Dissertations / Theses on the topic '060802 Animal Cell and Molecular Biology'

Caulobacter crescentus divides asymmetrically to produce two different progeny, a swarmer progeny that is replication incompetent, and a stalked cell progeny that is replication competent. Upon cell division, a cell cycle regulator protein (CtrA) was identified only in the swarmer cells, and additional circumstantial evidence links this protein to being a repressor of chromosome replication. For example, this response regulator protein binds to five specific sites in the replication origin (Cori) designated [a-e]. We carefully studied the binding characteristics of both phosphorylated (CtrA~P) and unphosphorylated forms of the protein to the five binding sites [a-e] in the replication origin (Chapter 2) and upstream of the ctrA gene (Chapter 3). We showed that phosphorylation significantly enhances binding affinity in the replication origin (Chapter 2) but not upstream of ctrA (Chapter 3). In addition a "pseudo-active" protein form (CtrA D51E) that resembles the phosphorylated form in vivo did not improve the binding characteristics (Chapter 3). These results suggest that enhanced binding on phosphorylation is not the only signal achieved on phosphorylation. In fact, CtrA half-site mutation binding studies shows that phosphorylation stimulates protein/protein interaction and cooperative binding between sites [a] and [b] in the replication origin (Chapter 2). We show that CtrA binding site [b] is the major contributor in the cooperative CtrA binding between [a] and [b] (Chapter 4). We demonstrate that cooperative binding of CtrA~P to sites [a] and [b] repress transcription from a strong promoter (Ps), which in turn blocks plasmid replication. In addition, mutating site [b] to block CtrA binding to [a] and [b] has a deleterious effect on chromosome replication (Chapter 4).
This cooperative CtrA binding at [a] and [b] is independent from the upstream binding sites [c-e] (Chapter 2). CtrA∼P binding in the origin is altered in the presence of the histone-like protein (IHF) that also binds and overlaps CtrA binding site [c] (Chapter 5). In-fact, IHF binds and overlaps binding site [c] (Chapter 5). We propose a replication model in the stalked cell were IHF binding hinders active CtrA binding in the replication origin and regulates cooperative transcription that coincides with replication initiation.

The Interferon Regulatory Factors (IRFs) are a family of interferon-inducible proteins which play distinct roles in diverse processes such as pathogen response, cytokine signalling, cell growth regulation and hematopoietic development. The objective of this research was to investigate the mechanisms by which IRF-1 and IRF-2 regulate gene expression and cell growth. Structure-function analyses of the IRF-2 protein demonstrate that transcriptional repression by IRF-2 is contained within the first 125 N-terminal amino acids and correlates directly with IRF-2 DNA binding. Overexpression of functional IRF-2 deletion mutant proteins in NIH3T3 cells results in oncogenic transformation and tumorigenesis, suggesting that IRF-2 oncogenicity correlates directly with transcriptional repression. Similar structure-function analyses localize IRF-1 transactivation to the C-terminus. Like IRF-1, hybrid constructs which fuse the DNA binding domain of IRF-1 and IRF-2 to the transactivation domain of NF-kappaB RelA(p65) are transcriptional activators. Inducible expression of IRF-1 and IRF/RelA in NIH3T3 cells results in reduced cellular growth and induction of apoptosis. Furthermore, expression of the PKR, STAT1(p91), and WAF1 growth regulatory proteins are elevated following induction of IRF-1 or IRF/RelA, correlating transactivation function and tumor suppressor activity of IRF-1 or IRF/RelA. By RNA fingerprinting, the secretory leukocyte protease inhibitor (SLPI) was identified as the first gene whose expression is downregulated by IRF-1 or IRF-1/RelA. A region in the SLPI promoter was identified that bound IRF-1, suggesting a direct mechanism for IRF-1 regulation of SLPI expression.

HOX proteins are homeodomain-containing transcription factors essential for embryonic patterning. Despite amino acid differences, all HOX homeodomains recognize highly similar sites on DNA. One mechanism by which HOX proteins achieve specificity is through interaction with cofactors of the PBX and MEIS/PREP1 families. Higher order complexes between HOX, PBX and MEIS/PREP1 proteins form in vivo and are essential for target recognition and transcriptional regulation. Another level of control of HOX function is the nuclear availability of its cofactors. This thesis addresses the regulation of the nuclear availability of the PBX protein by MEIS/PREP1 family members. We identified two nuclear localization signals (NLS) in the PBX homeodomain and showed that the NLS are masked in the absence of MEIS/PREP1. Upon a conformational change in PBX induced by MEIS/PREP1 binding, the NLS are exposed and a receptor-mediated active transport of PBX into the nucleus is allowed. This thesis also investigates the mechanisms of transcriptional regulation by the HOX·PBX complexes. We show that HOX·PBX complexes repress transcription and are switched to transcriptional activators in response to cell signaling. We demonstrate that PBX mediates the repression function by recruiting histone deacetylases (HDACs) to HOX target promoters. Inhibition of HDAC activity or stimulation of protein kinase A (PKA) signaling converts the HOX·PBX complex into a net activator of transcription. The activation function is mediated by the HOX protein through its recruitment of CREB-binding protein (CBP), a coactivator with histone acetyl-transferase (HAT) activity. We propose a model whereby HOX·PBX transcriptional activity is determined by cell signaling, and is mediated by the local modification of chromatin structure in the promoter of downstream targets.

Retinoblastoma (RB) is a tumour-suppressing protein involved in many cellular functions including: cell cycle regulation, cellular differentiation and cell death. In the past, its function has been intrinsically linked to its role in the cell cycle, wherein it acts as a key regulator of the G1 to S-phase transition checkpoint. However, evidence has emerged in support of cell cycle-independent roles for RB. In particular, it has been demonstrated that RB plays an important role in promoting normal animal development and survival to adulthood, a function that is conserved between mammals and metazoans. This role has been extensively studied using RBF1, the Drosophila melanogaster homolog of mammalian RB. An outstanding question in this field is whether or not the mutation of RB results in tissue-specific biological outcomes that ultimately affect adult viability. The results of this research thesis suggest that the mutation RBF1 can lead to tissue-specific developmental defects; however, the defects observed did not affect the ability of animals in surviving to adulthood. For example, a male germline-specific phenotype was observed when RBF1 expression was absent from germ cells. Adult male flies in which RBF1 expression was absent from the germline were sterile. Upon closer inspection, changes in germ cell number and patterning in the testes were observed. As well, an increase in cyclin E expression in RBF1-null mutants suggested that germ cells were being arrested at an early stage of spermatogenesis. A role for RBF1 in the fly germline has not been previously investigated. Therefore, this research project has identified a novel function of RBF1 with respect to germline regulation. Additionally, this research thesis also aimed to investigate whether RB interacted with a subset of known RB-associating factors in a tissue-specific manner. While this question was not completely addressed by the research presented herein, phosphorylation of Serine 728 on RBF1 was detected in vivo. This site was previously investigated in vitro and was suggested to be a putative phosphorylation target by cyclin/Cdk complexes. Therefore, the data of this research thesis have demonstrated the potential for this site to be phosphorylated in vivo. Further experimentation is required in order to verify the authenticity of this site as an endogenous target of phosphorylation. Overall, the data presented in this research thesis provide new insight into the various roles of RBF1 and the manner in which it is regulated.
Le rétinoblastome (RB) est une protéine classifiée comme suppresseur de tumeur. Elle joue plusieurs rôles importants dans la cellule, incluant: la régulation du cycle cellulaire, la différentiation cellulaire et l'apoptose. Dès sa découverte, le rétinoblastome a été caractérisé par son rôle primaire en règlant le cycle cellulaire dans lequel RB fonctionne comme régulateur de la transition entre la phase G1 et la phase S. Depuis ce temps, d'autres rôles pour RB indépendents de son fonctionnement dans le cycle cellulaire ont été identifiés. Notamment, il a été démontré que RB joue un rôle important dans la promotion de processus de dévelopement normal dans l'animal et la survie jusqu'à l'âge d'adulte. La plupart des recherches concentrant sur ce rôle ont été faites en utilisant la Drosophile, une espèce modèle qui convient à la manipulation génétique et en laquelle l'homologue de RB se nomme RBF1.Il reste plusieurs questions à rechercher à propos du rôle de RBF1 concernant la survie de l'animal jusqu'à l'âge d'adulte. Cette thèse essaie de répondre à la question suivante; est-ce que les effets biologiques résultant de mutater RBF1 sont spécifiques à des tissus particuliers, et est-ce qu'ils ont un effet sur l'abilité de la Drosophila de survivre jusqu'au stage d'adulte? Les résultats de cette thèse indiquent qu'en mutant RBF1, il est possible de produire des effets biologiques unique dans des tissus spécifiques. Quand même, aucun de ces effets ont affecté la survie de l'animal. Par example, il a été démontré que RBF1 joue un rôle unique dans le tissu gonade des mâles. Dans des Drosophiles qui n'expriment pas RBF1 dans les cellules de la lignée germinale, les adultes mâles étaient stériles. En examinant les testicules, le phénotype des cellules de la lignée germinale était différent en comparaison au phenotype sauvage. Aussi, le nombre de cellules da la lignée germinale qui expriment la cycline E avait augmenté. Précédemment, il n'y avait pas d'études qui ont recherché un rôle pour RBF1 dans la lignée germinale des Drosphiles mâles; donc, les résultats de cette thèse ont démontré un rôle unique pour RBF1 en régularisant la spermatogenèse dans les Drosophiles.En plus, cette thèse essaie de répondre à une autre question liée à celle ci-haut: est-ce que RB s'associe avec des macromolécules particulières (dont il est déjà connue à faire des associations), mais d'une manière unique à chaque tissu de la Drosophile? Les données de cette thèse ne donnent pas une réponse complète à cette question, mais elles ont identifié in vivo un site de phosphorylation (Sérine 728) sur RBF1. Ce site a déjà été recherché et a été suggéré in vitro d'être une cible de phosphorylation par les complexes Cdk-cyclines. Alors, les données présentées ci-haut démontrent que Sérine 728 peut être phosphorylé in vivo aussi. Plus de recherche est requis pour vérifier si ce site est un cible authentique de phosphorylation endogène in vivo. En conclusion, les données de cette thèse démontrent de nouvelles façons de régler le fonctionnement de RBF1, et elles présentent des informations nouvelles à propos des rôles variés de RBF1 dans la Drosophile.

The identification of a multi-potent stem cell-like population (SP) within the adult murine heart infers new methods of cardiac repair, i.e. stem cells compensate for damage by generating new cardiomyocytes. Several studies have emphasized the dominance of the tissue microenvironment on the differentiation & functional properties of stem cells. Of particular interest was Cardiotrophin-1 (CT-1), a member of the Interleukin-6 (IL-6) family of cytokines. To assess whether CT-1 functioned as an activator of cardiac SP cells in the murine heart, an adenovirus containing the full length CT-1 driven by a ubiquitous promoter was generated. The adenovirus was administered via intra-cardiac injections and the effects of CT-1 were primarily assessed using fluorescence-activated cell sorting (FACS) analysis. Analysis showed a temporary increase in the cardiac SP of CT-1 injected hearts. A closer look at the SP cells in other tissues (liver, skeletal muscle and bone marrow) demonstrated that this phenomenon was cardiac specific. Interestingly the cardiac SP expressed the Leukemia Inhibitory Factor (LIF) receptor, which is required for CT-1 signaling through the 130 pathway. Protein analysis also showed that STAT3, a downstream member of the 130 pathway becomes activated in the heart during CT-1 injections. Preliminary results from co-culture experiments suggested that this increase was also accompanied by SP cell differentiation. These results proposed that CT-1 not only increased the cardiac SP size but may have also activated a cardiac differentiation program. The existence of a potential biologic stimulant (CT-1) for cardiac stem cells is an exciting prospect and offers support to the notion that cardiac repair may become a viable therapeutic option in the not too distant future.

Follicle stimulating hormone (FSH) is an important survival factor in the ovarian follicular development. Inhibitor of apoptosis proteins (IAPs) is a family of intracellular anti-apoptosic proteins. X-linked IAP (XIAP) has been shown to be involved in multiple biological activities (e.g. inhibition of caspase activities, promotion of ubiquitin-proteasome-mediated protein degradation, regulation of cell signaling pathways). The present thesis research project examines: (1) the role and gonadotropic regulation of XIAP expression in rat granulosa cells during ovarian follicular development and atresia; (2) the possible involvement of intra-ovarian factors such as transforming growth factor alpha (TGFalpha) in the FSH-induced XIAP expression and follicular development; and (3) the signal pathways involved in the gonadotropic up-regulation of XIAP during follicular development in vitro. A follicle culture system coupled to an adenoviral gene manipulation procedure has been established. FSH significantly increased follicular growth as evident by increases in follicular size, cell number and DNA contents in vitro. While cultured pre-antral or early-antral follicles showed a low XIAP content and evidence of apoptosis in the absence of FSH, gonadotropin addition increased XIAP content and suppressed apoptosis. At low FSH concentration, adenoviral XIAP sense cDNA expression increased follicular cell XIAP and DNA contents, reduced apoptosis, and enhanced follicular growth, while XIAP antisense elicited opposite responses. FSH-induced XIAP up-regulation appeared mediated, in part, by the secretion and action of follicular TGFalpha. In cultured rat follicles, FSH-stimulated estradiol production, TGFalpha secretion, XIAP expression and follicular growth were suppressed by intra-follicular injection of a neutralizing anti-TGFalpha antibody or addition of the estradiol antagonist ICI 182780 to the culture media. These results support my hypothesis that the FSH induces follicular growth by stimulating granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis. Since the promoter region of XIAP gene has nuclear kappa B (NFkappaB) binding site, it is possible that the transcription of XIAP is mediated via NFkappaB activation. FSH increased rat granulosa cell XIAP mRNA abundance and protein content. While the gonadotropin induced granulosa cell NFkappaB translocation from cytoplasm to nucleus and increased NFkappaB-DNA binding activity, pretreatment with an NFkappaB translocation inhibitor suppressed FSH-stimulated XIAP expression. (Abstract shortened by UMI.)

Prosaposin is a multifunctional glycoprotein with different molecular masses and dual destinations. A 65 kDa form of prosaposin is targeted to lysosomes and converted by partial proteolysis, into four smaller non-enzymatic saposin A-D required for the hydrolysis of glycosphingolipids. However, the 65 kDa protein may be further glycosylated to a 70 kDa secretory form that is found in various biological fluids and suspected to have a trophic activity. Mutations of the prosaposin gene are linked to several lysosomal disorders. This thesis examines various aspects of the synthesis, targeting and function of prosaposin, and for practical purposes, the results and discussion were divided in three main sections. The first part deals with the cloning of the mouse prosaposin gene, the analysis of its transcribed mRNA and translation products. The second section examines the mechanism of targeting of the 65 kDa protein to lysosomes using mutagenic analyses. The third part deals with the effect of the inactivation of the prosaposin gene on the development of the male reproductive system. Sequence analysis revealed that the mouse prosaposin gene is over 20 kb in length and composed of 15 exons and 14 introns. Two forms of alternatively spliced mRNA (including or excluding exon 8) were found by RT-PCR in a tissue specific manner. Structure analysis and secondary structure predictions among mouse, rat and human prosaposins illustrated a common framework of amino acids forming amphiphatic helices enclosing an internal hydrophobic core implicated on their interaction with lipids. Mutagenic deletions of functional domains of prosaposin demonstrated that its C-terminus was required for the lysosomal targeting of this protein. Further evidence from chimeric constructs of albumin attached to various functional domains of prosaposin, suggested that the C-terminus plus at least one saposin domain are necessary for the targeting of albumin to lysosomes. Investigation of the effect of p

The hemopoietic microenvironment of the bone marrow is an essential regulator of in vivo hemopoiesis. In addition to supporting the growth of normal blood cells, it also influences the growth of leukemia. This thesis describes the use of a rat model to examine three aspects of the function of the hemopoietic microenvironment. First, using a myeloid leukemia cell line (BNML), we showed that the pattern of growth of these cells differed in the bone marrow and spleen, that their presence was associated with a relocalization of normal hemopoietic stem cells from marrow to spleen, and that factors (yet to be defined) released from spleen cells altered the pattern, possibly to create a more permissive environment. Second, we showed that the "ST3" marker of marrow fibroblasts was associated with the Thy-1 molecule, and either directly or indirectly contributed to the in vitro adhesion reaction between marrow fibroblastoid cells and normal and leukemic myeloid precursors. Third, we showed that ectopic bony ossicles induced by subcutaneous implantation of recombinant human bone morphogenetic protein-2 contained marrow expressing the full range of hemopoiesis, including stem cells with a potential for long-term repopulation (demonstrated using a rat Y-chromosome specific DNA probe that we developed), and contained fibroblastoid cells differentiated to express the ST3 antigen in a manner similar to those from femoral bone marrow. These results provide further evidence for, although not final proof of, the hypothesis that the ST3 antigen participates in the function of the rat hemopoietic microenvironment, and points the way to future experiments on the interactions between stromal elements and normal and leukemic myeloid precursors.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2008.
Title from PDF t.p. (viewed on Jul 23, 2009). Source: Dissertation Abstracts International, Volume: 69-10, Section: B, page: 5924. Adviser: Christine Quirk.

4-vinylcyclohexene diepoxide (VCD) is an environmental xenobiotic formed as a by-product in the manufacture of rubber and therefore potential human exposure is likely. VCD destroys half of the small pre-antral (25-100 μm) follicles in ovaries of rats following 15 days of dosing. The overall goal of this research, was to determine the mode and earliest time for identification of follicular destruction and examine the specificity of the response for 25-100 μm follicles. The particular involvement of protein synthesis and gene expression in this ovotoxic response was also examined. After daily dosing with VCD (80 mg/kg), the rate of protein synthesis in 25-100 μm follicles was inhibited following 3, 6, and 10 hr of in vitro incubation with VCD; whereas, the inhibition in the rate of protein synthesis at 3 hr in 25-100 μm follicles from untreated animals was reversed at 6 and 10 hr. Furthermore, follicular viability was compromised to a greater extent in 25-100 μm follicles from dosed versus untreated animals. Following 10 days of daily dosing with VCD, there was an increase in random DNA fragmentation in 25-100 μm follicles; however, there was not a reduction in the numbers of primordial and primary (25-100 μm) follicles. Morphological analysis showed changes characteristic of an apoptotic-like form of cell death in oocytes and granulosa cells of primordial and primary follicles 4 hr following 10 days of daily dosing. There was an increase in levels of mRNA for bax, manganese superoxide dismutase (MnSOD) and microsomal epoxide hydrolase (mEH) in 25-100 μm follicles following 10 days dosing with VCD, but the increase was not observed in large pre-antral (100-250 μm) follicles or liver. However, decreases in levels of mRNA for bax in liver and mEH in 100-250 μm follicles were observed. These results suggest that repeated dosing makes 25-100 μm follicles more susceptible to VCD-induced cellular changes and that VCD-induces an apoptotic-like form of cell death which is mediated through changes in levels of expression of genes associated with death of the follicular cells.

Stress is implicated in various diseases such as cancer, ischemia, and inflammatory diseases. Many different forms of stress may result in impaired physiological functions. Therefore, it is important to understand the mechanisms by which cells compensate and adapt to stress. Defining the changes that occur in nuclear structure and transport between the nucleus and cytoplasm in a cell are of specific interest and will contribute significantly to our understanding of stress response.
I have demonstrated that classical nuclear import is inhibited by oxidative stress in living HeLa cells as a result of relocalization and degradation of nuclear factors important for the nuclear transport apparatus. Specifically, hydrogen peroxide redistributes Ran, the GTPase important for the directionality of transport, importin-beta, a subunit of the nuclear import receptor, and Nup153, a component of the nuclear pore complex. Moreover, the stress-induced relocalization and degradation does not rely on the activation of MAPK pathways.
Heat shock proteins have established roles in normal cellular homeostasis as well as in stress response. In unstressed conditions, proteins of the hsp70/hsc70 family shuttle between the nucleus and the cytoplasm. Upon stress, cytoplasmic hsp70s/hsc70s accumulate in the nucleus. I have further characterized the effect of stress on hsc70s localization in HeLa cells. Heat-induced nuclear concentration of hsc70s depends on cell density. Moreover, protein phosphorylation negatively regulates hsc70 nuclear accumulation in response to heat. During recovery from heat stress hsc70s redistribute as they are exported into the cytoplasm. Hsc70 export is temperature- and energy-dependent, but is independent of the Crm1/exportin1-mediated pathway. Moreover, export of hsc70 is inhibited by depolymerization of nuclear actin.
In higher eukaryotes, lamins and other lamina-associated proteins provide links between the nuclear envelope and chromatin. The protein circumferin is located at the nuclear periphery in yeast and higher eukaryotes. I have demonstrated that stress relocalizes circumferin, which is released from the nuclear periphery into the nucleoplasm upon heat shock. These studies are the first to demonstrate that nuclear envelope organization in yeast and mammalian cells is modulated by stress.
Taken together, these results indicate that both nuclear transport and nuclear organization are affected by stress.

Sensory cells located in the pancreas, the liver, the stomach and the intestines sense nutrient and peptide levels. A change in blood glucose concentration is one of the primary signals monitored by this cell type and alterations in the sensing capabilities of these glucose sensing cells may underlie the pathophysiology associated with obesity and diabetes. To understand how these glucose sensing cells function and what components are essential for maintaining glucose homeostatic mechanisms in the body, we characterized these cells by isolating and characterized the responses of these cells to glucose using functional assays. The principal glucose sensing cells of interest for our research were the neurons located within the hypothalamus, which are believed to integrate signals from sensory cells throughout the body to maintain energy homeostasis. We proposed that the mechanisms by which hypothalamic neurons sense glucose are similar to those used by pancreatic beta cells. By suggesting that hypothalamic neurons and pancreatic beta cells use similar mechanisms to sense and respond to changes in glucose levels, we hoped to identify the shared components to help us learn more about this unique and rare cell type. We found that the enzyme glucokinase (GK) was expressed throughout all stages of development along with the GLUT-1, GLUT-3 and GLUTX-1 glucose transporters, but that the liver form of GKRP was not found in the hypothalamus. The enzyme GK was also found in tissues of the distal stomach and proximal intestine of adult rats. Each of the 3 methods we employed, RT-PCR, in vitro functional assays and the development of a transgenic animal enabled us to initiate a line of research that may one day lead to a further understanding of how the body maintains energy homeostasis.

MEQC (mvc embryonic quail cardiomyocytes) is a permanent cell line derived from cardiac tumors produced by infection of 3-day quail with the MC29 myelocytoma virus, which contains the v-myc proto-oncogene. This cell line can be induced to differentiate as evidenced by expression of muscle specific markers upon co-culture with NIH 3T3 fibroblasts. When MEQC are treated with low concentrations of BrdU before co-culture, they can no longer be induced to express phenotypic markers. In the current study, I have isolated BrdU-sensitive transcripts from MEQC cells by subtractive hybridization and investigated their distribution in developing chick embryos. In all, 29 transcripts were isolated, 14 of which could be identified by sequence comparison with the Genbank data base. Complete sequences were obtained for two of the remaining transcripts, pX19 and pX27. pX19 encodes a protein of 23 kDa that contains an amphipathic alpha-helix previously described only in plant seed embryos. By in situ hybridization pX19 was identified mainly in hemopoietic tissues; it was also found in cardiac cushion mesenchyme by PCR. The clone pX27 was identified as the avian homologue of mammalian glypican core proteins and was localized in early stages of development to the cephalic regions of the neural folds, rostral paraxial mesoderm, and newly formed somites. Later, glypican transcripts were found in the apical epidermal ridge of the limb buds, mantle zone of the telencephalon, and endocardial cushions of the atrioventricular canal and aortopulmonary outflow tract. An antibody raised against the glypican core protein was localized to the cell membrane in MEQC cells. Furthermore, upon withdraw of serum from cultures of MEQC expression of glypican transcripts was enhanced and the cells tended to clump. Administration of a glypican antisense oligonucleotide prevented cell clumping and blocked the migration of endocardial endothelial cells over collegen gel. Taken together, these results suggest that MEQC express transcripts unique to either myocardium or endocardium and provide a useful system from which transcripts expressed during development can be isolated.

In this thesis the regulation of gene expression of rat parathyroid hormone related peptide (PTHRP), the biosynthesis and processing of this peptide, and its role in cell growth and tumor progression were studied. It was found that PTHRP gene expression was increased in cultured rat H-500 Leydig tumor cells by serum and growth factors; in contrast, gene expression was decreased by glucocorticoids, 1,25-dihydroxyvitamin D, and androgens. These effects on PTHRP were mostly accounted for by an alteration of gene transcription. The biosynthesis and processing of PTHRP were assessed by transfection of PTHRP cDNA into COS-7 cells, a cell line which does not endogenously produce PTHRP and which lacks the major prohormone convertases except furin. Analysis of the molecular forms of PTHRP after transfection revealed that the main species generated was an amino-terminal fragment PTHRP (1-36). Antisense furin cotransfection with PTHRP into COS-7 cells resulted in the production of bioinactive pro-PTHRP. Antisense furin cDNA was also introduced into H-500 cells by transfection. As a result, pro-PTHRP processing was blocked and bioinactive pro-PTHRP was also produced in these cells. In addition, these H-500 cells now exhibited a reduced growth rate in vitro. When these tumor cells were inoculated into Fisher rats, tumor growth in vivo was very much attenuated and the animals did not develop the syndrome of hypercalcemia which occurred in control animals; moreover, the animals displayed an extended survival time. These results indicate that furin is a processing enzyme for the conversion of pro-PTHRP to mature PTHRP and PTHRP not only induces hypercalcemia when it is over produced but also may promote tumor progression.

The mammary gland cell growth and differentiation are under the control of both systemic hormones and locally produced growth factors. Among all these important hormones and growth factors, estrogen plays a central role in mammary gland development. The biological function of estrogen is mediated by estrogen receptor α (ERα) and estrogen receptor β (ERβ). Both ERα and ERβ are expressed in the mammary gland, but with distinct expression patterns. In the mammary gland, ERα has been proved to be the estrogen receptor that mediates the mitogenic function of estrogen. However the function of ERβ in mammary cell proliferation is less understood and there remains some controversy. Accumulating evidence indicates that ERβ, unlike ERα, is a negative regulator of mammary epithelial cell proliferation. In this dissertation, ERα and ERβ were evaluated for their expression patterns in the mammary gland. In the proestrus phase, ERα was detected in about 20% of mammary epithelial cells; in the diestrus phase, no ERα staining was detected in the mammary gland. ERβ was expressed in more than 50% of mammary epithelial cells and ERβ staining was detected in some stromal cells in the proestrus phase. In the diestrus phase, ERβ staining cells were very limited and the staining intensity was very weak. These data suggest that the expression levels of both ERα and ERβ undergo dynamic changes during the estrous cycle. In the ovariectomised (OVX) rats, both ERα and ERβ were detected in more than 50% of mammary epithelial cells. Compared with the ovary-intact rats, the mammary gland of the OVX rats showed more cells with ERα expression, but the staining intensity was weaker. Taken together, the expression of ERα and ERβ is regulated by estrogen in normal mammary gland, while without estrogen stimulation in the OVX rats, more mammary cells showed ERα expression, but at a lower level in these cells. The effects of ERα and ERβ on mammary cell proliferation were studied by two different approaches, activation of endogenous ERα and ERβ via selective agonists, and overexpression of ERα and ERβ via lentiviral infection. In the first approach, we used ERα and ERβ selective agonists, propylpyrazole-triol (PPT) and diarylpropionitrile (DPN) respectively, to activate endogenous ERα and ERβ in the OVX rats. We found that ERβ selective agonist DPN counteracts the proliferative effect of ERα selective agonist PPT in the mammary gland. In the second approach, ERα and ERβ were ectopically overexpressed in the mammary gland of mature virgin rats by lentivirus infection. We found that ERβ overexpression significantly decreased mammary cell proliferation rate in both the proestrus and diestrus phases, indicating that ERβ, unlike ERα, is a negative regulator for mammary cell proliferation. Collectively, these data supports that in contrast to ERα, ERβ activation or overexpression is able to inhibit mammary cell proliferation.

CDKs são proteínas responsáveis pela ativação e progressão do ciclo celular e também apresentam função na ativação e alongamento na transcrição. Dentre elas, CDK7 atua em ambas as funções, fosforilando as CDKs do ciclo celular e fazendo parte do fator geral de transcrição TFIIH como subunidade catalítica. A partir de uma biblioteca de ESTs de glândula salivar, construído com mRNAs presentes durante o período em que ocorre o início do último ciclo replicativo da politenização e início da amplificação gênica, foram encontradas poucas mensagens relacionadas diretamente com o ciclo celular, correspondendo a 3.11% do ESTs. Dentre elas, foram encontradas as mensagens de Cdc2-like e Cdk7; portanto, foi realizada a caracterização do gene Cdk7 e a análise de sua atuação durante o desenvolvimento larval de Rhynchosciara americana. O gene Cdk7 apresenta 4 éxons, mais que em vertebrados. A sequência completa do mRNA foi obtida a partir de RACE, apresentando 1230 bases e uma ORF de 1020 bases. Perfis de expressão foram determinados por RT-PCR e Western blots. Modificações pós-traducionais foram analisadas por imunoblots em 2D. Seu perfil de expressão de mRNA e proteína apresentam variações durante o ciclo celular e entre os tecidos analisados; os imunoblots mostram a presença de uma fosforilação e possíveis modificações na cadeia lateral de alguns aminoácidos. O estudo de proteínas relacionadas ao ciclo celular nesse modelo é importante para um melhor entendimento dos ciclos celulares incomuns presentes em diferentes tecidos de insetos.
CDKs are proteins responsible for activation and progression of cell cycle and also work on the activation and elongation of transcription. Among them, CDK7 acts in both functions, phosphorylating cell cycle CDKs and as a part of general transcription factor TFIIH as its catalytic subunit. From an EST library of salivary gland, constructed with mRNAs present during the period when the beginning of the last polyteny replicative cycle occurs, it was found only few messages directly related to cell cycle, corresponding to 3.11% of the ESTs. Among them, it was found Cdc2-like and Cdk7; therefore, it was performed the characterisation of Cdk7 gene and the analysis of its role during the larval development of Rhynchosciara americana. Cdk7 gene presents 4 exons, more than in vertebrates. Complete mRNA sequence was obtained via RACE, presenting 1230 bases and an 1020 bases ORF. Expression profiles were determined by RT-PCR and Western blots. Posttranslational modifications were analysed by 2D immunoblots. Its mRNA and protein expression profiles presented variations during cell cycle and between the studied tissues; immunoblots showed the presence of one phosphorylation and possible modifications on the side chain of some amino acids. The study of proteins related to cell cycle in this model is important for a better understanding of uncommon cell cycles in different insect tissues.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
FAPESP:00/08457-0

Molting in crustaceans is a highly complex physiological process involving negative regulation by two paired endocrine glands, the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). The XO/SG complex is responsible for making molt-inhibiting hormone (MIH) which negatively regulates synthesis of the molting hormones, ecdysteroids, by the YO. Analysis of gene expression in the XOs and YOs has led to the development of a proposed molecular signaling pathway which regulates ecdysteroidogenesis and subsequent molting in crustaceans. In this study, changes in protein abundance in the YO were characterized over the course of a molt cycle (intermolt, early premolt, mid premolt, and late premolt) induced by multiple leg autotomy (MLA) in the blackback land crab, Gecarcinus lateralis. In all, 457 distinct protein spots were detected in the molting gland using two-dimensional gel electrophoresis, of which 230 (50%) changed significantly in abundance over the course of the molt cycle (one-way permutation ANOVA, p≤0.05). Changes in protein abundance were most notable between the intermolt and the three premolt stages, indicative of a biological ‘on-off’ switch in the Y-organ. Several hemolymph species proteins, including hemocyanin, cryptocyanin, and transglutaminase, were identified which characterized physiological changes associated with molting beyond the Y-organ. An abundance of cytoskeletal proteins were identified which correspond with glandular hypertrophy and are indicative of vesicular-mediated exocytosis, possibly of ecdysteroids. Further, several proteins involved in the immune, proteostasis, and oxidative stress response are characteristic of supporting the dynamic and demanding cellular changes associated with ecdysteroidogenesis and the transition of the Y-organ from the basal to the highly active state. Many proteins involved in energetic pathways including glycolysis, the citric acid cycle, amino acid metabolism, and one-carbon metabolism changed in abundance in response to both the higher energy demands and the requirement for precursors of macromolecular synthesis of the YO over the molt cycle. Taken together, these changes in diverse physiological pathways represent the complexity involved with regulation of the Y-organ, even with just the single proposed physiological purpose of ecdysteroidogenesis.

Environmental conditions imposed onto organisms during certain phases of their life cycles such as embryogenesis or puberty can not only impact the organisms’ own health, but also affect subsequent generations. The underlying mechanisms causing intergenerational phenotypes are not encoded in the genome, but the result of reversible epigenetic modifications. This work investigates in a mouse model the impact of paternal nicotine exposure on the next generation regarding addictive behavior modulation, metabolic changes, and molecular mechanisms. It provides evidence that male offspring from nicotine-exposed fathers (NIC offspring) is more resistant to lethal doses of nicotine. This phenotype is gender-specific and depends on short-term environmental challenges with low doses of nicotine prior to the LD50 application. The observed survival phenotype is not restricted to nicotine as drug of abuse, but also presents itself, when NIC offspring is challenged with a cocaine LD50 after acclimatization to low doses of either nicotine or cocaine. Functionally, NIC offspring metabolizes nicotine faster than control. Mechanistically, NIC offspring livers show global up-regulation of xenobiotic processing genes (XPG), an effect that is even more pronounced in primary hepatocyte cultures. Being known targets of Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR), these XPGs show higher baseline expression in naïve NIC offspring livers. Nicotine’s action on the brain’s reward circuitry does not appear to be of biological significance in our model system. Taken together, paternal nicotine exposure leads to a non-specific and conditional phenotype in male NIC offspring that may provide a general survival advantage against xenobiotic challenges.

Environmental conditions imposed onto organisms during certain phases of their life cycles such as embryogenesis or puberty can not only impact the organisms’ own health, but also affect subsequent generations. The underlying mechanisms causing intergenerational phenotypes are not encoded in the genome, but the result of reversible epigenetic modifications. This work investigates in a mouse model the impact of paternal nicotine exposure on the next generation regarding addictive behavior modulation, metabolic changes, and molecular mechanisms. It provides evidence that male offspring from nicotine-exposed fathers (NIC offspring) is more resistant to lethal doses of nicotine. This phenotype is gender-specific and depends on short-term environmental challenges with low doses of nicotine prior to the LD50 application. The observed survival phenotype is not restricted to nicotine as drug of abuse, but also presents itself, when NIC offspring is challenged with a cocaine LD50 after acclimatization to low doses of either nicotine or cocaine. Functionally, NIC offspring metabolizes nicotine faster than control. Mechanistically, NIC offspring livers show global up-regulation of xenobiotic processing genes (XPG), an effect that is even more pronounced in primary hepatocyte cultures. Being known targets of Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR), these XPGs show higher baseline expression in naïve NIC offspring livers. Nicotine’s action on the brain’s reward circuitry does not appear to be of biological significance in our model system. Taken together, paternal nicotine exposure leads to a non-specific and conditional phenotype in male NIC offspring that may provide a general survival advantage against xenobiotic challenges.

In murine and human brown adipose tissue (BAT), mitochondria are powerful generators of heat. Emerging evidence has suggested that the actions of mitochondria extend beyond this conventional biochemical role. In mouse BAT and cultured brown adipocytes, impaired mitochondrial respiratory capacity is accompanied by attenuated expression of Ucp1, a key thermogenic gene, implying a mitochondrial retrograde signaling. However, few have investigated this association in the context of mitochondria-nucleus communication. Using mice with adipose-specific ablation of LRPPRC, a regulator of respiratory capacity, we show that respiration-dependent retrograde signaling from mitochondria to nucleus contributes to transcriptional and metabolic reprogramming of BAT. Impaired respiratory capacity triggers down-regulation of thermogenic and oxidative genes, promoting a storage phenotype in BAT. This retrograde regulation functions by interfering with promoter-specific recruitment of PPARg. In addition, cytosolic calcium may mediate the retrograde signal from mitochondria to nucleus. These data are consistent with a model whereby BAT connects its respiratory capacity to thermogenic gene expression, which in turn contributes to determining its metabolic commitment. Additionally, we find that augmented respiratory capacity activates the thermogenic gene program in inguinal (subcutaneous) white adipose tissue (IWAT) from adipose-specific LRPPRC transgenic mice. When fed a high-fat diet at thermoneutrality, these mice exhibit metabolic improvements as shown by reduced fat mass and improved insulin sensitivity. Furthermore, there is increased recruitment of brown-like adipocytes in IWAT and thus energy expenditure is significantly increased, providing a potential explanation for protection from obesity. These data suggest that augmented respiratory capacity promotes ‘browning’ of IWAT, which has beneficial effects on obesity and diabetes.

Pregnancy loss remains a major source of economic cost to the equine industry. Frequently, the exact causes of pregnancy loss remain unknown. It has been shown, in other species, that increased dietary protein leading to elevated blood urea nitrogen concentrations (BUN) can be a factor in decreased survival of the early embryo. Our studies provided novel information regarding the effects of elevated BUN on endometrium and embryos from mares as well as insights on changes in their gene expression. Our first objective was to develop an experimental model to elevate BUN during diestrus using intravenous urea infusion. We analyzed the effects of an acute elevation in BUN on uterine and vaginal pH along with changes in the endometrial transcriptome of mares with RNA sequencing. There was a significant increase in BUN and a decrease in uterine pH in the urea group compared to the control group. A total of 193 genes were differentially expressed (DEG) between the urea and control groups. The DEG were predicted to be related to cell pH, ion homeostasis, changes in epithelial tissue, fatty acid metabolism, and solute carriers. Our second objective was to evaluate the effects of elevated BUN in the endometrium of mares using a chronic oral urea administration to elevate BUN in mares. Uterine and vaginal pH were evaluated and RNA sequencing of the endometrium was again performed. There was an increase in BUN in the urea-fed mares, but no significant change in uterine or vaginal pH between the groups. A total of 60 DEG were characterized, with prediction of transcriptomic changes in the endometrium of mares related to cell death (necrosis) and cellular movement (invasion of cells). Our third objective was to determine the effects of a high BUN on the transcriptome of day-14 embryos. There was a positive correlation between plasma BUN and blastocoele fluid urea nitrogen concentration. Changes in embryo transcriptome were related to survival of organism, angiogenesis, adhesion, and quantity of cells. Our final objective was to evaluate the correlation between BUN and follicular fluid urea nitrogen and evaluate the survival of embryos collected from donor mares with high BUN concentrations. Urea nitrogen concentration was positively correlated between the plasma and follicular fluid of mares. Additionally, there was a higher pregnancy rate when embryos were collected from mares with lower BUN. Overall, these results further elucidate the mechanisms through which urea affects endometrial and embryonic transcriptome of mares with high BUN, serving to identify effects of a high BUN in the reproductive tract of mares that might lead to decreased fertility.

Ovarian cancer is the fifth leading cause of cancer death in women between the ages of 35 and 74. With 22 thousand new cases and 15 thousand deaths annually ovarian cancer is among the most deadly cancers with a death to incidence ratio of 68%. With 70% of cases High Grade Serous Ovarian Carcinoma (HGSOC) is the most common type of ovarian cancer and causes 90% of ovarian cancer deaths. 80% of patients have reoccurrence within five years and only 15-30% of patients with recurrent metastatic ovarian cancer respond to current therapies, chemotherapy and surgery. One reason for the high reoccurrence rate is thought to be linked to the heterogeneity of tumors: there is evidence that, among tumor cells, a subpopulation is cancer stem cells (CSCs). Since CSCs are frequently drug resistant, when the patient undergoes chemotherapy many of the cells may die but the CSCs are left behind and the tumors can therefore regrow. CSCs are also more likely to undergo epithelial-mesenchymal transition which gives these cells the ability to more readily migrate and invade through the extracellular matrix, leaving the primary tumor to form metastases. One key inducer of EMT and therefore possibly of metastasis of particular interest in this project is SNAI1 (Snail). It is therefore the goal of this project to understand the growth, makeup and metastatic ability of HGSOC cell lines to test possible strategies to decrease growth of cancer and prevent metastasis. In this thesis project the phenotype, CSC population make up, and functionality of various HGSOC cell lines was examined. The cell lines assessed were A2780, Kuramochi, OVSAHO, COV318, SKOV3 and OVCAR8. A Snail knockdown OVCAR8 cell line was also assessed as described above and in a xenograft model. It was determined that the cell lines show varying phenotype from epithelial like to mesenchymal like morphology and the cell lines have varying concentrations of cancer stem cells. It was also determined that the CSC population of the HGSOC cell lines were positive for both epithelial and mesenchymal markers in the same cells. OVCAR8 stood out as a hybrid line with both epithelial and mesenchymal characteristics and was therefore chosen for the Snail knockdown model. In the Snail knockdown we observed that CSC markers were reduced, however no change between control and knockdown was seen in the in vitro functional experiments. There was a difference seen between Snail knockdown and control in the in vivo mouse xenograft model. Snail knockdown showed a trend for decreasing tumor burden in both primary and metastatic tumors and showed a significant decrease in growth of metastatic tumor at day 43. Based on these results Snail may be an important target for cancer therapy.

Introdução: O câncer de próstata (CaP) é o tumor mais comum do homem nos países ocidentais e a segunda causa de óbito por câncer em homens nos EUA, Europa e Brasil. O câncer localizado tem sobrevida câncer especifica elevada quando tratado adequadamente, porém a doença metastática ainda apresenta tratamentos pouco eficientes com sobrevida global de 28%. Os microRNAs (miRNAs) são um grupo de moléculas pequenas de RNA que contém entre 19 a 25 nucleotídeos não codificantes de proteína, com ação fundamental na regulação da expressão gênica. Eles estão envolvidos em processos essenciais nas células normais e neoplásicas como ciclo celular, proliferação, apoptose, metabolismo energético, invasão e metastatização. Objetivos: Realizar estudos in vitro e in vivo usando miRNA em um modelo de câncer de próstata metastático inédito no nosso meio com intuito de analisar o seu potencial como agente terapêutico dessa neoplasia. Métodos: Nos estudos in vitro, três linhagens celulares foram utilizadas (PC3, DU145 e LNCaP). Essas linhagens foram transfectadas com os miRNAs 100, 145 e 373 e seus respectivos antiMiRs utilizando-se lipofectamina. Analisamos a expressão dos genes alvo mTOR, SMARCA5, KRAS, CMYC, MMP9, CD44 por PCR quantitativo em tempo real (qRT-PCR). Foram realizados também estudos de apoptose, ciclo celular e ploidia utilizando o citômetro de fluxo. Alterações no potencial de invasão foram avaliadas pela técnica do matrigel. O modelo in vivo pré-clínico foi desenvolvido pela injeção intra-cardíaca da linhagem PC-3M-Luc-C6 em camundongos NUDE com 9 semanas. O crescimento tumoral foi avaliado com o sistema de bioluminescência in vivo. Após o pleno estabelecimento das metástases no dia 21, os animais foram tratados com três injeções na veia da cauda contendo o miRNA conjugado com o atelocolágeno. Os animais foram sacrificados e no dia 48 para análise dos tecidos. Resultados: miR-100 aumenta a apoptose na LNCaP, e reduz a apoptose na DU145. Na linhagem DU145 o miR 100 inibiu a proliferação. Na análise da expressão gênica o miR-100 inibe SMARCA5 na DU145 e PC3 e mTOR na LNCaP, o anti-miR 100 estimula mTOR e SMARCA5 na LNCaP. O miR-145 promoveu aumento da apoptose em 24% na DU145. Na linhagem PC3 o miR-145 age inibindo a proliferação, com uma diferença absoluta de 18% em relação ao seu controle. O miR-145 inibe KRAS e CMYC nas três linhagens e o anti-miR-145 estimula CMYC na DU145 e RAS nas três linhagens O miR-373 reduziu a apoptose em 29% na DU145 e diminui a proliferação com uma diferença absoluta de 13% em relação ao controle. O miR- 373 estimula a MMP9 na DU145 e na LNCaP e inibe o CD44 na PC3. O antimiR- 373 inibe MMP9 na DU145 e LNCaP. Nos estudos in vivo de CaP metastático o miR-100 apresenta tendência a redução no crescimento tumoral (p=0,23) e o miR-145 reduz o crescimento de forma significativa no dia 34 (p=0,02). Após esses dias o tumor volta a crescer de forma agressiva. Os animais tratados com anti-miR-373 não apresentaram alterações em relação aos controles. Conclusões: O miR-100 é um miRNA contexto dependente, com papel supressor tumoral em linhagens de tumor de próstata agressivo e o miR- 373 age in vitro como oncomiR. O miR-145 age como supressor tumoral in vitro e em modelo animal de CaP metastático apresentando resposta terapêutica consistente, podendo ser utilizado no arsenal terapêutico contra essa neoplasia. Estudos futuros devem avaliar o uso dos miRNAs isoladamente ou de forma adjuvante no tratamento do CaP metastático
Introduction: Prostate cancer (PCa) is the most common neoplasia of man in Western countries and the second cause of death by cancer in men in the US, Europe and Brazil. The localized cancer has high cancer-specific survival when treated properly, however metastatic disease still presents low effective treatments with 28% of global survival. microRNAs (miRNAs) are a group of small RNA molecules containing from 19 to 25 nucleotides of noncoding protein with fundamental action in the regulation of gene expression. They are involved in key processes in normal and neoplastic cells as cell cycle, proliferation, apoptosis, energy metabolism, invasion and metastasis. Objectives: To carry out studies in vitro and in vivo using miRNA in a novel model of metastatic prostate cancer in our country in order to evaluate its potential as a therapeutic agent of this neoplasia. Methods: In the in vitro studies, three cell lines were used (PC3, DU145 and LNCaP). These cell lines were transfected with miRNAs 100, 145 and 373 and their antiMiRs using lipofectamine. We analyzed the gene expression of mTOR, SMARCA5, KRAS, CMYC, MMP9, CD44 by real-time polymerase chain reaction (qRT-PCR). We also performed studies of apoptosis, cell cycle and ploidy using flow cytometer. Changes in the invasion potential were evaluated by the technique of matrigel. The pre-clinical model in vivo was developed by intracardiac injection of PC-3MLuc-C6 cell line in NUDE mice with 9 weeks. Tumor growth was evaluated with an in vivo image system (IVIS). After the full establishment of metastases on day 21, the animals were treated with three injections into the tail vein containing the miRNA plus atelocollagen. The animals were sacrificed on day 48 for tissues analysis. Results: MiR-100 increases apoptosis in LNCaP and reduces apoptosis in DU145. The anti-miR-100 increased apoptosis in 14% in PC3. In cell line DU145, miR-100 inhibited proliferation. In the analysis of gene expression, the miR-100 inhibits SMARCA5 in DU145 and PC3 and mTOR in LNCaP, anti-miR-100 stimulates mTOR and SMARCA5 in LNCaP. The miR-145 promoted an increased in apoptosis by 24% in DU145. In PC3 cell line miR-145 acts by inhibiting the proliferation, with an absolute difference of 18% compared to control. MiR-145 inhibits KRAS and CMYC in the three cell lines and anti-miR-145 stimulates CMYC in DU145 and KRAS in the three cell lines. The miR-373 reduced apoptosis by 29% in DU145 and reduces proliferation with an absolute difference of 13% relative to control. MiR-373 stimulates MMP9 in DU145 and LNCaP cells and inhibits CD44 in PC3. The anti -miR-373 inhibits MMP9 in DU145 and LNCaP. In the in vivo studies of metastatic PCa, miR-100 shows a tendency to decrease tumor growth (p=0.23) and miR-145 reduces tumor growth on day 34 (p=0.02). After those days, the tumor grows back aggressively. Animals treated with anti-miR-373 showed no changes relative to controls. Conclusion: The miR-100 is a context-dependent miRNA, with tumor suppressor role in aggressive tumor cell lines. The miR-373 acts in vitro as oncomiR and miR-145 acts as a tumor suppressor in vitro and in an animal model with consistent therapeutic response and can be used in the therapeutic arsenal against this neoplasia. Future studies should evaluate the use of miRNAs alone or adjuvant in the treatment of metastatic prostate cancer

cJun NH2-terminal kinase (JNK) is a member of the MAPK (mitogen- activated protein kinase) signaling family that responds to various extracellular stimuli, such as stress, growth factors, cytokines, or UV radiation. JNK activation can lead to cellular responses including gene expression, growth, survival, and apoptosis. JNK has been implicated in normal developmental processes, including tissue morphogenesis, as well as pathological processes, such as cellular transformation and cancer. JNK exists in three isoforms, and knockout mice have been generated for each isoform; the ubiquitously expressed Jnk1 and Jnk2 have been studied independently, however, the two isoforms are partially functionally redundant. Jnk1-/- Jnk2-/-mice are nonviable, therefore studies of compound JNK-deficiency have been limited to mouse embryonic fibroblasts (MEF). Understanding the role of JNK in epithelial cells is now possible with the creation of conditional JNK knockout animals. I sought to elucidate the role of JNK in cellular transformation, cancer, and normal development. I employed both in vitro and in vivo approaches. First, I evaluated the role of JNK in cellular transformation using p53-/- Jnk1-/- Jnk2-/- MEF transduced with oncogenic Ras. To extend this study, I examined JNK-deficiency in a Kras-induced model of lung tumorigenesis. Second, I investigated JNK1- and JNK2-deficiency in a p53-mediated model of mammary tumorigenesis. Finally, I examined the role of JNK in mouse mammary gland development by establishing JNK-deficient primary mouse mammary epithelial cells and evaluating JNK-deficient mammary gland transplants. Taken together, this work provides evidence of context-dependent roles for JNK in both normal and pathological cell biology.

Previous research regarding aquatic acidification has examined the protonation of the carbonate and does not consider calcium to be a limiting factor. This is the first study to suggest that pH may affect the uptake of calcium in crustacean gills. This project describes ion transport mechanisms present in the cell membranes of white river shrimp Litopenaeus setiferus gill epithelium, and the effects of pH on the uptake of calcium by these means. Partially purified membrane vesicles (PPMV) of shrimp gills were prepared through a homogenization process that has been used previously to define ion transport in crab and lobster gill tissues. In the current study, shrimp gill PPMV calcium uptake at 50 µM, and 250 µM was greatest at pH 7.0 (p=0.01, p=0.0001). A valinomycin/K+ induced membrane potential (PD) at pH 7.0 significantly increased (p=0.003) calcium uptake from that observed in the absence of a PD. An induced PD at pH 8.0 significantly increased (p=0.003) calcium uptake from that observed in the absence of a PD, however, was not significantly greater than uptake at pH 7.0 in the presence of a PD (p=0.05). Amiloride (2mM) treatments, and amiloride (2mM) + verapamil (100µM) cocktail treatments showed significant decrease in calcium uptake from the control (p=0.03), however, they were not different from each other. This indicates an electrogenic carrier with two driving forces: calcium concentration, and asymmetric exchange stoichiometry.

L'utilisation accrue des methodes d'imagerie microscopique dans le domaine de la cancerologie, et les developpements recents des marqueurs specifiques utilises dans ce contexte ont fait naitre un besoin urgent en ce qui concerne le developpement de procedures de multi-marquages enzymatiques ou fluorescents, et leur quantification cellule a cellule. C'est dans ce contexte de mise au point methodologique qu'ont ete realises les travaux presentes dans cette these. Ils incluent les divers aspects de la standardisation de methodes de multi-marquages, l'adaptation des methodes d'imagerie pour la quantification du marquage in situ du recepteur a l'egf, et la normalisation des mesures. Les techniques developpees pour la quantification par cytometrie en image sont detaillees, faisant apparaitre les limites et les problemes eventuels rencontres, ainsi que la facon de les contourner ou de les resoudre. Ces developpements methodologiques ont ete appliques a l'etude de l'analyse quantitative in situ des recepteurs a l'egf dans les tissus animaux et humains (tumeurs mammaires), en relation avec la cinetique de proliferation de chacune des cellules (marquages ki67, brdu, agnor). L'etude de marquages simultanes des proteines ki67-agnors-egfr associes au marquage de l'adn montre qu'il devrait etre possible de preciser pour chaque cellule en cycle, dans un tissu normal ou tumoral, la relation entre la vitesse du cycle et l'expression des recepteurs a l'egf

Traumatic brain injury (TBI) is a leading cause of disability worldwide. Annually, 150 to 200/1,000,000 people become disabled as a result of brain trauma. Axonal degeneration is a critical, early event following TBI of all severities but whether axon degeneration is a driver of TBI remains unclear. Molecular pathways underlying the pathology of TBI have not been defined and there is no efficacious treatment for TBI. Despite this significant societal impact, surprisingly little is known about the molecular mechanisms that actively drive axon degeneration in any context and particularly following TBI. Although severe brain injury may cause immediate disruption of axons (primary axotomy), it is now recognized that the most frequent form of traumatic axonal injury (TAI) is mediated by a cascade of events that ultimately result in secondary axonal disconnection (secondary axotomy) within hours to days. Proposed mechanisms include immediate post-traumatic cytoskeletal destabilization as a direct result of mechanical breakage of microtubules, as well as catastrophic local calcium dysregulation resulting in microtubule depolymerization, impaired axonal transport, unmitigated accumulation of cargoes, local axonal swelling, and finally disconnection. The portion of the axon that is distal to the axotomy site remains initially morphologically intact. However, it undergoes sudden rapid fragmentation along its full distal length ~72 h after the original axotomy, a process termed Wallerian degeneration. Remarkably, mice mutant for the Wallerian degeneration slow (Wlds) protein exhibit ~tenfold (for 2–3 weeks) suppressed Wallerian degeneration. Yet, pharmacological replication of the Wlds mechanism has proven difficult. Further, no one has studied whether Wlds protects from TAI. Lastly, owing to Wlds presumed gain-of-function and its absence in wild-type animals, direct evidence in support of a putative endogenous axon death signaling pathway is lacking, which is critical to identify original treatment targets and the development of viable therapeutic approaches. Novel insight into the pathophysiology of Wallerian degeneration was gained by the discovery that mutant Drosophila flies lacking dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously recapitulated the Wlds phenotype. The pro-degenerative function of the dSarm gene (and its mouse homolog Sarm1) is widespread in mammals as shown by in vitro protection of superior cervical ganglion, dorsal root ganglion, and cortical neuron axons, as well as remarkable in-vivo long-term survival (>2 weeks) of transected sciatic mouse Sarm1 null axons. Although the molecular mechanism of function remains to be clarified, its discovery provides direct evidence that Sarm1 is the first endogenous gene required for Wallerian degeneration, driving a highly conserved genetic axon death program. The central goals of this thesis were to determine (1) whether post-traumatic axonal integrity is preserved in mice lacking Sarm1, and (2) whether loss of Sarm1 is associated with improved functional outcome after TBI. I show that mice lacking the mouse Toll receptor adaptor Sarm1 gene demonstrate multiple improved TBI-associated phenotypes after injury in a closed-head mild TBI model. Sarm1-/- mice developed fewer beta amyloid precursor protein (βAPP) aggregates in axons of the corpus callosum after TBI as compared to Sarm1+/+ mice. Furthermore, mice lacking Sarm1 had reduced plasma concentrations of the phosphorylated axonal neurofilament subunit H, indicating that axonal integrity is maintained after TBI. Strikingly, whereas wild type mice exhibited a number of behavioral deficits after TBI, I observed a strong, early preservation of neurological function in Sarm1-/- animals. Finally, using in vivo proton magnetic resonance spectroscopy, I found tissue signatures consistent with substantially preserved neuronal energy metabolism in Sarm1-/- mice compared to controls immediately following TBI. My results indicate that the Sarm1-mediated prodegenerative pathway promotes pathogenesis in TBI and suggest that anti-Sarm1 therapeutics are a viable approach for preserving neurological function after TBI.

Basic fibroblast growth factor (bFGF) plays an instrumental role in the cascade of events leading to restenosis, vascular re-occlusion due to excessive smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) deposition following arterial intervention procedures such as balloon angioplasty. The mechanism of bFGF activation following vascular injury has remained elusive. bFGF is stored bound to heparan sulfate proteoglycans in the ECM of the arterial media; release from extracellular sequestration may activate bFGF and initiate SMC proliferation and migration. bFGF mobilization at injured sites may be induced by platelet degranulation products. We have carried out in vitro studies demonstrating that platelet-derived heparanase and platelet factor-4 (PF4) liberate bFGF from the ECM of vascular SMCs, resulting in the induction of SMC proliferation and migration. Increases in proliferation and migration were inhibited by treatment with a bFGF-neutralizing antibody, suggesting that proliferation and migration in response to heparanase or PF4 are mediated by bFGF activation. When platelets were seeded on top of SMCs, degranulation products were found to release bFGF from the ECM, increasing cell proliferation and cell migration. These increases in SMC proliferation and migration were completely inhibited by the addition of a bFGF-neutralizing antibody. In order to investigate the role of heparanase and PF4 in vivo, each was delivered to the uninjured rat carotid artery. Heparanase and PF4 were both found to release bFGF, induce substantial SMC proliferation and increase the expression of several growth factor receptors thought to promote restenosis. An antibody that neutralizes platelet-derived heparanase was developed and evaluated in a rat carotid balloon injury model. Perivascular delivery of anti-heparanase IgG was found to inhibit bFGF depletion from the arterial wall by approximately 60% (p < 0.001) at 4 days. This correlated with the reduction in intimal thickening observed at 14 days. Platelet degranulation products, such as heparanase and PF4, may liberate bFGF from extracellular sequestration, activating the growth factor and inducing the SMC proliferation and migration that contribute to luminal narrowing following vascular injury. In addition, platelet-derived heparanase is likely to play a key role in initiating events leading to restenosis via bFGF mobilization.

This dissertation tested the hypothesis that the mammotrophic pituitary tumor/somatotroph (MtT/S) cell line is a model of the normal somatotroph which may be used to study the integrated regulation of growth hormone (GH) mRNA levels by physiologically relevant hormones. Quantification of GH mRNA levels by ribonuclease protection assay indicated that MtT/S cells are stimulated in a dose-dependent manner by treatment with either triiodothyronine or dexamethasone. Although the GH-releasing hormone (GHRH)-receptor mRNA was present in MtT/S cells, GH mRNA levels were not increased by GHRH. Furthermore, GHRH did not stimulate cyclic adenosine 3',5' monophosphate (cAMP), but cAMP was chronically elevated. Consistent with constitutive activation, GH mRNA levels were only slightly elevated by forskolin and were unresponsive to 3-isobutyl-1-methylxanthine. However, MtT/S cells did not express endogenous GHRH or a constitutively activated mutant G-protein alpha s, subunit, the most common causes of elevated cAMP in type 2 acromegalic adenomas Increasing concentrations of insulin-like growth-factor (IGF-1) reduced MtT/S cell GH mRNA levels in a switch-like/ultrasensitive manner. These kinetics were IGF-1 specific, as insulin reduced GH in a hyperbolic fashion. IGF type 1 and insulin receptors were detected by western blot. Pit-1 mRNA levels were not altered in IGF-1-treated cultures. Selective inhibitor treatment of MtT/S cells with wortmannin, LY 294,002 and PD098059 suggested that phosphatidylinositol 3-kinase- and mitogen-activated protein kinase-dependent pathways are not required for IGF-1 effects GH mRNA quantification by in situ hybridization revealed that GH mRNA levels varied greatly among individual cells. Mathematical analysis indicated that IGF-1 does not act in an ultrasensitive manner in individual cells. Interestingly, thyroid hormone and dexamethasone diminished the ultrasensitivity of the average response to IGF-1. These data suggest that the ultrasensitivity observed in the average GH expression per cell is due to IGF-1 binding factors outside the cells In conclusion, these data suggest that MtT/S cells are a model of acromegalic type 2 adenoma cells of unknown etiology, rather than normal somatotrophs. With the clarification of the source of the elevated cAMP in MtT/S cells, it will be possible to use these cells to dissect the molecular mechanisms that lead to GH-producing adenomas
acase@tulane.edu

Cloned A-channel candidates (Kv1.4, 3.4, 4.1, 4.2 and 4.3) were initially expressed in Xenopus oocytes. However, functional discrepancies exist between native A-currents and currents resulting from Kv alpha-subunit expression in oocytes. The discrepancies may stem from heteromeric subunit assembly or may simply result from the different expression environments. Therefore, we compared native A-current (IA) from adult rat sympathetic neurons with those from cloned A-channel candidates expressed in HEK293T cells. Native and cloned IA were recorded under similar experimental conditions using the patch-clamp technique. IA of SCG neurons activated at more hyperpolarized voltages than cloned IA. Inactivation of native IA occurred at more negative potentials and the recovery from inactivation was more rapid than that of cloned IA. Native current was more sensitive to 4-aminopyridine than currents from the Kv 4 family. These results demonstrate that single cloned Kv-channel alpha-subunits do not duplicate the native A-current of sympathetic neurons M-channels can be modulated by a large array of Gq/11-coupled receptors. The histamine H1 receptor (H1R) also appears to be a Gq/11-coupled receptor, however, the involvement of H 1R in M-channel modulation has not been addressed. Therefore, we investigated if histamine could inhibit recombinant M-channels produced by KCNQ2/KCNQ3 channel subunit expression via H1R receptors expressed in HEK 293T and HeLa cells. Our studies showed that activation of H1R by histamine significantly inhibited recombinant M-currents (IM). This inhibition was not a peculiarity of the HEK 293T cell since IM inhibition by histamine also occurred in HeLa cells. Activation of a pertussis toxin-insensitive G protein was involved in histamine inhibition. Moreover, Galphaq/11 is a mediator of the inhibition since expression of the Galpha q/11 buffer significantly prevented histamine inhibition. However, expression of Gbetagamma buffers also strongly attenuated IM inhibition by histamine, indicating that the Gbetagamma dimer was also involved in IM modulation by histamine. The histamine inhibition of I M was not mediated by Ca2+ release from an intracellular Ca2+ store since thapsigargin pretreatment with could not prevent histamine inhibition. Pretreatment with nonspecific protein kinases inhibitors were also unable to prevent histamine inhibition, indicating activation of protein kinases was not involved in histamine inhibition of IM
acase@tulane.edu

During gestation, the maternal liver undergoes various adaptive changes to cope with the increasing physiological and metabolic demands from both maternal and fetal compartments. Among these changes are robust growth and changes in transcriptome profile. However, how these events happen, and other aspects of this physiological phenomenon remains unexplored. Therefore, we aimed at further understanding how maternal liver responds to pregnancy. We used BrdU labeling combined with a virus-based tracing approach to quantify the percentage of maternal hepatocytes undergoing DNA synthesis and division over the course of gestation in mice.

We found that ~50% maternal hepatocytes entered S-phase but, unexpectedly, did not undergo cytokinesis. This strongly suggests that maternal hepatocytes in fact undergo endoreplication instead of hyperplasia, as believed previously. Pericentral Axin2⁺ hepatocytes were reported to behave as liver stem cells responsible for liver homeostasis and turnover. We generated an in vivo fate-tracing mouse model to monitor the behavior of these cells in the maternal liver. Our results showed that they did not proliferate during pregnancy, homeostasis, and following partial hepatectomy. Curiously, we uncovered that, hepatocytes exhibit developmental phenotypes at mRNA level pre-pregnancy and at both mRNA and protein level during pregnancy. In the non-pregnant state, hepatocytes reserved mRNA expression of liver progenitor marker genes Cd133 and Afp, which are localized in the nuclei, without protein translation. During gestation, maternal hepatocytes displayed cytoplasmic translocation of Cd133 and Afp transcripts, concomitant with corresponding protein expression.

Overall, all maternal hepatocytes became CD133⁺, and a subset of them express AFP. Additionally, in non-pregnant livers, mRNA of Epcam, another liver progenitor marker, was expressed within majority of hepatocytes, whereas its protein was solely translated in the pericentral region. In contrast, by end-gestation, EPCAM protein expression switched to the periportal region. These observations indicate that maternal hepatocytes exhibit heterogeneous developmental phenotypes, partially resembling fetal hepatocytes. It is intriguing why mature hepatocytes dedifferentiate into a progenitor state in response to pregnancy. AFP is considered to be produced primarily from fetal liver and thus is used to evaluate fetal development health.

A potential clinical relevance of our data is that we identified maternal liver as a new source of AFP. The hippo signaling pathway has been shown to potently control liver growth and hepatocyte heterogenicity. Surprisingly, we found that pregnancy neither altered the expression nor activities of the components of this pathway and its effector YAP1/TAZ. This finding indicates that pregnancy-induced maternal liver growth is not driven by hippo-YAP1 pathway. However, we demonstrate that the presence of YAP1 is essential for CD133 protein expression in maternal hepatocytes. Collectively, we revealed that, as pregnancy advances, maternal hepatocytes likely undergo endoreplication and display developmental phenotypes. Mechanistically, YAP1 dictates the expression of CD133, contributing to the pregnancy-dependent phenotypic changes of maternal hepatocytes.

TDP43 is an RNA and DNA binding protein that has been shown to play an integral role in disease mechanisms that underlie ALS. In fact, a common feature of the vast majority of ALS cases is the presence of TDP-43 aggregates in postmortem tissue from the brain and spinal cord. This finding has spurred research to understand the physiological roles of TDP-43 in the absence of disease, and how these roles are affected by disease. Current TDP-43 mouse models fail to faithfully and reproducibly recapitulate key aspects of ALS, possibly due to the transgenic approaches used. To address these concerns, we generated a targeted, conditional mouse model, and embryonic stem cell lines expressing either human WT or M337V mutant TDP-43 at equivalent levels. We show that expression of mutant hTDP-43 in mice with a mixed genetic background leads to selective motor neuron loss, muscle weakness and premature death. However this disease phenotype is not observed with the same TDP43 mutation in a pure Bl6 background. We next sought to identify alterations in the biochemistry of the mutant protein that may underlie its toxicity, such as its interactions with RNA and protein. By creating a library of RNAs bound by TDP-43 in the mouse spinal cord, we found that the M337V mutation does not compromise the ability of TDP43 to bind to target mRNA transcripts, although the mutation does lead to changes in expression of genes known to be involved in inflammation. In addition, we identified 22 proteins that bind to TDP43 in an RNA-dependent manner, and found that the M337V mutation does not alter these interactions. This work establishes novel mouse and cellular models that provide insights into the functions of normal and ALS-causing mutant TDP43 protein.

This study was undertaken to elucidate the synthesis site or sites of the major yolk protein, vitellin (Vt), in the red swamp crayfish, Procambarus clarkii. Vitellin was purified from late vitellogenic ovaries (containing oocytes $\ge$456 $\mu$m in diameter). The protein was purified by ultra centrifugation, gel filtration, and finally native polyacrylamide gel electrophoresis. Once the purified form of the protein was attained it was used to create polyclonal and monoclonal antibodies by inoculating rabbits and mice, respectively. The specificity and sensitivity of the antisera were each determined by titering using sandwich enzyme-linked immunosorbent assay (ELISA). The antisera were used to design a competitive quantitative ELISA for Vt and its precursor, vitellogenin (Vg), which is immunologically identical in many crustaceans. The concentrations of Vt in ovaries and Vg in hemolymph were determined in relation to the stage of reproductive maturity as determined by oocyte diameters. The total aqueous protein concentration in developing ovaries ranged from 1% to 5% and consisted of 23% to 55% Vt. The Vt concentrations from ovaries of sixteen mid-vitellogenic females averaged 38% of the total aqueous protein. The competitive ELISA was sensitive to standard Vt concentrations as low as 62 ng/ml and was capable of distinguishing concentrations as high as 8 $\mu$g/ml. Hemolymph Vg concentrations, determined from diluted hemolymph samples (1:20), ranged from 10 $\mu$g/ml in early vitellogenic females to 2880 $\mu$g/ml in late and post-vitellogenic females. There was a correlation between ovary maturity and specific hemolymph Vg concentrations after the onset of vitellogenesis. Hemolymph Vg concentrations are attributed to leakage and reabsorption of Vg and Vt, respectively, through the thin ovarian wall. Polyclonal antibody preabsorbed to male hemolymph was used for fluorescent (FITC) and enzymatic (DAB) immunohistochemistry for Vt or Vg localization in ovary and hepatopancreas sections from females of all reproductive stages and hepatopancreata of males (controls). Immunohistochemical analysis indicated no presence of Vg in males or female hepatopancreata at any reproductive stage. Ovaries showed Vg presence in ovarian stroma associated with follicle cells or a possible subset of which we call vitellocytes. Presence of Vt in oocytes corresponded closely to timing of folliculogenesis and concentrations of Vt can be related to relative numbers of secondary follicle cells present. Finally, our results indicated that the ovary may be the sole site of Vg synthesis and that the follicle cells are responsible for the production and transport of yolk protein to developing oocytes
acase@tulane.edu

Breast cancer is a leading cause of cancer in women and the second leading cause of cancer death. Lifetime exposure to estrogen contributes to this risk but high dose estrogen has been used to induce apoptosis as treatment for breast cancer. These opposing tumorigenic and anti-tumorigenic effects of estrogen may be regulated differently by the two Estrogen Receptors (ER), Estrogen Receptor alpha (ERα) and Estrogen Receptor beta (ERβ). Although the receptors share a 96% homology in their DNA binding domain, they are unique in the ligand-binding domain with 53% amino acid homology. Previous studies have shown that ERα drives cell proliferation in the mammary gland. We propose that ERβ mediates genomic surveillance in the mammary gland to restrict proliferation. To test this hypothesis we first characterized each of our reference breast cancer cell lines to determine the ERα and ERβstatus. We found that ERβ transcript and protein are expressed in some breast cancer cell lines that are considered to be “triple-negative” (HCC1937 and MDA MB 231). Using specific ER agonists, we were able to demonstrate that amphiregulin, a secreted protein and a marker of ERα activation, is upregulated by ERα agonists in a dose dependent manner in cell lines that have ERα (T47D & MCF7). ERα agonists do not enhance AREG expression in cell lines that primarily expresses ERβ (HCC1937). Instead, CEBPd, a tumor suppressor, is expressed at high levels in this cell line. In conclusion, targeting ERβ has the potential to selectively activate tumor suppressor pathways without stimulating proliferation and may provide a treatment option for patients for whom inhibition of ERa is not an option.

TNF-alpha has been proposed as a link between insulin resistance and obesity, however the molecular mechanism whereby TNF-alpha attenuates insulin signaling is not well understood. The aim of this thesis was to map the in-vivo mechanism by which TNF-alpha contributes to the pathogenesis of impaired insulin signaling, using obese Zucker rats in which TNF-alpha activity was inhibited through adenovirus (AdV)-mediated expression of a TNF inhibitor (TNFi). Control animals consisted of obese Zucker rats infected with the same titer of AdV carrying the lac-z cDNA. Both peripheral and hepatic insulin sensitivity were improved in obese Zucker rats following neutralization of TNF-alpha. Additionally, TNF-alpha neutralization led to a 2.5 fold increase in tyrosine phosphorylation (pY) of IR in skeletal muscle while this was unchanged in liver, suggesting that TNF-alpha is a mediator of insulin resistance in obesity and may modulate IR signaling in skeletal muscle and liver through different pathways. Further examination showed that pY of pp125FAK was significantly higher in TNF-alpha neutralized animals, indicating that TNF-alpha may induce insulin resistance in the liver by preventing insulin-stimulated pY of pp125FAK. Thus, TNF-alpha may induce insulin resistance in the liver by dephosphorylating pp125FAK or preventing its pY response to insulin treatment To explore the cellular mechanism whereby TNF-alpha prevents insulin-stimulated pY of pp125FAK in the liver, we measured c-Src kinase activity and the abundance of 3 major protein tyrosine phosphatases (PTPs)---PTP-1B, LAR and SH-PTP2---in liver homogenates from obese Zucker rats following TNF-alpha blockade. TNF-alpha neutralization did not alter hepatic c-Src kinase activity, but led to a 75% reduction in LAR protein levels while PTP-1B and SH-PTP2 levels remained unchanged. To further verify that TNF-alpha exerts direct effects on LAR expression, we treated HepG2 with TNF-alpha and found that LAR protein levels were elevated in a TNF-alpha dose-dependent manner while PTP-1B and SH-PTP2 abundance remained constant. Furthermore, treatment of HepG2 cells with TNF-alpha (0.8 mM, 72 hr) attenuated insulin stimulation of pp125FAK pY. Taken together, our data suggest that TNF-alpha may attenuate insulin action in the liver by preventing insulin-mediated pY of pp125FAK through increased LAR expression and that this probably represents the critical interface between TNF-alpha and insulin signaling
acase@tulane.edu

Leptin, the product of the LEP gene, may play an important role in pregnancy, specifically in the regulation of placental endocrinology and angiogenesis. In trophoblasts collected early in gestation, leptin increases hormone biosynthesis and metalloproteinase secretion and activation, suggesting leptin's involvement in placental function and development. Therefore, we hypothesize that: (1) estrogen regulates the expression of the leptin receptor in the placental syncytiotrophoblast and leptin, in turn, modulates the secretion of human chorionic gonadotropin (hCG), human growth hormone (hGH), and progesterone; (2) leptin, in combination with factors previously known to modulate placental angiogenesis and vascular permeability, regulates placentation by enhancing vascular endothelial cell proliferation Studies in the baboon (Papio sp) demonstrated leptin receptor (Ob-R) expression. Furthermore, use of in situ hybridization demonstrated the localization of two leptin receptor isoforms to the leptin-producing, placental syncytiotrophoblast, suggesting the possibility for autocrine/paracrine action. Subsequently, leptin's effect upon placental endocrinology was assessed in vitro using third trimester human syncytiotrophoblast cultures. Leptin did not affect hCG, progesterone or hGH elaboration, and estrogen administration had no effect on leptin secretion or Ob-R transcript abundance in this cell type. Collectively, these results differed from studies using first trimester cells, suggesting that leptin's role(s) in early pregnancy differs from that in late gestation. Leptin may potentially play roles in processes that are pertinent in early pregnancy such as angiogenesis and implantation After validating leptin's angiogenic potential using a murine corneal pocket model assay, cultured human umbilical vein endothelial cells (HUVECs) were utilized to test whether leptin affected HUVEC proliferation induced by angiogenic factors pertinent to placentation. Despite Ob-R expression on the cell membrane of HUVECs, leptin did not stimulate cell proliferation. Vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor alpha induced HUVEC proliferation, but leptin failed to enhance or decrease the effects of any of these factors. These results indicate that the angiogenic effects of leptin may be mediated by a mechanism other than the induction of vascular endothelial cell proliferation. Overall, early in gestation, leptin may mediate placental endocrinology and angiogenesis, a role that may change later in pregnancy
acase@tulane.edu

The production of developmentally competent in vitro derived embryos is necessary to decreasing both economic and emotional losses. Epigenetic abnormalities/insults have been shown to occur at a higher incidence in in vitro embryos. An increased prevalence of epigenetic derived disorders such as Parkinson’s disease, Prader-Willi syndrome, and α-thalassemia as well as elevated preimplantation embryo arrest and reduced developmental rates are theorized to be caused by errors in the mediation of chromatin remodeling. Chromatin remodeling refers to the restructuring of packaged DNA so that transcription factors are either given more or less access to specific sequences. This can be done by covalent modification through histone methylation, acetylation, and phosphorylation as well as noncovalent modifications which employ ATP dependent chromatin remodeling complexes. The purpose of this thesis was to characterize two structurally integral core subunits, BAF155 and BAF170, of the SWI/SNF chromatin remodeling complex in porcine oocytes and preimplantation embryos.

The first study concentrated on the transcript abundance of BAF155 and BAF170 in porcine oocytes and embryos. First, BAF155 and BAF170 transcript sequences were identified in porcine muscle and heart tissues. Those sequences were used to create quantitative polymerase chain reaction (qPCR) primers. mRNA from pools of GV oocytes (100-800) was converted to cDNA for transcript abundance measurements. However, transcript abundance remained too low for either BAF155 or BAF170 to be accurately quantified.

The second study focused on developmental competency of embryos post interfering RNA (RNAi) knockdown of BAF155, BAF170, or both BAF155/BAF170 combined. After 7 days of culture, an analysis of variance (ANOVA) was performed to determine differences in mean nuclei numbers and morphological blastocyst percentages across the three groups. No significant difference was seen between means of treatment groups vs. both control groups. Significant differences were seen between siRNA and Non-Injected groups as well as Non-Injected and Scramble RNA groups. However this indicates that loss of BAF155, BAF170, or a combination of the two transcripts is not the driving force of the significant differences, rather the microinjection itself caused the differences.

The third study examined the process by which BAF155 and BAF170 proteins are imported from the cytoplasm into the nucleus. It was hypothesized that karyopherin α 7 (KPNA7), a nuclear importer known to be prevalent in the porcine oocyte and early embryo, is the main importer of both subunits. A dominant-negative KPNA7 construct missing the importin beta binding (IBB) domain was microinjected into parthenogenetically activated embryos to outcompete competent wild-type KPNA7. No change in protein localization was seen at the 4-cell stage of development (48 hours post-injection) for either BAF155 or BAF170. To reinforce these results, an RNAi targeting KPNA7 was also microinjected into parthenogenetically activated embryos. Again, no change was shown in protein localization at the 4-cell stage (48 hours post-injection), indicating that KPNA7 was not the main nuclear importer of either BAF155 or BAF170.

Further study is necessary to determine transcript abundance and the mechanism of nuclear import of both BAF155 and BAF170.

In mammals, calcium influx is required for oocyte maturation and egg activation, as it supports the persistent calcium oscillations induced by fertilization. These oscillations are required for the initiation of embryo development. The molecular identities of the plasma membrane calcium-permeant channels that underlie calcium influx are not established. Among these channels, Transient Receptor Potential Vanilloid, member 3 (TRPV3) allows divalent cations, namely strontium (Sr2+) and calcium (Ca2+) with high permeability, into cells, and its expression pattern seems to predict an essential role in the initiation of development. Another channel that was identified to be expressed in oocytes/eggs is the low-voltage-activated T-type channel, CaV3.2. However, the ability to accurately probe the expression and function of these channels on Ca2+ homeostasis in mouse eggs is hindered by the lack of specific and known pharmacological agents and antibodies for these channels. Here, we simultaneously knockout out these two Ca2+ influx channels in the mouse to explore the effects on Ca2+ homeostasis. We examined fertility rates, development, and morphological defects that arose from the double null pups. Next, we investigated the consequences on Ca2+ store content in immature and mature oocytes and eggs. We also examined the effects on fertilization-induced Ca2+ oscillations in response to in vitro fertilization and PLCz cRNA microinjection. We found that female mice null for these channels display drastic subfertility compared to the single knockout mice for these channels. Additionally, the Ca2+ store content is significantly diminished in double knockout eggs versus controls, as was the frequency of the fertilization-induced Ca2+ oscillations. These results suggest that these channels play a crucial role in Ca2+ influx during maturation and contribute to maintain Ca2+ oscillations post-fertilization. These null oocytes and eggs will be an important tool to perform electrophysiological studies to accurately measure the native current(s) of a specific channel(s) in eggs, and to identify the channel(s) that mediate Ca2+ during fertilization.

A saugeye is the progeny of a female walleye (Sander vitreus) and male sauger (Sander canadensis). In the United States, hybrid saugeyes are considered important for recreational fisheries and as a potential food source. Saugeyes grow exceptionally faster than their non-hybrid parents and are more tolerant of a broader range of water conditions. They are also of interest to anglers due to their increased growth rate and ease to catch. Rather unexpectedly, biologists have recently observed fish that they believe to be saugeye in the Fort Wayne Rivers even though only walleye have been stocked in the area. The fish in Hurshtown Reservoir are believed to be walleye and the identification of those in the Three Rivers is unknown. A potential source for saugeye in the Fort Wayne Rivers is St. Marys State Fish Hatchery in Ohio. This research aims to determine if the fish found in the Fort Wayne Rivers are walleye or saugeye using microsatellite analysis. Microsatellites at seven loci were genotyped for 20 reference walleye, sauger, and saugeye as well as 21 unknown fish caught near Fort Wayne. Of the fish caught near Fort Wayne, three are from Hurshtown Reservoir and 18 are from the Three Rivers. Assignment tests of genotypes were completed using model and non-model based cluster analysis. Genotypic variation clearly resolved the two parent species from their hybrid offspring. Sixteen of eighteen Sander (unknown species) caught in Fort Wayne Rivers between 2018 and 2019 were determined to be first generation saugeye. The other two were walleye found in the Maumee River downstream of Hosey Dam. The three Sander caught in Hurshtown Reservoir were verified to be walleye. Sauger have never been stocked in the Fort Wayne Rivers and connecting waterways. Therefore, it is not likely that the saugeye found in the analysis are from natural reproduction. It is speculated that saugeye are swimming to Fort Wayne from hatcheries within the Maumee watershed. There are many potential sources for walleye in the Fort Wayne Rivers.

Down syndrome (DS) is a complex genetic disorder caused by the triplication of human chromosome 21 (Hsa21). The presence of an extra copy of an entire chromosome greatly disrupts the copy number and expression of over 350 protein coding genes. This gene dosage imbalance has far-reaching effects on normal development and aging, leading to cognitive and skeletal defects that emerge earlier in life than the general population.

The present study begins by characterizing skeletal development in young male Ts65Dn mice to test the hypothesis that skeletal defects in male Ts65Dn mice are developmental in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical defects were present generally throughout development, but trabecular defects emerged at P30 and persisted until P42.

The gene Dual-specificity tyrosine-regulated kinase 1a (Dyrk1a) is triplicated in both DS and in Ts65Dn mice and has been implicated as a putative cause of both cognitive and skeletal defects. To test the hypothesis that trisomic Dyrk1a is related to the emergence of trabecular defects at P30, expression of Dyrk1a in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown to fluctuate throughout development and overexpression generally aligned with the emergence of trabecular defects at P30.

The growth rate in trabecular measures between male Ts65Dn and euploid littermates was similar between P30 and P42, suggesting a closer look into cellular mechanisms at P42. Assessment of proliferation of BMSCs, differentiation and activity of osteoblasts showed no significant differences between Ts65Dn and euploid cellular activity, suggesting that the cellular microenvironment has a greater influence on cellular activity than genetic background.

These data led to the hypothesis that reduction of Dyrk1a gene expression and pharmacological inhibition of DYRK1A could be executed during a critical period to prevent the emergence of trabecular defects at P30. To tests this hypothesis, doxycycline-induced cre-lox recombination to reduce Dyrk1a gene copy number or the DYRK1A inhibitor CX-4945 began at P21. The results of both genetic and pharmacological interventions suggest that trisomic Dyrk1a does not influence the emergence of trabecular defects up to P30. Instead, data suggest that the critical window for the rescue of trabecular defects lies between P30 and P42.

The Keap1-Nrf2 pathway regulates a wide range of cytoprotective genes, and has been found to serve a protective and beneficial role in many body systems. There is limited information available, however, about its role in bone homeostasis. While Nrf2 activation has been suggested as an effective method of increasing bone mass and quality, there have been conflicting reports which associate Keap1 deficiency with detrimental phenotypes. As Keap1 deletion is a common method of Nrf2 activation, further study should address the impacts of various methods of regulating Nrf2 expression. Also, little research has been conducted on the specific pathways by which Nrf2 activation improves bone quality. In this study, the effects of alterations to Nrf2 activation levels were explored in two specific and varied scenarios. In the first experiment, moderate Nrf2 activation was achieved via partial deletion of its sequestering protein, Keap1, in an aging mouse model. The hypothesis tested here is that moderate Nrf2 activation improves bone quality by affecting bone metabolism and response to mechanical loading. The results of this first experiment suggest a subtle, sex-specific effect of moderate Nrf2 activation in aging mice which improves specific indices of bone quality to varying degrees, but does not affect loading-induced bone formation. It is likely that the overwhelming phenotypic impacts associated with aging or the systemic effects of global Keap1 deficiency may increase the difficulty in parsing out significant effects that can be attributed solely to Nrf2 activation. In the second experiment, a cell-specific knockout of Nrf2 in the osteocytes was achieved using a Cre/Lox breeding system. The hypothesis tested here is that osteocyte-specific deletion of Nrf2 impairs bone quality by affecting bone metabolism and response to mechanical loading. The results of this experiment suggest an important role of Nrf2 in osteocyte function which improves certain indices of bone quality, which impacts male and female bones in different 7 ways, but did not significantly impact loading-induced bone formation. Further studies should modify the method of Nrf2 activation in an effort to refine the animal model, allowing the effects of Nrf2 to be isolated from the potential systemic effects of Keap1 deletion. Future studies should also utilize other conditional knockout models to elucidate the effects of Nrf2 in other specific cell types.

Cerebral radiation necrosis as a consequence of radiation therapy is often observed in patients several months to years after treatment. Complications include painful headaches, seizures, and in the worst-case death. Radiation necrosis is an irreversible condition with the options available to manage it all having noticeable downsides. As such, there is a critical need for better ways of either preventing the onset of necrosis and/or managing its symptoms. As radiation necrosis cannot be induced in humans for ethical reasons, a mouse model that mirrors the features of radiation necrosis observed in patients would allow for new techniques to be tested before being used in human clinical trials. This thesis will explain how our lab designed a murine model of cerebral radiation necrosis that uses a 320 keV cabinet irradiator to produce radiation necrosis and MRI and histology to evaluate the development of radiation necrosis at multiple time points.

Our model required the development of a mouse positioning apparatus that could be used in the cabinet irradiator used as well as the machining of lead shields so that focal semi-hemispheric irradiations could be conducted with other critical structures spared. The MRI scans used as well as the algorithm used to draw radiation necrosis lesions were based off what has been used in previous Gamma Knife models of radiation necrosis. Our initial work showed that since the cabinet irradiator has a relatively flat dose distribution unlike the Gamma Knife, the radiation lesion volumes produced in the former either plateaued or decreased, unlike in the case of the latter where lesion volumes tended to decrease over time. Further work analyzed the effects of fractionation and found minimal sparing using four different fractionation schemes. The effects of strain and sex on the development of radiation necrosis were also analyzed, with strain being found to be a statistically significant parameter while sex was not. Future research should focus on testing the effects of new drugs and techniques for better dealing with radiation necrosis.

External representations (ERs) used in science education are multimodal ensembles consisting of design elements to convey educational meanings to the audience. As an example of a dynamic ER, an animation presenting its content features (i.e., scientific concepts) via varying the feature’s depiction over time. A production team invited the dissertation author to inspect their creation of a biochemistry animation about the role of the actin cytoskeleton in cell motility and the animation’s implication on learning. To address this, the author developed a four-step methodology entitled the Multimodal Variation Analysis of Dynamic External Representations (MVADER) that deconstructs the animation’s content and design to inspect how each content feature is conveyed via the animation’s design elements.

This dissertation research investigated the actin animation’s educational value and the MVADER’s utility in animation evaluation. The research design was guided by descriptive case study methodology and an integrated framework consisting of the variation theory, multimodal analysis, and visual analytics. As stated above, the animation was analyzed using MVADER. The development of the actin animation and the content features the production team members intended to convey via the animation were studied by analyzing the communication records between the members, observing the team meetings, and interviewing the members individually. Furthermore, students’ learning experiences from watching the animation were examined via semi-structured interviews coupled with post- storyboarding. Moreover, the instructions of MVADER and its applications in studying the actin animation were reviewed to determine the MVADER’s usefulness as an animation evaluation tool.

Findings of this research indicate that the three educators in the production team intended the actin animation to convey forty-three content features to the undergraduate biology students. At least 50% of the student who participated in this thesis learned thirty-five of these forty-three (> 80%) features. Evidence suggests that the animation’s effectiveness to convey its features was associated with the features’ depiction time, the number of identified design elements applied to depict the features, and the features’ variation of depiction over time.

Additionally, one-third of the student participants made similar mistakes regarding two content features after watching the actin animation: the F-actin elongation and the F-actin crosslink structure in lamellipodia. The analysis reveals the animation’s potential design flaws that might have contributed to these common misconceptions. Furthermore, two disruptors to the creation process and the educational value of the actin animation were identified: the vagueness of the learning goals and the designer’s placement of the animation’s beauty over its reach to the learning goals. The vagueness of the learning goals hampered the narration scripting process. On the other hand, the designer’s prioritization of the animation’s aesthetic led to the inclusion of a “beauty shot” in the animation that caused students’ confusion.

MVADER was used to examine the content, design, and their relationships in the actin animation at multiple aspects and granularities. The result of MVADER was compared with the students’ learning outcomes from watching the animation to identify the characteristics of content’s depiction that were constructive and disruptive to learning. These findings led to several practical recommendations to teach using the actin animation and create educational ERs.

To conclude, this dissertation discloses the connections between the creation process, the content and design, and the educational implication of a biochemistry animation. It also introduces MVADER as a novel ER analysis tool to the education research and visualization communities. MVADER can be applied in various formats of static and dynamic ERs and beyond the disciplines of biology and chemistry.