OVERVIEW: Hyperactivation and amplification of the oncogenic transcriptional co-factors YAP and TAZ are common in breast cancer. However, it is unknown if breast cancer cells are dependent on YAP/TAZ for growth and survival. In addition, key transcriptional targets of YAP/TAZ that enable breast cancer growth have yet to be defined. To address these unresolved questions, we will define YAP/TAZ-dependencies across a large cohort of breast cancer cells and generate gene expression signatures for both YAP/TAZ-dependent and YAP/TAZ-independent lines. We aim to identify YAP/TAZ-target genes that are essential for the growth and survival of YAP/TAZ-dependent breast cancer cells. This approach may reveal genetic dependencies in breast cancer that can then be therapeutically exploited. METHODS: A comprehensive cohort of breast cancer cells (45 cell lines) was obtained from the American Type Culture Collection (ATCC). The majority of time allotted for this thesis was spent culturing and expanding the cell lines. Five complete breast cancer cell line libraries were successfully generated and annotated. These libraries will be a useful resource for the Boston University School of Medicine Cancer Research Community. Protein and RNA extracts were collected from all cell lines. RNA extraction was performed in all cell lines with the Qiagen RNase Kit as per the manufacturer’s instructions. Protein extracts were collected from the cell lines with RIPA lysis buffer. Protein lysates were then run on an acrylamide gel and the relative abundance of YAP and TAZ was quantified. RNA extracts were sent for microarray analysis to obtain gene expression profiles. Cell lines were also fixed and stained for YAP and TAZ at subconfluence (50%) and confluence (90%) and visualized through immunofluorescence to assess the relative subcellular localization of YAP and TAZ. RESULTS: Our results indicate that YAP/TAZ levels and activity are highly variable across breast cancer cell lines. Seven cell lines were found to overexpress only YAP, nineteen cell lines were found to overexpress only TAZ, and two cell lines (BT-474 and HCC 1599) were found to overexpress both YAP and TAZ. Two cell lines (MDA-MB-134-VI and DU4475) had negligible protein expression levels of YAP/TAZ. We were also able to identify a subset of cells as being resistant to Hippo pathway activation, as seen in MCF 10A, MCF 10F, and MCF-12A cells, which maintained nuclear YAP and TAZ even under confluent conditions, and with MDA-MB-231 cells, which maintained only nuclear YAP under confluence. Given the importance of YAP and TAZ in cellular proliferation and survival, these results suggest that these Hippo pathway inactive cell lines may be dependent on YAP and TAZ for survival, which will be assessed at a future time point. We plan to complete our analysis of the subcellular localization of YAP and TAZ for all 45 breast cancer cell lines. Microarray profiling and gene expression signature analysis of all 45 cell lines are also ongoing. DISCUSSION: We surmise that increased levels/activity of YAP/TAZ will predict increased dependency on these oncogenes for growth and survival. This prediction will be directly tested by assessing cell viability following YAP/TAZ knockdown experiments. We also hypothesize that YAP/TAZ-dependent cells will be dependent on the transcription of specific YAP/TAZ target genes for survival. Current work detailed in this thesis will form the foundation for future work focusing on therapy-relevant YAP/TAZ target genes that are critical to breast cancer pathogenesis and disease progression. Our long-term aim is to identify pharmacologically-tractable YAP/TAZ target genes with the ultimate goal of finding novel chemotherapeutics that will improve prognosis for breast cancer patients.

Analysis of sexual assault evidence in a forensic laboratory setting requires accurate analysis of deoxyribonucleic acid (DNA) utilizing forensic DNA typing. This process can be complicated since most sexual assault samples are comprised of DNA from a female contributor as well as one or more male contributors. Generally female epithelial cells (e-cells) are in excess, making it more difficult to determine the short tandem repeat (STR) profile of the male contributor from the significantly fewer sperm cells present in the mixture. This complexity requires that the two contributing sources of DNA be separated in order to obtain a probative single source profile. Separation of the two fractions is accomplished by differential extraction. The most common protocol for a differential extraction involves preferentially lysing epithelial cells while leaving the sperm cells intact, separating the released female DNA from the sperm cells and finally lysing the sperm cells to obtain the sperm fraction. Research has shown that a Trypsin-ZyGEM extraction method produces higher yields of DNA compared to the standard differential extraction method. A previous study examined whether substrate type had an effect on the efficiency of the Trypsin-ZyGEM extraction. It was found that the Trypsin-ZyGEM protocol worked well at extracting DNA from aqueous semen, dried semen in a microfuge tube and semen dried on white cotton. However, results were inconclusive on the method’s ability to extract DNA from semen dried on denim fabric due to inhibition of the quantitative polymerase chain reaction (qPCR) used to quantify the samples. In this study, a subset of the denim samples was amplified and processed through capillary electrophoresis to verify that the extraction protocol had successfully recovered DNA from this substrate type and that the DNA could be amplified to create a STR profile. Full profiles were obtained for all three of the denim samples that were amplified to a target mass of 1 nanogram (ng). One full profile and two partial profiles were obtained for the same samples at a target mass of 0.25ng. Allelic dropout for the partial profiles varied with one profile having 3 alleles dropout while the other had 11 dropout. Peak heights and peak height ratios were more variable compared to the other substrate types but were still within an acceptable range for probative profiles. The results are promising and suggest that the Trypsin-ZyGEM protocol could possibly replace the current differential extraction technique. Further research is required to understand if this extraction protocol would be efficient on these substrate types with mixture samples of semen and e-cells. This research also builds upon the exploration of the effectiveness of the Trypsin-ZyGEM extraction on dried and aqueous semen samples compared to a modified Qiagen differential extraction. Samples that had been previously quantified were amplified and separated by size using capillary electrophoresis. Peak heights and peak height ratios (PHR) from the STR profiles were examined to assess profile quality of samples extracted using the Trypsin-ZyGEM protocol and the Qiagen protocol. Peak heights and peak height ratios (PHR) were found to be consistent between the two methods. / 2017-11-03T00:00:00Z

Osteocytes are the most abundant cell type in bone. Although our understanding of the function of osteocytes has progressed over the last decade, these cells remain the least understood cell in bone. Osteocytes are considered to be orchestrators of bone remodeling, mineral homeostasis, and hematopoiesis. The sympathetic nervous system (SNS) regulates almost every system in the body including bone. It is known that SNS suppresses bone formation but the mechanism is still not fully elucidated. Beta 2 adrenergic receptor (β2AR) is a G-protein coupled receptor that, upon binding to norepinephrine, activates the stimulatory subunit of the heterotrimeric G protein (Gαs) and the enzyme adenylate cyclase. The function of β2AR in osteocytes has not been studied. Therefore, the goal of this study is to delineate the role of β2AR signaling in osteocytes. To investigate that, an osteocyte-like cell line (Ocy454) was used. Ocy454 cells were treated with three different agents: norepinephrine (NE), isoproterenol (ISO), and butoxamine. Knock down of β2AR was achieved by using short hairpin RNA. Consequently, treatment with norepinephrine (NE) was done as well as fluid flow shear stress (FFSS) experiment. Taken together, this study shows that osteocytes do express β2AR and that β1AR and β2AR are almost equally expressed in osteocytes. Secondly, we have demonstrated that upon treatment of Ocy454 with NE and isoproterenol, there is a significant upregulation of Rankl, whereas Sost is not significantly regulated. Lastly, preliminary data suggest that β2AR might be involved in osteocyte’s mechanosensation.

The American Diabetes Association has determined that the number of people with diabetes in America was 30.3 million in 2015, and it is becoming increasingly more prevalent. Diabetes is a condition characterized by chronic hyperglycemia due to either insufficient insulin or reduced insulin sensitivity. Diabetes comes with a host of complications; one major complication is osteopenia, which increases fracture risks in both type 1 and type 2 diabetics14. The changes in diabetic bone may be due to reductions in lysyl oxidase (LOX) levels leading to decreased amounts of insoluble type 1 collagen fibers, which are necessary for bone strength28. LOX catalyzes oxidative deamination of the hydroxylysine and lysine side chains of the collagen molecules to create reactive aldehyde groups9,14,17. These aldehyde groups rapidly react with the lysine or hydroxylysine on the helical region of neighboring molecules of collagen fibrils, creating crosslinks between molecules leading to a mature cross-linked collagen matrix9,15,18. Our lab has now linked the LOX reductions in diabetes to incretins such as glucose-dependent insulinotropic peptide (GIP), which directly increases LOX, and anti-incretins such as dopamine, which reduces LOX by inhibiting GIP. Incretins and anti-incretins are gastric hormones released by the intestine in response to nutrient consumption4. Generally, incretins are hormones that stimulate insulin secretion from the pancreas where as anti-incretins inhibit that insulinotropic effect13,23. GIP is an incretin that also has an anabolic effect on bone in addition to its insulinotropic effect23. In diabetes there is an impaired cellular response to GIP in the pancreas, but its effects on the bone as related to diabetes are unknown. Additionally, it was observed in our lab that bone-derived LOX levels were decreased in diabetes while the serum level of anti-incretin gut-derived dopamine was vastly increased. Dopamine is known to inhibit GIP in the pancreas, but its effect on the bone was unknown. Prior to this experiment our lab had already started exploring a potential mechanism for diabetic osteopenia, where diabetes leads to an increase in gut-derived anti-incretin (dopamine), which interferes with GIP-stimulated LOX in osteoblasts. The decreased levels of LOX lead to lowered collagen integrity and a loss in trabecular bone structure. The current work is a sub-study within the larger aim to elucidate the mechanism for diabetic osteopenia. The aim of this study was to further verify the effects of dopamine by using amisulpride, a dopamine receptor antagonist, to determine if amisulpride could restore bone health in streptozotocin (STZ) induced diabetic mice. For this study C57BL/6 wild type mice were used. The mice were divided at random into three groups (N=10 per group) as follows: control (no STZ, no amisulpride), STZ diabetic (STZ, no amisulpride), and STZ + amisulpride. Twenty of the mice had low dose intraperitoneal injections of STZ for five days to induce diabetes and ten mice received vehicle injections for the control group. The mice were maintained diabetic or normal for 8 weeks, after which ten of the STZ mice received intraperitoneal injections of amisulpride daily for a month. It was found that STZ induced diabetic mice had a significant decrease in LOX levels and insulin levels. Amisulpride was able to rescue the decreases in LOX levels but did not alter insulin levels. Furthermore, micro-CT analysis and picrosirius red histology of long bones indicated that there is a decrease in bone volume, impaired trabecular structure, and disorganized collagen matrix in the STZ diabetic group. These impairments could be rescued by amisulpride administration, giving further evidence to the new mechanism for diabetic osteopenia. The increase in anti-incretin gut-derived dopamine signaling could be the cause of diabetic osteopenia. Inhibition of the anti-incretin gut-derived dopamine may possibly provide a therapeutic target for diabetic osteopenia.

<p> The regulation and mechanism of outer membrane (OM) lysis during spore wall assembly is not well understood. Although the timing of OM lysis has been established in previous studies, this event has never been assayed for. To this end, a new method was developed to assay the lysis of the OM during sporulation in <i>Saccharomyces cerevisiae</i>, the split GFP assay. By expressing complementary fragments of GFP in the ascal cytoplasm and the lumen between the OM and the spore plasma membrane (SPM), the lysis of the OM could be detected by the reconstitution of a functional GFP protein in the ascal cytoplasm as a result of these two compartments merging. A screen of several mutant strains with sporulation defects with published TEM images showing whether or not the lyse the OM then verified the split GFP assay for OM lysis as an effective method for detecting this event. This assay was then used to further investigate the sporulation defects associated with mutations in <i>FKS</i> genes, which all have 1,3-&beta;-glucan synthase homology. The results of this assay showed that mutation in <i> FKS2</i> or <i>FKS3</i> leads to a reduction on observed OM lysis. This provides further evidence for the current model that proper completion of the &beta;-glucan layer needs to occur before the OM will lyse. In addition, it was shown via dissection that <i>FKS3</i> is not able to substitute for <i>FKS1</i>, as <i>FKS2</i> has been shown to do. Although <i>FKS3</i> is very similar to the other 1,3-&beta;-glucan synthases homologically, the data suggest it has a function distinct from that of <i>FKS1</i> and <i>FKS2</i>.</p><p>

<p> Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42&bull;GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment.</p><p>

M.Sc. (Computer Science) / Astudy of one- and two-dimensional cellular automata was made. Two research projects were undertaken; these are discussed in depth. One- and two-dimensional cellular automata are defined. These automata are discussed with respect to the various characteristics which they exhibit. Practical applications for one- and two-dimensional cellular automata are given, as well as examples of existing systems. These systems make use of the theory on which cellular automata is based to solve practical problems. An overview of work done in the field of one- and two-dimensional cellular automata and formal languages. is given. Equivalence of cellular automata and other formal languages is discussed. As a first research project, the possible equivalence of two-dimensional cellular automata and array automata, and two-dimensional cellular automata and table matrix L-systems. are investigated. Another research project suggests a methodology for the shrinking of two-dimensional cellular automata. A software system called S.O.S. was developed to simulate cellular automata. and support the research done in this field. In the last part of this thesis, an in depth discussion of the S.O.S. system is given.

<p>Many cell types are remarkably adept at tracking chemical gradients, but they use different mechanisms in order to properly migrate or grow up-gradient. Bacteria use a temporal sensing mechanism to determine if they are swimming up-gradient. In contrast, eukaryotes are thought to use spatial sensing mechanisms where they compare the chemical concentration on one side of the cell to the other. In the present study, we utilized budding yeast <italic>(Saccharomyces cerevisiae) </italic>mating as a model for gradient tracking. Yeast cells are thought to use a spatial gradient tracking mechanism to grow up the pheromone gradient created by their mating partners. However, yeast cells polarize their receptors towards the direction of growth thereby reducing the distance that they can use to compare pheromone concentrations. </p><p>Yeast cells grow towards their mating partners by establishing a polarity patch that concentrates the master regulatory GTPase Cdc42 and its associated polarity factors on the membrane. The Cdc42 polarity patch orients actin cables so vesicles trafficking along these cables fuse at the polarity patch. Therefore, the location of the polarity patch determines the direction of growth. During mating, the pheromone gradient is thought to bias the polarity patch to the up-gradient side of the cell, but especially in shallow gradients, sometimes yeast cells initially establish the polarity patch on the wrong side of the cell. Work from our lab has found that the polarity patch wanders along the cell cortex during pheromone gradient tracking, and suggests that wandering behavior could serve as a mechanism of reorientation for cells whose polarity patch is misaligned with the gradient. </p><p>In order for yeast cells to properly track the pheromone gradient, their polarity patch must spend more time on the up-gradient side of the cell. How does the pheromone gradient bias wandering of the polarity patch to achieve this? We suggest that by polarizing their receptors and G proteins, yeast cells create a sensitized zone of the plasma membrane that can locally influence wandering of the polarity patch. As the polarity patch wanders along the cell cortex, so too does this zone of polarized receptors. If the patch wanders to a side of the cell with higher pheromone concentration, then more active receptors near the polarity patch could slow its wandering and allow more growth to occur in that direction.</p> / Dissertation

Ischemic injury is a common and debilitating outcome of natural illness and as a complication of commonly performed medical procedures. Whereas naturally occurring ischemic insults are often the result of unpredictable events, such as in the case of stroke or heart attack, the risk of operative and perioperative ischemia is somewhat better characterized in the clinical setting. Given the prevalence and severity of outcomes in ischemic injury, there is significant interest in developing better pharmacological and procedural approaches to improve patient outcomes. One approach that has shown significant promise in the laboratory setting, particularly in the context of planned medical procedures, is the use of delayed anesthetic preconditioning. Delayed anesthetic preconditioning is a phenomenon whereby a prior exposure to clinical concentrations of commonly used inhaled anesthetics, including isoflurane, induces the production of endogenous protective proteins that are able to provide robust protection against subsequent, potentially toxic, ischemic insults. Although many aspects of delayed anesthetic preconditioning have been previously described, a complete understanding of preconditioning mechanism has yet to emerge. The studies described in this dissertation aim to further our understanding of molecular mechanisms involved in delayed anesthetic preconditioning. In the first project, I used DNA microarray to identify genes that were differentially expressed in adult rat liver, kidney and heart following a clinically relevant exposure to the inhaled anesthetic isoflurane. By selecting those genes that were differentially expressed in multiple tissues, I was able to identify a small group of interesting genes for further study. In my second study, I chose from our list two related genes, metallothioneins I + II, to analyze for a role in anesthetic-mediated protection. Using a combination of approaches, I was able to establish that metallotioneins I + II play an essential role in delayed anesthetic preconditioning. In the final study of this dissertation I explore a possible role for metallothioneins I + II as sensor molecules, involved in detecting cellular oxidative stress. Taken together, these three studies represent an important contribution to our understanding of the mechanisms of delayed anesthetic preconditioning and how they might contribute to protecting against ischemic stroke.

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 β chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function. Looking upstream the kinase cascade, the identity of the kinase (s) that brings about the phosphorylation of the tyrosine residues remains to be elucidated. It has been suggested that the non receptor tyrosine kinase Src family is involved in the capacitation associated phosphorylation cascade. Using an immunological approach we show that the only Src family member present in mouse sperm extract is Src. The capacitation associated tyrosine phosphorylation is greatly reduced in the presence of Src specific inhibitors (SU6656 and SKI606) in vivo. As a means of control for the activity of Src inhibitors in our system, parallel experiments assaying the activity of PKA both in vivo and in vitro were realized. Surprisingly, Src inhibitors down regulates the phosphorylation of serine/threonine residues that correlate on earlier events in the capacitation, as assayed by western blot with PKA substrates antibody. However, in vitro kinase activity of PKA showed no effect of Src inhibitors in the phosphorylation of the PKA specific substrate, kemptide.