Ovarian cancer is the most deadly gynecological malignancy in the US. Once thought to be a single disease arising from the ovarian surface epithelium, we now understand that it is in fact a heterogenous disease with origins across the female reproductive tract. Ovarian cancers can be broken down into type I and type II tumors. While type I tumors are typically slow growing and arise from the ovary, type II tumors are highly aggressive, particularly the most common and deadliest subtype, High Grade Serous Ovarian Cancer (HGSOC), which arises from the fallopian tube epithelium. While our understanding of the complexity of ovarian cancer has evolved significantly over the last few decades, ovarian cancer is still primarily modeled by a few historical and robust cell lines which poorly recapitulate the genetics of HGSOC patient tumors. Here we characterize less studied cell lines with high genomic fidelity to HGSOC, KURAMOCHI and OVSAHO. Through in vivo xenograft experiments, we determined that these two cell lines form more diffuse tumors which better represent the HGSOC phenotype. In vitro, these lines are tractable to a variety of standard molecular biology techniques, but only express a subset of common markers of HGSOC, including PAX8 and stabilized TP53, while others were absent. These results indicate that it is important to take both molecular and genetic relevance into consideration when selecting cell lines for laboratory studies. PAX8 is a nuclear transcription factor critical to the development of the female reproductive tract, including the fallopian tube, and is conserved in both the adult fallopian tube and HGSOC. Recent studies have identified PAX8 as a differentially expressed, essential gene to ovarian cancer, making it a promising drug target for HGSOC. Here, we identify a subset of ovarian cancers that exhibit decreased proliferation when PAX8 expression is decreased, and determine that in KURAMOCHI cells this defect appears to be driven by cell cycle arrest. We also characterized the similarities and differences of PAX8 DNA binding and gene regulation through ChIPseq and RNAseq, respectively, in the context of the fallopian tube and HGSOC. We determined that while a small subset of PAX8 binding sites are conserved across the fallopian tube and ovarian cancer, there is a significant gain of novel binding sites in ovarian cancer that is unique to each cancer cell line. In contrast, different fallopian tube derived lines have very similar PAX8 binding sites and many of these are uniformly lost in the context of cancer. Understanding how the role of PAX8 changes from the fallopian tube to cancer is an important step to drug development targeting PAX8. Ultimately, this body of research seeks to improve ovarian cancer modeling techniques and to better understand fallopian tube pathogenesis, leading to more effective translation of ovarian cancer research from bench to bedside. / Biology, Molecular and Cellular

Germ cells are the unique source of gametes for multicellular organisms and are specified through different mechanisms. One such specification mechanism is inheritance-mediated, in which the molecular factors necessary and sufficient to impart germ cell fate (collectively referred to as “germ plasm”) are maternally synthesized and deposited during oogenesis or early embryogenesis. Incorporation of this germ plasm into newly formed cells results in primordial germ cells. This mechanism stands in contrast to the predominant metazoan (and likely ancestral) mode of germ cell specification termed induction; germ cells specified via induction are the result of extracellular signaling from one embryonic tissue to another, triggering the expression of germ plasm components in the recipient cell type. In Drosophila melanogaster, the gene oskar (osk) is necessary and sufficient for organizing germ plasm in the oocyte, leading to primordial germ cell specification. While many investigators have offered thorough developmental and genetic investigations of osk, an understanding of the specific molecular mechanisms by which its protein products accumulate germ plasm is presently lacking. Oskar protein is uncharacterized, but is predicted to contain two well-folded domains: an N-terminal winged helix-turn-helix domain (the LOTUS/OST-HTH domain) and a C-terminal domain bearing sequence similarity to SGNH hydrolases (the lipase-related domain). The function of both domains is unknown, but previously published mutational data demonstrate that the lipase-related domain is crucial for Osk protein function. It is currently not known if the LOTUS/OST-HTH domain is necessary for the accumulation of germ plasm at the posterior pole. Using X-ray crystallography, I solved four structures of the LOTUS domain (corresponding to residues 139-241) and found that it forms a homodimer. In contrast to published characterized winged helix-turn-helix structures, the LOTUS homodimer conformation results from two β-hairpins (one from each protomer) forming a completed β-sheet. Analytical size exclusion chromatography, bacterial two-hybrid, and multi-angle light scattering experiments confirm that the LOTUS domain exists as a dimer in solution and combined with site-directed mutagenesis, the dimerization interface is indeed formed by a completed β-sheet as revealed by the crystal structures. Using a GAL4/UAS inducible D. melanogaster transgenic osk reporter, deletion of the LOTUS domain was found to abrogate the accumulation of germ plasm components, indicating that this domain is necessary for Oskar protein function. Furthermore, substituting the LOTUS domain with exogenous dimerization domains does not restore Osk activity. It is then postulated that oligomerization as mediated by the LOTUS domain constitutes at least one crucial aspect of its function within the context of full length Oskar protein. / Biology, Molecular and Cellular

Dietary restriction (DR) or reduced food intake without malnutrition encompasses a variety of dietary interventions including reduction of calories or specific macronutrients such as amino acids or total protein. Even though DR is known to result in various beneficial health effects, including extended longevity and increased stress resistance, the underlying nutritional and genetic requirements remain incompletely understood. Previous studies in lower organisms, as well as in mammals, point to the importance of the restriction of dietary protein intake in DR benefits. Previously our lab has shown that short-term restriction of intake of individual essential amino acids protects against hepatic ischemia reperfusion injury (IRI) via activation of the amino acid deprivation sensing protein, general control non-derepressible 2 (GCN2). Interestingly, GCN2 was no longer required for the protective effects of total dietary protein restriction (PR) against hepatic IRI. We thus investigated the potential role of a distinct amino acid sensing pathway involving the mechanistic target of rapamycin complex 1 (mTORC1) kinase, which is normally repressed upon PR. To test the hypothesis that reduced mTORC1 signaling is required for benefits of PR against hepatic IRI, we used a mouse model with liver specific deletion of the mTORC1 repressor gene tuberous sclerosis complex 1 (TSC1), leading to constitutive hepatic mTORC1 activation (LTsc1KO). Although one week of PR was able to reduce circulating growth factors and amino acid levels and activate hepatic GCN2 signaling in both LTsc1KO and WT mice, LTsc1KO mice failed to gain the preconditioning benefits of PR against hepatic IRI. To understand the molecular mechanism underlying the genetic requirement for the TSC complex in PR benefits, we focused on the observation that PR improved hepatic insulin sensitivity in WT but not LTsc1KO mice. Additional data from liver specific insulin receptor knockout (LIrKO) mice and in WT mice using pharmacological PI3K inhibition by wortmannin indicated a partial requirement for post-reperfusion insulin/Akt signaling in PR-mediated protection from hepatic IRI. In addition to defects in insulin signaling important for PR action, LTsc1KOs also failed to upregulate hepatic production of another potent protective molecule, hydrogen sulfide (H2S), which was increased in WT mice upon PR and required for PR-mediated protection from hepatic IRI. Finally, we investigated the mechanisms of regulation of hepatic H2S production. Using an in vitro model of increased H2S production upon serum deprivation, we identified growth hormone (GH) as a negative regulator of H2S production through JAK/STAT signaling, and increased autophagy as the likely source of free cysteine, the substrate for cystathionine gamma lyase (CGL)-mediated H2S production. The mechanistic details of how in vivo PR translates into increased H2S production, and the potential role of mTORC1, GH signaling and autophagy in this process remain to be fully elucidated. / Biological Sciences in Public Health

The noncoding genome, or the portion of the genome that does not encode for proteins, encompasses >95% of the human genome. It has been found that the majority of disease-associated genetic variants identified by genome-wide association studies (GWAS) are located in this noncoding 95%, where they have the potential to affect regions that control transcription (promoters, enhancers) and noncoding RNAs that also can influence gene expression. The discovery of these alterations has already contributed to a better understanding of the etiology of human diseases and has begun to yield insight into the function of these noncoding loci I am interested in studying how the noncoding genome functions and contributes to human development and disease pathology, especially when it is considered that our understanding of human disease is almost entirely contained within the realm of the <5% of the genome that is protein coding. Toward this end, I have focused my studies on one part of the noncoding genome, long noncoding RNAs. In order to identify whether long noncoding RNAs are important for mammalian development and disease, our lab created a set of lincRNA knockout animal models in which a cassette expressing beta-galactosidase (lacZ) replaces the lincRNA DNA sequence. I have used these models for the in vivo characterization of several lincRNAs, including Fendrr in the lungs, Brn1b in the brain, Tug1 in the testes, and Cox2 in the innate immune system. Each of these studies reveals perturbations in development induced by loss of function of the respective lincRNA locus, and demonstrates promising potential for further examination of the role these molecules play in human disease. / Biology, Molecular and Cellular

In eukaryotes, 20-30 nt small RNAs (sRNAs) regulate many cellular processes by a process called RNA interference (RNAi). sRNAs serve as adaptable specificity factors to direct silencing at either the post-transcriptional or transcriptional level. On one hand, the ability to target genome-wide is a valuable feature; however, this can potentially be a double-edged sword, as spurious expression of sRNAs could lead to inappropriate silencing. Fission yeast have developed mechanisms to combat inappropriate sRNA-mediated silencing. In fission yeast, small interfering siRNAs (siRNAs) and silenced heterochromatin are mutually dependent at pericentromeric repeat sequences, and are required for proper centromere function. Euchromatic regions of the genome typically do not generate siRNAs and are refractory to RNAi-mediated silencing and heterochromatin formation. This dissertation aims to identify mechanisms by which the cell protects itself against inappropriate RNAi-induced silencing or heterochromatin formation. Specifically, we seek to define 1) factors that determine whether a locus can produce siRNAs and 2) factors that determine whether a locus can be targeted by siRNAs for silencing or heterochromatin formation. Using high throughput sequencing of small RNAs in cells overexpressing Dicer, I show that sites of overlapping transcription including convergent genes and centromeric repeats are potential substrates for Dicer activity, but at endogenous levels of Dicer only centromeric repeats generate siRNAs. RT-PCR and ChIP-seq experiments reveal that, at euchromatic loci, generation of siRNAs in cis does not correlate with reduced transcript levels or with methylation of H3K9, a conserved marker for heterochromatin. Thus, there are features of euchromatic loci that protect them from siRNA-mediated silencing. Genetic studies involving deletion or mutation of the 3’ UTR of an endogenous reporter gene in cells expressing complementary siRNAs identify transcriptional cleavage and polyadenylation signals as one of these protective factors. Direct sequencing of polyadenylated transcripts reveals a divergence in cleavage patterns between centromeric and mRNA-coding loci, supporting the idea that the ability of a nascent transcript to be targeted for silencing by siRNAs correlates with inefficient 3’ end processing. Finally, I show that Mlo3, a gene associated with 3’ ends of ORFs and involved in mRNA export, antagonizes siRNA-mediated heterochromatin formation genome-wide. ChIP experiments and growth assays show that mlo3∆ cells are capable of generating siRNA- mediated H3K9 dimethylation at a locus in trans, but surprisingly, this rarely correlates with silencing of the targeted locus. Enrichment in H3K9 dimethylation that is not associated with silencing is not heritable through meiosis. However, once siRNA-induced silencing is established, heterochromatin is stable and transmissible through meiosis even in the absence of the driver siRNA-producing locus. Silencing is better correlated with H3K9me3 than H3K9me2, and we propose that H3K9me3 is required for formation of a silenced and heritable heterochromatic state. Together, these results demonstrate that the cell has evolved several mechanisms to protect itself from spurious siRNA-mediated silencing or heterochromatin formation. First, limiting concentrations of Dicer restrict siRNA generation to repetitive sequences surrounding the centromeres. However, if siRNAs mapping to mRNAs are expressed, factors involved in efficient 3’ end processing antagonize silencing and H3K9 methylation directed by siRNAs, These factors include transcript cleavage and polyadenylation sequences and mRNA export factor mlo3+. / Medical Sciences

Runx2 is a transcription factor that has a crucial role in the development of bone; haploinsufficiency of Runx2 leads to the autosomal-dominant disorder, cleidocranial dysplasia (CCD) characterized by various skeletal abnormalities. Zinc finger protein 521 (Zfp521) is a transcription factor that is expressed in several cell types including bone. Recent studies demonstrated that Zfp521 interacts with Runx2 and regulates osteoblast maturation at least in part by repressing the transcriptional activity of Runx2; furthermore, it was demonstrated that the repression of Runx2 by Zfp521 involves the recruitment of HDAC3. The interaction of Runx2 with HDAC3 is strongly enhanced by Zfp521. Zfp521 may regulate osteoblast commitment and differentiation by modulating the Runx2 transcriptional activity by decreasing the levels of Runx2 acetylation. Objective: Runx2 is a key regulator of osteoblast differentiation; Zfp521 may regulate osteoblast commitment and differentiation, at least in part by decreasing the levels of Runx2 acetylation. We investigated 1) Effect of Zfp521 on Runx2 acetylation in HEK293 cells 2) The endogenous Runx2 acetylation levels in MC3T3-E1 cells during osteoblast differentiation. Results: These studies demonstrated that Runx2 acetylation is decreased when Zfp521 is stable expressed in HEK293 cells. Runx2 acetylation levels were detected using immunoprecitation analyses. Sodium butyrate (NaB) prevents protein deacetylation by inhibiting HDACs. The treatment of cells with NaB increased global protein acetylation levels. Importantly, stable expression of Zfp521 did not change global protein acetylation. Therefore, this study suggested that Zfp521 specifically influences Runx2 acetylation. In addition, our findings suggest that Zfp521 impairs Runx2 acetylation by HDACs. Preliminary results show that transient transfection of P300 in HEK293 increased Runx2 acetylation levels. However, stable expression of Zfp521 can still partially decrease Runx2 acetylation levels. This study suggests that Zfp521 function can be linked to P300. Runx2 acetylation levels were then assessed in MC3T3-E1 cells during osteoblast differentiation. While Runx2 protein levels increase by 7 days in culture, and gradually decreases by days 14, Runx2 acetylation was undetectable. Conclusion: Runx2 is the transcription factor that has essential role in osteoblast commitment and differentiation. Zfp521 represses the transcriptional activity of Runx2 by recruiting HDAC3. These studies suggest that Zfp521 modulates Runx2 activity by decreasing Runx2 acetylation level. These studies have extended our knowledge of the mechanisms by which Zfp521 regulates osteoblast differentiation and bone formation, which could have important implications for on the development of future osteo-anabolic treatments.

No description available.

Two naturally-occurring beta-lactamase producing plasmids from Neisseria gonorrhoeae, 7.2 kilobases (kB) (Asian-type, pJD4) and 5.1 kb (African-type, pJD5) in size, were analysed and mapped by restriction endonuclease analysis and DNA-DNA heteroduplex analysis. The two plasmids were similar except for a 2.1 kb fragment missing in pJD5, as compared to pJD4, which was located 1.74 kb counter-clockwise from the unique PstI site. In addition, a novel penicillinase-producing plasmid, 4.9 kb (pJD7, 'Toronto-type') in size was discovered during the study and was compared to pJD4 and pJD5. Except for a 2.3 kb fragment, pJD7 was shown to be similar to pJD4 and probably derived from pJD4. Plasmid pJD7 was compared to a plasmid of reportedly smaller size, pGO4717, isolated in the Netherlands and was found to be identical to it. Part of the large BamHI fragment of pJD4 was sequenced and compared to that of Tn3. It was shown that after base 79 from the BamHI site, the sequence starts to differ. The presence of two replication regions on pJD4 has been identified. Through the construction of mini-plasmids (pJD8, pJD9), the replication region of pJD4 was located on a 1.5 kb fragment, designated region 'a', which included the unique HindIII site. However, fragment 'a' was absent in pJD5. A 1.5 kb fragment (region 'b') was found to be required for the replication of this plasmid. In pJD4, the nucleotide sequence of region 'b' was interrupted by region 'a'. Incompatibility studies showed that, in vitro derived deletion-derivatives from pJD4 and pJD5, containing either region 'a' or region 'b', were compatible. Sequence analysis of region 'a' indicated that this region shares many characteristics of plasmids such as pSC101, P1, F, and R6K. Ten conjugative plasmids were tested for their ability to mobilize plasmid pJD4 between E. coli strains. Plasmid pJD4 was mobilized by RP4 (IncP), R124 (IncIV), pBG791 (IncI$\alpha)$ and R100 (IncFII), but not by R621a (IncI$\gamma$), R199 (IncN), R6K (IncX), R1drd16 (IncFII), TP124 (IncH$\sp{\rm e}$) and S-a (IncW). The 2.4 kb BamHI fragment of pJD4, containing the beta-lactamase gene and a 1.0 kb BamHI- HinfI fragment designated region 'M', was cloned into pACYC184, and was shown to be required in cis for the mobilization of the recombinant plasmid by pBG791. The entire DNA sequence of region 'M' was analysed, and contained sequences that were found to be essential for the conjugal transfer of other plasmids. Mobilization of deletion derivatives of the 7.2 kb and the 5.1 kb penicillinase-producing plasmids of N. gonorrhoeae by pBG791 indicated another origin of transfer, located within one of the two replication regions in pJD4.

The objective of this project was to construct a shuttle vector for the transfer of gonococcal genes between Escherichia coli and Neisseria gonorrhoeae using an in vitro-derived deletion derivative of the penicillinase-producing plasmid of N. gonorrhoeae. The ability of N. gonorrhoeae strain F62 to selectively acquire a 1.5 kb TagI fragment derived from the 4.2 kb cryptic plasmid was confirmed by uptake experiments. Neither the 1.5 kb fragment nor the oligonucleotide had any effect on the ability of chromosomal DNA to transform a strain that did not recognize the 1.5 kb fragment. However, the efficiency of transformation of the strain that did acquire the fragment was lowered by the presence of the two competing DNAs. Conjugation, using pUB307, an IncP conjugative plasmid, was the most efficient method for introducing plasmid DNA into recipient gonococci. Deletion derivatives of pJD8 (smallest deletion derivative of pJD4) were constructed in order to reduce the size of pJD8 to the smallest mobilizable plasmid. Plasmid pGRY4 was chosen as a basis for the construction of a shuttle vector. The lacZ complementation system of E. coli and the xy1E system from Pseudomonas putida were investigated as possible positive selection markers for the shuttle vector. Several problems were encountered when the proC gene of N. gonorrhoeae was cloned into pGCY1. Future transformation attempts should be performed in a restriction$\sp-$ and recA$\sp-$ strain. (Abstract shortened by UMI.)

We have designed an electrophoresis system that can purify plasmid DNA from a culture without centrifugation. This system is based on electrofiltration where bacterial cell lysates are loaded in one chamber and the purified plasmid DNA is recovered in an adjacent chamber. These two chambers are separated by a membrane made of regenerated cellulose, which allows plasmid DNA to migrate to the recovery chamber while retaining most contaminants in the loading chamber. Unfortunately, even with the optimization of the parameters involved in the electrofiltration, the only DNA that can pass through the middle membrane still has some contaminants, which prevent sequencing of the plasmid. Our results have shown that a pure plasmid cannot cross a membrane with pores small enough to prevent the migration of most of the contaminants. Only a plasmid complexed with some contaminants can cross a small pore membrane. In parallel, we have compared six direct sequencing methods that do not require any plasmid purification prior to the sequencing reaction. We compared the reliability, quality of sequences, time required, and cost of these six methods. We found that the best method was that of Zhang et al. (1999). This method is fast, reliable, produces good quality sequences and is inexpensive. The performance of this method is due to the amount of ABI's ready reaction mix used, the pre-sequencing heating step to lyse the cell, the large volume of the PCR sequencing reaction and the addition of BSA.