Browsing by Subject "High-throughput screening"

Understanding the differences between normal and malignant tissue is required to find vulnerabilities in cancer that can be exploited. One of the hallmarks of cancer is its ability to sustain proliferative signaling, leading to unbridled cellular replication. This puts an increased pressure on the cell’s ability to maintain genome integrity and creates a potential vulnerability to be targeted by cancer therapies. Targeting how a cancer cell prevents or responds to DNA damage is one way to take advantage of this vulnerability.

My dissertation work aims to better understand this DNA damage response in cancer and tests two hypotheses: The first is whether inhibition of the transcriptional regulator BRD4 leads to an increase in transcription-replication conflicts, DNA damage, and cell death. The second is whether the tumor microenvironment alters the way cancer cells respond to DNA damage induced by radiation therapy in glioblastoma.

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintain genomic integrity and cell survival. My work shows that BRD4, a BET bromodomain protein and known transcriptional regulator, is important for preventing dysregulation of these systems leading to conflicts between the transcription and replication machinery in S-phase. We demonstrate that inhibition or degradation of BET bromodomain proteins leads to an accumulation of RNA:DNA hybrids, a known cause of transcription-replication conflicts, and causes increased DNA damage and cell death in cancer cells actively undergoing replication. Furthermore,

over-expression of full-length BRD4, which contains a P-TEFb interacting domain known to activate efficient transcription, is necessary and sufficient to rescuing this effect. These results give mechanistic insight into chemotherapeutics that target BRD4 currently in clinical trials.

In complementary work, we explored the effect that the extracellular environment of cancer plays in its response to DNA damage caused by radiation therapy. Standard methods of culturing cancer cells, which do not replicate the extracellular environment of a native tumor, have led to an incomplete understanding of response to therapies such as ionizing radiation in vivo. To understand the role that the tumor environment plays on the radiation response, we used both human and murine glioblastoma cells to show that organotypic brain slice culture was better able to recapitulate the expression profiles of in vivo tumors. Specifically, we saw that pathways involved in multicellular processes, cell morphogenesis, and the extracellular matrix were not only significantly upregulated in glioblastoma cells cultured on brain slices compared to in vitro culture but were also critically important to radiation survival.

Collectively, this dissertation provides novel understandings of how cancer cells prevent and respond to DNA damage as well as a framework for future work in cancer biology.

The brain supports numerous complex processes ranging from signal processing and motor control to learning and memory. These processes rely on signal transduction between interconnected networks of neurons that form neural circuits. Understanding how neural circuits function requires non-invasive, genetically specific technologies to both record and manipulate neural activity. Recording neural activity establishes a correlative relationship between the activity and cognitive function, while manipulating neural activity establishes a causal relationship between the activity and behavioral or physiological processes. Genetically encoded protein tools facilitate neuroscience research in both experimental paradigms. Genetically encoded sensors enable optical recording of neural activity across a wide spatiotemporal range. These indicators detect diverse forms of neural activity, including calcium ion flux, membrane voltage potential, and neurotransmitter concentration. Conversely, optogenetic actuators enable targeted, optical excitation or inhibition of neurons upon activation with a specific wavelength of light.

Advancement of genetically encoded tools will allow researchers to access new experimental regimes of neuroscience. Enhancing the fluorescence response and temporal fidelity of genetically encoded sensors improves signal detection fidelity, enabling neuroscientists to access more neurons at once and more precisely analyze neural circuits. Expanding the spectral diversity of genetically encoded tools makes it possible to record from multiple neural populations simultaneously or to optogenetically excite one population with a specific wavelength of light while recording the activity of another in a distinct optical channel. Such multi-channel experiments enable neuroscientists to investigate the influence of the activity of an ensemble of neurons on the activity of another ensemble downstream in a neural circuit or feedback between neural circuits. However, expanding the palette of protein sensors and actuators for such multi-channel experiments has been challenging. Most state-of-the-art genetically encoded sensors fuse cyan-light-sensitive green fluorescent protein to a sensing domain, so the dual channel experiments described above require a complementary sensor or actuator that is sensitive to a spectrally distinct wavelength of light. However, the performance of red fluorescent genetically encoded tools typically lags relative to their green counterparts, and using cyan-light-activated sensors in conjunction with green-light-activated actuators introduces high optical crosstalk. Additionally, the dynamic properties and context-dependent performance of genetically encoded sensors make high-throughput screens of this class of tools labor intensive and time consuming. This constraint on the throughput of screens has limited development efforts to a miniscule fraction of the possible variants of each sensor.

In this dissertation, I expanded the spectral diversity of the tools described above and developed a novel strategy for high throughput evolution of genetically encoded sensors. First, I developed a red fluorescent genetically encoded voltage sensor by engineering the fluorescence resonance energy transfer (FRET) efficiency between a voltage sensitive domain and a red fluorescent protein. This red fluorescent sensor enabled high fidelity recordings of neural activity with sub-millisecond temporal resolution, dual-channel recordings in parallel with green fluorescent sensors, and simultaneous optogenetic excitation and voltage imaging with minimal optical crosstalk. Second, I developed an optogenetic actuator with a blue-shifted activation spectrum by employing this same FRET mechanism. I demonstrated that the activation spectrum of optogenetic tools could be tuned by engineering FRET efficiency between the actuator domain and a compatible fluorescent protein. This straightforward strategy represents a technical step forward for engineering the spectra of optogenetic actuators, which has been difficult to achieve without compromising functionality. Third, I developed a screening method that enabled pooled, high-throughput screens of diverse libraries containing genetically encoded sensor mutants. This method employed both experimental and computational advancements. I used in situ optical mRNA sequencing to determine the sequence of each screened protein variant and machine learning to predict the function of unscreened variants. I expanded the coverage of the possible sequence space by over an order of magnitude compared to traditional directed evolution.