ETD Search Results: instcode:ucin

Skip navigation

ETD Center Search

54 matches in the database. These are records: 1 - 30.
Did you mean instcode:ucii?

[1] [2]

1. ABLE, JESSICA ANN. MDMA ADMINISTRATION AFFECTS COGNITION IN THE RAT.

Degree: MS, Medicine : Cell and Molecular Biology, 2006, University of Cincinnati

► 3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine analog. MDMA causes cognitive impairment in humans.… (more)

▼ 3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine analog. MDMA causes cognitive impairment in humans. Work done in primates corroborates this evidence. A reliable rat model of cognitive deficits has not been established. Experiment 1 examined the effect of a single-day dose regimen of MDMA (4 x 15mg/kg) on Sprague-Dawley rats in spatial learning on the Morris water maze (MWM), path integration learning in the Cincinnati water maze (CWM), and novel object recognition (NOR). The MDMA-treated animals made more errors than controls on the last three days of CWM testing. MWM and NOR revealed no differences. At the end of behavioral testing, serotonin (5-HT) was depleted in the MDMA-treated group in the hippocampus, striatum and prefrontal cortex and dopamine (DA) was depleted in the striatum. Since human MDMA use likely involves multiple doses, Experiment 2 examined the effects of a multiple-day dose regimen of MDMA on behavior. There were 3 treatment groups: (1) a 1-day/week for 5 weeks regimen of MDMA (15 mg/kg x 4/day), (2) a 1-day/week for 5 weeks regimen of saline (SAL, 4/day), and (3) a 1-day/week for 4 weeks regimen of saline and a 1-day regimen of MDMA on the fifth week. Subjects were given the following behavioral tests: elevated zero maze (EZM), locomotor activity, marble burying, CWM, MWM (3 phases), NOR, locomotion with methamphetamine challenge, and a delayed MWM probe trial. There were no differences between controls and the MDMA-treated groups in MWM or NOR. In the CWM, both MDMA treated groups showed significant deficits in learning. The 5-dose MDMA group showed increased anxiety in EZM and more locomotor activity following methamphetamine challenge. Examination of monoamines had 5-HT and DA reductions, similar to Experiment 1. Experiment 3 was designed to determine the time-course (across 5 weeks) for the DA depletions resulting from a single-day of MDMA administration. MDMA treatment depleted DA in the striatum during the first week, but levels recovered in weeks 2 through 4, and then were decreased again at week 5. These experiments show that MDMA causes impairments in complex brain functioning. Advisors/Committee Members: Williams, Dr. Michael T.

Keywords: MDMA, learning, memory, rat, path integration, spatial learning

2. ANGUS, STEVEN PATRICK. THE MECHANISM OF RB-MEDIATED CELL CYCLE INHIBITION.

Degree: PhD, Medicine : Cell and Molecular Biology, 2003, University of Cincinnati

► The retinoblastoma tumor suppressor (RB) is a critical negative regulator of cellular… (more)

▼ The retinoblastoma tumor suppressor (RB) is a critical negative regulator of cellular proliferation. The importance of RB in controlling cell growth and division is underscored by the observation that greater than 60% of human tumors exhibit functional inactivation of the RB pathway. RB functions as a transcriptional repressor, able to bind to the E2F family of transcription factors and regulate the expression of genes required for S-phase entry and progression. In response to anti-mitogenic stress signals (e.g. DNA damage), RB becomes hypophosphorylated/activated, able to down-regulate these genes, and elicit cell cycle arrest. RB/E2F-regulated genes include cyclin-dependent kinase (CDK) and cyclins, components of the DNA replication machinery, and metabolic enzymes required for the production of deoxyribonucleotides (dNTPs). Here, we investigated the relevant targets of RB-mediated repression that contribute to the arrested phenotype. We describe functional signaling pathways of active RB that target the activity of CDK2/cyclin A to disrupt the function of PCNA, a late step in the DNA synthetic process. In a parallel pathway, RB represses the expression of dNTP synthetic enzymes to limit available DNA precursor molecules during cell cycle arrest. Under conditions of chronic RB activation, the expression of multiple DNA replication components is attenuated, leading to a replicative exit state. Loss of RB signaling permits cells to escape this long-term arrest state, demonstrating the requirement of RB in the maintenance of two, kinetically distinct checkpoints. RB has been shown to bind to over 100 cellular proteins. Using innovative imaging techniques, we demonstrate that E2F sites on chromatin are the principal targets of active RB during cell cycle arrest. These studies elucidate the critical targets and resultant phenotypes mediated by the retinoblastoma tumor suppressor protein to inhibit cellular proliferation. Advisors/Committee Members: Knudsen, Dr. Erik S.

Keywords: cell cycle; DNA replication; tumor suppressor; E2F

3. BENNETT, RICHARD A. The p53-p21-Cyclin E Pathway in Centrosome Amplification and Chromosome Instability.

Degree: PhD, Medicine : Cell and Molecular Biology, 2007, University of Cincinnati

► Cancer is characterized by cells that have many genetic mutations. Chromosome instability… (more)

▼ Cancer is characterized by cells that have many genetic mutations. Chromosome instability is a hallmark of cancer, because it promotes the acquisition of the many mutations necessary for malignancy. Centrosome amplification is a major factor in chromosome instability because it leads to multipolar spindles, which directs the unequal segregation of chromosomes during mitosis. Centrosome amplification occurs when the mechanisms that regulate centrosome duplication are disrupted. Although the mechanisms that regulate centrosome duplication are not clear, it is known that the p53-p21-cyclin E pathway plays a major role in regulating centrosome duplication. p53, a tumor suppressor, induces the expression of p21, a cell cycle inhibitor, which inhibits the activity of cdk2/cyclin E. Previous work has shown that disruption of this pathway (loss of p53 or p21, or overexpression of cyclin E) can induce centrosome amplification. In this work, we study various aspects of this pathway and its effect on centrosome duplication. We show that common anti-cancer drugs that inhibit S phase uncouple centrosome duplication and DNA synthesis, leading to an increase in centrosome amplification and chromosome instability. This may help to explain why recurrent tumors are more malignant than their primary counterpart is. If some cells receive low doses of these drugs, it is possible that they can reversibly arrest in S phase, but allow centrosomes to duplicate. Once the drugs are gone, cells can re-enter the cell cycle with amplified centrosomes. As a result, chromosome instability occurs and cells can invade and destroy nearby tissues and spread to other parts of the body. p53 probably controls centrosome duplication via multiple pathways. To examine further the role of the p53-p21-cyclin E pathway in drug-induced centrosome amplification, we show that cyclin E is required for centrosome amplification to occur in drug-treated cells. Cells lacking cyclin E show minimal centrosome amplification compared to cells that express cyclin E. These data indicate that treatment of p53-null cells probably exploits the inactive p53-p21-cyclin E pathway to induce centrosome amplification. Fibroblasts from cyclin E-knockout mice are viable and centrosome duplication occurs normally, thus questioning the necessity of cyclin E in regulating centrosome duplication. Since cyclin A is also a binding partner of cdk2, we show that cyclin A may substitute for the loss of cyclin E in regulating centrosome duplication. Expression of wild-type cyclin A in fibroblasts lacking cyclin E induces centrosome amplification, but not at the same level as exogenous wild-type cyclin E, indicating that cyclin E is the preferred binding partner of cdk2 in terms of centrosome duplication regulation. We also directly investigated the role p53 centrosome localization plays in centrosome duplication regulation. p53 is known to localize to centrosomes, but its functional significance is unknown. Using a p53 mutant that is unable to leave the nucleus, we show that both the transcriptional activity and the centrosome localization of p53 are required for p53 to fully regulate centrosome duplication, indicating that it may have non-transcriptional activities that regulate centrosome duplication. Lastly, p21 regulates centrosome duplication by inhibiting cdk2/cyclin E activity. However, p21 also binds and inhibits proliferating cell nuclear antigen (PCNA), but the effect on centrosome duplication is unknown. Therefore, we further evaluated the relative contribution of both binding regions of p21 (CDK and PCNA) in regulating centrosome duplication. We show that p21 regulates centrosome duplication through its ability to bind and inhibit cdk2/cyclin E, rather than PCNA. Advisors/Committee Members: Fukasawa, Dr. Kenji.

Keywords: Centrosome; Chromosome Instability; p53; p21; Cyclin E; Cancer; Centrosome amplification; Mitosis; Anticancer drugs

4. Bosco, Emily E. The Retinoblastoma Tumor Suppressor Modifies the Therapeutic Response of Breast Cancer.

Degree: PhD, Medicine : Cell and Molecular Biology, 2006, University of Cincinnati

► The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of… (more)

▼ The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of human cancers, and nearly half of all breast cancers. Here, we investigate the consequence of RB loss on the response to DNA damage and anti-estrogenic therapies used in the treatment of breast cancer. Initially, we demonstrate that downstream RB targets are severely mis-regulated following acute deletion in adult primary cells causing abrogation of the DNA damage checkpoint and consequently, accumulation of secondary DNA lesions upon treatment with chemotherapeutics. Additionally, we found that RB modifies the DNA repair response in adult primary fibroblasts, such that RB-deficient cells are able to repair UV-induced lesions at an accelerated rate. These initial studies reveal that RB loss in primary cells modifies the response to DNA damage by promoting aberrant replication and inappropriately accelerating repair, both of which may ultimately sensitize cells to DNA damaging therapies. To specifically recapitulate RB loss in breast cancer, we directed shRNA against RB in MCF7 cells. RB-deficiency resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the loss of RB compromised the short-term cell cycle inhibition following anti-estrogen, cisplatin, and ionizing radiation therapies. In the context of DNA damaging agents this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of anti-estrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of RB loss were reiterated by ectopic E2F expression, indicating that control of downstream target genes was important for the observed responses. Specific analyses of the RB/E2F gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB-pathway is a determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for informing therapy in the treatment of breast cancer. Advisors/Committee Members: Knudsen, Erik S.

Keywords: Retinoblastoma Tumor Suppressor; Breast Cancer; E2F; Tamoxifen; anti-estrogen resistance; DNA damage; cell cycle checkpoint

5. BRADEN, WESLEY A. EMERGING ROLES FOR THE RB-PATHWAY IN DNA REPLICATION CONTROL.

Degree: PhD, Medicine : Cell and Molecular Biology, 2007, University of Cincinnati

► The retinoblastoma tumor suppressor (RB) pathway is disrupted in over 70% of… (more)

Subjects: Biology, Cell

Keywords: tumor suppressor; DNA replication; RB; preRC; p16ink4a; CDK; cyclin

6. BURD, CRAIG J. PROSTATIC REGULATION OF THE ANDROGEN RECEPTOR BY CYCLIN D1: FUNCTION AND DYSFUNCTION.

Degree: PhD, Medicine : Cell and Molecular Biology, 2006, University of Cincinnati

► The role of androgens in the prostate is clear, as activation of… (more)

▼ The role of androgens in the prostate is clear, as activation of the androgen receptor (AR) is critical for development, survival, and function of this organ. Moreover, AR activity is necessary for the growth and survival of prostatic cancers and is the primary target of non-organ confined tumors. Unfortunately, androgen ablation therapy is ultimately circumvented by restoration of AR activity, therefore providing the impetus to understand the factors which govern androgen-dependent growth. Cyclin D1 plays a multifaceted role in the prostate in that it is a key regulator of both the AR and molecules controlling cellular proliferation. Hence, it exists as a positive and negative regulator of androgen dependent growth. Herein, we show that cyclin D1 utilizes at least two distinct mechanisms as a potent inhibitor of AR function in the context of prostatic cancer. First, cyclin d1 interacts with histone deacetylase 3 and utilizes its enzymatic activity to elicit repression. Second, cyclin D1 interacts with an important regulatory domain in the amino terminus of the AR to preclude changes required for receptor activity. We demonstrate these cyclin D1 co-modifier functions are contained within a central region of the protein termed the repressor domain (RD) and that this region is sufficient to inhibit androgen dependent proliferation in prostate cancer. Furthermore, we reveal that deregulation of these cyclin D1 functions has clinical implications to prostate cancer, as a splice variant of cyclin D1, termed cyclin D1b, is highly expressed in prostate tumors. While cyclin D1b retains some transcriptional coregulatory functions, its ability to repress certain AR targets is compromised. The biological consequence of this defect is it fails to limit androgen dependent growth. In fact, ectopic expression of cyclin D1b increased proliferation in an androgen dependent prostate cancer cell line. Together, these data demonstrate a functional role for cyclin D1 in regulating AR activity and provide evidence of altered regulation in the development of prostate cancer. It further demonstrates a complex role of cyclin D1 as a rheostat for prostatic growth Advisors/Committee Members: Knudsen, Dr. Karen E.

Keywords: prostate cancer; androgen receptor; cyclin d1

7. Buschmann, Mary McVey. Laminin-332-Mediated Proliferation Control: Mechanisms Regulating Formation of the Epithelium.

Degree: PhD, Medicine : Cell and Molecular Biology, 2010, University of Cincinnati

► Normal epithelial cells rely on spatial cues from the extracellular matrix to… (more)

▼ Normal epithelial cells rely on spatial cues from the extracellular matrix to proliferate, migrate, and survive. The extracellular matrix protein, laminin-332 (LM-332) seems to be particularly important in proliferation control during re-formation of a wounded epithelium. Inhibition of the LM-332-cell interaction prevents wound healing in keratinocytes and mammary epithelial cells. Treatment of normal renal epithelial cells with LM-332 rich medium increases the rate of proliferation, and inhibition of the LM-332-cellular interaction prevents proliferation in mammary epithelial and rat bladder carcinoma cells. Despite this information, the mechanism of LM-332-mediated proliferation control is largely unknown. The goal of the studies described here was to understand the requirement for LM-332 in proliferation control to form a polarized epithelium using the Madin Darby Canine Kidney (MDCK) cell model system. LM-332 expression was turned on at low cell density when cells were proliferative, and turned off and degraded upon re-formation of a quiescent epithelium. Furthermore, the suppression of LM-332 by expression of an shRNA targeted against the LMα3 subunit induced a G1 cell cycle arrest, likely through a mechanism mediated by p21waf1 inhibition of the cyclin E/cdk2 complex. The LMα3 shRNA-mediated proliferation arrest, however could not be validated, as the G1 block could not be rescued by plating cells on, or exposing cells to, endogenous LM-332, or by co-expression of human LMα3. Also, inhibition of the LM-332 receptors, integrins α3β1 and α6β4, did not cause proliferative arrest to a similar extent as cell expressing the shRNA, and last, expression of three additional siRNAs specific for the LMα3 chain did not alter proliferation. Instead, studies using the new siRNAs indicated that LM-332 is important for cell spreading and morphogenesis of the epithelium. All of the studies presented in this work collectively suggest that deposition of LM-332 plays an important role in the regulation of cell spreading, morphogenesis, and possibly proliferation, to establish a polarized epithelium. Advisors/Committee Members: Wells, Susanne.

Subjects: Cellular biology

Keywords: Laminin-332; Extracellular Matrix; Epithelia; MDCK; Proliferation

8. CARLSON, ERIC CURTIS. THE ROLE OF LUMICAN IN THE FORMATION OF BIO-GLASS: TRANSPARENT CORNEA.

Degree: PhD, Medicine : Cell and Molecular Biology, 2003, University of Cincinnati

► Corneal opacity leaves 1.5 million people visually impaired worldwide. One potential key… (more)

▼ Corneal opacity leaves 1.5 million people visually impaired worldwide. One potential key player in the formation and maintenance of corneal transparency is a keratan sulfate proteoglycan lumican. The role of lumican in the formation and maintenance of bio-glass, transparent cornea, was examined using three different experimental systems. First, a wound healing system was used to understand the expression of lumican by corneal cells during stromal matrix restructuring and remodeling. Three different types of wounds were generated on mouse corneas: partial epithelial debridement, total epithelial debridement, and alkali burn wounds. Lumican expression was analyzed over a 12 week time course. Second, cultured corneal fibroblasts were used in an ex vivo expression system to determine the impact of lumican on collagen fibrillogenesis. Cultured corneal fibroblasts were stably transfected with either a wild-type lumican (lumWT) or 41cysteine to serine mutated lumican (lumC/S). Under proper conditions stable transformants formed a three-dimensional extracellular matrix over a 4 or 6 week time period. Finally, DNA microinjection into the corneal stroma was used as an in vivo expression system. Our results demonstrate the following: (1) the amount of lumican expression by corneal stroma cells in a wound healing situation decreases during stromal remodeling and is also dependent on the severity of the wound. This finding indicates that the gene expression pattern of keratocytes is lost during the wound healing response. (2) Mutation of 41Cys to serine is adequate to impair the role of lumican in ex vivo collagen fibrillogenesis. The actual interaction of lumican with collagen is disrupted by this single amino acid mutation. The actual impact is possibly due to a structural change caused by this mutation. (3) Plasmid DNA microinjection into the corneal stroma is an effective tool to drive protein expression and is adequate to rescue the thin corneal stroma phenotype of lumican null mice. DNA microinjection into the corneal stroma is capable of being an efficient and safe way to serve as a gene therapy strategy in treating ocular surface diseases. This technique is simple, non-invasive, and effective in expressing a therapeutic protein of interest. Advisors/Committee Members: -Y.Kao, Dr. Winston W.

Keywords: cornea; lumican; keratocyte; wound; collagen fibillogenesis

9. Cervantes, Rachel Bolante. PATHWAYS TO MUTATION IN SOMATIC AND STEM CELLS.

Degree: PhD, Medicine : Cell and Molecular Biology, 2000, University of Cincinnati

► Embryonic stem (ES) cells are intrinsically different from somatic cells in that… (more)

▼ Embryonic stem (ES) cells are intrinsically different from somatic cells in that they retain the capacity to differentiate into multiple cell types (Nagy et al., 1993; Brook and Gardner, 1997). Damage to any cell can have serious consequences, but the consequences of mutation in early embryonic cells are increased as these cells will eventually give rise to numerous progeny. Here we have shown that mutation in murine ES cells, heterozygous at the selectable Aprt locus, differed from that in somatic cells. The mutation rate in ES cells was significantly lower than in mouse embryonic fibroblasts (MEFs) and the distribution of mutagenic events was remarkably different between the two cell types. Loss of the functional allele was the predominant mutation type in both cases, representing about 80% of all events. However, mitotic recombination accounted for all LOH events detected in somatic cells whereas chromosome loss/reduplication, leading to uniparental disomy (UPD) was the predominant mechanism of mutation in ES cells. Accumulation of UPD with continued culture will lead to reduction to homozygosity at multiple recessive disease loci and may limit the potential clinical use of ES cells. In addition to its usefulness as a reporter of mutation in vitro, Aprt can be used to detect mutation in situ. The ability to visually distinguish between APRT proficient and deficient cells forms the basis of an APRT mouse model to detect point mutations in vivo. The overall strategy is to create mice that are Aprt-/- and whose mutant alleles contain known inactivating point mutations that are revertible when exposed to a mutagenic compound or the environment. Radiolabeled adenine that is injected intraperitoneally is only incorporated into cells that have reverted the point mutation and subsequently gained APRT activity. To establish the sensitivity of the APRT mutagenesis model, the APRT null (Aprtneo/neo) mouse was used as a syngeneic mouse model for detecting transplanted APRT wild type donor hematopoietic cells. Rare APRT+ donor cells were easily detected in the blood and tissues of recipient mice by PCR and autoradiography. Interestingly, the pattern of engraftment of primitive hematopoietic cells differed between nonirradiated and sublethally irradiated mice. The findings indicate that the Aprt-/- mouse mutagenesis model currently being produced will provide a sensitivie and effective method of detecting mutation in situ. Advisors/Committee Members: Stambrook, Peter J.

Keywords: ES cell; mutation; genomic instability; loss of heterozygosity; aprt

10. DORNETTE, POLLY A. EFFECTS OF ESTROGEN ON THE EXPRESSION OF GPR41 AS DETECTED BY A NOVEL ANTIBODY.

Degree: MS, Medicine : Cell and Molecular Biology, 2004, University of Cincinnati

► Estrogen regulates gene expression through interactions with nuclear receptors. However, estrogen can… (more)

Subjects: Biology, Molecular

Keywords: GPR41; antisera; rapid effects of estrogen

11. FENG, YUXIN. MECHANISM OF ESTROGEN RECEPTOR-ALPHA ACTION AND THE CONSEQUENCE OF ITS CONDITIONAL DELETION ON MAMMARY GLAND DEVELOPMENT AND FUNCTION.

Degree: PhD, Medicine : Cell and Molecular Biology, 2007, University of Cincinnati

► ERá is a critical regulator in breast cancer and mammary gland development.… (more)

▼ ERá is a critical regulator in breast cancer and mammary gland development. Deregulation of ER signaling correlates with abnormal mammary gland development and breast cancer. However, the role of epithelial ER remains to be clarified in vivo and the mechanism of ER signaling regulation is far from comprehensive. We hypothesize that 1) mammary epithelial ER plays critical roles in mammary gland development during pregnancy and lactation and that 2) novel, as yet identified factors in ER transcriptional regulation are involved in breast cancer development. The loxP-Cre system was used to generate epithelial ERKO mice. The well characterized MMTV-Cre and WAP-Cre transgenic mice were used to delete ER in mammary epithelial cells at different developmental stages. Early expression of MMTV-Cre arrested mammary gland development at the neonatal stage. Successive pregnancy and lactation activated epithelial ER ablation, which compromised side-branching, alveolar development, and epithelial proliferation. Further analysis revealed a massive loss of luminal epithelial cells presumably caused by apoptosis. The abnormal mammary gland development decreased milk production, thereby, caused growth retardation in the offspring. Similar phenotypes were also observed in MMTV-ERKO females in lactation. Thus, we concluded that epithelial ER is essential for mammary gland development during pregnancy and lactation stages. To further pursue the molecular mechanism of ER signaling regulation, a human mammary gland cDNA library was screened to identify novel factors that interact with ER. One novel ERá binding protein identified in the screen contains two conserved LXXLL motifs (NR-box) and a coiled-coil domain. The protein product, which we named NRCC, consists of 3 isoforms that vary in their N-terminal region. NRCC isconserved in vertebrates and its mRNA was detected in human breast cancer cells and mouse breast tumors. We found that NRCC-A interacts with ERá and enhances ERá transcriptional activity in human cancer cells. Moreover, NRCC-A co-localized with ERá in the cell nucleus and was recruited to ER target gene promoters. SiRNA analysis indicated that NRCC proteins are important for endogenous ERá-mediated transcriptional activity and estrogen dependent cell proliferation. Taken together, these data indicate that NRCC-A is a novel coactivator for ERá. Advisors/Committee Members: Khan, Dr. Sohaib.

12. Gildea, Lucy Anne. INTERACTIONS OF HISTOPLASMA CAPSULATUM YEASTS WITH HUMAN DENDRITIC CELLS.

Degree: PhD, Medicine : Cell and Molecular Biology, 2000, University of Cincinnati

► Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of world-wide importance. As… (more)

Keywords: dendritic cells; Histoplasma capsulatum; host defense; antigen presentation

13. Guo, Ning. SIP30 (ZWINT1), a placental mammal specific gene, modulates stimulated vesicle exocytosis and neuropathic pain.

Degree: PhD, Medicine : Cell and Molecular Biology, 2009, University of Cincinnati

► SIP30 is a multifunctional protein that participates in different molecular and cellular… (more)

Keywords: SIP30; vesicle; neuropathic; neuropathic pain; exocytosis; PC12 cells; PC12

14. Hess-Wilson, Janet Katherine. Influence of the Nuclear Hormone Receptor Axis in the Progression and Treatment of Hormone Dependent Cancers.

Degree: PhD, Medicine : Cell and Molecular Biology, 2007, University of Cincinnati

► Due to its pivotal role in prostatic growth and survival, the androgen… (more)

▼ Due to its pivotal role in prostatic growth and survival, the androgen receptor (AR) is the primary target of disseminated prostate cancer (CaP), as achieved via androgen deprivation therapy (ADT). Unfortunately, ADT is circumvented by restoration of AR activity, resulting in ADT resistant tumors for which there is no alternate treatment option. Through multiple mechanisms, reactivation of the AR specifically underlies the progression to therapy resistant tumors. The environmentally prevalent endocrine disrupting compound (EDC), bisphenol A (BPA) is able to activate specific somatically mutated ARs commonly found in CaP, resulting in androgen-independent proliferation of CaP cells. To directly assess the effect of BPA on ADT, we used an in vivo xenograft model of CaP that expresses a BPA-sensitive mutant AR, and mimics standard human cytotoxic response to ADT, followed by subsequent tumor re-growth. When tumor-bearing animals were exposed to environmentally relevant levels of BPA during ADT, the tumors failed therapy more rapidly (compared to placebo controls), with AR re-activation and concomitant increased tumor cell proliferation. These data suggest that environmentally relevant exposure to EDCs may reduce the efficacy of mainline ADT for CaP. We next determined that the environmentally persistent pesticide, DDE, was able to activate select AR mutants, and in in vitro models of CaP, DDE induces cellular proliferation in the absence of androgen, demonstrating that this observed response was not unique to BPA. Strikingly, the mechanism of DDE impact on CaP cells was distinct from BPA, in that at low doses this agent also activated the MAPK pathway, and requires this activation for mitogenesis. Given the context specific impact of AR activation by EDCs, and the deleterious effect of exposure to BPA on ADT in vivo, we next addressed the impact of AR action on taxane-based therapy. These cytotoxic agents have recently been shown to improve survival outcome for patients with advanced CaP, and are potentially new second line therapies, however the impact of AR action on this cytotoxic modality had yet to be assessed. Contrary to the stigma of AR as a survival factor, we found that AR activation by both endogenous and exogenous agonists (i.e. DHT and BPA), synergized with taxanes to decrease cell survival, through p53-mediated, caspase dependent apoptosis. This synergistic action was attributed directly to the AR-dependent mitogenic capacity of these agents. Importantly, these data further support the conclusion that the environmental impact on AR action and CaP therapeutic outcome is context specific. We further evaluated the complexity of EDC affects by assessing the impact of these agents under clinically relevant conditions in another hormone-dependent tissue, breast cancer. BPA and the phytoestrogen, coumestrol (COU), were able to activate the estrogen receptor (ER-alpha), but this activation was restricted to estrogen-depleted conditions, and could be blocked by the standard ER antagonist, tamoxifen. Additionally, BPA and COU have disparate affects in multiple analyses including mutant ER-alpha activation, and specific coactivator deregulation. In conclusion, the environmental impact on nuclear receptor signaling and hormone dependent cancer progression and treatment is highly molecular context specific. Therefore, the impact of EDCs on hormone dependent cancer treatment needs to be identified under specific and well defined, clinically relevant, molecular contexts. Advisors/Committee Members: Knudsen, Dr. Karen E.

Keywords: nuclear hormone receptors; endocrine disrupting compounds; androgen receptor; bishphenol A; taxanes; DDE; prostate cancer; breast cancer

15. Hilty, Jeremy S. Iron Acquisition and Homeostasis in Histoplasma capsulatum.

Degree: PhD, Medicine : Cell and Molecular Biology, 2008, University of Cincinnati

► Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within… (more)

▼ Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (MΦ). Studies in human and murine MΦ have demonstrated that the intracellular growth of H. capsulatum yeasts is exquisitely sensitive to the availability of iron. As H. capsulatum produces hydroxymate siderophores, we sought to determine if siderophore production was required for intracellular survival in MΦ, and in a murine model of pulmonary histoplasmosis. UC7 SIDA silenced yeasts and SIDA knockout yeasts (UC8 ΔsidA) grew normally in rich medium, did not synthesize siderophores, and were unable to grow on apotransferrin-chelated medium. The intracellular growth of UC7 SIDA silenced and UC8 ΔsidA yeasts in MΦ was significantly decreased compared to wild type (WT) yeasts, but growth was restored to WT levels by the addition of exogenous iron. Compared to WT (G217B) yeasts, C57BL/6 mice infected with demonstrated significantly reduced growth in the lungs and spleens seven days after infection. Additionally, we sought to identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human MΦ. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron replete medium, but not on iron deficient media. On iron deficient medium, the growth of the vma1 mutant was restored in the presence of wild type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within MΦ was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28°C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis, and in fungal dimorphism and demonstrate that: SIDA expression is required for siderophore biosynthesis by H. capsulatum; that siderophore synthesis is required for optimum intracellular growth in MΦ and that in vivo, inhibition of siderophore synthesis reduces the virulence of H. capsulatum yeasts. Advisors/Committee Members: Newman, Simon.

Subjects: Biology

Keywords: iron; histoplasma; vma; siderophore; fungal; siderophores; virulence

16. Horn, Henning Friedrich. The Role of p21 CIP1/WAF1 and CDK2/Cyclin E in Regulating Centrosome Duplication.

Degree: PhD, Medicine : Cell and Molecular Biology, 2006, University of Cincinnati

► The centrosome plays an important role in directing the formation of the… (more)

▼ The centrosome plays an important role in directing the formation of the bipolar spindle during mitosis, a process that ensures the accurate segregation of chromosomes to daughter cells. Because of its importance in mitosis, centrosome duplication is highly controlled, and is under many of the same constraints as DNA replication. Duplication of both is initiated concomitantly, both initiate duplication in response to activation of cyclin-dependent kinase 2/cyclin E (CDK2/CycE), and the reduplication within the same cell cycle is suppressed in both. Many tumors exhibit an abnormal number of centrosomes, indicative of the loss of these regulatory mechanisms. The functional loss of the tumor suppressor p53 is one of the most common occurrences in tumor formation, and p53 has been shown to play an important role in regulating centrosome duplication by promoting proper initiation of centrosome duplication as well as by suppressing reduplication. CDK2/CycE has also been shown to play an important role in initiating centrosome duplication, and the centrosome hyperamplification of many tumors can be linked to a constitutive activation of this kinase complex. We have shown that the loss of p53 induces centrosome hyperamplification in both cultured cells as well as in spontaneously formed rodent tumors synergistically with the constitutive activation of CDK2/CycE. We have shown that p21Cip1/Waf1 (p21) plays an important role in coordinating the initiation of centrosome duplication with the initiation of DNA synthesis, and p21-null cells initiate centrosome duplication before initiation of DNA synthesis. We have also identified nucleophosmin (NPM) as a centrosomal target of CDK2/CycE, and have shown that the phosphorylation of NPM by CDK2/CycE is a necessary event for centrosome duplication. And finally, we have identified a novel interaction between the Mitogen Activated Protein Kinase (MAPK) and p21. The MAPK-mediated phosphorylation of p21 promotes nuclear localization of p21, at least in part by mediating an increase interaction between p21 and the nuclear import factor karyopherin α1. The MAPK-p21 interaction may have interesting implications for the regulation of centrosome duplication as well as for the migration of centrosomes to the spindle poles. Advisors/Committee Members: Fukasawa, Dr. Kenji.

Keywords: centrosome; CDK2/Cyclin E; p53; p21/Cip1/Waf1

17. Johansson, L. Gunnar. Identification of Targeted Therapeutics for Malignant Peripheral Nerve Sheath Tumors.

Degree: PhD, Medicine : Cell and Molecular Biology, 2008, University of Cincinnati

► Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant… (more)

Subjects: Cellular biology

Keywords: Neurofibromatosis type 1; NF1; MPNST; mTOR; Cancer

18. Kulkarni, Rishikesh Manohar. Regulation of Lymphatic Endothelial Development and Function in Lung.

Degree: PhD, Medicine : Cell and Molecular Biology, 2009, University of Cincinnati

► Lymphatics play a vital role in maintaining homeostasis by keeping the tissue… (more)

▼ Lymphatics play a vital role in maintaining homeostasis by keeping the tissue free of excess fluid. This function is important in lungs to facilitate gas exchange at the capillary alveolar barrier. Although lymphatic endothelial cells (LEC) arise from blood endothelial cells (BEC) in vivo, the molecular mechanisms of LEC lineage selection are poorly understood. Further, the role of pulmonary lymphatics in fluid clearance at birth and the contribution of lung lymphatic hypoplasia to development of lung diseases are not well elucidated. This is partly due to lack of an in vitro model to study the LEC lineage selection and lack of an in vivo model mimicking pulmonary lymphatic hypoplasia respectively. Our studies in human microvascular endothelial cells from lungs (HMVEC-L) have demonstrated that BEC in this population undergo a lineage selection into LEC. VEGF-A, a growth factor increased in injury, accelerated the BEC-LEC shift, making HMVEC-L an in vitro system to study mechanisms of lymphatic lineage selection in physiological and pathological lymphangiogenesis. Differential microarray analysis on enriched BEC and LEC populations from HMVEC-L identified differentially expressed transcription factors, including NFATc1. NFATc1 is critical for lineage selection in T-cell differentiation, cardiac valve morphogenesis and osteoclastogenesis. NFATc1 expression was higher in LEC over BEC in microarray studies and was selectively expressed on LEC in vitro. In vivo, NFATc1 colocalized with lymphatic markers Prox-1, VEGFR-3 and podoplanin as LEC were specified, formed lymph sacs and on mature lymphatics. In NFATc1 null mice coalescence of LEC into lymph sacs was impaired. NFAT activation requires the phosphatase calcineurin. Embryos treated in utero with cyclosporine-A (calcineurin inhibitor) showed cytoplasmic NFATc1, diminished podoplanin and FGFR-3 expression by lymphatics and irregular lymphatic patterning. We have previously demonstrated that VEGF-A over expression in the developing lungs, mimicking lung remodeling following injury, induced lymphangiogenesis. We found that NFATc1 was expressed on the VEGF-A-induced lung neolymphatics. Mice lacking the calcineurin-A-beta regulatory subunit, with diminished nuclear NFAT, failed to increase lymphangiogenesis in response to VEGF-A. NFATc1 siRNA significantly reduced endogenous and VEGF-A-induced VEGFR-3 and podoplanin expression in HMVEC-L. In reporter assays, NFATc1 activated lymphatic gene promoters. Our results demonstrate the role of calcineurin-NFAT pathway in physiological and pathological lymphangiogenesis, with NFATc1 being the key NFAT. VEGFR-3 signaling is essential for lymphangiogenesis. We demonstrated that selective expression of dominant negative VEGFR-3 (dnR-3) in lungs diminished pulmonary lymphatic development without affecting blood vascular development or lung organogenesis. In neonatal mice, dnR-3 expression caused pulmonary lymphatic hypoplasia, respiratory distress and increased mortality. Mice that survived until adulthood showed marked pulmonary lymphatic hypoplasia. Using morphometric analysis, we have established criteria to assess the effect of pulmonary lymphatic hypoplasia on neonatal lung fluid clearance. The dnR-3 model needs further characterization regarding normal lymphatic function and during acute lung injury. Taken together, our results demonstrate a role for NFATc1 in lymphangiogenesis and the importance of dnR-3 model to elucidate the role of lung lymphatics in perinatal fluid clearance and the contribution of lymphatic hypoplasia to development of lung diseases. Advisors/Committee Members: Akeson, Ann.

Subjects: Biomedical research; Cellular biology; Molecular biology

19. LaPensee, Elizabeth W. Mechanisms of Chemoresistance in Breast Cancer and Liposarcoma.

Degree: PhD, Medicine : Cell and Molecular Biology, 2008, University of Cincinnati

► Chemotherapy is mainstay treatment for cancer patients, but unfortunately many tumors are… (more)

▼ Chemotherapy is mainstay treatment for cancer patients, but unfortunately many tumors are resistant to therapy. Much research now focuses on identifying mechanisms that contribute to chemoresistance. The importance of such research lies in the fact that no other treatment has proven as effective in controlling cancer metastasis and preventing tumor recurrence. This thesis focuses on: 1) identifying a cellular model appropriate for studying chemoresistance in liposarcoma and 2) understanding the role of endogenous hormones, such as prolactin (PRL) and estradiol (E2), as well as the endocrine disruptor bishpenol A (BPA), in affecting anti-cancer drug efficacy in breast cancer cells. Project 1: Our first goal was to compare the responsiveness of our LS14 liposarcoma cell line and SW872 liposarcoma cells to anti-cancer drugs and explore the mechanism(s) underlying chemoresistance. Using complementary assays for cell viability and apoptosis, we found that LS14 cells are much less respsonsive to anti-cancer drugs and have higher expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. In addition, LS14 cells are also tumorigenic in nude mice. Project 2: The role of PRL as a survival hormone led to the hypothesis that PRL protects breast cancer cells from chemotherapy. We found that low dose PRL antagonized the cytotoxic effects of various anti-cancer drugs and prevented cisplatin-induced cell cycle arrest and apoptosis. γ-H2AX staining and mass spectroscopy reveal less DNA damage and less cisplatin bound to DNA in the presence of PRL. Glutathione s-transferase, which sequesters cisplatin in the cytoplasm, was dramatically increased by PRL; an effect abrogated by Jak and MAPK inhibitors. PRL did not antagonize cisplatin or prevent its binding to DNA in the presence of a GST inhibitor. Project 3: Our third study focused on the roles of BPA and E2 in chemoresistance in breast cancer cells. BPA and E2 confer resistance to various anti-cancer drugs in both ERα-positive and -negative cells. Antagonists for ERα and ERβ revealed that BPA and E2 unlikely mediate their effects via either of these receptors. Both cell lines express non-classical estrogen receptors, including GPR30 and members of the ERR family. The ability of BPA and estradiol to alter the expression of Bcl-2 and/or increase proliferation are likely mechanisms by which they confer chemoresistance. Overall Conclusions: The reduced responsiveness of LS14 cells to anti-cancer drugs more accurately reflects chemoresistance in patients. This, combined with their tumorigenicity, make LS14 cells an excellent model for exploring anti-cancer drug efficacy and resistance in liposarcoma. Ours is the first study showing PRL antagonizes cisplatin by preventing its entry to the nucleus. Future therapies that reduce PRL levels or block its actions should be beneficial to breast cancer patients undergoing chemotherapy and allow for expanded drug options. Ours is the first demonstration that BPA alters responsiveness to chemotherapy. Therapeutically, the measurement of BPA and E2 levels in breast cancer patients should allow for dosage adjustment and drug selection on a patient by patient basis. Advisors/Committee Members: Ben-Jonathan, Nira.

Subjects: Cellular biology

Keywords: chemoresistance; prolactin; estrogen; bisphenol A; apoptosis

20. LIBY, KAREN. PROLACTIN AS A LOCAL GROWTH FACTOR IN BREAST CANCER.

Degree: PhD, Medicine : Cell and Molecular Biology, 2002, University of Cincinnati

► Prolactin (PRL) is a 23 kDa hormone that targets the breast, but… (more)

▼ Prolactin (PRL) is a 23 kDa hormone that targets the breast, but its role in breast cancer is controversial. An N-terminal 16K PRL fragment suppresses endothelial cell proliferation, but its ability to inhibit tumor growth had not been tested. Our hypotheses were: 1) locally-produced PRL promotes breast cancer growth and 2) 16K PRL inhibits tumor vascularization and growth by suppressing angiogenesis. Using MDA-MB-435 (MDA) breast cancer cells as our model, the specific aims were to: 1) determine the effects of 23K human PRL (hPRL) on cell proliferation and PRL receptor (PRL-R) expression in vitro , 2) test whether 23K hPRL accelerates tumor growth in nude mice, 3) confirm the angiostatic activity of recombinant 16K hPRL in vitro and 4) compare the inhibitory properties of 16K hPRL and endostatin on angiogenesis and tumor growth in nude mice. Exogenous hPRL increased MDA cell proliferation and PRL-R mRNA expression. MDA clones overexpressing 23K hPRL were generated, and PRL production/secretion were confirmed by RT-PCR, Western blotting and the Nb2 assay. When incubated in charcoal-stripped serum, the PRL clones proliferated faster and expressed higher levels of the PRL-R than controls; PRL-overexpressing tumors grew 2-4 times faster than vector or wild type MDA tumors when injected into nude mice. Western analysis demonstrated increased hPRL, PRL-R, and bcl-2 proteins in the PRL-overexpressing tumors compared to controls. These data support a role for local PRL as a mitogenic/anti-apoptotic factor in breast cancer and suggest it may serve as a new therapeutic target for treating breast cancer.Recombinant 16K and 23K hPRL (rhPRL) were produced by baculovirus expression. 16K rhPRL failed to suppress the proliferation of endothelial cells unless contaminated with endotoxins. MDA clones overexpressing 16K hPRL or endostatin were generated. MDA clones overexpressing 16K hPRL did not inhibit angiogenesis or tumor growth in nude mice, but tumors from the endostatin clones were 4-5 fold smaller than vector tumors. The apoptotic index was significantly higher while the blood vessel density was lower in tumors expressing endostatin than in vector controls. These data confirm the potent anti-angiogenic activity of endostatin but do not support a similar function for 16K hPRL. Advisors/Committee Members: Ben-Jonathan, Dr. Nira.

Subjects: Biology, Cell

Keywords: prolactin; breast cancer; tumors; nude mice; growth factor

21. LINK, KEVIN A. BAF57 MODULATION OF ANDROGEN RECEPTOR ACTION AND PROSTATE CANCER PROGRESSION.

Degree: PhD, Medicine : Cell and Molecular Biology, 2008, University of Cincinnati

► Prostate cancer relies on androgens for growth and survival. Androgens elicit their… (more)

▼ Prostate cancer relies on androgens for growth and survival. Androgens elicit their function primarily through the androgen receptor (AR), a transcription factor that is required for the development and progression of prostate cancer. Therefore, current treatment options for disseminated disease are aimed at either inhibiting androgen synthesis or preventing activation of the receptor. These treatment options are initially effective; however, recurrent disease ultimately arises within two to three years that typically present as therapy resistant. From this, it is obvious that more effective means of targeting this pathway are necessary to treat prostate cancer. The present study exploited a specific aspect of the AR activation pathway and uncovered a novel means for potentially targeting AR action and subsequent prostate cancer proliferation. The SWI/SNF chromatin remodeling complex is one such necessary component involved in mediating AR activation. Specifically, the BRM core ATPase was demonstrated to exhibit a preferential response to activating AR target genes which was dependent on promoter context. Subsequent studies identified the BAF57 subunit of the SWI/SNF complex as the primary interacting AR subunit, thus indicating a possible role in complex recruitment to AR target DNA. BAF57 proved to be critical for AR mediated transcriptional activation, AR enhanced coactivator activation, and prostate cancer proliferation. Based on these findings, the potential for targeting BAF57 as a prostate cancer therapeutic option was examined. First, expression of BAF57 was verified in human prostate tissue specimens, with possible enhanced expression observed in metastatic samples. Subsequent molecular analyses determined that the primary region of interaction occurred between the DBD/hinge region of AR and the proline-rich/HMG domain of BAF57. The N-terminal binding region of BAF57 was then shown to reduce AR recruitment to AR target genes, inhibit AR target gene activity, and decrease prostate cancer cell proliferation. Together, the data herein establish the SWI/SNF chromatin remodeling complex, specifically BAF57, as a valid target for the treatment of prostate cancer. Advisors/Committee Members: Knudsen, Dr. Karen E.

Subjects: Biology, Molecular

Keywords: prostate cancer; androgen receptor; BAF57; SWI/SNF

22. Mallory, Bradford Paul. The Pulmonary Vasculature Mediates Differential Time-Sensitive Effects on Embryonic Lung Development.

Degree: PhD, Medicine : Cell and Molecular Biology, 2005, University of Cincinnati

► Lung development is analogous to a well orchestrated symphony, and depends on… (more)

▼ Lung development is analogous to a well orchestrated symphony, and depends on successful interactions between various cellular components within the lung. The pulmonary vasculature is essential for normal lung development and is a key member of the lung development ensemble. Impairment of pulmonary vascular development is the hallmark of neonatal diseases such as respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Members of the VEGF family of growth factors and receptors are critical mediators of vascular development. Two members of the VEGF family, VEGF-A and VEGF-D are highly expressed in developing lung. VEGF-A is known to play a role in pulmonary vascular development, but its role in vessel specification in the developing lung is poorly understood. In addition, we hypothesize that VEGF-D plays a role in pulmonary lymphatic development based on its high expression in developing lung mesenchyme and its ability to bind the lymphatic receptor VEGFR-3. Through conditional regulation of VEGF-A, we have characterized differential temporal effects of the pulmonary vasculature on lung organogenesis, unique to early (embryonic day E10.5-E12.5), middle (E12.5-E16.5), and late (E16.5-E18.5) phases of development. Induction of VEGF-A in distal lung from E14.5 to E18.5 resulted in disorganization of endothelial ultrastructure, increased endothelial cell proliferation and rearrangement of the established vascular plexus, altering alignment with the epithelium. Remarkably, VEGF-A induction caused a 3.3-fold increase in small lymphatic vessels and a 1.3-fold increase in arteries. This preferential induction indicates that VEGF-A influences lymphatic specification in late stages of lung development. In addition, we have characterized differential time-sensitive effects of VEGF-A on other aspects of embryonic lung development. VEGF induction during the perinatal period disrupted terminal branching morphogenesis, inhibited formation of primitive alveolar septae, and altered the established vascular and smooth muscle pattern. Further, VEGF-A induction during the period prior to birth increased postnatal mortality and morbidity. Taken together, these results demonstrate that VEGF-A regulates developmental programs for specification of pulmonary lymphatic and blood vessels during late stage lung development. Further, we show that the time-sensitive effects of VEGF on lung epithelial morphology and organogenesis are indirect, mediated through endothelial to epithelial signaling. Advisors/Committee Members: Akeson, Ann L.

Keywords: VEGF, VEGF receptor, lymphatic, lymphangiogenesis, lung development, arteriogenesis

23. MARKEY, MICHAEL PATRICK. TRANSCRIPTIONAL REGULATION BY THE RETINOBLASTOMA TUMOR SUPPRESSOR: NOVEL TARGETS AND MECHANISMS.

Degree: PhD, Medicine : Cell and Molecular Biology, 2004, University of Cincinnati

► The retinoblastoma tumor suppressor (RB) is a key regulator of the cell… (more)

Keywords: retinoblastoma; RB; cancer; cell cycle; bioinformatics; microarray

24. Marsh, Randall Glenn. Characterization of a new role for plakoglobin in suppressing epithelial cell translocation.

Degree: PhD, Medicine : Cell and Molecular Biology, 2001, University of Cincinnati

► Despite advances in the treatment of cancer patients, spread, or metastasis, of… (more)

▼ Despite advances in the treatment of cancer patients, spread, or metastasis, of tumor cells remains a major cause of death. Although metastasis is a complex process, an important component is cell motility. Acquisition of motility allows tumor cells to breach normal barriers and thereby disseminate. Regulation of motile behavior is poorly understood, but evidence suggests the cadherin family of adhesion molecules plays an important role. The goal of my thesis was to investigate regulation of epithelial cell translocation by cadherins. For my studies, I focused on PAM212 keratinocytes. These cells express high levels of E-cadherin and show suppression of translocation upon contact (STUC). Using repeated light trypsinization and assessment of colony morphology, I selected for variants having a scattered, motile phenotype. As hoped, some variants had reduced expression of E-cadherin (ED cells). Surprisingly, however, some variants retained relatively normal E-cadherin expression but had drastically reduced plakoglobin levels (PD cells). Re-expression of E-cadherin in ED cells, or plakoglobin in PD cells, restored STUC, demonstrating a causal role for each protein. Time-lapse analyses revealed that E-cadherin mediated initial cell-cell contacts, while plakoglobin mediated a subsequent long-term stabilization event. Plakoglobin has known adhesive and signaling functions and could therefore suppress translocation through an adhesive or signaling mechanism. Strong support for a signaling mechanism was provided by: (a) adhesion assays, which revealed no significant difference in adhesiveness of parental PAM212 versus PD cells; (b) time-lapse analyses, which revealed no difference in the ability of parental versus PD cells to form contacts, and; (c) analysis of a deletion-mutant of plakoglobin, which restored STUC despite being unable to participate in desmosome assembly or cadherin adhesion. The most likely candidates for mediating signaling downstream of plakoglobin were TCF/LEF transcription factors, but these were ruled out by: (a) analysis of a dominant-negative TCF/LEF, which failed to render parental cells motile, and; (b) the effects of a deletion-mutant of the plakoglobin-related protein ?-catenin, which restored STUC in PD cells despite being unable to bind these factors. These results reveal a new role for plakoglobin in suppressing epithelial cell translocation, and show that an undescribed signaling pathway underlies this suppression. Advisors/Committee Members: Brackenbury, Robert.

Keywords: cell motility; plakoglobin; cadherin; TCF/LEF signaling; cell movement; cell translocation

25. MCFARLAND, KEVIN LEE. CHARACTERIZATION OF THE REGULATORY REGION OF THE DISPERSED HOMEOBOX GENE gsh-1.

Degree: MS, Medicine : Cell and Molecular Biology, 2002, University of Cincinnati

► Gsh-1 , a dispersed homeobox gene, is expressed in a regionally restricted… (more)

▼ Gsh-1 , a dispersed homeobox gene, is expressed in a regionally restricted pattern in the developing mouse central nervous system. To better understand the temporal and spatial regulation of gsh-1 , we have isolated mouse genomic regions surrounding the ORF of the gsh-1 gene. This was used for reporter-gene analysis in transgenic mice, and in cell culture. First, a previously isolated 10 kb genomic region surrounding the ORF of the gsh-1 gene was fused to a Lac-Z gene and used for analysis of the cis acting regions of the gsh-1 gene promoter by generating transgenic mice. The expression pattern of the gsh-1/β-gal ;5.5/4.5 transgene construct was localized to the hypothalamus, thalamus, and pons, therefore the transgene showed a similar but distinct pattern compared to the gsh-1 pattern seen earlier by in situ hybridization. A 21 kb piece of the mouse genomic gsh-1 gene was then isolated, containing 14.5 kb of 5' and 6.5 kb of 3' the gsh-1 gene. A gsh-1/β-gal ;14.5/0 reporter construct was expressed diffusely throughout the midbrain and medial gaglionic eminence. Again this pattern was similar to, but did not completely recapitulate the distinct pattern of gsh-1 seen by in situ hybridization in the developing mouse embryo. Second, a SV-40 LTAg gene was inserted into the first exon of the gsh-1 gene, and this construct was used to generate transgenic mice. The mice developed brain tumors, which were developed into two cell lines that expressed gsh-1 mRNA. Wild type gsh-1 /SV-40 LTAg mice were bred to heterozygous gsh-1 knockout mice to create a gsh-1 null cell line. Third, in order to perform analysis of the cis acting regions of the gsh-1 promoter, gsh-1 constructs with a luciferase reporter gene were generated. These were then transfected into the gsh-1/SV-40 LTAg cell line clones. Transient transfection of these constructs showed a ~ 350 fold increase in activity in a gsh-1 expressing cell lines over a non-neuronal cell line. Also, luciferase activity for the gsh-1/luc ;14.5/6.5 construct is 4 fold higher than luciferase activity for the gsh-1/luc ;5.5/4.5 construct. Fourth, a test of the autoregulation of gsh-1 , showed no increase in luciferase expression. Advisors/Committee Members: Potter, Dr. S. Steven.

Keywords: gsh-i; homeobox; promoter; transient transfections; reporter genes

26. MCFARLAND-MANCINI, MOLLY MELINDA. PROLACTIN PRODUCTION BY HUMAN BREAST ADIPOSE TISSUE AND ADIPOCYTES.

Degree: PhD, Medicine : Cell and Molecular Biology, 2006, University of Cincinnati

► PRL is a 23 kDa hormone that is expressed at pituitary and… (more)

▼ PRL is a 23 kDa hormone that is expressed at pituitary and extrapituitary sites. Although the breast expresses PRL, it is not known what compartments as well as cell types within the breast are responsible for PRL production, how much PRL is released from these tissues and how PRL expression is regulated. Breast PRL can contribute to breast development, local adipose tissue metabolism as well as to breast cancer progression. By identifying the sources of breast PRL and how it is regulated, the function of PRL as an autocrine/paracrine factor can be determined. Our hypotheses were that PRL is produced by the major compartments of the breast and is regulated differently than pituitary PRL. Using human adipose and glandular explants and primary breast preadipocytes in vitro, the specific aims were to: 1) determine if biologically active PRL is produced by breast explants, 2) ascertain if PRL is regulated by ovarian steroids in these tissues, 3) verify whether breast PRL is transcribed from the superdistal promoter, 4) identify the adipose cell types responsible for PRL production, 5) identify putative ligand that may stimulate/inhibit PRL expression and release from adipocytes, and 6) compare major regions within the superdistal promoter that regulates PRL expression in primary preadipocytes, liposarcoma-derived adipocytes and breast cancer cells. We found that PRL expression increases in a time-dependent manner in both adipose and glandular explants in vitro. PRL release also increases over time in culture and is 10-12 fold higher in adipose than glandular tissue. PRL release is decreased in glandular, but not adipose, tissue by progesterone, while estradiol has no effect on either tissue. Within adipose tissue, PRL is expressed at low levels in both primary preadipocytes and freshly isolated mature adipocytes. During differentiation PRL expression increases transiently during the commitment phase of adipogenesis. PRL expression/release in preadipocytes is stimulated by the cAMP activators IBMX, isoproterenol and PACAP. PRL transcription is driven by the superdistal promoter in primary preadipocytes, SW872, LS14 liposarcoma cells and Jurkat lymphoblast cells, but with notable differences in the relative activity of the various regions of the promoter. Advisors/Committee Members: Ben-Jonathan, Dr. Nira Melinda.

Subjects: Biology, Cell

Keywords: Prolactin; Breast; Adipose; Adipogenesis

27. Memmott, Regan. Inhibition of mTOR for the treatment and prevention of lung cancer.

Degree: PhD, Medicine : Cell and Molecular Biology, 2010, University of Cincinnati

► Activation of the mTOR pathway is an important and early event in… (more)

▼ Activation of the mTOR pathway is an important and early event in lung tumorigenesis, and therapies that target mTOR could be effective in the treatment or prevention of lung cancer. The prototypic mechanism of mTOR regulation in cells is through activation by the PI3K/Akt pathway, but mTOR receives input from multiple signaling pathways. In particular, the LKB1/AMPK pathway negatively regulates mTOR. To validate mTOR as a therapeutic target in lung cancer, we evaluated three drugs that inhibit the mTOR pathway, namely, the lipid-based Akt inhibitors phosphatidylinositol ether lipid analogues (PIAs), the AMPK activator metformin, and the mTOR inhibitor rapamycin. PIAs activated AMPK independently of Akt in NSCLC cells, which contributed to the cytotoxicity of these compounds and their ability to inhibit mTOR. AMPK activation by PIAs also occurred independently of the tumor suppressor LKB1, but required another upstream kinase of AMPK, CaMKKβ. Treatment of LKB1-mutant NSCLC xenografts with PIA decreased tumor volume by ~50%. These studies suggest that PIAs might have utility in the treatment of LKB1-mutant lung cancers, which are characterized by aberrant activation of the mTOR pathway. Another AMPK activator, metformin, was also effective in preventing tumor growth in a mouse model of tobacco carcinogen-induced lung tumorigenesis. Administration of the tobacco carcinogen NNK induces K-Ras mutations that activate the mTOR pathway and promote lung tumorigenesis in A/J mice. Metformin decreased tumor burden in this model by as much as 72%. The ability of metformin to prevent NNK-induced lung tumorigenesis was likely due to indirect effects on tumor cells. Although metformin inhibited the mTOR pathway in lung tissue and tumors, this occurred independently of AMPK activation and was associated with decreases in the phosphorylation of IGF-1R/IR and Akt. These data suggest that metformin might prevent NNK-induced lung tumorigenesis by decreasing the response of lung tissue to insulin or IGF-1. Inhibitors of the mTOR pathway might also prevent tobacco carcinogen-induced lung tumorigenesis by affecting the tumor microenvironment. Administration of NNK increased lung-associated Foxp3+ regulatory T cells (Treg) weeks prior to tumor development in A/J mice, and both metformin and rapamycin prevented this increase. Treg are a subset of CD4+ T cells that can limit the development of an effective immune response against cancer. Using a variety of techniques, we demonstrated that Foxp3+ Treg are required for K-Ras driven lung tumorigenesis. Therefore, the fact that metformin and rapamycin decrease lung- and tumor-associated Treg could contribute to their abilities to prevent NNK-induced lung tumorigenesis. Collectively, our studies demonstrate that inhibitors of the mTOR pathway are effective in both the treatment and prevention of lung cancer. Also, because metformin and rapamycin are both FDA-approved drugs, our studies could provide rationale for clinical prevention trials with these agents in patients at high risk to develop lung cancer. Advisors/Committee Members: Plas, David.

Subjects: Cellular biology

Keywords: mTOR; AMPK; lung cancer; metformin; rapamycin; Treg

28. MENON, SHEKAR. INTERACTION BETWEEN REDOX CHAPERONES AND RDW MUTANT THYROGLOBULIN.

Degree: PhD, Medicine : Cell and Molecular Biology, 2004, University of Cincinnati

► Inherited as an autosomal recessive, the WIC-rdw rat exhibits congenital dwarfism (rdw).… (more)

▼ Inherited as an autosomal recessive, the WIC-rdw rat exhibits congenital dwarfism (rdw). Genetic linkage analysis revealed that the rdw gene locus was identical to the thyroglobulin (Tg) gene locus. Complete sequencing of both wild type and the WIC-rdw rat Tg cDNA’s revealed a single missense mutation G6958C, causing a G2320R amino acid substitution at a position that is highly conserved. Further, transient expression of intact Tg cDNA containing the rdw mutation in COS-7 cells showed no detectable Tg in the secreted media confirming a defect in the export of rdw Tg. Despite the many similar features to other animal models for congenital hypothyroidism, the one major difference was that the rdw rat thyroid glands displayed marked hypoplasia. To examine the role of multiple ER chaperones in the processing of both wild type and rdw Tg, we followed their interactions by co-immunoprecipitation after pulse-chase. In COS-7 cells transfected with wild type Tg, the kinetics of dissociation of the chaperone BiP from Tg closely followed the kinetics of folding and ER exit of Tg, whereas, BiP association with the mutant Tg’s did not diminish over the same early chase periods. The oxidoreductases ERp72 and PDI but not ERp57 displayed little or no interaction with wild type Tg but did display significant stable binding to the mutant Tg. At long chase times up to 24 hrs, it was observed that majority of intracellularly retained mutant Tg was associated with BiP, ERp72 and PDI. These interactions were found to be both covalent and non-covalent. The classic non-covalent Tg – chaperone interaction diminished in parallel with the intracellular degradation of the rdw-mutant Tg but the covalent interaction with the redox chaperones remained constant throughout the chase period. During extended periods of chase, the covalently linked fraction remained unchanged. Investigations of both wild type and rdw rat thyroid tissue sections revealed the presence of foci that were positive for apoptosis. Thus, it is possible that the loss-of-function rdw mutation that results in accumulation of mutant Tg in the thryocyte ER may also be responsible for cellular toxicity leading to apoptosis, accounting for the observed thyroid gland hypoplasia. Advisors/Committee Members: Kim, Dr. Paul S.

Keywords: rdw,; thyroglobulin,; ERp72

29. Meyer, Sara. The Ron Receptor Tyrosine Kinase in Tissue Morphogenesis.

Degree: PhD, Medicine : Cell and Molecular Biology, 2009, University of Cincinnati

► The Ron receptor tyrosine kinase is overexpressed in many human cancers including… (more)

▼ The Ron receptor tyrosine kinase is overexpressed in many human cancers including colorectal and breast, and studies have established Ron as a predictor of disease outcome and as a therapeutic target. Ron overexpression and constitutive activation contributes to the tumorigenic properties of human colon cancer cells. Moreover, metastatic dissemination of colon cancer cells from primary orthotopic tumors in mice can be reduced upon Ron knockdown. The majority of hereditary and sporadic colorectal cancers harbor aberrant Apc/β-catenin signaling, however, the relationship between Ron, Apc, and β-catenin signaling in intestinal tumorigenesis is not well understood. We sought to test the requirement of Ron tyrosine kinase signaling for initiation of intestinal tumors in vivo using a well-characterized mouse model of mutant Apc-driven intestinal tumorigenesis. By generating (ApcMin/+ mice with a targeted deletion of the tyrosine kinase domain of Ron (RonTK-/-), we found that Ron is not required for intestinal adenoma formation, and that Ron loss increases tumor burden in a large fraction of mice. Unexpectedly, the loss of Ron in non-transformed intestinal epithelium significantly increases crypt cell proliferation, which may lead to an increased susceptibility to tumor initiation in this model. β-catenin localization and target gene expression were not significantly altered in ApcMin/+;RonTK-/- mouse tumors or normal intestine compared to controls, suggesting that Ron is not required for β-catenin signaling in this model. Like in colon cancer, Ron overexpression has also been observed in approximately half of human breast cancers. Mammary-specific overexpression of Ron in mice results in mammary carcinomas in 100% of mice that metastasize to the lungs and liver, supporting the conclusion that Ron overexpression is a causal oncogenic factor in breast cancer. Interestingly, mammary glands from virgin mice with aberrant Ron expression have dilated mammary ducts and sparse ductal branches. Based on these observations, and that molecules deregulated in breast cancers often have important roles in development, we hypothesized that Ron is a novel regulator of mammary gland morphogenesis. To study Ron in mammary development, RonTK-/- mice were utilized. We found that Ron tyrosine kinase domain-deficient mice had enhanced ductal morphogenesis during puberty, which was also evident when the mice were ovariectomized. Increased ductal elongation and earlier regression of terminal end buds was also observed in RonTK-/- mammary glands. Interestingly, accelerated pubertal mammary development was accompanied by increased phosphorylated MAPK, which was necessary for enhanced RonTK-/- epithelial branching morphogenesis in vitro. Together, these studies identified novel roles for Ron tyrosine kinase in the regulation of normal intestinal tissue homeostasis and normal mammary gland development. Interestingly, these studies identified important new roles for the Ron receptor in normal tissues. These studies not only further our knowledge of Ron receptor biology, but also provide meaningful insight into the potential consequences of Ron loss that might result from a cancer therapy directed at this receptor. Advisors/Committee Members: Waltz, Susan.

Subjects: Cellular biology

Keywords: colon cancer; mammary gland; development; receptor tyrosine kinase; morphogenesis

30. Monk, Kelly R. Consequences of Mast Cell Signaling in Peripheral Nerve.

Degree: PhD, Medicine : Cell and Molecular Biology, 2006, University of Cincinnati

► Expression of the human epidermal growth factor receptor (EGFR) in murine Schwann… (more)

Subjects: Biology, Neuroscience

Keywords: Neurofibromatosis; Mast cell; Schwann cell; Fibrosis

[1] [2]