imbalance as these can affect the number of peaks observed at each locus. Allelic dropout can also have an impact on the number of peaks observed. Dropout occurs when an allele is not detected due to technical, biochemical, or sampling issues, and predominantly affects the level of ambiguity associated with low-template DNA interpretation. As the NOC to a sample may be underestimated in the presence of dropout, it is essential to reasonably predict the probability that an allele has dropped out. This work has two aims: first, to evaluate different characterizations of allelic dropout rates and, second, to determine the impact allelic dropout has on estimating the NOC to a DNA sample. Two different types of dropout characterization were examined – ‘indirect’ models based on observed peak heights and ‘direct’ models using observed dropout frequencies of single-source calibration data. The indirect models predicted allelic dropout based on the peak height distribution of the data at a specific target amount and locus using a fitted or non-fitted cumulative Gaussian curve. For the direct models, a logistic or exponential regression of the observed dropout frequencies versus target amount for each locus was used to predict dropout rates. The impact that allelic dropout has on estimating the NOC was assessed by varying the probability of dropout (Pr(D)) in simulated mixtures with up to six contributors in the presence or absence of a major contributor. Simulations for the short tandem repeat (STR) loci consistent with the AmpFℓSTR® Identifiler® Plus (Applied Biosystems®, Foster City, CA) and GlobalFiler™ (Applied Biosystems®, Foster City, CA) amplification kits were completed to explore the impact additional polymorphic loci have on estimating the NOC. The NOC for each profile was determined using the maximum allele count (MAC) method. An exponential or logistic regression of observed frequencies of dropout (Fr(D)) was found to be an appropriate characterization of allelic dropout rates. In general, the peak height based methods overestimated dropout at higher target levels and underestimated it at lower target amounts. The underestimation suggests that other factors beyond detection and polymerase chain reaction (PCR) variation contribute to dropout. Across all loci, the Fr(D) increased as target amount decreased and as molecular weight increased. Estimating the actual NOC using MAC was found to be unreliable for mixtures with greater than three contributors or with one or more minor contributors present at low levels. While a high level of dropout did not affect correctly identifying two-person mixtures, it greatly increased the number of misidentifications with three or more contributors. The number of misidentifications was reduced for mixtures when 21 STR loci plus amelogenin were used to evaluate the NOC. These higher accuracies were frequently attributable to the highly polymorphic locus SE33. The presence or absence of a major contributor did not appear to substantially affect the results. Forensic laboratories using MAC to determine the NOC of mixed samples should be aware of the tendency to underestimate the NOC using this method. It is also important to understand the impact that allelic dropout has on correctly estimating the NOC. The probability that allele dropout may have occurred in a sample should be considered when evaluating the NOC that explains the evidentiary profile.

Transmembrane and immunoglobulin domain-containing 1 (TMIGD1) is a newly identified cell adhesion molecule that mediates cell-cell interactions and is mainly expressed in kidney and colon epithelial cells. In renal epithelial cells, TMIGD1 regulates cell proliferation and migration. Human tissue panels showed expression of TMIGD1 in placenta; however, the potential function of TMIGD1 in placenta is not known. We elected to study the expression and function of TMIGD1 during placentation. This is of interest because dysregulation of placental invasion is linked to obstetrical complications such as preeclampsia and intrauterine growth restriction (IUGR). We hypothesized that TMIGD1 is expressed in trophoblast cells and regulates cell migration during placental invasion. Placental tissues were subjected to immunofluorescence (IF) staining using anti-TMIGD1 antibody and TMIGD1 localization in trophoblast was visualized using a fluorescence microscope. Additionally, we overexpressed TMIGD1 in the immortalized trophoblast cell line, HTR8/SVneo, via a retroviral system. Transduction was verified using IF, Western blot, and qPCR to compare the modified and original cell lines. Migration of TMIGD1-overexpressing HTR8/SVneo cells was assessed using wound-healing and transwell migration assays. We observed TMIGD1 localization in the apical region of syncytiotrophoblasts. TMIGD1 mRNA expression in the transduced HTR8/SVneo cells was 3-fold greater than that in the control line, and 400-fold greater in first trimester whole placenta. TMIGD1-overexpressing HTR8/SVneo cells exhibited a 30±5% decrease in migration in the wound-healing assay, compared to the untransduced cells. Similarly, TMIGD1 overexpression in HTR8/SVneo suppressed migration by 36%, compared to control cells in transwell assays. Fluorescent staining showed that increased TMIGD1 expression modifies actin cytoskeleton by redistributing filaments to the peripheries. Additionally, cells overexpressing TMIGD1 exhibit a distinct morphology that lacks filopodia or other motility structures. Our study demonstrates for the first time that TMIGD1 is expressed in trophoblast cells and acts to inhibit cell migration. The evidence presented in this study supports the idea that TMIGD1 expression in trophoblast may play an important function in regulating placental invasion, and that perturbations in its activity may be associated with obstetrical complications such as preeclampsia and intrauterine growth restriction.

Although severe lung disorders, including cystic fibrosis, asthma, and chronic obstructive pulmonary disease (COPD), represent a significant global disease burden, little is known about the molecular pathways by which the cells of the lung develop, respond to damage, or become diseased. Consequently, there are few treatment options for patients. Improving lung disease outcomes therefore relies on both refining the current understanding of the normal development of the lung epithelium and developing new model systems to provide mechanistic insight into disease biology. In this thesis, I describe a multifaceted approach using both in vivo models and novel mouse and human pluripotent stem cell reporter systems to explore this important topic, focusing primarily on the hypothesis that canonical Wnt signaling is a key stage-dependent inhibitor of proximal lung development. To address this hypothesis, I developed new tools allowing for the precise manipulation of developmental pathways and access to rare cell populations in vitro. This toolkit included both mouse and human pluripotent stem cell (mPSC/hPSC) lines with reporters for specific airway lineages. In parallel, I built on our lab’s previous work in directed differentiation of hPSCs to lung progenitors. I found that canonical Wnt signaling regulates proximodistal epithelial patterning in human NKX2-1+ lung progenitors. While canonical Wnt activation is required for lung specification, withdrawal of Wnt activation leads to emergence of a proximal airway program and loss of distal identity. This finding culminated in the development of a novel protocol to differentiate epithelial-only airway organoids from hPSCs. These organoids are derived from purified NKX2-1+ lung progenitors, contain functional airway cell types including secretory, goblet, and basal cells, and can be further expanded and differentiated to multiciliated epithelia in air-liquid interface culture. To provide a proof of principle for the clinical utility of this platform, I generated airway organoids from cystic fibrosis patient-derived hPSC lines pre- and post-correction of the dF508 mutation in the CFTR gene. These organoids respond in a CFTR-dependent manner to epithelial forskolin swelling assays, highlighting the potential utility of this approach for disease modeling and drug screening for a variety of genetic and acquired airway disorders.

Successful reproduction in mammals requires the interaction of the reproductive and neuroendocrine systems. Nhlh2 is a basic helix-loop-helix transcription factor that is expressed in both the developing and adult neuroendocrine hypothalamus. Targeted deletion (N2KO) of Nhlh2 results in hypogonadism and obesity. In addition, male N2KO mice exhibit micropenis and reduction of male accessory sex organs and half of the female N2KO mice have thread-like uteri and small pale ovaries. Male N2KO mice have reduced levels of testosterone and FSH, and are infertile, while female N2KO are not completely infertile, but show a decline in reproductive potential as they age. In order to determine if the mechanisms underlying reduced fertility in N2KO male and female mice, we examined gamete number and competence, hormone concentrations and sexual behavior. We found that although sperm from KO mice is as functional as sperm from normal mice in IVF assays, N2KO mice have a 50% reduction in the number of mature sperm isolated from the caudal epididymis. Oocyte numbers in N2KO female is decreased with aging, but were competent for fertilization and development. Like N2KO males who are unresponsive to sexually receptive female mice, N2KO females show reduced lordosis when placed with an experienced male. Based on these findings we examined mRNA levels of estrogen receptor alpha (ERα) and progesterone receptor (PR), as they are important in the regulation of female and male sexual behavior. In wildtype (WT) female mice, a decline in ERα production is seen in response to the addition of estrogen. In N2KO females this decline is not observed; ERα levels are increased compared to WT females during proestrus. In male N2KO there is an increase in progesterone receptor mRNA levels. These data suggest that Nhlh2 may play a role in sexual behavior possibly via the regulation of ERα and progesterone receptor.

Proper regulation of the p53 protein plays a critical role in suppressing tumor formation in a variety of tissues including the mammary gland. Activity of the p53 pathway is diminished in the mammary epithelium of nulliparous mice in comparison to mid-pregnant mice. P53 was shown to be regulated by estrogen and progesterone. The aim of this dissertation is to determine the molecular mechanism estrogen and progesterone utilize to sensitize p53 to radiation induced DNA damage in the mouse mammary epithelium. The first component of this work identified the minimal hormonal treatment to restore p53 activity in response to radiation. Results demonstrate that estrogen and progesterone in combination are necessary to sensitize p53 to radiation. Neither hormone alone was as effective. The effects of estrogen and progesterone were blocked by receptor antagonists. Tamoxifen in combination with progesterone was as effective as estrogen and progesterone treatments in restoring p53 responsiveness to radiation. However, the proliferation was much less in this combination than observed in treatments with estrogen and progesterone. In addition, comparison of the effects of single hormone treatments on cell cycle recruitment and p53-dependent responses revealed that theses processes were separable. The second part of this dissertation was to identify growth factors and hormones which may augment the effects of estrogen and progesterone. Results from this experiment demonstrate that transforming growth factor-β (TGF-β) is required for estrogen and progesterone to restore p53 responsiveness to radiation, as TGF-β neutralizing antibodies blocked the effects of estrogen and progesterone. However, TGF-β alone was unable to sensitize p53 to radiation. This suggests that hormone- and TGF-β-dependent pathway cooperate to restore p53 responsiveness to radiation.

Eukaryotic ribosomal RNAs (rRNAs) contain scores of 2′-O-methylated nucleotides that are formed by a large family of small RNA:protein machines called snoRNPs. The snoRNPs contain a small nucleolar RNA (snoRNA) and several proteins. Nucleotides to be modified are selected by the snoRNA component through base pairing with a long (10–21 nucleotides) guide sequence, and methylation is catalyzed by one of the snoRNP proteins. Modifications can be targeted to new sites by outfitting a snoRNA with a new guide sequence. In this thesis, targeting ribosome methylation to new sites in vivo was used to interfere with yeast rRNA function for the purpose of functional mapping. New snoRNAs were: (1) targeted to individual nucleotides known or predicted to have important roles, or (2) used in a gene library format to target all nucleotides in the reaction center of the ribosome. For several sites examined in detail, interference was shown not to be due to a simple anti-sense phenomenon. The results indicate that this new strategy is, indeed, effective for identifying important rRNA regions. The deleterious effects of novel methylations on rRNA function were analyzed at different levels for sensitive nucleotides located in the peptidyl transferase center (PTC). Detailed analyses were conducted on: cell growth, global translation, rDNA transcription, pre-rRNA processing, and ribosome production. The results show that the interfering snoRNPs can cause severe growth defects by two general means: (1) disrupting ribosome production, causing a low yield or; (2) creating ribosomes that are functionally defective. This novel approach should also be useful for functional mapping of other RNAs in Saccharomyces cerevisiae, and for use in other organisms as well including humans.

According to the National Cancer Institute, breast cancer is second only to lung cancer in cancer-related deaths for women in the United States (American Cancer Society). Despite a gradual decline in deaths due to breast cancer (likely attributable to increased screening), there is a rise in the incidence of newly-diagnosed breast cancer. Thus protective therapies for breast cancer are a new arena which will become of increasing importance. The work presented in this dissertation demonstrated the possible treatments which might be effective in the fight against breast cancer and the pathways involved in the prevention and therapy. An early full-term pregnancy imparts a significant protection for women from getting breast cancer. In animals, this protection can be mimicked by a short-term exposure to physiological doses of ovarian hormones. In my dissertation, I investigated the ability of retinoids, such as 9-cis retinoic acid, all-trans retinoic acid, and N-4-hydroxyphenylretinamide (4-HPR), to sensitize the ductal epithelial cells of virgin mammary glands to DNA damage responses using a whole-organ culture system. My data suggest that sensitization of the mammary epithelium to p53-dependent apoptosis is a common pathway, which is engaged by retinoids as well as ovarian hormones. I went on to investigate the effect and mechanism of another possible chemopreventive and chemotherapeutic agent, Rhodiola Crenulata. Rhodiola is a perennial plant which grows in the high Tundra regions of Tibet and Siberia. Though it has been used for a long time in the eastern traditional medicine, Rhodiola has never been used in the treatment of breast cancer. In this dissertation, I showed that dietary Rhodiola is effective in increasing the survival time of mice bearing tumor grafts. In vitro analysis shows that Rhodiola is capable of reducing proliferation and increasing death of certain breast cancer cells. Rhodiola is demonstrated to inhibit the motility and invasion of breast cancer cells. Caspase-dependent and-independent pathways as well as Akt and p53 pathways were shown to be involved in the chemoprevention and chemotherapeutic action of Rhodiola on breast cancer.

Small nucleolar RNAs (snoRNAs) can be divided into two large families based on conserved sequence elements. These two classes are the box C/D snoRNAs and the box H/ACA snoRNAs. At the onset of this thesis study, a small number of snoRNAs were known to be required for processing (cleavage) of pre-ribosomal RNA; the function of majority of snoRNAs remained an enigma. The thesis research revealed two important and exciting discoveries: (1) the box C/D snoRNAs are mainly involved in guiding the formation of 2$\sp\prime$-O-methylation in rRNA, and; (2) most box HACA snoRNAs direct $\Psi$ modification of rRNA. Taken together, I demonstrated that the main function of the snoRNAs is nucleotide modification of ribosomal RNA.