BECN1 is a haploinsufficient tumor suppressor gene that is monoallelically deleted or epigenetically silenced in many human cancers. In breast cancer, 40% of tumors exhibit monoallelic deletion of Beclin 1. Additionally, low Beclin 1 mRNA expression is observed in aggressive breast cancer subtypes and reduced expression is an independent predictor of overall patient survival. The role of Beclin 1 in cancer has almost exclusively been attributed to its function in autophagy. However, our lab demonstrated an alternative role for Beclin 1 in the regulation of growth factor receptor signaling that could contribute to cancer. The goal of my thesis project was to investigate the molecular basis by which Beclin 1 regulates breast tumor growth and progression in vivo. Using in vivo models, I discovered that Beclin 1 promotes endosomal recruitment of hepatocyte growth factor tyrosine kinase substrate (HRS), which is necessary for sorting receptors to intraluminal vesicles for signal silencing and degradation. Beclin 1-dependent recruitment of HRS results in the autophagy-independent regulation of endocytic trafficking and degradation of the epidermal growth factor (EGFR) and transferrin (TFR1) receptors. When Beclin 1 expression is low, endosomal HRS recruitment is reduced and receptor function is sustained to drive tumor proliferation. An autophagy-independent role for Beclin 1 in regulating tumor metabolism was also observed. Collectively, my results demonstrate a novel role for Beclin 1 in impeding tumor growth by coordinating the regulation of growth promoting receptors. These data provide an explanation for how low levels of Beclin 1 facilitate tumor proliferation and contribute to poor cancer outcomes, independently of autophagy.

In this study, we investigated differential activation and the role of two mTOR complexes in cell migration of prostate cancer cells. Specific knock-down of endogenous RAPTOR and RICTOR by siRNA resulted in decreased cell migration in LNCaP, DU145, and PC3 cells indicating that both mTORC1 and mTORC2 are required for cell migration. EGF treatment induced the activation of both mTORC1 and mTORC2 as determined by complex-specific phosphorylation of mTOR protein. Specific knock-down or inhibition of Rac1 activity in PC3 cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the over-expression of constitutively active Rac1 (Rac1Q61L) resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Constitutively active Rac1 (Rac1Q61L) in PC3 cells was localized in the plasma membrane and was found to be in a protein complex which contained mTOR and RICTOR proteins, but not RAPTOR. In conclusion, we suggested that EGF-induced activation of Rac1 causes the phosphorylation/activation of mTORC2 via RICTOR, specific regulator of mTORC2 activation in numerous cancer cells. The major role played by mTOR in a wide array of cancers has in the recent decades led to the development of numerous mTOR inhibitors. One of the drawback of these first generation mTOR inhibitors are that m TORC1 activity is inhibited but effect on mTORC2 activity require high dosages and prolonged exposure in different cancer cell types including HeLa, PC3, LNCaP, and A549. High dosage of rapamycin and its associated rapalogs required for mTORC2 inhibition is clinically unsuitable. Studies have shown that the dual mTORC1/C2 inhibitors trigger feedback loops causing metastasis and affect the cell viability of normal tissues in vitro and in vivo. There is a need for specific mTORC1 and mTORC2 inhibitor, which overcome the disadvantages of the previously developed mTOR inhibitors. The Rac1-RICTOR axis suggested in this study could be used as a potential target for the development of mTORC2 inhibitor and lead to a potential therapeutic treatment for aggressive prostate cancer.

The largest U.S. cancer health disparity exists in prostate cancer, with African American men having the highest incidence and mortality rates. The present study evaluated the effects of ZIC2 and the underlying mechanisms in the E006 parental African-American cell line that produces tumors at accelerated growth rates because of the increase of ZIC2 genes in African-American males. We analyzed the experimental research that the overexpression of ZIC2 contributes to progression of prostate cancer. E006AA cells with overexpressed or suppressed ZIC2 were analyzed to determine phenotypic differences, PCR, cell proliferation and immunoblot assays. The expression levels of ZIC2 were analyzed by CRISPR-Cas9, Western blot and proliferation growth curves. We discovered using these experimental techniques to knockout ZIC2, reduced cell proliferation occurred. This research investigated the role of ZIC2 in prostate cancer progression and the effects of the loss or gain of function of ZIC2 by using CRISPR-Cas 9 genome editing technology.

This investigation was an analysis of the interaction of adolescent exposure to methylphenidate (MPH; trade name: Ritalin) on nicotine sensitization and conditioned place preference (CPP) in a rodent model and underlying mechanisms of this effect. Animals were treated IP with 1 mg/kg MPH or saline using a ―school day‖ regimen of five days on, two days off, from postnatal day (P) 28-50. During the final two weeks of MPH treatment, animals were either behaviorally sensitized to nicotine (0.5 mg/kg free base) or saline for 10 days, or conditioned to nicotine or saline using the CPP behavioral paradigm. In addition, three days after behavioral sensitization was complete, animals were analyzed for stress behavior using the forced swim stress behavioral test. In addition, 24 hours after post-test conditioning animals were analyzed for the effect of a clinically relevant dose of pre-exposed MPH (1mg/kg) and nicotine treatment on the expression of BDNF in the nucleus accumbens and dorsal hippocampus. Behavioral results revealed that adolescent pre-exposure to MPH blunted nicotine behavioral sensitization in both male and female rats during the first week of testing. However, MPH enhanced nicotine CPP in both adolescent male and female rats. Interesting, animals administered MPH demonstrated a significantly decreased latency to immobility in the forced swim stress behavioral test. In addition, pre-exposure to a 1 mg/kg dose of MPH appears to have sensitized the BDNF response to nicotine in females as compared to all other groups.

Protein identification using tandem mass spectrometry (MS/MS) has proven to be an effective way to identify proteins in a biological sample. An observed spectrum is constructed from the data produced by the tandem mass spectrometer. A protein can be identified if the observed spectrum aligns with the theoretical spectrum. However, data generated by the tandem mass spectrometer are affected by errors thus making protein identification challenging in the field of proteomics. Some of these errors include wrong calibration of the instrument, instrument distortion and noise. In this thesis, we present a pre-processing method, which focuses on the removal of noisy data with the hope of aiding in better identification of proteins. We employ the method of binning to reduce the number of noise peaks in the data without sacrificing the alignment of the observed spectrum with the theoretical spectrum. In some cases, the alignment of the two spectra improved.

This present study was intended to identify the reliability and validity of the Vert™ device when compared to a Switch mat. Vert is a wireless device intended to measure jump count and jump height through an application on a smartphone or tablet and the Switch mat provides jump height using wireless sensors. Jump height is an important factor in many sports such as volleyball and basketball and it is important to have devices that coaches and trainers can use for testing that they can rely on. If this device is found to be valid and reliable, coaches and trainers could potentially use it in more practical settings such as practice and games due to the portability and small size. This study consisted of 6 subjects who volunteered. The switch mat was connected to the device to display the jump height immediately after the jump. The Vert sensor was clipped onto the subject’s hip near the center of mass and the jump count and height were then displayed on an app. The subjects completed a series of warm-ups followed by 3 sets of 5 repetition countermovement jumps while using both devices to collect the data. The total 15 jump heights from these 3 sets of 5 were then analyzed using Pearson correlation analysis as well as a paired sample T-test. The jump height recorded from the Vert was consistently about 10 cm off from the jump height of the switch mat, which for a volleyball player, could be the difference between blocking and missing the ball. The results of this study showed that the Vert device is reliable but not practically valid. If technical improvements were made to the device to correct the height components the device could potentially be used in place of a force plate or switch mat when conducting athlete testing but the device is not currently valid for practical use.

Heart failure (HF) induced by aortic pressure over-load is associated with increased coronary artery stiffness. Perivascular adipose tissue (PVAT) and advanced glycation end products (AGE) both promote arterial stiffness. However, the mechanisms by which coronary PVAT promotes arterial stiffness and the efficacy of exercise to prevent coronary stiffness are unknown. The present study hypothesized both chronic continuous and interval exercise training would prevent coronary artery stiffness associated with inhibition of PVAT secreted AGE. Yucatan mininature swine were divided into four groups: control-sedentary (CON), aortic-banded sedentary heart failure (HF), aortic-banded HF continuous exercise trained (HF+CONT), and aortic-banded HF interval exercise trained (HF+IT). Coronary artery stiffness was assessed by ex vivo mechanical testing and coronary artery elastin, collagen and AGE-related proteins were assessed by immunohistochemistry. HF promoted coronary artery stiffness with reduced elastin content and greater AGE accumulation which was prevented by chronic continuous and interval exercise training. HF PVAT secreted higher AGE compared with CON and was prevented in the HF+CONT and HF+IT groups. Young healthy mouse aortas cultured in HF PVAT conditioned media had increased stiffness, lower elastin content and AGE accumulation compared with CON, which was prevented by PVAT from the HF+CONT and HF+IT groups. HF coronary PVAT secreted greater interleukin-6 (IL-6) and IL-8 compared to CON which was prevented by both continuous and interval exercise training regimens. We conclude chronic continuous and interval exercise is a potential therapeutic strategy to prevent coronary artery stiffness via inhibition of PVAT-derived AGE secretion in a pre-clinical mini-swine model of pressure overload-induced HF.

Protein–lipid binding interactions play crucial roles in various physiological and pathological processes, making it very important to study these interactions at the molecular level. However, investigation of these interactions is complicated by several issues, including the inherent complexity of membranes as well as the diverse mechanisms by which proteins interact with the membrane surfaces. As a result, many of these interactions remain poorly characterized. Synthetic probes are useful tools employed for studying protein–lipid binding interactions. This thesis will detail the design and synthesis of metabolically stabilized analogues of various signaling lipids, which mimic the natural species and are not easily modified by enzymes present in biological systems. A modular approach is employed for synthesizing these lipid probes, giving access to a wide range of derivatized lipid probes that can then be used for several studies. Although a wide variety of metabolically stabilized lipid analogues have been synthesized, their activity has not yet been characterized and quantified in detail. So, there is a great need to synthesize biologically active phosphorothioate and phosphonate analogues of various signaling lipids in order to properly characterize and compare the binding affinities and activity of these analogues. Synthesis of metabolically stabilized lipid analogue would take us one step closer towards understanding protein–lipid interactions in biological systems and in trying to find answers to the myriad of questions pertaining to these systems.

The alternative fuel biodiesel is produced from the transesterification of vegetable oils or animal fat to fatty acid methyl esters. Pomona has a reactor on campus that can be used to run this reaction and produce biodiesel. The use of biodiesel has been found to lower air pollutant and greenhouse gas emissions, but can be potentially harmful to the engines if it contains impurities. This paper proposes a method using high-performance liquid chromatography to test the quality of biodiesel. This method utilizes instrumentation and materials that are available in Pomona College's Chemistry Department, requires very little sample preparation, and is relatively safe, as long as general lab safety practices are followed. This method can also be used to optimize the procedure used to make the biodiesel. An optimized production procedure and a test method to assess the final product will ensure high quality fuel that can be used with confidence in diesel engines. This will likely add strength to proposals to increase the use of the on-campus reactor and produce biodiesel for campus grounds equipment from waste vegetable oil.

The purpose of this investigation was to examine whether a single 10 minute bout of exercise, performed at multiple intervals throughout the day to equal 30 minutes, can effectively elicit post-exercise hypotension (PEH). Secondly, it is important to explore whether a light (40% VO2R) or moderate (70% VO2R) intensity is required to elicit PEH within 10 minutes. Subjects (N=11) completed a VO2max test utilizing the Bruce Treadmill protocol. Each subject returned within 3 – 5 days to complete two separate exercising trials. A counter balanced system was employed so that each subject did not perform the same intensity rotation (Counter Balance 1 = 40% VO2R and 70% VO2R for session 1 and session 2, respectively; Counter Balance 2 = 70% VO2R and 40% VO2R for session 1 and session 2). The first session consisted of 3 sessions (morning, noon, evening) separated by an average of 3.5 hours at one of two intensities (40% VO2R or 70% VO2R). The second group of sessions were performed identical to the first, however, the intensity was altered depending upon counter balance. Baseline BP was measured prior to exercising. After each session, BP was measured at 2 intervals for the morning and noon sessions (immediately following and 20 minutes post-exercise); and at 3 intervals for the evening sessions (60 minutes post-exercise added) for both intensities. At 40% VO2R, BP decreased significantly at the morning (p = 0.007), noon (p = 0.018) and evening (p = 0.010) sessions at the 20 minute post-exercise interval. Although not significantly different, BP was observed to be lower at 60 minutes post-exercise interval. During the 70% VO2R session, BP was significantly lower at the morning 20 minute (p = .029) and evening 60 minute post-exercise measurements (p = .006) when compared to baseline. There was no significant difference noted between 40% and 70% VO2R intensities at eliciting a drop in BP at any interval at any time point. Although not statistically significant, 70% VO2R appeared to produce a further decrease at the 60 minute post-exercise measurement (102 mmHg) than did the 40% session (106 mmHg). The results of this study indicate that PEH may be elicited after a single 10 minute exercise session. Furthermore, multiple bouts of 10 minutes produce an accumulated decrease in BP that can be observed at the completion of the day.