Innate immune responses of lumpfish (Cyclopterus lumpus) : Transcriptome analysis and characterization of pro-inflammatory cytokines

In recent years, lumpfish and different species of wrasse are used as cleaner fishes for removal of sea lice from farmed Atlantic salmon in Europe and Canada. The production is successful, but there are challenges with high mortality due to bacterial infections. In-depth knowledge of the immune system in lumpfish, particularly immune responses upon bacterial infections and identification of immune genes, will make an important basis for development of immune-prophylactic measures. Prior to this study, there were no available sequences of immune genes in lumpfish in public databases. To obtain sequence information from lumpfish, RNA sequencing of head kidney leukocytes (HKLs) exposed to Vibrio anguillarum O1, followed by de novo transcriptome assembly, was performed. The transcriptome encompassed 221659 trinity genes, 9033 differentially expressed genes (DEG) at 6 hours post exposure (hpe) and 15225 DEG at 24 hpe. The DEG analysis of the lumpfish transcriptome clearly showed that the alternative pathway of the complement cascade is one of the chief mechanisms in the Vibrio anguillarum response of lumpfish HKLs. Furthermore, the DEG analysis also clearly showed that the TLR signaling through the canonical NFκB pathway was the major pathway in the adaptation of the innate immune response. Further, a wider MAPK pathway regulation was observed, involving all of its sub pathways, however, at much less potent regulation levels compared with NFκB. Globally, the DEG analysis displayed a picture of a pro-inflammatory reaction, initiated by toll-like receptor TLR5S, and the alternative complement pathway, and resulting in high up-regulation of the cytokines interleukin (IL)-1β, IL-6, CXCL8 (also known as IL-8) and tumor necrosis factor (TNF)-α. Further characterization of the lumpfish IL-1 family, both ligands and receptors (IL-1R), were performed. Downstream signaling pathways were also characterized. Full-length sequences of the ligands IL-1β, IL-18, and the fish-specific IL-1 family members nIL-1F and IL-1Fm2, the receptors IL-1R1, IL-1R2, IL18-R1, suppression of tumorgenicity (ST2), and partial sequences of double immunoglobulin IL-1R related molecule (DIGIRR) and IL- receptor accessory protein (IL-Ra) were identified. In vitro stimulation of lumpfish leukocytes with a selection of PAMPs, showed that lumpfish IL-1β and nIL-1F were upregulated, most potently by flagellin. The phylogenetic analysis of the IL-1 family ligands showed that IL-1β, nIL-1F1 and IL1Fm2 are more similar to each other than to IL-18. Furthermore, it placed sequences from lobe-finned fish and shark within the nIL-1F clade, suggesting that nIL-1F, together with IL-1β are ancestral genes. This is the first report describing the occurrence of nIL-1F in non-teleost species. Characterization of lumpfish TNF-α revealed that it contained the hallmark properties of TNF-family at nucleotide and peptide levels. Characterization of lumpfish IL-6 revealed a novel gene arrangement, with one less exon, than other described IL-6 teleost sequences. Furthermore, the predicted peptide sequence of lumpfish IL-6 contained a signal peptide first revealed after proteolytic cleavage. Caspase 1 was predicted to cleave in this region of lumpfish IL-6. It is therefore hypothesized that lumpfish IL-6 may be dependent on caspase 1 processing. In the normal tissues and unstimulated leukocytes, the levels of TNF-α transcripts were higher than IL-6, except for eye and brain where the transcript levels of IL-6 were unexpectedly high. Both genes displayed a similar induction pattern to PAMP stimulation in vitro and both were most potently stimulated by flagellin. IL-6 was more potently stimulated that TNF-α. In conclusion, the lumpfish innate immune responses are potent and consist of most of the molecules for a modern teleost. The alternative complement pathway and TLR signaling pathway are fundamental to the in vitro response to Vibrio anguillarum O1 by lumpfish leukocytes. The identification and characterization of the major pro-inflammatory cytokines and design of qPCR assays in lumpfish provides a valuable tool to measure innate immune responses in lumpfish e.g. upon immune modulation, such as vaccination, microbial disease or physiological trials

Chemical communication in a declining North American anuran amphibian, the northern leopard frog (lithobates pipiens)

Chemical signaling is known to be an important communication mechanism for amphibians. However, very few studies have directly investigated chemical signaling in anuran groups (frogs and toads), particularly adult anurans. Previous work has focused primarily on salamander and larval anuran behavioral responses. Additionally, chemoreceptor (CR) genes have only been identified for Xenopus species with no previous identifications for any other adult anuran species. Here, I examine the evolutionary and functional implications of northern leopard frog (Lithobates pipiens) CRs, identified through collaboration, by conducting a phylogenetic analysis for each CR type identified using a Maximum Likelihood approach (Chapter 1). I incorporated amino acid CR sequences spanning aquatic, semiaquatic, and terrestrial vertebrate species, and the resulting trees indicate split divergence patterns across CR types. More specifically, olfactory receptors (ORs) and trace amine associated receptors (TAARs) appear to be most closely related to transitional vertebrate and terrestrial species’ orthologs, and vomeronasal receptors (VRs Types I and II) appear to be most closely related to transitional vertebrate and aquatic vertebrate orthologs, hinting at both volatile and aquatic (water soluble and nonvolatile) detection of chemicals. In this study, I also investigate terrestrial, sex-biased anuran behavioral responses to conspecific chemical cues in adult L. pipiens, comparing time spent on cue and non-cue sides of an experimental terrarium (Chapter 2) to expand on current knowledge of chemical detection in a declining, North American species. Results show nuanced response profiles for males and females, based on familiarity (odors of individuals housed together and those that were housed in separate enclosures) and same-sex vs. opposite-sex cues. Females were more responsive overall (P <0.005), investigating unfamiliar, opposite sex and familiar, same-sex odors. Though not statistically significant (P=0.40), the data (see Figure 7.) suggest that male investigation of cue and non-cue sides differed slightly for familiar, same-sex conspecifics. Neither sex appeared to respond to unfamiliar, same-sex odors. Intuitively, ecological benefits associated with familiar odor detection and mate localization are likely responsible for these trends. Collectively, my results expose CR divergence patterns across aquatic, semiaquatic, and terrestrial species and help elucidate conspecific ligand functional roles that influence L. pipiens behavior

An acoustic-phonetic approach in automatic Arabic speech recognition

In a large vocabulary speech recognition system the broad phonetic classification technique is used instead of detailed phonetic analysis to overcome the variability in the acoustic realisation of utterances. The broad phonetic description of a word is used as a means of lexical access, where the lexicon is structured into sets of words sharing the same broad phonetic labelling. This approach has been applied to a large vocabulary isolated word Arabic speech recognition system. Statistical studies have been carried out on 10,000 Arabic words (converted to phonemic form) involving different combinations of broad phonetic classes. Some particular features of the Arabic language have been exploited. The results show that vowels represent about 43% of the total number of phonemes. They also show that about 38% of the words can uniquely be represented at this level by using eight broad phonetic classes. When introducing detailed vowel identification the percentage of uniquely specified words rises to 83%. These results suggest that a fully detailed phonetic analysis of the speech signal is perhaps unnecessary. In the adopted word recognition model, the consonants are classified into four broad phonetic classes, while the vowels are described by their phonemic form. A set of 100 words uttered by several speakers has been used to test the performance of the implemented approach. In the implemented recognition model, three procedures have been developed, namely voiced-unvoiced-silence segmentation, vowel detection and identification, and automatic spectral transition detection between phonemes within a word. The accuracy of both the V-UV-S and vowel recognition procedures is almost perfect. A broad phonetic segmentation procedure has been implemented, which exploits information from the above mentioned three procedures. Simple phonological constraints have been used to improve the accuracy of the segmentation process. The resultant sequence of labels are used for lexical access to retrieve the word or a small set of words sharing the same broad phonetic labelling. For the case of having more than one word-candidates, a verification procedure is used to choose the most likely one

Lagrangian acceleration measurements via PTVA (Particle Tracking Velocimetry and Accelerometry) on a turbulent-like flow

Quasi-two-dimensional turbulence (Q2D) has both a practical and theoretical relevance. Following the topological approach, turbulent-like flows have the aim to model and study some of the aspects of turbulence. With this target, in the laboratory of the Imperial College London, Q2D electromagnetically-controlled multiple-scale flows are experimentally studied. To study those flows, an accurate Lagrangian technique to measure acceleration is needed: a novel technique, Particle Tracking Velocimetry and Accelerometry (PTVA), based on Image Analysis techniques, has been designed, developed and validated. PTVA is a self-adaptive methods which relies on a number of positions to approximate the trajectories that adapts to the topology and acceleration of the flow and grants much more precise measurements compared to previous methods. PTVA has allowed to experimentally measure all the Navier-Stokes equation’s terms over the entire investigation field, to classify zero acceleration points, to extract the map of power input and output along with the one of the divergence of acceleration (stirring intensity), measured for the first time, obtaining the tools to work on mixing efficiency. The pdf of the acceleration components has turned out to be very similar to the one usually measured in real turbulence: as this pdf has been often considered as the signature of turbulence, this result makes the question of which characteristics of turbulence generate which features arise

Lagrangian acceleration measurements via PTVA (Particle Tracking Velocimetry and Accelerometry) on a turbulent-like flow

Quasi-two-dimensional turbulence (Q2D) has both a practical and theoretical relevance. Following the topological approach, turbulent-like flows have the aim to model and study some of the aspects of turbulence. With this target, in the laboratory of the Imperial College London, Q2D electromagnetically-controlled multiple-scale flows are experimentally studied. To study those flows, an accurate Lagrangian technique to measure acceleration is needed: a novel technique, Particle Tracking Velocimetry and Accelerometry (PTVA), based on Image Analysis techniques, has been designed, developed and validated. PTVA is a self-adaptive methods which relies on a number of positions to approximate the trajectories that adapts to the topology and acceleration of the flow and grants much more precise measurements compared to previous methods. PTVA has allowed to experimentally measure all the Navier-Stokes equation’s terms over the entire investigation field, to classify zero acceleration points, to extract the map of power input and output along with the one of the divergence of acceleration (stirring intensity), measured for the first time, obtaining the tools to work on mixing efficiency. The pdf of the acceleration components has turned out to be very similar to the one usually measured in real turbulence: as this pdf has been often considered as the signature of turbulence, this result makes the question of which characteristics of turbulence generate which features arise

Dendrimer Nanoconjugates And Their Oral Inhalation Formulations For Lung Cancer Therapy

Lung cancers are leading cause of cancer death for both men and women in the world. Lungs are also one of the primary organs to which almost all cancer can spread. Chemothepray plays a crucial role in the fight against both primary lung cancers and lung metastases. Doxorubicin (DOX) is a potent anticancer drug that has been approved for treating many cancers including lung cancers. However, only a few percent of systemically administered DOX can be found in the lungs. The issue is further complicated by dose-limiting toxicity of DOX. Another major challenge in the use of DOX is its cardiac toxicity. Free DOX in bloodstream can accumulate in cardiac tissues, thus leading to fatal heart damages. On the other hand, the lungs are considered as a portal to external environment and suitable for local delivery. Additionally, oral inhalation (OI) has long been accepted as the preferred mode of administration of therapeutics to the lungs. To address these discussed challenges, we used a strategy — polymeric nanocarriers (PNCs) to achieve the spatial and temporal drug release and local delivery of drug to the lungs. In this work, we desgined polyamidoamine dendrimer (PAMAM) conjugates which can only release DOX intracellularly, while being stable in physiological environment. We observed the PAMAM-DOX conjugates upon local delivery to the lungs can significantly inhibit tumor growth both in vitro and in vivo. We also successfully prepared the PAMAP-DOX conjugates into propellant-based aerosol formulations which is conducive to deep lung areas. To our knowledge, it is for the first time that polymeric nanocarrier-based drug delivery system has been formulated into propellant-based aerosol formulations

The Unnaming of Aliass

The Unnaming of Aliass performs a paradoxical quest for wildly “untold” stories in the company of one special donkey companion, a femammal of the species Equus asinus and, significantly, a registered “American Spotted Ass.” Beast of burden that she is, this inscrutable companion helped carry a ridiculous load of human longings and quandaries into a maze of hot, harrowing miles, across the US South from Mississippi to Virginia, in the summer of 2002 -- all the while carrying her own onerous and unreckoned burdens and histories. Over two decades, the original journey evolved -- from the cracking-open of a quasi-Western novel-that-never-was by an implosive pun, into an ongoing philosophical and assthetic adventure: a hybrid roadside- and barnyard-based living-art practice, wherein “Aliass” un/names something much harder to grasp than the body of a lovely little ass: protagonist, setting, and traditional Western narratives turn inside-out around this “name-that-ain’t.” Through a deeply dug-in questioning of its own authorial assumptions, The Unnaming of Aliass makes space for untold autobiographies and bright dusty lacunae, tracing ineffable tales through the tangled shapes and shadows that interweave in any environment