Katarina Yacuk “The role of dilp8 in Drosophila oogenesis and ovulation” (PI: Sun)

Major: Physiology and Neurobiology

Class: 2024

Abstract: Drosophila insulin-like peptide 8 (dilp8) has been extensively studied in Drosophila larval development, where it is known to ensure symmetrical growth of the insect. In adults, the peptide is highly expressed in stage-14 follicle cells of the ovarioles. These cells rupture to release a mature oocyte into the oviduct, and recent work suggests that dilp8 plays an important role in regulating ovulation. I will conduct several ovulation assays to determine dilp8’s role in the follicle rupture process and ovulation. Because dilp8 modulates ecdysone levels in the developing larva, and ecdysone levels control the stage-8 checkpoint of the ovarioles, I suspect that dilp8 produced from the mature follicles could be regulating oogenesis. Therefore, I will further investigate the role of dilp8 in this context using genetically manipulated flies, and then I will dissect them to analyze the distribution of eggs within the ovarioles. Finally, dilp8 has a role in metabolism and in inducing cancer anorexia-cachexia syndrome. I will conduct feeding assays to learn how follicle-derived dilp8 regulates proper allocation of nutrients to the ovaries and how it pauses oogenesis when nutrients are not available to produce eggs. The diversity of roles that dilp8 plays in adult Drosophila mirrors the many roles that other drosophila insulin-like peptides play, and my project will uncover the importance of dilp8 in ovulation, oogenesis, and feeding patterns.

James He “Identifying the Cell Composition and Clonal Diversity of Supratentorial Ependymoma Using Single Cell RNA-Sequencing” (PI: LoTurco)

Major: Physiology and Neurobiology

Class: 2021

Abstract: Ependymoma is the third most common type of pediatric brain tumor with very limited treatment options. One subtype, supratentorial ependymoma (ST-EPN), is particularly aggressive and has been found to be driven by an oncogenic fusion mutation between the C11orf95 and RELA genes. Molecular characterization of this mutation has allowed the LoTurco Lab to develop a new mouse model of ST-EPN, which can be used to study the different mechanisms of tumorigenesis. Experimental results in the lab suggest that there are different cell types susceptible to transformation by the C11orf95-RELA fusion and the presence of distinct tumor subclones in the same sample. Like many tumors, ST-EPN appear to be highly heterogeneous, which may contribute to its malignancy and lack of effective therapeutic strategies. Recently developed single-cell RNA-sequencing (scRNA-Seq) technology is capable of unraveling both inter- and intratumoral heterogeneity by parsing out distinct cell populations based on gene expression patterns. My project involves a combined computational and experimental approach that makes use of scRNA-Seq and CRISPR technology to define the molecular signatures of ST-EPN cells and to better characterize the heterogeneous cell populations present in ST-EPN tumors. Ultimately, this will help identify the specific mechanisms by which ST-EPN tumors develop to inspire novel efficacious treatment regimens.

Madison Britting “Investigating Sexual Dimorphisms in Thalamic Anatomy and Thalamo-amygdaloid Projections” (PI: Ostroff)

Major: Physiology and Neurobiology

Class: 2021

Abstract: The thalamus and the amygdala are critical brain regions involved in the fear circuit, which contains a multitude of projections that take part in the response to fear and anxiety. However, there are conflicting findings in the literature regarding rat thalamic anatomy, specifically two nuclei within the posterior thalamus, the posterior intralaminar nucleus (PIN) and the medial division of the medial geniculate (MGm), which project to the lateral amygdala (LA). In order to determine the location and delineate the borders of these nuclei I will analyze the expression of specific cell type markers, such as parvalbumin (PV), calretinin (CR), and calbindin (CB) qualitatively and quantitatively. I will also use Fluro-Gold, a retrograde tracer, to map projection patterns from MGm and PIN to the LA to further characterize these two nuclei. Additionally, I will take a novel approach by investigating any sex and estrous differences across the cell types and projection patterns in thalamic sections from both male and female rats. The female rat estrous cycle will be tracked using vaginal cytology, and their brains will be collected during specific phases of the estrous cycle. These methods will result in the determination of thalamic anatomy and sexual dimorphisms in thalamoamygdala circuitry.

Venkata Patchigolla “Engineering New Assays to Reveal the Mechanisms of Centromere Function” (PI: Mellone)

Major: Physiology and Neurobiology

Class: 2022

Abstract: The centromere is a specialized part of the chromosome defined by the presence of nucleosomes containing the histone H3 variant centromere protein CENP-A. It provides the foundation for kinetochore assembly, which ultimately serves as a site for sister chromatid attachment/segregation during mitosis/meiosis to allow for faithful cell division. Proper centromere function and propagation through cell generations is vital as errors could lead to abnormal cell division along with chromosomal instability. Both of these would likely lead to the loss or disruption of vital genomic information, potentially resulting in inviability and disease.
In multicellular organisms, centromere-associated proteins and histone variants have been widely studied and characterized in their role for proper centromere function/propagation. However, the role of centromeric DNA in this process remains largely unknown.The goal of this project is to uncover the role of centromeric DNA by forming a centromere on an acentric chromosome and observing its propagation through cell generations.
I will be using the Drosophila melanogaster as the model for this project because of its fast-reproductive time, easy genetic manipulation and its well studied biology. I plan to use the restriction enzyme, I-CreI, to form an acentric X chromosomal fragment along with the LacI/lacO tethering system under various drivers to form a centromere on this acentric fragment and observe its behavior using fluorescence microscopy.
I anticipate to see a new self-propagating centromere on this X chromosomal fragment. This would mean that centromeric DNA might not be required for centromere function and identity.

Ekaterina Skaritanov “Investigating the ETS transcription factor Pnt and its inhibitor Yan for their role in Drosophila ovulation ” (PI: Sun)

Major: Physiology and Neurobiology

Class: 2020

Abstract: Our previous work has shown that akin to mammalian ovulation, Drosophila also exhibit a follicle rupture process: posterior somatic follicle cells of stage 14 preovulatory egg chambers in Drosophila break down to allow extrusion of the oocyte from the ovary into the oviduct. The importance of matrix metalloproteinase 2 (MMP2) activity for ovulation has been established in both Drosophila and mammals, however it still remains unknown what transcriptional mechanisms regulate its expression and enzymatic activity. Based on their importance in other developmental processes of Drosophila such as oogenesis, the E26 transformation-specific transcription factor, Pointed (Pnt), in conjunction with its inhibitor, Yan, have become interesting candidates for direct regulation of MMP2 expression due to their connection to the Ras/MAPK pathway. In this study we hope to investigate whether and how Pnt and Yan are involved in ovulation and the follicle rupture process.

Jeffrey Rasmussen “Investigating the Role of Adipose Tissue in the Prognosis of Amyotrophic Lateral Sclerosis ” (PI: Kanadia)

Major: Physiology and Neurobiology

Class: 2020

Abstract: My long term objective is to understand the molecular and physiological changes underlying the pathogenesis and progression of amyotrophic lateral sclerosis (ALS) caused by mutation in FUS. Specifically, mutations such as P525L and others that disrupt the nuclear localization FUS. These mutations are often inherited in an autosomal dominant fashion, which suggests that the mutant proteins might gain a toxic function. In this regard, FUSP525L protein has been shown to form cytoplasmic aggregates that are now thought to be toxic as they sequester many RNA-binding proteins and small nuclear RNAs (snRNAs) such as U11, which is a crucial component of the minor spliceosome. Thus, ALS pathology in this case could be caused by the indirect loss of minor spliceosome function, which is responsible for splicing less than 0.5% of the introns that are often found as isolated introns in genes that mostly consist of introns spliced by the canonical spliceosome. To directly test the hypothesis that the sequestration of U11 snRNA in the cytoplasmic FUSP525L-aggregatres results in the inhibition of the minor spliceosome and aberrant expression of minor intron-containing genes causes ALS, the Kanadia lab has produced a U11 conditional knockout mouse. Here we have systemically ablated U11 at postnatal day 15/16 to inhibit the minor spliceosome without any disruption in FUS function. This mouse model develops an ALS-like phenotype including, increased hindlimb clasping during tail suspension tests, kyphosis, decreased life-span, and an inability to gain weight after four weeks post-Tamoxifen injection. My objective for the proposed project is to test the significance of recent reports that have shown that ALS patients that are overweight at the time of diagnosis have a better prognosis in regards to the severity and longevity. Since the U11-cKO mouse does show failure to gain weight along with severe phenotype, my first aim is to determine whether the lack of weight gain in the mutant mice is due to aberrant feeding behavior or increased physical activity. I will employ the Noldus Ethovision mouse tracking system in conjunction with our customized liquid feeding system to monitor feeding behavior, amount of food consumed and physical activity. Upon determining the root cause of lack of weight gain, I will explore whether there is a general shift in basal metabolism. Finally, I will investigate whether increasing adipose tissue in the mutant mice by feeding them fat-rich diet can ameliorate the age of onset and severity of the ALS-like phenotype.

Ericka Randazzo “Targeting the Genetic and Molecular Mechanisms of Supratentorial Ependymoma Using Mice Models” (PI: LoTurco)

Major: Pursuing a dual degree in PNB and Pathobiology. Minor:Creativity, Innovation, and Entrepreneurship

Class: 2019

Abstract:

Sarah Ferrigno

“Serotonin receptor mechanisms mediating the anergia/fatigue induced by fluoxetine (Prozac): Focus on the 5-HT_1B receptor” (PI: Salamone)

Major: Psychology

Class: 2019

Abstract: Major depressive disorder (MDD) is a common psychiatric disorder, with roughly 7.6% of Americans over 12 suffering from MDD in a given 2-week period. Because of its prevalence and severity as a health concern, the need for effective treatments is extremely great. The most commonly prescribed antidepressants are serotonin selective reuptake inhibitors (SSRIs) such as fluoxetine (Prozac). While these drugs treat a variety of depressive symptoms, including mood, rumination, and anxiety, motivational symptoms, which impact several aspects of a patient’s life, they tend to be much more treatment-resistant. Not only has fluoxetine been found to be relatively ineffective in reversing motivational deficiencies in clinical studies and preclinical rodent models, but it has even been found to exacerbate motivational deficits in rats and humans. Because SSRIs elevate extracellular levels of serotonin (5-HT) by blocking 5-HT reuptake, it is likely that the motivational dysfunctions induced by fluoxetine are due to an overstimulation of one or more 5-HT receptors. 5-HT_1B receptors in the ventral tegmental area (VTA) play a role in the modulation of impulsive behaviors, possibly by acting on the DA neurons that originate in VTA and project to the nucleus accumbens. For this reason, there is particular interest in the motivational functions of 5-HT_1B receptors. Therefore, the aim of this study is to understand whether the 5-HT_1B receptor has a role in mediating this deficiency in motivational behavior through a number of behavioral, pharmacological and neurochemical methods in a rat model.

Eric Beltrami “Understanding the Function of Inhibitory Lateral Hypothalamic Neurons and their Contribution to Generating Complex Behavioral States” (PI: Jackson)

Major: Physiology and Neurobiology, Molecular and Cell Biology

Class: 2019

Abstract: The neuronal diversity of the lateral hypothalamic area (LHA) is poorly defined. The heterogeneity of the LHA may contribute to its role in several critical homeostatic functions, including feeding, arousal and stress. A large inhibitory neuron population in the LHA, defined by the expression of the vesicular transporter for GABA (VGAT), has been shown to be implicated in mediating some of the functions attributed to the LHA, such as vigorous feeding, but these inhibitory neurons and any putative subpopulations of those neurons have yet to be parsed out anatomically or functionally. My project seeks to elucidate the role of transcriptionally-distinct inhibitory LHA neurons in mediating complex behavioral states. Using Cre-lox recombinant mouse lines, along with optogenetic and chemogenetic techniques of neuronal activation, novel cell populations may be selectively targeted based on their expression of specific neuropeptides to determine their behavioral effects in vivo. Additionally, specific neuronal projections may be activated in vivo using optogenetics to better understand how the LHA coordinates with other brain regions and contributes to generating aspects of behavior. Paired with behavioral video-tracking software, the development of a manual scoring protocol will allow for quantification of the behavioral outputs of the inhibitory neuron population and its subpopulations. This project offers the first glimpse at the function of newly described cell populations in the LHA and their potential role in maladaptive behaviors and neuropsychiatric disorders.

Elizabeth Rodier “Validation of a cover blocker for epilepsy associated KNCQ2 channels.” (PI: Tzingounis)

Major: Physiology and Neurobiology; Minor: Spanish

Class: 2018

Abstract: KCNQ2 potassium channels are critical controllers of neonatal brain activity. A growing number of gain-of-function pathogenic KCNQ2 variants have been reported in patients with severe neonatal epilepsy. Currently there are no specific KCNQ2 channel inhibitors to block the activity of these channels. The goal of my project is to provide insight into the drug “HN38” as a potential treatment for the gain-of-function variants of KCNQ2 potassium channels. I will determine the selectivity of HN38 on KCNQ channel family members, as well as determine whether this drug can inhibit gain-of-function variants identified from pediatric epilepsy patients.

Jessica Young “The role of Jun kinase (JNK) signaling pathway in Drosophila ovulation." (PI: Sun)

Major: Physiology and Neurobiology

Class: 2019

Abstract: The Jun Kinase (JNK) signaling pathway has been thoroughly investigated within cell apoptosis and cancer metastasis; however, its role in ovulation has not been studied. I will investigate the role of the JNK signaling pathway using the novel Drosophila ovulation model, which utilizes a conserved cellular and molecular mechanism. Preliminary research performed implicates that the transcription factor Jun is involved in ovulation. Further investigation of other components in the JNK pathway will establish a Drosophila model for understanding the mechanism by which the JNK signaling pathway regulates ovulation and where it exists within the established ovulation signaling pathway.

Brock Chimileski “The neurochemical phenotype of lateral hypothalamic hypocretin/orexin and melanin-concentrating hormone neurons identified through single-cell gene expression profiling and manipulations to metabolic state” (PI: Jackson)

Major: Physiology and Neurobiology/ Molecular and Cell Biology

Class: 2017

Abstract: One essential, yet poorly understood, brain region is the lateral hypothalamic area (LHA). The LHA integrates diverse physiological signals to regulate hunger, metabolism, arousal, and motivation. It is comprised of a heterogeneous cell population, but two cell types are of particular significance; the hypocretin/orexin and melanin-concentrating hormone (MCH) neurons, which are named as such because they release either hypocretin/orexin or MCH as their primary neuropeptide. For both of these cell types, many questions still pertain to their precise neurochemical properties and subsequent roles in the neural circuitry that regulate the function of the LHA as a whole. This project attempts to better define the molecular characteristics of these neurons by addressing three questions: 1) What neuropeptides and fast neurotransmitters are co-expressed in these populations, 2) what is the diversity in gene expression within these populations, and 3) are these properties dependent on physiological state?

To investigate these questions, we optimized a single cell isolation method and performed gene expression profiling for 48 key genes in many single hypocretin/orexin and MCH cells. In addition, in-situ hybridization and immunohistochemistry were performed for key markers in coronal brain sections containing the LHA. Additional trials were conducted using mice that underwent 24 hours of food deprivation directly prior to experimentation. This analysis revealed that both of these cell populations have extensive co-expression with other neuropeptides in addition to their primary neuropeptide. Furthermore, they express markers for both excitatory and inhibitory neurotransmitter components, creating the possibility of a dual excitatory/inhibitory fast neurotransmitter phenotype. Experiments with manipulations to physiological state through food deprivation are on-going. Through this detailed characterization of the neurochemical phenotype of Hcrt/Ox and MCH neurons, we hope to further our understanding of their unique roles in the neural circuitry of the LHA modulating behavioral and physiological homeostasis.