Maintenance of the integrity of the genome is vital to survival and the small non-histone protein Heterochromatin protein 1 (HP1) plays an important role in the process. Here we demonstrate two roles for HP1 in regulating transcription in the early Drosophila embryo. HP1, together with its telomeric binding partner, HOAP, play a role in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the master switch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, SxlPe, is essential to females as it provides SXL protein to initiate the autoregulatory feedback loop, which ensures productive female-specific splicing of the Sxl transcripts that are transcribed from the maintenance promoter (SxlPm), which is active in both sexes. HOAP mutants show inappropriate SxlPe expression in males and incorrect splicing of SxlPm-derived transcripts, while females show premature activation of SxlPe. These results implicate HOAP in regulating the promoter. HP1 mutants, by contrast, display SxlPm splicing defects in both sexes, suggestive of inappropriate expression of SxlPe in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with SxlPe sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II (RNAPII) recruitment to SxlPe are severely compromised. Our genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles of HP1 at SxlPe. These findings prompted us to further investigate the role of chromatin modifying proteins in regulating the sex determination decision. Consistent with important role for HP1 in regulating transcription, we also find that HP1 collaborates with Polycomb/trithorax Group (PcG/trxG) proteins to regulate gene expression in the early embryo. PcG/trxG proteins were first identified in Drosophila as repressors (PcG) and activators (trxG) of the homeotic genes, serving to pattern the body axis. Mutations in PcG/trxG genes can antagonize or synergize with those in the HP1 chromatin modifying system, suggesting the two systems work together to set the chromatin state and influence transcription. These proteins regulate the chromatin by placing either repressive H3K27me3 (PcG) or activating H3K4me3 (trxG) marks. In early embryogenesis, we find that promoters have both H3K27me3/H3K4me3 marks suggesting that they are bivalent. Both marks appear to be dependent on HP1 in the early embryo, which is not the case in later larval stages. We speculate that as the early embryo organizes its genome into heterochromatin and euchromatin, a transition between the HP1/H3K9me silencing system to the PcG/H3K27me3 system takes place. This transition is sensitive to changes in HP1. This work reveals a novel complex process in which two distinct chromatin systems collaborate to regulate gene expression. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2014. / April 10, 2014. / Drosophila, Heterochromatin Protein 1, Polycomb, Sex Determination / Includes bibliographical references. / Jamila Horabin, Professor Directing Dissertation; Wumin Deng, University Representative; Yoichi Kato, Committee Member; Yanchang Wang, Committee Member; Akash Gunjan, Committee Member.

The 14-3-3 family of proteins is implicated in the regulation of a broad range of key neuronal processes. Previous human and animal studies have provided evidence for an association between 14-3-3 proteins and schizophrenia. Here, I report the characterization of the 14-3-3 functional knockout (FKO) mice that perinatally express an isoform-independent 14-3-3 inhibitor peptide in the brain. Fluorescent microscopy was first used to determine the specific transgene expression pattern in the brain for each 14-3-3 FKO founder line. Results from initial behavioral screenings of the 14-3-3 FKO founder lines identified a variety of behavioral changes in different lines and these alterations are also correlated with specific transgene expression patterns. Further behavior characterization revealed that certain 14-3-3 FKO mice exhibit a variety of behavioral changes that correspond to the core symptoms of schizophrenia. I show that these behavioral deficits may be attributed to alterations in multiple neurotransmission systems in the 14-3-3 FKO mice. In addition, administration of dopamine receptor antagonist drugs, such as clozapine or haloperidol, are sufficient to attenuate certain schizophrenia-associated behavioral deficits of the 14-3-3 FKO mice. Disruption of 14-3-3 proteins has previously been linked to improper neuronal development. Consistently, we identify global and region-specific reductions in brain volume and corresponding wet weights. Particularly, inhibition of 14-3-3 proteins results in a reduction of dendritic complexity and spine density in forebrain excitatory neurons, which may underlie an altered synaptic activity in the 14-3-3 FKO mice. Together, my data provide a link between 14-3-3 dysfunction, synaptic alterations, and schizophrenia-associated behavioral deficits. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2014. / July 10, 2014. / 14-3-3 Proteins, Antipsychotic Drugs, Dendritic Spines, Dopamine, Mouse Model, Schizophrenia / Includes bibliographical references. / Yi Zhou, Professor Directing Dissertation; Debra Ann Fadool, University Representative; Cathy Levenson, Committee Member; Charles Ouimet, Committee Member.

Genome analysis in several eukaryotes shows a surprising number of transcripts that do not encode conventional messenger RNAs. Once considered noise, these non-coding RNAs (ncRNAs) appear capable of controlling gene expression by various means. We find Drosophila sex determination, specifically the master-switch gene Sex-lethal (Sxl), is regulated by long ncRNAs (>200 nt). The lncRNAs influence the dose sensitive establishment promoter of Sxl, Sxl Pe, which must be activated to specify female sex. They are primarily from two regions, R1 and R2, upstream of Sxl Pe and show a dynamic developmental profile. Of the four lncRNA strands only one, R2 antisense, has its peak coincident with Sxl Pe transcription, suggesting it may promote activation. Indeed, its expression is regulated by the X chromosome counting genes, whose dose determines whether Sxl Pe is transcribed. Transgenic lines which ectopically express each of the lncRNAs show they can act in trans, impacting the process of sex determination but also altering the levels of the other lncRNAs. Generally, expression of R1 is negative whereas R2 is positive to females. This ectopic expression also results in a change in the local chromatin marks, affecting the timing and strength of SxlPe transcription. The chromatin marks are those deposited by the Polycomb and Trithorax groups of chromatin modifying proteins, which we find bind to the lncRNAs. We suggest the increasing numbers of non-coding transcripts being identified are a harbinger of interacting networks similar to the one we describe. The control of the activity of RNA polymerase II (RPII) is vital to proper gene expression. At many gene promoters, particularly regulatory genes, RPII remains associated with nascent RNA without productively elongating, known as pausing. Developmental genes, such as Sxl, utilize this pausing to achieve rapid activation of expression. Recently, proteins involved in the RNA silencing pathway have been implicated in regulation of this pausing. Drosophila elongator protein 1 (D-elp1) , part of the RPII core elongator complex, has been found to interact with RNA silencing proteins. The involvement of D-elp1 with RNA silencing proteins and their role in pausing and gene regulation remains unclear. We find a non-conanical role for D-elp1 in gene repression, opposite to its role as an elongator, through potential interactions with pausing factors, RNA silencing proteins, and chromatin modifiers. Various mechanisms of gene expression control are required to protect the integrity of the genome and thus the survival of an organism. Elucidation of these mechanisms and understanding cellular processes is important to aid our ability to improve human health. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2014. / April 8, 2014. / Drosophila, lncRNAs, Sex Determinatio, Sex-Lethal, Transcription / Includes bibliographical references. / Jamila Horabin, Professor Directing Dissertation; Wu-min Deng, University Representative; Yoichi Kato, Committee Member; Timothy Megraw, Committee Member; Yanchang Wang, Committee Member.

Alzheimer's disease (AD) currently underlies dementia for tens of millions of people worldwide, and its occurrence is set to double in the next 20 years. The currently approved drugs for treating AD only marginally ameliorate cognitive deficits, and provide limited symptomatic relief, while newer substances under therapeutic development are potentially years away from benefiting patients. Melatonin (MEL) is a potent antioxidant, can prevent toxic aggregation of Alzheimer's beta-amyloid (Aβ) peptide and, when taken long term, can protect against cognitive deficits in APP transgenic mice. To study the effects of melatonin on brain mitochondrial function in an AD model, APP/PS1 transgenic mice were treated for one-month with melatonin. Analysis of isolated brain mitochondria from mice indicated that melatonin treatment decreased mitochondrial Aβ levels by two to fourfold in different brain regions. This was accompanied by a near complete restoration of mitochondrial respiratory rates, membrane potential, and ATP levels in isolated mitochondria from the hippocampus, cortex, or striatum. When isolated mitochondria from untreated young mice were given melatonin, a slight increase in respiratory rate was observed. No such effect was observed in mitochondria from aged mice. In APP-expressing neuroblastoma cells in culture, mitochondrial function was restored by melatonin or by the structurally related compounds indole-3-propionic acid or AFMK. This restoration was partially blocked by melatonin receptor antagonists indicating melatonin receptor signaling is required for the full effect. Therefore, melatonin receptor signaling may be beneficial for restoring mitochondrial function in AD, and preservation of mitochondrial function may an important mechanism by which long-term melatonin treatment delays cognitive dysfunction in AD mice. However, while melatonin is presumed to provide neuroprotection via activation of the two membrane-bound, G-protein-coupled melatonin receptors (GPCR; MTNRs), some data indicate that MEL acts intracellularly to protect mitochondria and neurons by scavenging reactive oxygen species. Therefore, I sought to determine whether the genetic deletion of the MT1 and MT2 receptors (encoded by the MTNR1a and MTNR1b genes respectively) obviates MEL's neuroprotective actions in the AβPPswe/PSEN1dE9 mouse model of AD (2xAD). Beginning at 4 months of age, both AD and control mice either with or without both MTNR receptors were administered either MEL or vehicle in their drinking water for 12 months. Behavioral and cognitive assessments of 15-month-old AD mice revealed receptor-dependent effects of MEL on spatial learning and memory (Barnes Maze, Morris Water Maze), but receptor-independent neuroprotective actions of MEL on non-spatial cognitive performance (Novel Object Recognition Test). Similarly, hippocampal and frontal cortex amyloid plaque load and serum Aβ1-42 levels were significantly reduced by MEL in a receptor-independent manner, while MEL reduced cortical antioxidant gene expression in a receptor-dependent manner. These findings demonstrate that long-term MEL significantly reduces AD neuropathology and associated cognitive deficits in a manner primarily independent of the two GPCR melatonin cell surface receptors. Furthermore, melatonin receptor activation combined with non-receptor dependent mechanisms provides the clearest benefit, both cognitively and molecularly. Future identification of direct molecular targets for MEL action in the brain should open new vistas for the development of better AD therapeutics. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2014. / July 1, 2014. / Alzheimer's disease, Amyloid-Beta, Learning and Memory, Melatonin, Melatonin Receptors, Transgenic Mice / Includes bibliographical references. / James Olcese, Professor Directing Dissertation; Colleen Kelley, University Representative; Charles Ouimet, Committee Member; Akash Gunjan, Committee Member; Richard Hyson, Committee Member.

Aim: The aim of the study was to design an upper body phantom to mimic the movement of the lesion inside the lungs during a breathing cycle. Phantom design included an assessment of the motion observed for lung lesions, identification of suitable phantom materials as well as design of a motorized arm to mimic the movements observed inside the lung area of the phantom. Introduction: Expansion margins are added to clinical target volumes contoured by Oncologists in order to safeguard against under- or over-treatment of the target volume. They are designed to account for errors during setup, inaccuracies on the linear accelerator, and movement of targets inside the patient. If the margins are too small, there is a risk that the lesion/target may not receive the necessary dose, due to being partially missed. On the other hand, if the margins are too wide, the lesion will be covered, but normal tissue may receive unnecessary dose, resulting in additional side effects to the patient. Assessment of the impact of these margins is not possible in a static phantom and the availability of a low-cost motorized phantom would assist in the validation of these margins. Method: Previously treated patients' 4D CT scanning data were used to quantify the amount of movement seen for lesions within the lung. A phantom was then designed and built in an attempt to mimic both patient anatomy and movement. Materials were identified to replicate anatomical shape and densities of various organs in the thorax, as seen on CT scan data. Two treatment planning systems (Monaco, (Elekta) and Eclipse (Varian)) were used to determine the dosimetric characteristics of the materials. This was compared to actual dose as delivered by a linear accelerator (Elekta Synergy). Results: Paths were calculated from the breathing cycles during the 4D-CT scan sets and templates designed to mimic these movements. A thorax phantom was built with the appropriate materials suitable and matched densities to replicate a human thorax. Comparing transmission for these materials on a linear accelerator for 6MV and 10MV energy, the deviation from planned versus measured dose varied between 1.67% to 3.32% and 0.45% to 2.30%, respectively for the silicon material and between 0.77% to 3.22% and 0.17% to 2.57% for the 3D printed bone for 6MV and 10MV. iv Conclusion: The measurements done on the linear accelerator matched closely with the calculated values on the treatment planning system for transmission through the materials in the customised phantom. Various proposals were put forward to mimic the movement of the targets within the lung regions. However, it was not possible to manufacture a mechanically based working model due to the small movements observed (<5mm). It is recommended that a robotic solution be investigated as alternative to mimic these small movements.

Background: Plasmodium falciparum malaria is an infectious disease considered to have great impact on public health due to its associated high mortality rates especially in sub Saharan Africa. Falciparum drugresistant strains, notably, to chloroquine and sulfadoxine-pyrimethamine in Africa is traced mainly to Southeast Asia where artemisinin resistance rate is increasing. Although careful surveillance to monitor the emergence and spread of artemisinin-resistant parasite strains in Africa is on-going, research into new drugs, particularly, for African populations, is critical since there is no replaceable drug for artemisinin combination therapies (ACTs) yet. Objective: The overall objective of this study is to identify potential protein targets through host–pathogen protein–protein functional interaction network analysis to understand the underlying mechanisms of drug failure and identify those essential targets that can play their role in predicting potential drug candidates specific to the African populations through a protein-based approach of both host and Plasmodium falciparum genomic analysis. Methods: We leveraged malaria-specific genome wide association study summary statistics data obtained from Gambia, Kenya and Malawi populations, Plasmodium falciparum selective pressure variants and functional datasets (protein sequences, interologs, host-pathogen intra-organism and host-pathogen inter-organism protein-protein interactions (PPIs)) from various sources (STRING, Reactome, HPID, Uniprot, IntAct and literature) to construct overlapping functional network for both host and pathogen. Developed algorithms and a large-scale data-driven computational framework were used in this study to analyze the datasets and the constructed networks to identify densely connected subnetworks or hubs essential for network stability and integrity. The host-pathogen network was analyzed to elucidate the influence of parasite candidate key proteins within the network and predict possible resistant pathways due to host-pathogen candidate key protein interactions. We performed biological and pathway enrichment analysis on critical proteins identified to elucidate their functions. In order to leverage disease-target-drug relationships to identify potential repurposable already approved drug candidates that could be used to treat malaria, pharmaceutical datasets from drug bank were explored using semantic similarity approach based of target–associated biological processes Results: About 600,000 significant SNPs (p-value< 0.05) from the summary statistics data were mapped to their associated genes, and we identified 79 human-associated malaria genes. The assembled parasite network comprised of 8 clusters containing 799 functional interactions between 155 reviewed proteins of which 5 clusters contained 43 key proteins (selective variants) and 2 clusters contained 2 candidate key proteins(key proteins characterized by high centrality measure), C6KTB7 and C6KTD2. The human network comprised of 32 clusters containing 4,133,136 interactions between 20,329 unique reviewed proteins of which 7 clusters contained 760 key proteins and 2 clusters contained 6 significant human malaria-associated candidate key proteins or genes P22301 (IL10), P05362 (ICAM1), P01375 (TNF), P30480 (HLA-B), P16284 (PECAM1), O00206 (TLR4). The generated host-pathogen network comprised of 31,512 functional interactions between 8,023 host and pathogen proteins. We also explored the association of pfk13 gene within the host-pathogen. We observed that pfk13 cluster with host kelch–like proteins and other regulatory genes but no direct association with our identified host candidate key malaria targets. We implemented semantic similarity based approach complemented by Kappa and Jaccard statistical measure to identify 115 malaria–similar diseases and 26 potential repurposable drug hits that can be 3 appropriated experimentally for malaria treatment. Conclusion: In this study, we reviewed existing antimalarial drugs and resistance–associated variants contributing to the diminished sensitivity of antimalarials, especially chloroquine, sulfadoxine-pyrimethamine and artemisinin combination therapy within the African population. We also described various computational techniques implemented in predicting drug targets and leads in drug research. In our data analysis, we showed that possible mechanisms of resistance to artemisinin in Africa may arise from the combinatorial effects of many resistant genes to chloroquine and sulfadoxine–pyrimethamine. We investigated the role of pfk13 within the host–pathogen network. We predicted key targets that have been proposed to be essential for malaria drug and vaccine development through structural and functional analysis of host and pathogen function networks. Based on our analysis, we propose these targets as essential co-targets for combinatorial malaria drug discovery.

Purpose of the Study: The primary aim of this research is to demonstrate the safety and efficacy, or lack thereof, of percutaneous cholecystostomy placement as a management option in patients with acute cholecystitis (AC), not suitable for cholecystectomy and not responding to best medical management. The secondary aim of this research is to investigate the feasibility and complexities of interval cholecystectomy in this cohort of patients, with respect to the conversion rate to open, operating time and performing a subtotal cholecystectomy. Background: Acute cholecystitis is a complication of cholelithiasis (gallstones) and one of the most common admission diagnoses in Acute Care Surgery Units. The standard of care, according to the Tokyo Guidelines (1-4), for the management of acute cholecystitis, includes the immediate use of empiric antimicrobial drugs and index-admission laparoscopic cholecystectomy. A (>72 hour) delay between the onset of symptoms and presentation and initiation of medical care, as well as high operative risk patients are the two main reasons for diversion from this protocol of care. In the case of delay, the guidelines suggest the use of interval (six week) cholecystectomy as appropriate care. Index admission cholecystectomy in the setting of delayed presentation has been associated with increased morbidity. As inflammation of the gallbladder progresses, the tissues become more oedematous, with anatomic distortion and therefore increased difficulty in identifying important structural landmarks during LC. This difficulty increases the risk of operative complications, including bleeding and common bile duct injury, the most feared complication of LC. In addition to this distortion, adjacent surrounding organs may be involved in this inflammatory complex, thereby also being placed at risk of injury during dissection. In such circumstances, alternative methods of controlling disease progression may be necessary. 7 According to the Tokyo guidelines (1-4), AC can be classified into three grades of severity, namely mild (grade I), moderate (grade II) and severe (grade III). The grading system takes into account clinical and laboratory parameters, with organ dysfunction representing more advanced disease. Percutaneous cholecystostomy tube placement has been described as a method to achieve sepsis control in patients with severe AC, in which case LC may not be safe, owing to operative and high anaesthetic risk. The use of percutaneous cholecystostomy is well established in critically ill patients with acalculous cholecystitis and its safety and efficacy have been reported in many studies (5-11). Early LC has recently been shown to reduce the rate of major complications as compared to PC, even in high risk patients (15) The management of one subset of patients with acute cholecystitis remains unclear. This group comprises those with delayed presentation, in whom index-admission surgery is not advised, but who subsequently do not respond to best medical therapy. They have traditionally undergone urgent cholecystectomy but suffer higher rates of both morbidity and mortality (12- 14). In the current setting, patients often present with a delay since the onset of symptoms, rendering index-admission cholecystectomy unsafe. This problem is exacerbated by the lack of urgent operating theatre time, often with more urgent cases taking preference, thus delaying operative care beyond what is deemed safe by the Tokyo guidelines. The vast majority of patients are managed by interval cholecystectomy, leaving only the mentioned unresponsive subset. Recent reports have established the safety of the use of percutaneous cholecystostomy tube placement in patient groups that include this subset (severe sepsis, septic shock, local gallbladder rupture, progressive intolerant pain and persistent fever) (5-11).

Pancreatic cancer is a lethal disease with very poor prognosis as it is one of the leading causes of cancer related deaths worldwide. Pancreatic cancer may manifest in different ways and there are a number of different genetic mutations that can lead to carcinomas of the pancreas. This study reviewed some of the genetic alterations seen in pancreatic cancer and how they appear in the context of disease progression. While progress has been made in identifying genetic mutations that may contribute to pancreatic cancer, more work has to be done to solidify biomarkers and potentially contribute to early detection of the disease. Pancreatic cancer is often asymptomatic until late stages of disease, which is why it is often diagnosed at such a progressed state. Late detection contributes to its poor prognosis as it unlikely to have curative potential at such a late stage. Approach to treatment generally depends on the stage at diagnosis. This study reviewed a number of different treatment options including surgical resection, chemotherapy, and targeted therapy. Surgical resection is currently considered the only cure for pancreatic cancer. The other treatment options may be helpful in reducing recurrence of cancer and/or increasing survival. Targeted therapy is a very recent approach that is currently used as a treatment to manage pancreatic cancer with fairly positive outcomes. Hopefully, with further exploration into this individualized approach and modification of current targeted agents we are able to discover a cure for this devastating disease.

The research project aims at locating regions of interest (ROIs) on human subjects prior to X-ray imaging on the Lodox Statscan whole body imaging system.

The purpose of this work was to partition the 6 MV photon beam of a Philips SL75-5 linear accelerator into primary and scattered dose components in water. The two quantities that are necessary to define the primary beam component are a reference dose DR and a primary linear attenuation coefficient &Icirc;¼ 0. DR describes the magnitude of the primary dose as a fraction of the total dose in a reference field at a reference depth, while &Icirc;¼ 0 describes how the primary dose changes with depth in a medium. The scattered component is the difference between the primary and total dose. &Icirc;¼ 0 for the beam in water was determined in four different ways, namely through the extrapolation of measured TMRs to zero field size, through linear attenuation measurements, through the fit of a convolution model to CAPDD data and through a method involving a central axis attenuator. The primary dose component was determined in two ways, namely by the extrapolation of the phantom scatter correction factor to zero field size and also by the central axis attenuator method. &Icirc;¼ 0 varied from 0.0445 cm-1 to 0.0469 cm-1 with an average of 0.0455 +- 0.0012 cm-1. DR for a 10 cm x 10 cm field at the depth of maximum dose was found to vary between 0.933 Gy/ 100 MU and 0.935 Gy/ 100 MU, with an average of 0.934 Gy/ 100 MU. These values agree very well with values published in the literature. It has thus been shown that the 6 MV photon beam is separable into primary and scattered dose components.