My doctoral dissertation consists of three fundamental studies: (1) synthesis of biocompatible materials that can be used as microfluidic substrates, (2) characterizing these materials with respect ...to properties important to microfluidic fabrication, biochemical separations and concentration enrichment, and (3) employing these novel devices for real world applications in bioanalytical chemistry. The surface properties of a substrate will dramatically affect the resolution and efficiency that can be obtained for a specific CE separation. Thus, the ability to modify the surface is very useful in tailoring a microfluidic chip to a specific separation mode. The substrates we have synthesized for microfluidic devices include metal oxide modified poly(dimethylsiloxane) (PDMS), poly(ethyleneoxide)-PDMS (PEO-PDMS) coblock polymers, and surfactant coated PDMS. The metal oxide modified PDMS materials we synthesized include silica-PDMS, titania-PDMS, vanadia-PDMS and zirconia-PDMS. The surfaces of these materials were characterized using contact angle, X-ray photoelectron spectroscopy (XPS), Raman, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM) and electroosmotic mobility (EOM) measurements. All of the metal oxide modified PDMS surfaces were significantly more hydrophilic than native PDMS, suggesting potential application in separations of biopolymers. In addition to being more hydrophilic the EOF and zeta potential of the channels were stable and quite durable with aging. Well characterized silane chemistry was used to derivitize the surface of the PDMS metal oxide surfaces allowing a number of different functionalities to be placed on the surface. This method has the potential for wide applicability in many different fields, but specifically for the fabrication of microstructures that need specific surface chemistries. We have also made a number of advancements using sol-gel chemistry and laminar flow within microfluidic channels to fabricate nanoporous membranes. Sol-gel patterned membranes are a simple and facile method of incorporating nanoscale diameter channels within a microfluidic manifold. These membranes have been used to perform preconcentration of amino acids, proteins and small particles for further analysis and separation using CE. We are also using these membranes for further study in desilanization and protein recrystallization studies.
Doctor of Philosophy
Department of Chemistry
Christopher T. Culbertson
My doctoral dissertation consists of three fundamental studies: 1) synthesis of biocompatible materials that can be used as ...microfluidic substrates, 2) characterizing these materials with respect to properties important to microfluidic fabrication, biochemical separations and concentration enrichment, and 3) employing these novel devices for real world applications in bioanalytical chemistry.;
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The surface properties of a substrate will dramatically affect the resolution and efficiency that can be obtained for a specific CE separation. Thus, the ability to modify the surface is very useful in tailoring a microfluidic chip to a specific separation mode. The substrates we have synthesized for microfluidic devices include metal oxide modified poly(dimethylsiloxane) (PDMS), poly(ethyleneoxide)-PDMS (PEO-PDMS) coblock polymers, and surfactant coated PDMS. The metal oxide modified PDMS materials we synthesized include silica-PDMS, titania-PDMS, vanadia-PDMS and zirconia-PDMS. The surfaces of these materials were characterized using contact angle, X-ray photoelectron spectroscopy (XPS), Raman, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM) and electroosmotic mobility (EOM) measurements. All of the metal oxide modified PDMS surfaces were significantly more hydrophilic than native PDMS, suggesting potential application in separations of biopolymers. In addition to being more hydrophilic the EOF and zeta potential of the channels were stable and quite durable with aging. Well characterized silane chemistry was used to derivitize the surface of the PDMS metal oxide surfaces allowing a number of different functionalities to be placed on the surface. This method has the potential for wide applicability in many different fields, but specifically for the fabrication of microstructures that need specific surface chemistries. ;
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We have also made a number of advancements using sol-gel chemistry and laminar flow within microfluidic channels to fabricate nanoporous membranes. Sol-gel patterned membranes are a simple and facile method of incorporating nanoscale diameter channels within a microfluidic manifold. These membranes have been used to perform preconcentration of amino acids, proteins and small particles for further analysis and separation using CE. We are also using these membranes for further study in desilanization and protein recrystallization studies.
The transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that ...promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLCγ2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.
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► CD14 is a receptor for LPS that promotes TLR4 endocytosis and interferon expression ► CD14-induced endocytosis occurs independently of TLR4 signaling ► CD14-induced endocytosis is an ITAM-mediated process dependent on Syk and PLCg2 ► Mature dendritic cells exhibit enhanced CD14-dependent responses to bacteria
The pattern recognition receptor CD14 not only delivers bacterial lipopolysaccharide to TLR4 on the plasma membrane of dendritic cells but also regulates TLR4 endocytosis and trafficking to the endosomes to trigger interferon production.
The evolution of animal behaviour is poorly understood
. Despite numerous correlations between interspecific divergence in behaviour and nervous system structure and function, demonstrations of the ...genetic basis of these behavioural differences remain rare
. Here we develop a neurogenetic model, Drosophila sechellia, a species that displays marked differences in behaviour compared to its close cousin Drosophila melanogaster
, which are linked to its extreme specialization on noni fruit (Morinda citrifolia)
. Using calcium imaging, we identify olfactory pathways in D. sechellia that detect volatiles emitted by the noni host. Our mutational analysis indicates roles for different olfactory receptors in long- and short-range attraction to noni, and our cross-species allele-transfer experiments demonstrate that the tuning of one of these receptors is important for species-specific host-seeking. We identify the molecular determinants of this functional change, and characterize their evolutionary origin and behavioural importance. We perform circuit tracing in the D. sechellia brain, and find that receptor adaptations are accompanied by increased sensory pooling onto interneurons as well as species-specific central projection patterns. This work reveals an accumulation of molecular, physiological and anatomical traits that are linked to behavioural divergence between species, and defines a model for investigating speciation and the evolution of the nervous system.
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bstract
It is well-known that unitary irreducible representations of groups can be usefully classified in a 3-fold classification scheme: Real, Complex, Quaternionic. In 1962 Freeman Dyson pointed ...out that there is an analogous 10-fold classification of irreducible representations of groups involving both unitary and antiunitary operators. More recently, it was realized that there is also a 10-fold classification scheme involving superdivision algebras. Here we give a careful proof of the equivalence of these two 10-fold ways.
Abstract
Artificial Intelligence (AI) can support diagnostic workflows in oncology by aiding diagnosis and providing biomarkers directly from routine pathology slides. However, AI applications are ...vulnerable to adversarial attacks. Hence, it is essential to quantify and mitigate this risk before widespread clinical use. Here, we show that convolutional neural networks (CNNs) are highly susceptible to white- and black-box adversarial attacks in clinically relevant weakly-supervised classification tasks. Adversarially robust training and dual batch normalization (DBN) are possible mitigation strategies but require precise knowledge of the type of attack used in the inference. We demonstrate that vision transformers (ViTs) perform equally well compared to CNNs at baseline, but are orders of magnitude more robust to white- and black-box attacks. At a mechanistic level, we show that this is associated with a more robust latent representation of clinically relevant categories in ViTs compared to CNNs. Our results are in line with previous theoretical studies and provide empirical evidence that ViTs are robust learners in computational pathology. This implies that large-scale rollout of AI models in computational pathology should rely on ViTs rather than CNN-based classifiers to provide inherent protection against perturbation of the input data, especially adversarial attacks.
Inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) delay progression of metastatic breast cancer. However, complete responses are uncommon and tumors eventually relapse. Here, we show that ...CDK4/6i can enhance efficacy of T cell-based therapies, such as adoptive T cell transfer or T cell-activating antibodies anti-OX40/anti-4-1BB, in murine breast cancer models. This effect is driven by the induction of chemokines CCL5, CXCL9, and CXCL10 in CDK4/6i-treated tumor cells facilitating recruitment of activated CD8+ T cells, but not Tregs, into the tumor. Mechanistically, chemokine induction is associated with metabolic stress that CDK4/6i treatment induces in breast cancer cells. Despite the cell cycle arrest, CDK4/6i-treated cells retain high metabolic activity driven by deregulated PI3K/mTOR pathway. This causes cell hypertrophy and increases mitochondrial content/activity associated with oxidative stress and inflammatory stress response. Our findings uncover a link between tumor metabolic vulnerabilities and anti-tumor immunity and support further development of CDK4/6i and immunotherapy combinations.
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•Breast cancer cells treated with CDK4/6 inhibitor secrete chemokines CCL5 and CXCL10•Chemokine induction is associated with deregulated mTOR, metabolic stress, and ROS•Chemokines induced by CDK4/6 inhibitor facilitate T cell infiltration into tumors•Chemokines induced by CDK4/6 inhibitor augment adoptive T cell therapy
Inhibitors of cell cycle kinases CDK4/6 delay progression of metastatic breast cancer; however, they do not eliminate tumors. Uzhachenko et al. report that metabolic changes in CDK4/6 inhibitor-treated cancer cells make them vulnerable to T cell therapies. These data highlight potential utility of CDK4/6 inhibitors to overcome immunotherapy resistance.
Individuals use both passive and active defensive responses to environmental threats. Much is known about the neural circuits of passive defensive responses (e.g., freezing), but less is known about ...the substrates of active defensive responses (e.g., avoidance). We developed an active avoidance task in which rats learn to avoid a tone-signaled footshock by stepping onto a nearby platform. An advantage of this task is that freezing, which can interfere with avoidance, is reduced, thereby facilitating comparison of the effects of manipulations on avoidance versus freezing. After 10 d of avoidance training, rats were infused with muscimol to pharmacologically inactivate the prelimbic cortex (PL), infralimbic cortex (IL), ventral striatum (VS), or basolateral amygdala (BLA). Inactivating PL, VS, or BLA all impaired avoidance expression, but these areas differed with respect to freezing. Inactivating BLA decreased freezing consistent with loss of the tone-shock association, whereas inactivation of VS increased freezing consistent with loss of avoidance memory. Inactivation of PL had no effect on freezing. Inactivation of IL did not impair avoidance expression but did impair avoidance extinction. Our findings suggest that active avoidance is mediated by prefrontal-striatal circuits, which may be overactive in individuals suffering from trauma-related disorders.
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