Deep learning for facial emotion recognition

The ability to perceive and interpret human emotions is an essential as-pect of daily life. The recent success of deep learning (DL) has resulted in the ability to utilize automated emotion recognition by classifying af-fective modalities into a given emotional state. Accordingly, DL has set several state-of-the-art benchmarks on static affective corpora collected in controlled environments. Yet, one of the main limitations of DL based intelligent systems is their inability to generalize on data with nonuniform conditions. For instance, when dealing with images in a real life scenario, where extraneous variables such as natural or artificial lighting are sub-ject to constant change, the resulting changes in the data distribution commonly lead to poor classification performance. These and other con-straints, such as: lack of realistic data, changes in facial pose, and high data complexity and dimensionality increase the difficulty of designing DL models for emotion recognition in unconstrained environments. This thesis investigates the development of deep artificial neural net-work learning algorithms for emotion recognition with specific attention to illumination and facial pose invariance. Moreover, this research looks at the development of illumination and rotation invariant face detection architectures based on deep reinforcement learning. The contributions and novelty of this thesis are presented in the form of several deep learning pose and illumination invariant architectures that offer state-of-the-art classification performance on data with nonuniform conditions. Furthermore, a novel deep reinforcement learning architecture for illumination and rotation invariant face detection is also presented. The originality of this work is derived from a variety of novel deep learning paradigms designed for the training of such architectures

Efficient targeting to storage granules of human proinsulins with altered propeptide domain.

In neuronal and endocrine cells, peptide hormones are selectively segregated into storage granules, while other proteins are exported continuously without storage. Sorting of hormones by cellular machinery involves the recognition of specific structural domains on prohormone molecules. Since the propeptide of insulin is known to play an important role in its three-dimensional structure, it is reasonable to speculate that targeting of proinsulin to storage granules would require a functional connecting peptide. To test this hypothesis, we constructed two mutations in human proinsulin with different predicted structures. In one mutation, Ins delta C, the entire C peptide was deleted, resulting in an altered insulin in which the B and the A chains are joined contiguously. In the other mutation, Ins/IGF, the C peptide of proinsulin was replaced with the unrelated 12-amino acid connecting peptide of human insulin-like growth factor-I; this substitution should permit correct folding of the B and A chains to form a tertiary structure similar to that of proinsulin. By several biochemical and morphological criteria, we found that Ins/IGF is efficiently targeted to storage granules, suggesting that the C peptide of proinsulin does not contain necessary sorting information. Unexpectedly, Ins delta C, which presumably cannot fold properly, is also targeted to granules at a high efficiency. These results imply that either the targeting machinery can tolerate changes in the tertiary structure of transported proteins, or that the B and A chains of insulin can form a relatively intact three-dimensional structure even in the absence of C peptide

DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES

We have biochemically characterized the bacterial-like DnaG primase contained within the hyperthermophilic crenarchaeon Sulfolobus solfataricus (Sso) and compared in vitro priming kinetics with those of the eukaryotic-like primase (PriS&L) also found in Sso. SsoDnaG exhibited metal- and temperature-dependent profiles consistent with priming at high temperatures. The distribution of primer products for SsoDnaG was discrete but highly similar to the distribution of primer products produced by the homologous Escherichia coli DnaG. The predominant primer length was 13 bases, although less abundant products of varying sizes are also present. SsoDnaG was found to bind DNA cooperatively as a dimer with a moderate dissociation constant. Mutation of the conserved glutamate in the active site severely inhibited priming activity, showing functional homology with E. coli DnaG. SsoDnaG was also found to have a greater than four-fold faster rate of DNA priming over that of SsoPriS&L under optimal in vitro conditions. The presence of both enzymatically functional primase families in archaea suggests that the DNA priming role may be shared on leading or lagging strands during DNA replication. DNA replication polymerases have the inherent ability to faithfully copy a DNA template according to Watson Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in Sso is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by SsoDpo1 allowed for template-directed synthesis at temperatures more than 30 °C above the melting temperature of naked DNA. Surprisingly, SsoDpo1 also displays a terminal deoxynucleotide transferase (TdT) activity unlike any other B-family DNA polymerases. SsoDpo1 is shown to elongate single stranded DNA in template-dependent and template-independent manners. The multiple activities of this unique B-family DNA polymerase make this enzyme an essential component for DNA replication and DNA repair for the maintenance of the archaeal genome at high temperatures. Preliminary results of primer transfer studies in Sso show that SsoDpo1 can elongate DNA primers de novo synthesized by SsoPriS&L, but elongation of RNA primers synthesized by both SsoPriS&L and SsoDnaG was not observed for SsoDpo1. Future studies to untangle the primer transfer mechanism in archaea are discussed

Nano/biosensors Based On Large-Area Graphene

Two dimensional materials have properties that make them ideal for applications in chemical and biomolecular sensing. Their high surface/volume ratio implies that all atoms are exposed to the environment, in contrast to three dimensional materials with most atoms shielded from interactions inside the bulk. Graphene additionally has an extremely high carrier mobility, even at ambient temperature and pressure, which makes it ideal as a transduction device. The work presented in this thesis describes large-scale fabrication of Graphene Field Effect Transistors (GFETs), their physical and chemical characterization, and their application as biomolecular sensors. Initially, work was focused on developing an easily scalable fabrication process. A large-area graphene growth, transfer and photolithography process was developed that allowed the scaling of production of devices from a few devices per single transfer in a chip, to over a thousand devices per transfer in a full wafer of fabrication. Two approaches to biomolecules sensing were then investigated, through nanoparticles and through chemical linkers. Gold and platinum Nanoparticles were used as intermediary agents to immobilize a biomolecule. First, gold nanoparticles were monodispersed and functionalized with thiolated probe DNA to yield DNA biosensors with a detection limit of 1 nM and high specificity against noncomplementary DNA. Second, devices are modified with platinum nanoparticles and functionalized with thiolated genetically engineered scFv HER3 antibodies to realize a HER3 biosensor. Sensors retain the high affinity from the scFv fragment and show a detection limit of 300 pM. We then show covalent and non-covalent chemical linkers between graphene and antibodies. The chemical linker 1-pyrenebutanoic acid succinimidyl ester (pyrene) stacks to the graphene by Van der Waals interaction, being a completely non-covalent interaction. The linker 4-Azide-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester (azide) is a photoactivated perfluorophenyl azide that covalently binds to graphene. A comparison is shown for genetically engineered scFv HER3 antibodies and show a low detection limit of 10 nM and 100 pM for the pyrene and azide, respectively. Finally, we use the azide linker to demonstrate a large-scale fabrication of a multiplexed array for Lyme disease. Simultaneous detection of a mixture of two target proteins of the Lyme disease bacterium (Borrelia burgdorferi), this is done by separating the antibodies corresponding to each target in the mixture to different regions of the chip. We show we can differentiate concentrations of the two targets

Involvement of Vav2 in the EphA4 signaling pathway of \u3ci\u3eXenopus laevis\u3c/i\u3e as a possible Rho regulator

The purpose of this research is to shed light on the involvement of the Vav2 guanine nucleotide exchange factor in the Xenopus laevis EphA4 signaling pathway. In the first phase of the project, the presence of Vav2 mRNA within seven different Xenopus laevis embryogenic stages was detected via embryogenic stage collection, retrotranscription, PCR, using forward and reverse Vav2 primers, agarose gel electrophoresis, and UV gel visualization. In the second phase of the project, Western Blotting techniques were used to determine the presence of Vav2 protein in Xenopus laevis embryogenic stages

Interactions between the Translation Machinery and a Translational preQ1 Riboswitch.

Gene expression is highly regulated with a diversity of regulation at the RNA level. In bacteria, regulation of mRNA translation into protein often occurs through RNA sequence features such as the Shine-Dalgarno (SD) sequence and local structural features. Translational riboswitches in bacteria exemplify such cis-acting regulation. This work look at how structural features of a preQ1 riboswitch effect regulation through interactions with the translation machinery. Broader questions about how individual translational machinery components, such as ribosomal protein S1 and the 30S ribosomal subunit, interact with structured RNAs are also addressed. We sought a more detailed mechanistic view of the interplay between the translational preQ1 riboswitch found in the 5′ UTR of an mRNA from T. tengcongensis, its ligand preQ1, and the SD sequence accessibility. To this end, we developed SiM-KARTS, a generalized strategy to interrogate site-specific structural dynamics of RNA molecules based on probe hybridization kinetics. Intriguingly, we found that the riboswitch expression platform alternates between conformations with differing SD accessibility, which are distinguished by “bursts” of probe binding, the pattern of which is modulated by ligand. This challenges the assumption that riboswitches behave in simple ON/OFF fashion and thus has broader implications for how we think about translational riboswitch regulation. The folding and unfolding of RNA structure influences other cellular processes besides translation. Ribosomal protein S1 performs other roles outside of the context of translation, which are related to its RNA binding or unfolding capacity. We used the well-characterized preQ1 riboswitch as a model pseudoknot to study how S1 interacts with defined, stable tertiary structure. S1 is able to bind and at least partially unfold this pseudoknot in a manner that is limited by RNA structural stability. Lastly, we investigated the influence of S1 on translation of preQ1 riboswitch-containing mRNAs and found that the effects of ligand on translation are not potentiated by the loss of S1. There is, however, a dramatic effect on translational coupling, invoking a role for S1 in polycistronic mRNA translation. These results highlight the need for additional techniques, such as assays at the single molecule level, to monitor early 30S-mRNA interactions during translation.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116677/1/palund_1.pd