Mechanisms of substrate selection by the signal recognition particle

The signal recognition particle (SRP) targets membrane and secretory proteins to their correct cellular destination with remarkably high fidelity. Previous studies have shown that multiple checkpoints exist within this targeting pathway that allows ‘correct cargo’ to be quickly and efficiently targeted and for ‘incorrect cargo’ to be promptly rejected. In this work, we delved further into understanding the mechanisms of how substrates are selected or discarded by the SRP. First, we discovered the role of the SRP fingerloop and how it activates the SRP and SRP receptor (SR) GTPases to target and unload cargo in response to signal sequence binding. Second, we learned how an ‘avoidance signal’ found in the bacterial autotransporter, EspP, allows this protein to escape the SRP pathway by causing the SRP and SR to form a ‘distorted’ complex that is inefficient in delivering the cargo to the membrane. Lastly, we determined how Trigger Factor, a co-translational chaperone, helps SRP discriminate against ‘incorrect cargo’ at three distinct stages: SRP binding to RNC; targeting of RNC to the membrane via SRP-FtsY assembly; and stronger antagonism of SRP targeting of ribosomes bearing nascent polypeptides that exceed a critical length. Overall, results delineate the rich underlying mechanisms by which SRP recognizes its substrates, which in turn activates the targeting pathway and provides a conceptual foundation to understand how timely and accurate selection of substrates is achieved by this protein targeting machinery.

Mechanisms of Transposable Element Repression by Piwi Proteins in the piRNA Pathway of Drosophila Germ Cells

The ability to reproduce is a defining characteristic of all living organisms. During reproduction, the integrity of genetic material transferred from one generation to the next is of utmost importance. Organisms have diverse strategies to ensure the fidelity of genomic information inherited between generations of individuals. In sexually reproducing animals, the piRNA pathway is an RNA-interference (RNAi) mechanism that protects the genomes of germ cells from the replication of ‘selfish’ genetic sequences called transposable elements (TE). When left unabated, the replication of TE sequences can cause gene disruption, double-stranded DNA breaks, and germ cell death that results in sterility of the organism. In Drosophila, the piRNA pathway is divided into a cytoplasmic and nuclear branch that involves the functions of three Piwi-clade Argonaute proteins—Piwi, Aubergine (Aub) and Argonaute-3 (Ago3)—which bind piwi-interacting RNA (piRNA) to form the effector complexes that represses deleterious TE sequences.

The work presented in this thesis examines the function and regulation of Piwi proteins in Drosophila germ cells. Chapter 1 presents an introduction to piRNA biogenesis and to the essential roles occupied by each Piwi protein in the repression of TE. We discuss the architecture and function of germ granules as the cellular compartments where much of the piRNA pathway operates. In Chapter 2, we present how Piwi in the nucleus co-transcriptionally targets genomic loci expressing TE sequences to direct the deposition of repressive chromatin marks. Chapter 3 examines the cytoplasmic function of the piRNA pathway, where we find that the protein Krimper coordinates Aub and Ago3 in the piRNA ping-pong pathway to adaptively target and destroy TE transcripts. Chapter 4 explores how interactions of Piwis with associated proteins are modulated by arginine methylation modifications. Lastly, in Chapter 5 I present evidence that the cytoplasmic branch of the piRNA pathway can potentially ‘cross-talk’ with the nuclear branch to transfer sequence information to better target and co-transcriptionally silence the genomic loci coding active TE sequences. Overall, the work presented in this thesis constitutes a part of the first steps in understanding the molecular mechanisms that protect germ cells from invasion by TE sequences.

Design of antenna-coupled lumped-element titanium nitride KIDs for long-wavelength multi-band continuum imaging

Many applications in cosmology and astrophysics at millimeter wavelengths including CMB polarization, studies of galaxy clusters using the Sunyaev-Zeldovich effect (SZE), and studies of star formation at high redshift and in our local universe and our galaxy, require large-format arrays of millimeter-wave detectors. Feedhorn and phased-array antenna architectures for receiving mm-wave light present numerous advantages for control of systematics, for simultaneous coverage of both polarizations and/or multiple spectral bands, and for preserving the coherent nature of the incoming light. This enables the application of many traditional "RF" structures such as hybrids, switches, and lumped-element or microstrip band-defining filters.

Simultaneously, kinetic inductance detectors (KIDs) using high-resistivity materials like titanium nitride are an attractive sensor option for large-format arrays because they are highly multiplexable and because they can have sensitivities reaching the condition of background-limited detection. A KID is a LC resonator. Its inductance includes the geometric inductance and kinetic inductance of the inductor in the superconducting phase. A photon absorbed by the superconductor breaks a Cooper pair into normal-state electrons and perturbs its kinetic inductance, rendering it a detector of light. The responsivity of KID is given by the fractional frequency shift of the LC resonator per unit optical power.

However, coupling these types of optical reception elements to KIDs is a challenge because of the impedance mismatch between the microstrip transmission line exiting these architectures and the high resistivity of titanium nitride. Mitigating direct absorption of light through free space coupling to the inductor of KID is another challenge. We present a detailed titanium nitride KID design that addresses these challenges. The KID inductor is capacitively coupled to the microstrip in such a way as to form a lossy termination without creating an impedance mismatch. A parallel plate capacitor design mitigates direct absorption, uses hydrogenated amorphous silicon, and yields acceptable noise. We show that the optimized design can yield expected sensitivities very close to the fundamental limit for a long wavelength imager (LWCam) that covers six spectral bands from 90 to 400 GHz for SZE studies.

Excess phase (frequency) noise has been observed in KID and is very likely caused by two-level systems (TLS) in dielectric materials. The TLS hypothesis is supported by the measured dependence of the noise on resonator internal power and temperature. However, there is still a lack of a unified microscopic theory which can quantitatively model the properties of the TLS noise. In this thesis we derive the noise power spectral density due to the coupling of TLS with phonon bath based on an existing model and compare the theoretical predictions about power and temperature dependences with experimental data. We discuss the limitation of such a model and propose the direction for future study.