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Heat Shock-Inducible Model to Study Tumor Environment In Drosophila

Description

The goal of this project was to design and create a genetic construct that would allow for tumor growth to be induced in the center of the wing imaginal disc of Drosophila larvae, the R85E08 domain, using a heat shock. The resulting transgene would be combined with other transgenes in…

The goal of this project was to design and create a genetic construct that would allow for tumor growth to be induced in the center of the wing imaginal disc of Drosophila larvae, the R85E08 domain, using a heat shock. The resulting transgene would be combined with other transgenes in a single fly that would allow for simultaneous expression of the oncogene and, in the surrounding cells, other genes of interest. This system would help establish Drosophila as a more versatile and reliable model organism for cancer research. Furthermore, pilot studies were performed, using elements of the final proposed system, to determine if tumor growth is possible in the center of the disc, which oncogene produces the best results, and if oncogene expression induced later in development causes tumor growth. Three different candidate genes were investigated: RasV12, PvrACT, and Avli.

ContributorsSt Peter, John Daniel (Author) / Harris, Rob (Thesis director) / Varsani, Arvind (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)

Created2021-05

Genetic Ablation Systems of the Drosophila Imaginal Wing Disc: Building and Testing the TASER System

Description

Drosophila melanogaster, commonly known as the fruit fly, is widely used in genetic research due to its ease of genetic manipulation and well characterized genome. One of its structures, the imaginal wing disc, has played a key role in uncovering the mechanisms underlying tissue regeneration. Damage is typically induced via…

Drosophila melanogaster, commonly known as the fruit fly, is widely used in genetic research due to its ease of genetic manipulation and well characterized genome. One of its structures, the imaginal wing disc, has played a key role in uncovering the mechanisms underlying tissue regeneration. Damage is typically induced via physical, irradiation, or genetic ablation to study these regenerative processes. Recently, genetic techniques have been developed to cause targeted damage in situ by triggering cell death through the expression of pro-apoptotic or necrotic genes. The GAL4/UAS system allows for precise, tissue-specific cell death by the induction of a heat shift, an exposure of a higher temperature for an extended period of time. However, this 1st system is unable to express genes of interest in the wing disc to determine how other factors influence the regenerative process. Therefore, another approach was created, known as DUAL (Duration and Location) control, to allow the induction of both cell death as well as gene expression in the surrounding cells. The DUAL control system is regulated with exposure to higher temperature for a shorter period, referred to as a heat shock. The limitation of this 2nd system is its inability to temporally separate the timing of cell death from gene expression. This lack of control over timing highlights the need for a newer ablation system. In this work, we chose to create a novel system, termed the Temporal Ablation with Separation of Expression Regulation (TASER) system. The TASER system uses both heat shift and heat shock to provide refined control over the timing of cell ablation and gene expression. The system was developed by crossing specific Drosophila stocks that include genetic components like GAL80ts and GAL4tp, allowing for differing temporal regulation. We confirmed that the system is functional at separating the gene expression from ablation when the heat shock precedes the heat shift. However, initiating the heat shift first led to not only ablation, but also premature gene expression due to prolonged high temperatures. Despite this drawback, the TASER system holds significant potential in providing a more precise temporal control in regeneration studies, making it a valuable tool for future research.

ContributorsRogler, Isabel (Author) / Harris, Rob (Thesis director) / Bean, Heather (Committee member) / Huijben, Silvie (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of Psychology (Contributor) / School of International Letters and Cultures (Contributor)

Created2024-12

Directed Differentiation of human Embryonic Stem Cells to form Colon Organoids: A Model of Development in an Obesogenic Environment

Description

Early life exposures play a significant role in shaping health and disease susceptibility. Maternal obesity influences the offspring’s long-term risk of colorectal cancer, yet the understanding of the impact on developmental programming of intestinal stem cells and later risk of colorectal cancer remains limited. This has highlighted the growing need…

Early life exposures play a significant role in shaping health and disease susceptibility. Maternal obesity influences the offspring’s long-term risk of colorectal cancer, yet the understanding of the impact on developmental programming of intestinal stem cells and later risk of colorectal cancer remains limited. This has highlighted the growing need for a model that studies the influence of the maternal environment on the developing colon. The goal is to optimize a platform that generates human colon organoids (hCOs) from embryonic stem cells (hESCs) reliably and reproducibly and use it as a model to study colonic developmental patterning in an obesogenic environment. The organoids are derived by multi-step directed differentiation trajectory of embryonic stem cells, definitive endoderm, mid/hind gut, to fetal colon. The protocol was optimized to ensure consistent production of colonic organoids in contrast to small intestinal or urothelial cells which follow similar differentiation paths. To model an obesogenic environment on the platform, both the intrinsic and extrinsic properties of the developing colon were leveraged: 1) pharmacologically activating the transcription factor Peroxisome Proliferator-Activated Receptors Delta (PPARd) to mimic the cell-autonomous response to a pro-obesity maternal high fat diet, and 2) exposing the developing colon organoids to pro-inflammatory cytokine IL-17a to mimic the pre-pathological inflammatory state. The hESC-to-HCO model demonstrates the utility of ESC-derived organoids in studying the impact of maternal environmental factors on colonic development, generating a platform to explore factors that affect human development and maturation.

ContributorsSankar, Swathi (Author) / Mana, Miyeko (Thesis advisor) / Bartelle, Benjamin (Committee member) / Andrews, Madeline (Committee member) / Harris, Rob (Committee member) / Arizona State University (Publisher)

Created2025

A Novel Split-Input Genetic System for Tissue Regeneration Research in Drosophila melanogaster

Description

The model organism Drosophila melanogaster, commonly known as the fruit fly, has been a cornerstone of genetics research for over a century. Due to its extensively characterized genome, highly conserved genes and mechanisms, and relatively short life cycle, Drosophila have become critical to the advancement of research in the field…

The model organism Drosophila melanogaster, commonly known as the fruit fly, has been a cornerstone of genetics research for over a century. Due to its extensively characterized genome, highly conserved genes and mechanisms, and relatively short life cycle, Drosophila have become critical to the advancement of research in the field of regenerative medicine. This study introduces a novel split input genetic system to address the drawbacks in two existing methods. By separating the activation of tissue ablation and gene manipulation into two distinct inputs, this third system offers spatial and temporal control over experimental conditions in the imaginal wing disc. The system combines components from binary activation systems such as the GAL4/UAS and LexA/LexAop pathways along with modifications to prevent cross-activation. Initial experiments identified challenges in its functionality. Issues with stop cassette stability, phenotypic markers, and environmental factors prevented a thorough characterization of the system. Despite these concerns, this system successfully induced apoptosis through the activation of pro-apoptotic gene reaper after a 40-hour heat shift at 30℃, as shown through caspase activity. However, phenotypic analysis of adult wings revealed inconsistencies with expected regeneration outcomes. Interestingly, PCR and gel electrophoresis suggested recombination events involving the phenotypic marker sternopleural. These findings highlight the need for further refinement, such as redesigning the system to incorporate a more stable balancer on the second chromosome and exploring alternative activation inputs such as optogenetics or drug-induced gene expression. This novel system provides a strong base for more precise studies of tissue regeneration in Drosophila, which can offer insights into conserved pathways relevant to human health.

ContributorsNott, Chloe (Author) / Harris, Rob (Thesis director) / Varsani, Arvind (Committee member) / Barrett, The Honors College (Contributor) / School of Ocean Futures (Contributor) / School of Art (Contributor) / School of Molecular Sciences (Contributor)

Created2025-05

The Molecular Impact of PTEN Heterozygosity on mTOR Signaling in ASD-Megalencephaly Brain Organoids

Description

The mammalian target of rapamycin (mTOR) signaling pathway plays a vital role in the development of the human cortex. Neurodevelopmental disorders associated with mTOR dysfunction are termed “mTORopathies” often resulting in clinical phenotypes, including cortical malformations, intellectual disability, and epilepsy. The PTEN gene is a negative regulator of mTOR signaling.…

The mammalian target of rapamycin (mTOR) signaling pathway plays a vital role in the development of the human cortex. Neurodevelopmental disorders associated with mTOR dysfunction are termed “mTORopathies” often resulting in clinical phenotypes, including cortical malformations, intellectual disability, and epilepsy. The PTEN gene is a negative regulator of mTOR signaling. Heterozygous mutations in PTEN have been found in a subset of patients with Autism Spectrum Disorder (ASD) and co-morbid megalencephaly. While PTEN is a negative regulator of mTOR, the discrete changes in mTOR complex activity and signaling dynamics, as a result of PTEN heterozygosity, are unclear. To investigate the contribution of PTEN changes in human brain development, we use forebrain organoids derived from induced pluripotent stem cells (iPSCs) collected from ASD patients with PTEN mutations, which are a robust model to temporally study and perturb human neurodevelopment. We used quantitative real time polymerase chain reaction (q-RT-PCR) and immunofluorescent (IF) analyses of organoids collected during peak neurogenesis to measure the temporal expression of mTOR signaling genes. We observed changes in the expression of downstream targets of the mTOR signaling complexes 1 and 2 (mTORC1/C2). Our findings reveal distinct changes in mTOR complex activation and gene expression changes as a consequence of PTEN heterozygosity. Identifying vulnerable cell types and defining the relevant signaling mechanisms regulating changes offer valuable insights into potential therapeutic targets and possible biological markers for ASD to allow for early diagnosis.

ContributorsChowdhury, Madhumanti (Author) / Andrews, Madeline (Thesis director) / Harris, Rob (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)

Created2025-05