<p> Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) whereby cells adopt a motile and invasive phenotype through loss of epithelial markers, namely Cadherin 1/E-Cadherin (CDH1), and acquisition of mesenchymal markers, such as vimentin (VIM) and Cadherin 2/N-Cadherin (CDH2). While MAPK/ERK1/2 kinase inhibitors (MEKi) have shown promise as antitumor agents in the preclinical setting, application has had limited success clinically. Activation of compensatory signaling, potentially contributing to the emergence of drug resistance, has shifted the therapeutic strategy to combine MEK1/2 inhibitors with agents targeting oncoproteins (RAF) or parallel growth pathways (PI3K). </p><p> Conventional MAPK family members have been well-characterized in modulation of cellular processes involved in tumor initiation and progression, yet the role of MEK5-ERK5 in cancer biology is not completely understood. Recent studies have highlighted the importance of the MEK5 pathway in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast. Furthermore, elevated levels of ERK5 expression and activity observed in breast carcinomas are linked to worse prognosis in TNBC patients. The purpose of this work is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. </p><p> Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide rationale for combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.</p>

No description available.

Tendons are robust structures, made of cellular and extracellular components, that transmit force efficiently between muscle and bone that is essential for permitting strength and mobility in vertebrates. Although the tendon is mainly comprised of extracellular matrix, resident tendon cells – tenoblasts and tenocytes – have been established as responsible for constructing the fibrotic structure of the tendon during development. Various forms of tendinopathy impact a broad demographic range, yet effective treatment modalities remain rather limited due to our lack of understanding of the molecular mechanisms that drive tendon healing, which has made this a critical area of tendon research. Recently, the presence of a stem cell/progenitor-like tendon cell population within the tendon was identified, implicating their potential role in tendon regeneration and providing course for further research on this novel tendon cell population (Bi et al., 2007) . Several genetic markers, such as Scleraxis (Scx), collagen type I (Col I) and Mohawk (Mkx), have been shown to trace the tendon cell lineage (Edom‐Vovard & Duprez, 2004; Ito et al., 2010; Schweitzer et al., 2001). However, they do not provide specificity towards these tendon-derived stem cell/progenitor cells (TSPCs), nor do they give much insight into the interactions between the resident cells that govern tendon biology. Through the use of an Axin2 marker, previous stem cell research has suggested the Wnt/beta-Catenin signaling pathway to be involved in regulating the self-renewing capacity of these cells within the intestine, liver, epidermis and brain (Bowman, van Amerongen, Palmer, & Nusse, 2013; Lim et al., 2013; Wang, Zhao, Fish, Logan, & Nusse, 2015). Therefore, this study aims to apply the Axin2 marker to the previously identified TSPC population to illustrate the heterogeneity of these cells and implicate that their proliferative potential is controlled through Wnt/beta-Catenin signaling. By using an Axin2-CreERt2;Rosa-LSLTdTomato mouse model in an injured state, due to the disruption of the tendon matrix during TSPC isolation, we have demonstrated through RT-qPCR analysis that there are differences in gene expression between Axin2+/- cells, particularly in Mkx and Col II. Furthermore, we utilized cell counting and FACS analysis to show that the Axin2+ cells have a greater propensity to proliferate than Axin2- cells. Our findings suggest that the Wnt/beta-Catenin pathway is involved in regulating tendon cell fate and may be an underlying mechanism behind tendon repair.

We have identified a region in the coding sequence (CRAS) of two replication-dependent histone genes, a H2a.2 and a H3.2, the deletion of which causes a 20-fold drop in expression. In vivo expression data obtained after transfection of in-frame oligonucleotide-directed deletions in the H3.2 CRAS show that there are multiple areas in the CRAS which act to increase expression of the intact gene. DNAase I protection of a sequence designated the CRAS alpha box is present in the CRAS of four classes of histone genes--H2a, H2b, H3 and H4. Deletion of the alpha box causes a 5-fold drop in expression of the H3.2 gene. The comparable "CRAS" region of a H3.3 gene, a nonreplication-dependent variant, was used as a non-specific competitor. Although the coding region is highly conserved among classes of histone genes, the H3.3 "alpha box" sequence contains 5 base changes out of 7. UV crosslinking of the protein bound H3.2 CRAS alpha sequence identifies a protein doublet migrating at 80 and 90 kD on SDS-polyacrylamide gels. Binding to the alpha box is regulated by the phosphorylation state of the nuclear protein that binds the alpha sequence. Dephosphorylation of the CRAS alpha protein is necessary for binding to the target sequence. The role of the CRAS-binding proteins in cell cycle regulation of histone gene expression was studied by preparing G1 and S phase nuclear extracts from synchronous populations of CHO cells. Binding of CRAS alpha protein to duplex oligonucleotides containing the consensus sequence is greatly elevated in S phase compared to G1. / Source: Dissertation Abstracts International, Volume: 55-04, Section: B, page: 1295. / Major Professor: William F. Marzluff. / Thesis (Ph.D.)--The Florida State University, 1994.

The CH asymmetric stretching region of the methanol spectrum has been measured from 2900 to 3200 cm$\sp{-1}$ using the newly constructed Fourier transform spectrometer (FTS). The nominal resolution, the reciprocal of twice the maximum optical path difference, is 0.004 cm$\sp{-1}$. / The objectives of this investigation were to identify, assign, and analyze the torsion-rovibrational transitions of the CH$\sb3$ asymmetric stretching fundamentals $v\sb2$ and $v\sb9$. The theory used in the investigation is principally that used by Lees and Baker with the modifications described by Y. Y. Kwan. It is here assumed that this model is suitable for fundamentals other than the torsion rotation. / A total of 13 P branch and 11 R branch series were assigned (13 series representing 6 excited states belonging to the $v\sb2$ fundamental and 11 series representing 5 excited states belonging to the $v\sb9$ fundamental). A partial nonlinear least squares analysis of the series origins yields a band center of 2999.44 cm$\sp{-1}$, a barrier height of 405.62 cm$\sp{-1}$, and a value of 5.29 cm$\sp{-1}$ for the moment of inertia of the methyl group about the symmetry axis for the $v\sb2$ fundamental. The corresponding values for the $v\sb9$ fundamental are 2970.18 cm$\sp{-1}$, 529.71 cm$\sp{-1}$, and 5.34 cm$\sp{-1}$ respectively. These parameters give a quality of fit with rms deviations of 1.15 cm$\sp{-1}$ and 1.26 cm$\sp{-1}$ for the $v\sb2$ and $v\sb9$ bands respectively. / A criterion was used to divide the assignments between two separate bands. A comparison between the asymmetric stretch data of methyl fluoride, the OH and CO stretch data of methanol indicates that our assignments are reasonable. / Tentative assignments of several series observed in the spectra based on calculations, using the fitted parameters and normal state parallel combination differences, are also given. / Source: Dissertation Abstracts International, Volume: 53-10, Section: B, page: 5260. / Major Professor: Robert H. Hunt. / Thesis (Ph.D.)--The Florida State University, 1992.

Sequences homologous to a putative origin of DNA replication from Xenopus laevis have been cloned and sequenced. The prototype, Xori, has been demonstrated to enhance the replication of vector molecules microinjected into Xenopus eggs. Thus, it was of interest to isolate additional clones and to compare their sequences in order to determine if common sequence motifs exist, which might provide some insight into gene regulation and DNA replication. Including Xori, six clones were compared. Two of the clones lie in the globin gene cluster; one 5' to the adult (alpha)1 gene, and the other in the second intron of the tadpole (alpha)1 gene. In all clones, the homologous region begins at the same site, but the lengths of the common regions vary from 142 bp to over 300 bp due to heterogeneous 3' ends. Some of the repeats are bracketed by direct and/or inverted repeats, and relatively large palindromes were found 5' to the common region in some clones. These characteristics, and the presence of a repeat 5' to one of a pair of duplicated (alpha)1 genes suggests that Xori may be capable of transposition. The homologous regions are 20 to 30 percent divergent, but contain a number of sequences of interest. Five clones contain 17 elements which are highly homologous to the yeast autonomously replicating sequence, as well as a region of 25 bp which is similar to the SV40 enhancer. However, following transfection of cultured cells, plasmids harboring repeats are progressively lost, and apparently do not replicate. Although the repeats are transcribed at gastrulation, they are not transcribed in oocytes, blood, or in kidney cells in culture, nor are they associated with the nuclear matrix. Implications of these data are discussed. / Source: Dissertation Abstracts International, Volume: 47-06, Section: B, page: 2319. / Thesis (Ph.D.)--The Florida State University, 1986.

Genes coding for replication-dependent, partially replication-dependent, and replication-independent histones have been isolated from mouse. These genes were isolated from libraries of mouse DNA in lambda bacteriophage. Three previously-isolated mouse histone genes, several heterologous histone genes, and synthetic oligonucleotides were used to identify the clones. The four replication-dependent and two partially replication-dependent genes have been sequenced. Transcripts of these genes from mouse myeloma cells, from C127 mouse fibroblasts, and from differentiating murine erythroleukemia cells have been characterized using the S1 nuclease assay. Transcripts of several of the replication-independent genes from differentiating murine erythroleukemis cells have been studied, and one of these genes has been sequenced. There are no intervening sequences in the coding region of this gene, but there may be intervening sequences in the 5' flanking region. Primer extension studies have been used to determine the length of the transcript from the genes for replication-independent histones. The replication-dependent and partially replication-dependent histone genes are clustered, and the clusters contain members of only one expression class. Only genes coding for the core histones have been found in these clusters. The replication-independent H3 genes appear to be isolated from other histone genes. / Source: Dissertation Abstracts International, Volume: 47-01, Section: B, page: 0083. / Thesis (Ph.D.)--The Florida State University, 1986.

A detailed study of the OH stretching vibration of the hydrogen peroxide molecule was conducted covering the wavelength region from 3410 cm('-1) to 3794 cm('-1). Five data runs using 98% pure hydrogen peroxide were made, one of them of low intensity. Three calibration runs using 90% pure H(,2)O(,2) were carried out thus introducing water as a calibrant. In addition several runs were made with slightly heated or slightly cooled H(,2)O(,2) to enhance certain vibrational states. / Analysis of the data allowed extension of the series assignments for the perpendicular n = 0(--->)0' transitions to higher J values. New assignments were made for the P and R branches of the perpendicular n = 1(--->)1' transition. In addition tentative assignments were made for a few series belonging to the perpendicular n = 2(--->)2' transition and to parallel transitions identified in the band center region. Additional series were seen but were not identified though some may belong to the symmetric OH stretch vibration. / Assignments of series allowed the calculation of a more accurate set of values for the ground state and first excited state parameters. A hindering potential for the excited state was established with values for the cis barrier, trans barrier, and (zeta)(,o) of 2480.45 cm('-1), 426.77 cm('-1), and 108.808(DEGREES), respectively.(' ) / Source: Dissertation Abstracts International, Volume: 46-01, Section: B, page: 0209. / Thesis (Ph.D.)--The Florida State University, 1984.

Control of the timing of replication within S-phase of chromosomes or chromosome bands has long been suspected to be a general mechanism of compartmentalizing the genome for differential gene expression. I have developed a method that provides both a direct probe for active and inactive regions of the chromosome and a means to determine the time of replication of these individual sites. By using a retrovirus shuttle vector which contains a selectable marker gene (HSV-tk), to infect tissue culture cells, it is possible to introduce the same single copy sequence in many different chromosomal locations. I have isolated and characterized a series of cell clones which have single or a small number of either active or cryptic copies of the marker gene. To determine the time of replication of the marker sequence, asynchronous or synchronized cell cultures were pulse labeled with bromo-deoxy-uridine. Cells were fractionated according to their position in S-phase by mitotic collection or separately pulse labeled cultures (for synchronized cells). BrdU containing DNA was then degraded selectively by exposure to Hoechst 33257 dye, long wave UV light and S1 nuclease, which makes it possible to analyze each cell fraction with regard to BrdU substitution in the marker sequence. One can thus infer the time of replication of a specific single copy sequence. All clones analyzed, both expressing and cryptic clones, replicate the SW 272 sequence in early S-phase. The findings suggest, that this retrovirus does not integrate randomly into the cellular genome with respect to replication compartments. In one clone the cryptic copy of the viral Tk-gene, could be activated by treatment with 5-aza-cytidine, implying a role of cytosine methylation in regulation of expression in this case. / Source: Dissertation Abstracts International, Volume: 47-07, Section: B, page: 2773. / Thesis (Ph.D.)--The Florida State University, 1986.

X chromosome inactivation (XCI), the mammalian form of dosage compensation, is a canonical example of epigenetic regulation and involves the transcriptional repression of nearly an entire chromosome (Xi) while preserving the transcriptional activity of its homologue (Xa) in females. Since the initial report describing a dense nuclear cytological feature in female feline neurons and Mary Lyon’s subsequent hypothesis of random X chromosome inactivation as a means of compensating for the lack of two X chromosomes in males, XCI has yielded decades of insights into the mechanisms of epigenetic regulation. This dissertation focuses on the three-dimensional organization of the Xi and the functional potential of large tandem repeats. The large X-linked tandem repeat, DXZ4, adopts a euchromatic conformation on the Xi in contrast to the largely heterochromatic chromosome and is able to form CTCF-dependent interactions with other euchromatic repeats exclusively on inactive X chromosome in females. We demonstrate here that DXZ4 has a critical role in maintaining the three-dimensional organization of the Xi as well as the separation of multi-megabase domains containing different types of heterochromatin. While characterizing the genomic interval of DXZ4, we uncovered transcriptional activity corresponding to two novel, long non-coding RNAs (lncRNAs) which originate on opposite sides of the DXZ4 and are transcribed antisense to one another. Both of these lncRNAs traverse the array in human embryonic stem cells (hESCs). Developmentally associated transcription suggests a potential connection between their transcription activity and maintenance of a heterochromatic DXZ4 on the Xa and male X prior to differentiation. Mouse and human genomes largely share the same gene content in accordance with Ohno’s law; however, the mouse genome has undergone rearrangements involving large syntenic blocks and has acquired several multi-megabase, lineage-specific regions of repetitive DNA that are absent in human. To further our understanding of the mouse inactive X chromosome and highlight another difference between human and mouse XCI, we characterized an approximately 20-Mb repeat that, similar to DXZ4, displays marks of euchromatin on the Xi and conversely displays marks of heterochromatin on the Xa. Overall, this work gives new insight into the function and epigenetic regulation of macrosatellites as well as the relationship between higher-order chromatin organization and heterochromatin maintenance of the Xi. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester 2017. / December 4, 2017. / Includes bibliographical references. / Brian P. Chadwick, Professor Directing Dissertation; Myra M. Hurt, University Representative; Karen M. McGinnis, Committee Member; Wu-Min Deng, Committee Member; Hank W. Bass, Committee Member.