Exploring protein-RNA interactions with site-directed mutagenesis and phage display

The importance of RNA as a mediator of genetic information is widely appreciated. RNA molecules also participate in the regulation of various post-transcriptional activities, such as mRNA splicing, editing, RNA stability and transport. Their regulatory roles for these activities are highly dependent on finely tuned associations with cognate proteins. The RNA recognition motif (RRM) is an ancient RNA binding module that participates in hundreds of essential activities where specific RNA recognition is required. We have applied phage display and site-directed mutagenesis to dissect principles of RRM-controlled RNA recognition. The model systems we are investigating are U1A and CUG-BP1. In this dissertation, the molecular basis of the binding affinity of U1A-RNA beyond individual contacts was investigated. We have identified and evaluated the contributions of the local cooperativity formed by three neighboring residues (Asn15, Asn16 and Glu19) to the stability of the U1A-RNA complex. The localized cooperative network was mapped by double-mutant cycles and explored using phage display. We also showed that a cluster of these residues forms a “hot spot” on the surface of U1A; a single substitution at position 19 with Gln or His can alter the binding properties of U1A to recognize a non-cognate G4U RNA. Finally, we applied a deletion analysis of CUG-BP1 to define the contributions of individual RRMs and RRM combinations to the stability of the complex formed between CUG-BP1 and the GRE sequence. The preliminary results showed RRM3 of CUG-BP1 is a key domain for RNA binding. It possibly binds to the GRE sequence cooperatively with RRM2 of CUG-BP1. RRM1 of CUG-BP1 is not required for GRE recognition, but may be important for maintaining the stability of the full-length CUG-BP1.

An auditory, acoustic, articulatory and sociophonetic study of Swedish Viby-i

The study investigates the acoustic, articulatory and sociophonetic properties of the Swedish /iː/ variant known as 'Viby-i' in 13 speakers of Central Swedish from Stockholm, Gothenburg, Varberg, Jönköping and Katrineholm. The vowel is described in terms of its auditory quality, its acoustic F1 and F2 values, and its tongue configuration. A brief, qualitative description of lip position is also included. Variation in /iː/ production is mapped against five sociolinguistic factors: city, dialectal region, metropolitan vs. urban location, sex and socioeconomic rating. Articulatory data is collected using ultrasound tongue imaging (UTI), for which the study proposes and evaluates a methodology. The study shows that Viby-i varies in auditory strength between speakers, and that strong instances of the vowel are associated with a high F1 and low F2, a trend which becomes more pronounced as the strength of Viby-i increases. The articulation of Viby-i is characterised by a lowered and backed tongue body, sometimes accompanied by a double-bunched tongue shape. The relationship between tongue position and acoustic results appears to be non-linear, suggesting either a measurement error or the influence of additional articulatory factors. Preliminary images of the lips show that Viby-i is produced with a spread but lax lip posture. The lip data also reveals parts of the tongue, which in many speakers appears to be extremely fronted and braced against the lower teeth, or sometimes protruded, when producing Viby-i. No sociophonetic difference is found between speakers from different cities or dialect regions. Metropolitan speakers are found to have an auditorily and acoustically stronger Viby-i than urban speakers, but this pattern is not matched in tongue backing or lowering. Overall the data shows a weak trend towards higher-class females having stronger Viby-i, but these results are tentative due to the limited size and stratification of the sample. Further research is needed to fully explore the sociophonetic properties of Viby-i.

An Exploration of Auditory Brainstem Encoding of Stop Consonants in Infants and Implications for Language Outcomes

Current trends in speech-language pathology focus on early intervention as the preferred tool for promoting the best possible outcomes in children with language disorders. Neuroimaging techniques are being studied as promising tools for flagging at-risk infants. In this study, the auditory brainstem response (ABR) to the syllables /ba/ and /ga/ was examined in 41 infants between 3 and 12 months of age as a possible tool to predict language development in toddlerhood. The MacArthur-Bates Communicative Development Inventory (MCDI) was used to assess language development at 18 months of age. The current study compared the periodicity of the responses to the stop consonants and phase differences between /ba/ and /ga/ in both at-risk and low-risk groups. The study also examined whether there are correlations among ABR measures (periodicity and phase differentiation) and language development. The study found that these measures predict language development at 18 months.

Vocal differentiation parallels development of auditory saccular sensitivity in a highly soniferous fish

Vocal differentiation is widely documented in birds and mammals but has been poorly investigated in other vertebrates, including fish, which represent the oldest extant vertebrate group. Neural circuitry controlling vocal behaviour is thought to have evolved from conserved brain areas that originated in fish, making this taxon key to understanding the evolution and development of the vertebrate vocal-auditory systems. This study examines ontogenetic changes in the vocal repertoire and whether vocal differentiation parallels auditory development in the Lusitanian toadfish Halobatrachus didactylus (Batrachoididae). This species exhibits a complex acoustic repertoire and is vocally active during early development. Vocalisations were recorded during social interactions for four size groups (fry: <2 cm; small juveniles: 2-4 cm; large juveniles: 5-7 cm; adults >25 cm, standard length). Auditory sensitivity of juveniles and adults was determined based on evoked potentials recorded from the inner ear saccule in response to pure tones of 75-945 Hz. We show an ontogenetic increment in the vocal repertoire from simple broadband-pulsed 'grunts' that later differentiate into four distinct vocalisations, including low-frequency amplitude-modulated 'boatwhistles'. Whereas fry emitted mostly single grunts, large juveniles exhibited vocalisations similar to the adult vocal repertoire. Saccular sensitivity revealed a three-fold enhancement at most frequencies tested from small to large juveniles; however, large juveniles were similar in sensitivity to adults. We provide the first clear evidence of ontogenetic vocal differentiation in fish, as previously described for higher vertebrates. Our results suggest a parallel development between the vocal motor pathway and the peripheral auditory system for acoustic social communication in fish.

A atitude dos internautas face à Publicidade Display

Atualmente, com o aumento do consumo de internet e com a afirmação deste meio como veículo de comunicação e transmissão de publicidade, as marcas cada vez mais escolhem a internet para canalizar o seu investimento em publicidade, deste modo têm vindo a aumentar o seu investimento publicitário na internet. Neste canal existem vários tipos de publicidade, sendo que neste estudo académico será analisada a Publicidade Display, tendo como objetivo perceber a atitude e recordação face a este tipo de publicidade. Na metodologia foram utilizadas escalas que procuram analisar a atitude e a recordação dos internautas face à Publicidade Display, escortinando as dimensões Informação, Diversão, Irritação e Confiança bem como os fatores mais importantes para a recordação deste tipo de publicidade. A recolha de dados, teve como base um questionário online no qual foram inquiridos 190 participantes. Para analisar as hipóteses de estudo, foi feita uma regressão linear múltipla e correlações. Os principais resultados confirmam que as dimensões Informação, Entretenimento, Confiança e Irritação são preditores significativos das atitudes relacionadas com a publicidade display.

TRANSFORMATIONS OF TASK-DEPENDENT PLASTICITY FROM A1 TO HIGHER-ORDER AUDITORY CORTEX

Everyday, humans and animals navigate complex acoustic environments, where multiple sound sources overlap. Somehow, they effortlessly perform an acoustic scene analysis and extract relevant signals from background noise. Constant updating of the behavioral relevance of ambient sounds requires the representation and integration of incoming acoustical information with internal representations such as behavioral goals, expectations and memories of previous sound-meaning associations. Rapid plasticity of auditory representations may contribute to our ability to attend and focus on relevant sounds. In order to better understand how auditory representations are transformed in the brain to incorporate behavioral contextual information, we explored task-dependent plasticity in neural responses recorded at four levels of the auditory cortical processing hierarchy of ferrets: the primary auditory cortex (A1), two higher-order auditory areas (dorsal PEG and ventral-anterior PEG) and dorso-lateral frontal cortex. In one study we explored the laminar profile of rapid-task related plasticity in A1 and found that plasticity occurred at all depths, but was greatest in supragranular layers. This result suggests that rapid task-related plasticity in A1 derives primarily from intracortical modulation of neural selectivity. In two other studies we explored task-dependent plasticity in two higher-order areas of the ferret auditory cortex that may correspond to belt (secondary) and parabelt (tertiary) auditory areas. We found that representations of behaviorally-relevant sounds are progressively enhanced during performance of auditory tasks. These selective enhancement effects became progressively larger as you ascend the auditory cortical hierarchy. We also observed neuronal responses to non-auditory, task-related information (reward timing, expectations) in the parabelt area that were very similar to responses previously described in frontal cortex. These results suggests that auditory representations in the brain are transformed from the more veridical spectrotemporal information encoded in earlier auditory stages to a more abstract representation encoding sound behavioral meaning in higher-order auditory areas and dorso-lateral frontal cortex.

The effect of notch filtering on the waveform of the newborn auditory brainstem response

OBJECTIVE: To identify whether the use of a notch filter significantly affects the morphology or characteristics of the newborn auditory brainstem response (ABR) waveform and so inform future guidance for clinical practice. DESIGN: Waveforms with and without the application of a notch filter were recorded from babies undergoing routine ABR tests at 4000, 1000 and 500 Hz. Any change in response morphology was judged subjectively. Response latency, amplitude, and measurements of response quality and residual noise were noted. An ABR simulator was also used to assess the effect of notch filtering in conditions of low and high mains interference. RESULTS: The use of a notch filter changed waveform morphology for 500 Hz stimuli only in 15% of tests in newborns. Residual noise was lower when 4000 Hz stimuli were used. Response latency, amplitude, and quality were unaffected regardless of stimulus frequency. Tests with the ABR stimulator suggest that these findings can be extended to conditions of high level mains interference. CONCLUSIONS: A notch filter should be avoided when testing at 500 Hz, but at higher frequencies appears to carry no penalty.

Does cochlear implantation improve speech recognition in children with auditory neuropathy spectrum disorder? A systematic review

OBJECTIVE: Cochlear implantation (CI) is a standard treatment for severe-profound sensorineural hearing loss (SNHL). However, consensus has yet to be reached on its effectiveness for hearing loss caused by auditory neuropathy spectrum disorder (ANSD). This review aims to summarize and synthesize current evidence of the effectiveness of CI in improving speech recognition in children with ANSD. DESIGN: Systematic review. STUDY SAMPLE: A total of 27 studies from an initial selection of 237. RESULTS: All selected studies were observational in design, including case studies, cohort studies, and comparisons between children with ANSD and SNHL. Most children with ANSD achieved open-set speech recognition with their CI. Speech recognition ability was found to be equivalent in CI users (who previously performed poorly with hearing aids) and hearing-aid users. Outcomes following CI generally appeared similar in children with ANSD and SNHL. Assessment of study quality, however, suggested substantial methodological concerns, particularly in relation to issues of bias and confounding, limiting the robustness of any conclusions around effectiveness. CONCLUSIONS: Currently available evidence is compatible with favourable outcomes from CI in children with ANSD. However, this evidence is weak. Stronger evidence is needed to support cost-effective clinical policy and practice in this area.

Motivating the use of public transit through a mobile glanceable display

With increasing concerns about the impact of global warming on human life, policy makers around the world and researchers have sought for technological solutions that have the potential to attenuate this process. This thesis describes the design and evaluation of an information appliance that aims to increase the use of public transportation. We developed a mobile glanceable display that, being aware of the user’s transportation routines, provides awareness cues about bus arrival time, grounded upon the vision of Ambient Intelligence. We present the design process we followed, from ideation to building a prototype and conducting a field study, and conclude with a set of guidelines for the design of relevant personal information systems. More specifically we seek to test the following hypotheses: 1) That the tangible prototype that provides ambient cues will be used more frequently than a similar purpose mobile app, 2) That the tangible prototype will reduce the waiting time at the bus stop, 3) That the tangible prototype will result to reduced anxiety on passengers, 4) That the tangible prototype will result to an increase in the perceived reliability of the transit service, 5) That the tangible prototype will enhance users’ efficiency in reading the bus schedules and 6) That the tangible prototype will make individuals more likely to use public transit. In a field study, we compare the tangible prototype against the mobile app and a control condition where participants were given no external support in obtaining bus arrival information, other than their existing routines. Using qualitative and quantitative data, we test the aforementioned hypotheses and explore users’ reactions to the prototype we developed.

Prototype and proof of concept for a display system in retail stores

Selling devices on retail stores comes with the big challenge of grabbing the customer’s attention. Nowadays people have a lot of offers at their disposal and new marketing techniques must emerge to differentiate the products. When it comes to smartphones and tablets, those devices can make the difference by themselves, if we use their computing power and capabilities to create something unique and interactive. With that in mind, three prototypes were developed during an internship: a face recognition based Customer Detection, a face tracking solution with an Avatar and interactive cross-app Guides. All three revealed to have potential to be differentiating solutions in a retail store, not only raising the chance of a customer taking notice of the device but also of interacting with them to learn more about their features. The results were meant to be only proof of concepts and therefore were not tested in the real world.

The influence of actions on auditory perception: cognitive and neural mechanisms

It is well known that self-generated stimuli are processed differently from externally generated stimuli. For example, many people have noticed since childhood that it is very difficult to make a self-tickling. In the auditory domain, self-generated sounds elicit smaller brain responses as compared to externally generated sounds, known as the sensory attenuation (SA) effect. SA is manifested in reduced amplitudes of evoked responses as measured through MEEG, decreased firing rates of neurons and a lower level of perceived loudness for self-generated sounds. The predominant explanation for SA is based on the idea that self-generated stimuli are predicted (e.g., the forward model account). It is the nature of their predictability that is crucial for SA. On the contrary, the sensory gating account emphasizes a general suppressive effect of actions on sensory processing, regardless of the predictability of the stimuli. Both accounts have received empirical support, which suggests that both mechanisms may exist. In chapter 2, three behavioural studies concerning the influence of motor activation on auditory perception were presented. Study 1 compared the effect of SA and attention in an auditory detection task and showed that SA was present even when substantial attention was paid to unpredictable stimuli. Study 2 compared the loudness perception of tones generated by others between Chinese and British participants. Compared to externally generated tones, a decrease in perceived loudness for others generated tones was found among Chinese but not among the British. In study 3, partial evidence was found that even when reading words that are related to action, auditory detection performance was impaired. In chapter 3, the classic SA effect of M100 suppression was replicated with MEG in study 4. With time-frequency analysis, a potential neural information processing sequence was found in auditory cortex. Prior to the onset of self-generated tones, there was an increase of oscillatory power in the alpha band. After the stimulus onset, reduced gamma power and alpha/beta phase locking were found. The three temporally segregated oscillatory events correlated with each other and with SA effect, which may be the underlying neural implementation of SA. In chapter 4, a TMS-MEG study was presented investigating the role of the cerebellum in adapting to delayed presentation of self-generated tones (study 5). It demonstrated that in sham stimulation condition, the brain can adapt to the delay (about 100 ms) within 300 trials of learning by showing a significant increase of SA effect in the suppression of M100, but not M200 component. Whereas after stimulating the cerebellum with a suppressive TMS protocol, the adaptation in M100 suppression disappeared and the pattern of M200 suppression reversed to M200 enhancement. These data support the idea that the suppressive effect of actions on auditory processing is a consequence of both motor driven sensory predictions and general sensory gating. The results also demonstrate the importance of neural oscillations in implementing SA effect and the critical role of the cerebellum in learning sensory predictions under sensory perturbation.

Motivating the use of public transit through a mobile glanceable display

With increasing concerns about the impact of global warming on human life, policy makers around the world and researchers have sought for technological solutions that have the potential to attenuate this process. This thesis describes the design and evaluation of an information appliance that aims to increase the use of public transportation. We developed a mobile glanceable display that, being aware of the user’s transportation routines, provides awareness cues about bus arrival time, grounded upon the vision of Ambient Intelligence. We present the design process we followed, from ideation to building a prototype and conducting a field study, and conclude with a set of guidelines for the design of relevant personal information systems. More specifically we seek to test the following hypotheses: 1) That the tangible prototype that provides ambient cues will be used more frequently than a similar purpose mobile app, 2) That the tangible prototype will reduce the waiting time at the bus stop, 3) That the tangible prototype will result to reduced anxiety on passengers, 4) That the tangible prototype will result to an increase in the perceived reliability of the transit service, 5) That the tangible prototype will enhance users’ efficiency in reading the bus schedules and 6) That the tangible prototype will make individuals more likely to use public transit. In a field study, we compare the tangible prototype against the mobile app and a control condition where participants were given no external support in obtaining bus arrival information, other than their existing routines. Using qualitative and quantitative data, we test the aforementioned hypotheses and explore users’ reactions to the prototype we developed.