13 resultados para Chiropractic Manipulation

em Brock University, Canada


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The manipulation of large (>10 kb) plasmid systems amplifies problems common to traditional cloning strategies. Unique or rare restriction enzyme recognition sequences are uncommon and very rarely located in opportunistic locations. Making site-specific deletions and insertions in larger plasmids consequently leads to multiple step cloning strategies that are often limited by time-consuming, low efficiency linker insertions or blunt-end cloning strategies. Manipulation ofthe adenovirus genome and the genomes ofother viruses as bacterial plasmids are systems that typify such situations. Recombinational cloning techniques based on homologous recombination in Saccharomyces cerevisiae that circumvent many ofthese common problems have been developed. However, these techniques are rarely realistic options for such large plasmid systems due to the above mentioned difficulties associated with the addition ofrequired yeast DNA replication, partitioning and selectable marker sequences. To determine ifrecombinational cloning techniques could be modified to simplify the manipulation of such a large plasmid system, a recombinational cloning system for the creation of human adenovirus EI-deletion rescue plasmids was developed. Here we report for the first time that the 1,456 bp TRP1/ARS fragment ofYRp7 is alone sufficient to foster successful recombinational cloning without additional partitioning sequences, using only slight modifications of existing protocols. In addition, we describe conditions for efficient recombinational cloning involving simultaneous deletion of large segments ofDNA (>4.2 kb) and insertion of donor fragment DNA using only a single non-unique restriction site. The discovery that recombinational cloning can foster large deletions has been used to develop a novel recombiliational cloillng technique, selectable inarker 'kilockouf" recombinational cloning, that uses deletion of a yeast selectable marker coupled with simultaneous negative and positive selection to reduce background transformants to undetectable levels. The modification of existing protocols as described in this report facilitates the use of recombinational cloning strategies that are otherwise difficult or impractical for use with large plasmid systems. Improvement of general recombinational cloning strategies and strategies specific to the manipulation ofthe adenovirus genome are considered in light of data presented herein.

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The operational sex ratio has long been considered an important constraint on the structure of mating systems. The effects of an experimentally manipulated sex ratio on mating behavior and selection were investigated in a polygynous species, Gryllus pennsylvanicus, where the potential exists for spatial/temporal fluctuations in sex ratio of field populations. Four different sex ratios (males: females, 5:0, 5:2, 5:5, 5:10) were investigated. Observations were conducted in late summer over two field seasons, from 2400 h , to 1000 h EST. Several male characters thought to be associated with male reproduc.tive success were studied: calling duration, searching distance, weight, fighting behavior, courtship frequency, and mating success. Variance in male mating success was used as the indicator for the opportunity for sexual selection. Total selection was estimated as the univariate regression coefficient between relative fitness and the character of interest, while direct selection was estimated as standardized partial regression coefficients generated from a multiple regression of relative fitness on each character. The opportunity for sexual selection was highest at 5:2 and lowest at 5:10. The frequency of fighting behavior was highest at 5:2 and 5:5. Fighting ability (% wins) was determined to be an important correlate of male body weight. Direct selection for increased male body weight was detected at 5:2, while total selection for body weight was seen at 5:5. Selection on male body weight was not detected at 5: 10. Calling duration decreased as sex ratio became more female-biased. Total and direct selection were detected for increased calling at 5:2, only total selection for calling was seen at 5:5, whereas direct selection against calling was detected at 5: 10. Searching distance also decreased as sex ratio became more female-biased, however no form of selection was detected for searching at any of the sex ratios. Data are discussed in terms of sexual selection on male reproductive tactics, the mating system and maintenance of genetic variation in male reproductive behavior.

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The relative ease to concentrate and purify adenoviruses, their well characterized mid-sized genome, and the ability to delete non-essential regions from their genome to accommodate foreign gene, made adenoviruses a suitable candidate for the construction of vectors. The use of adenoviral vectors in gene therapy, vaccination, and as a general vector system for expressing foreign genes have been documented for some time. In this study, the objective was to rescue a BAV3 E1 or E3 recombinant vector carrying the kanamycin resistant gene, a dominant selectable marker with useful applications in studying vectored gene expression in mammalian cells. To accomplish the objective of this study, more information about BAV3 DNA sequences was required in order to make the manipulation of the virus genome accessible. Therefore, sequencing of the BAV3 genome from 1 1 .7% to 30.8% was carried out. Analysis of the determined sequences revealed the primary structure of important viral gene products coded by E2 including BAV3 DNA pol and precursor to terminal protein. Comparative analysis of these proteins with their counterparts from human and non human adenoviruses revealed important insights as to the evolutionary lineage of BAV3. In order to insert the kanamycin resistance gene in either E1 or E3, it was necessary to delete BAV3 sequences to accommodate the foreign gene so as not to exceed the limit of the packaging capacity of the virus. To construct a recombinant BAV3 in which a foreign gene was inserted in the deleted E1 region, an E1 shuttle vector was constructed. This involved the deletion from the viral sequences a region between 1.3% to 9% and inserting the kanamycin resistance gene to replace the deletion. The E1 shuttle vector contained the left (0%- 53.9%) segment of the genome and was expected to generate BAV3 recombinants that can be grown and propagated in cells that can complement the missing E1 functions. To construct a similar shuttle vector for E3 deletion, DNA sequences extending from 78.9% to 82.5% (1281 bp) were deleted from within the E3 region that had been cloned into a plasmid vector. The deleted region corresponds to those that have been shown to be non-essential for viral replication in cell culture. The resulting plasmid was used to construct another recombinant plasmid with BAV3 DNA sequences extending from 37.1% to 100% and with a deletion of E3 sequences that were replaced by kanamycin resistance gene. This shuttle plasmid was used in cotransfections with digested viral DNA in an attempt to rescue a recombinant BAV3 carrying the kanamycin resistance gene to replace the deleted E3. In spite of repeated attempts of transfection, El or E3 recombinant BAV3 were not isolated. It seems that other approaches should be applied to make a final conclusion on BAV3 infectivity.

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Recent dose-response sleep restriction studies, in which nightly sleep is curtailed to varying degrees (e.g., 3-, 5-, 7-hours), have found cumulative, dose-dependent changes in sleepiness, mood, and reaction time. However, brain activity has typically not been measured, and attentionbased tests employed tend to be simple (e.g., reaction time). One task addressing the behavioural and electrophysiological aspects of a specific attention mechanism is the Attentional Blink (AB), which shows that the report accuracy of a second target (T2) is impaired when it is presented soon after a first target (Tl). The aim of the present study was to examine behavioural and electrophysioiogical responses to the AB task to elucidate how sleep restriction impacts attentional capacity. Thirty-six young-adults spent four consecutive days and nights in a sleep laboratory where sleep, food, and activity were controlled. Nightly sleep began with a baseline sleep (8 hours), followed by two nights of sleep restriction (3,5 or 8 hours of sleep), and a recovery sleep (8 hours). An AB task was administered each day at 11 am. Results from a basic battery oftests (e.g., sleepiness, mood, reaction time) confirmed the effectiveness of the sleep restriction manipulation. In terms of the AB, baseline performance was typical (Le., T2 accuracy impaired when presented soon after Tl); however, no changes in any AB behavioural measures were observed following sleep restriction for the 3- or 5-hour groups. The only statistically significant electrophysiological result was a decrease in P300 amplitude (for Tl) from baseline to the second sleep restriction night for the 3-hour group. Therefore, following a brief, two night sleep restriction paradigm, brain functioning was impaired for the TI of the AB in the absence of behavioural deficit. Study limitations and future directions are discussed.

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Strain improvement of the insect pathogenic fungus Metarhizium anisopUae is necessary to increase its virulence towards agricultural pests and thus improve its commercial efficacy. Nevertheless, the release of genetically modified conidia in crop fields may negatively affect the ecosystem. Controlling conidiation is a potential means of limiting the release of engineered strains since conidia are the infective propagules and the means of dispersal. The purpose of this study was to research the colony development of M. anisopUae to identify potential targets for genetic manipulation to control conidiation. Following Agrobacterium tumefaciem insertional mutagenesis, phenotypic mutants were characterized using Y-shaped adaptor dependent extension PCR. Four of 1 8 colony development recombinants had T-DNA flanking sequences with high homology to genes encoding known signaling pathway proteins that regulate pathogenesis and/or asexual development in filamentous fungi. Conidial density counts and insect bioassays suggested that a Serine/Threonine protein kinase COTl homolog is not essential for conidiation or virulence. Furthermore, a choline kinase homolog is important for conidiation, but not virulence. Finally, the regulator of G protein signaling CAG8 and a NADPH oxidase NoxA homolog are necessary for conidiation and virulence. These genes are candidates for further investigation into the regulatory pathways controlling conidiation to yield insight into promising gene targets for biocontrol strain improvement.

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Recombinant Adenoviruses (Ads) have been shown to have potential applications in three areas: gene therapy, high level protein expression and recombinant vaccines.' At least three different locations within the Ad genome can be deleted and subsequently used for the insertion of foreign sequences. These include the Early 3 (E3), Early 1 (E1) and Early 4 (E4) regions. Viral vectors of this type have been well studied in Human Ads 2 and 5, however one has not yet been constructed for Bovine Adenovirus Type 2 (BAV2). The E3 region is located between 76.6 and 86 m.u. on the r-strand and is transcribed in a rightward direction. The gene products of the Early 3 region (E3) have been shown to be non-essential for viral replication, in vitro, but are required for host immunosurveillance. This study represents the cloning and reconstitution of a BAV2 E3 deletion mutant. A deletion of 1800bp was made within the E3 region of BAV2 and the thymidine kinase gene was subsequently inserted in the deleted area . . The plasmid pdlE3-4tk1 (23.4Kbp) was constructed and used to to facilitate homologous recombination with the wild type BAV2 to produce a mutant. Southern Blotting and Hybridization results suggest the presence of a BAV2 E3 deletion mutant with thymidine kinase sequences present. The E4 region of Human Adenovirus types 2 and 5 is located at the extreme right end of the genome (91.3 map units - 99.1 map units) and is transcribed in a leftward direction giving rise to a complicated set of differentially spliced mRNAs. Essentially there are 7 open reading frames (ORFs) encoding for at least 7 polypeptides. The gene products encoded by the E4 region have been shown to be essential for the expression of late viral genes, host cell shutoff and normal viral growth. We have cloned and sequenced the right end segment between 90.5 map units and 100 map units of the BAV2 genome. The results show several open reading frames which encode polypeptides exhibiting homology to three polypeptides encoded by the E4 region of human adenovirus type 2. These include the 14kDa protein encoded by ORF1, the 34kDa protein encoded by ORF6 and the 13kDa protein encoded by ORF3. The nucleotide sequence, restriction enzyme map, and ORF map of the E4 region could be very useful in future molecular manipulation of this region and could possibly explain the slow growth rate of BAV2 in MDBK cells.

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Human adenoviruses (Ads), members of the family adenoviridae, are medium-sized DNA viruses which have been used as valuable research tools for the study of RNA processing, oncogenic transformation, and for the development of viral vectors for use in gene delivery and immunization technology. The left 12% of the linear Ad genollle codes for products which are necessary for the efficient replication of the virus, as well as being responsible for the forlllation of tumors in animallllodels. The establishlllent of the 293 cell line, by immortalization of human embryonic kidney cells with th~ E1 region of Ad type S (AdS), has facilitated extensive manipulation of the Ads and the development of recombinant Ad vectors. The study of bovine adenoviruses (BAVs), which cause mild respiratory and gastrointestinal infections in cattle has, on the other hand, been limited primarily to that of infectivity, immunology and clinicallllanifestations. As a result, any potential as gene delivery vehicles has not yet been realized. Continued research into the molecular biolo~gy of BAVs and the development of recolllbinant vectors would benefit from the development of a cell line analogous to that of the 293 cells. In an attelllpt to establish such a cell line, the recombinant plaslllid pKC-neo was constructed, containing the left 0-19.7% of the BAV type 3 (BAV3) genome, and the selectable marker for resistance to the aminoglycoside G418, a neomycin derivative. The plasmid construct was then used to transfect both the Madin-Darby bovine kidney (MDBK) -iicell line and primary bovine lung cells, after which G418-resistant foci were selected for analysis. Two cell lines, E61 (MDBK) and E24 (primary lung), were subsequently selected and analysed for DNA content, revealing the presence of the pKC-neo sequences in their respective genomes. In addition, BAV3 RNA transcripts were detected in the E61 cells. Although the presence of E1 products has yet to be confirmed in both cell lines, the E24 cells exhibit a phenotype characteristic of partial transformation by E1. The apparent immortalization of the primary lung cells will permit exploitation of their ability to take up exogenous DNA at high efficiency.

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This study examined the effects that a training program in phonological awareness had on the early writing skills of children in a Grade One class in the Lincoln County Separate school system. The intent of the training program was to provide consistent and systematic practice in the manipulation of the phonological structure of language. The games and activities of the training program were related to a framework of developmental phonological skills and practised in a group setting during an unstructured period of the regular classroom schedule. The training program operated three days in a six-day cycle for approximately twenty minutes a day, from November until mid-March. All children were tested at the outset and conclusion of the study to determine level of functioning in letter identification, word recognition, verbal intelligence, phonological awareness and spelling. Results of the pre-tests and post-tests were compared to determine differences between the experimental and control groups over time. In addition, a systematic analysis of the children's writing looked at the development of the spelling of regular and irregular words. The results of this study provided strong support for the hypothesis that the treatment group would progress through the stages of early writing development more quickly than children without such training. On the basis of differences between the groups over time, it was evident that training in phonological awareness had a direct positive effect on the spelling of regular words for children during the early stages of writing. The training program did not have a significant effect on the spelling of irregular words. Test results evaluating phonological awareness indicated a significant difference within each group over time but no significance between the groups during the experimental period. It would appear that the results of these tests reflect maturational changes in the child rather than causal effects of the training program. Nor did the effects of the training program transfer significantly to other aspects of language. Although some of the hypotheses considered were not supported by the study, the results do indicate that children during the early stages of writing development can benefit from a training program in phonological awareness. The theoretical direction for effective programming as a result of this study is discussed. The educational implications of training phonological awareness concurrent to beginning efforts in writing are considered.

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Daytime napping improves well-being and performance for young adults. The benefits of napping in older adults should be investigated because they have fragmented nocturnal sleep, cognitive declines, and more opportunity to nap. In addition, experience with napping might influence the benefits of napping. Study 1 examined the role of experience with napping in young adults. Habitual (n = 23) and non-habitual nappers (n = 16) were randomly assigned to a 20-minute nap or a 20- minute reading condition. Both groups slept the same according to macro architecture. However, microarchitecture showed greater theta, alpha, and beta power during Stage 1, and greater delta, alpha, and sigma power during Stage 2 for habitual nappers, for the most part indicating better sleep. Both groups felt less sleepy after the nap. P2 latency, reflecting information processing, decreased after the nap for habitual nappers, and after the control condition for non-habitual nappers. In sum, both groups who slept felt better, but only the habitual nappers who napped gained a benefit in terms of information processing. Based on this outcome, experience with napping was investigated in Study 2. Study 2 examined the extent to which daytime napping enhanced cognition in older adults, especially frontal lobe function. Cognitive deficits in older adults may be due to sleep loss and age-related decline in brain functioning. Longer naps were expected to provide greater improvement, particularly for older adults, by reducing sleep pressure. Thirty-two adults, aged 24-70 years, participated in a repeated measures dose-response manipulation of sleep pressure. Twenty- and sixty-minute naps were compared to a no-nap condition in three age groups. Mood, subjective sleepiness, reaction time, working memory, 11 novelty detection, and waking electro physiological measures were taken before and after each condition. EEG was also recorded during each nap or rest condition. Napping reduced subjective sleepiness, improved working memory (serial addition / subtraction task), and improved attention (reduced P2 amplitude). Physiological sleepiness (i.e., waking theta power) increased following the control condition, and decreased after the longer nap. Increased beta power after the short nap, and seen with older adults overall, may have reflected increased mental effort. Older adults had longer latencies and smaller amplitudes for several event-related potential components, and higher beta and gamma power. Following the longer nap, gamma power decreased for older adults, but increased for young adults. Beta and gamma power may represent enhanced alertness or mental effort. In addition, Nl amplitude showed that benefits depend on the preceding nap length as well as age. Since the middle group had smaller Nl amplitudes following the short nap and rest condition, it is possible that they needed a longer nap to maintain alertness. Older adults did not show improvements to Nl amplitude following any condition; they may have needed a nap longer than 60 minutes to gain benefits to attention or early information processing. Sleep characteristics were not related to benefits of napping. Experience with napping was also investigated. Subjective data confirmed habitual nappers were happier to nap, while non-habitual nappers were happier to stay awake, reflecting self-identified napping habits. Non-habitual nappers were sleepier after a nap, and had faster brain activity (i.e., heightened vigilance) at sleep onset. These reasons may explain why non-habitual nappers choose not to nap.

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The presence of rewards has been found to undermine intrinsic motivation (Deci & Ryan, 1999). This conclusion is primarily based on research conducted in non-sporting environments. The purpose of this study was to examine perceived motivational changes resulting from the hypothetical manipulation of a reward (i.e., athletic scholarships). Differences in "present" motivation between scholarship and non-scholarship athletes were also assessed. Gender, life roles, and sport experience were also examined in relation to scholarship status. Basketball players from four Ontario (n = 70) and seven U.S. Division I universities (n = 46) were examined. All athletes completed a set of demographic questions, as well as questions from the Sport Motivation Scale (SMS; Pelletier, Fortier, Vallerand, Tuson, Briere, & Blais, 1995) which assessed their "present" motivation. Athletes also completed the SMS to evaluate their "perceived future" motivation based on a hypothetical manipulation of the scholarship status. For Ontario non-scholarship athletes, extrinsic regulation (an extrinsic motive) increased with the introduction of a scholarship and the intrinsic motive to experience stimulation decreased. For U.S. scholarship athletes, the intrinsic motive to accomplish things decreased when scholarships were removed. When the two scholarship status groups were compared across "present" levels of motivation, U.S. scholarship males reported significantly higher levels of introjected regulation compared to Ontario non-scholarship males. Ontario non-scholarship females reported significantly higher levels of introjected regulation compared to U.S. scholarship females. U.S. scholarship athletes reported significantly higher levels of external regulation compared to Ontario non-scholarship athletes. Results offer partial support for self-determination theory. Implications for future research are discussed.

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The ease of production and manipulation has made plasmid DNA a prime target for its use in gene transfer technologies such as gene therapy and DNA vaccines. The major drawback of plasmid however is its stability within mammalian cells. Plasmid DNA is usually lost by cellular mechanisms or as a result of mitosis by simple dilution. This study set out to search for mammalian genomic DNA sequences that would enhance the stability of plasmid DNA in mammalian cells.Creating a plasmid based genomic DNA library, we were able to screen the human genome by transfecting the library into Human Embryonic Kidney (HEK 293) Cells. Cells that contained plasmid DNA were selected, using G418 for 14 days. The resulting population was then screened for the presence of biologically active plasmid DNA using the process of transformation as a detector.A commercially available plasmid DNA isolation kit was modified to extract plasmid DNA from mammalian cells. The standardized protocol had a detection limit of -0.6 plasmids per cell in one million cells. This allowed for the detection of 45 plasmids that were maintained for 32 days in the HEK 293 cells. Sequencing of selected inserts revealed a significantly higher thymine content in comparison to the human genome. Sequences with high A/T content have been associated with Scaffold/Matrix Attachment Region (S/MAR) sequences in mammalian cells. Therefore, association with the nuclear matrix might be required for the stability of plasmids in mammalian cells.

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The effects of a complexly worded counterattitudinal appeal on laypeople's attitudes toward a legal issue were examined, using the Elaboration Likelihood Model (ELM) of persuasion as a theoretical framework. This model states that persuasion can result from the elaboration and scrutiny of the message arguments (i.e., central route processing), or can result from less cognitively effortful strategies, such as relying on source characteristics as a cue to message validity (i.e., peripheral route processing). One hundred and sixty-seven undergraduates (85 men and 81 women) listened to eitller a low status or high status source deliver a counterattitudinal speech on a legal issue. The speech was designed to contain strong or weak arguments. These arguments were 'worded in a simple and, therefore, easy to comprehend manner, or in a complex and, therefore, difficult to comprehend manner. Thus, there were three experimental manipulations: argument comprehensibility (easy to comprehend vs. difficult to comprehend), argumel11 strength (weak vs. strong), and source status (low vs. high). After listening to tIle speec.J] participants completed a measure 'of their attitude toward the legal issue, a thought listil1g task, an argument recall task,manipulation checks, measures of motivation to process the message, and measures of mood. As a result of the failure of the argument strength manipulation, only the effects of the comprehel1sibility and source status manipulations were tested. There was, however, some evidence of more central route processing in the easy comprehension condition than in the difficult comprehension condition, as predicted. Significant correlations were found between attitude and favourable and unfavourable thoughts about the legal issue with easy to comprehend arguments; whereas, there was a correlation only between attitude and favourable thoughts 11 toward the issue with difficult to comprehend arguments, suggesting, perhaps, that central route processing, \vhich involves argument scrutiny and elaboration, occurred under conditions of easy comprehension to a greater extent than under conditions of difficult comprehension. The results also revealed, among other findings, several significant effects of gender. Men had more favourable attitudes toward the legal issue than did women, men recalled more arguments from the speech than did women, men were less frustrated while listening to the speech than were ,vomen, and men put more effort into thinking about the message arguments than did women. When the arguments were difficult to comprehend, men had more favourable thoughts and fewer unfavourable thoughts about the legal issue than did women. Men and women may have had different affective responses to the issue of plea bargaining (with women responding more negatively than men), especially in light of a local and controversial plea bargain that occurred around the time of this study. Such pre-existing gender differences may have led to tIle lower frustration, the greater effort, the greater recall, and more positive attitudes for men than for WOlnen. Results· from this study suggest that current cognitive models of persuasion may not be very applicable to controversial issues which elicit strong emotional responses. Finally, these data indicate that affective responses, the controversial and emotional nature ofthe issue, gender and other individual differences are important considerations when experts are attempting to persuade laypeople toward a counterattitudinal position.

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Research implies that there ~ay be an association between attitudes toward margil1alized human outgroups and non-human animals. Very few studies, however, have specifically tested this relation empirically. The general purpose of the present research was to determine if such a relation exists and if perceptions of human-animal similarity avail as a common predictor of both types of attitudes. Ideological orientations associated with prejudiced attitudes (Social Dominance Orientation, Right-Wing Authoritarianism, and Universal Orientation) were also examined as individual differences in predicting perceptions of human-animal similarity. As predicted, people who endorsed prejudiced attitudes toward human outgroups (Study 1) and immigrants in particular (Studies 2 and 3), were more likely to endorse prejudiced attitudes toward non-human animals. In Study 2, perceptions that humans are superior (versus similar) to other animals directly predicted higher levels of prejudice toward non-human animals, whereas the effect of human superiority beliefs on immigrant prejudice was mediated by dehumanization. In other words, greater perceptions of humans as superior (versus similar) to other animals "allowed for" greater dehumanization of immigrants, which in turn resulted in heightened immigrant prejudice. Furthermore, people higher in Social Dominance Orientation or Right-Wing Authoritarianism were particularly likely to perceive humans as superior (versus similar) to other animals, whereas people characterized by a greater Universal Orientation were more likely to perceive humans and non-human animals as similar. Study 3 examined whether inducing perceptions of human-animal similarity through experimental manipulation would lead to more favourable attitudes toward non-human animals and immigrants. Participants were randomly assigned to read one of four 11 editorials designed to highlight either the similarities or differences between humans and other animals (i.e., animals are similar to humans; humans are similar to animals;~~nimals are inferior to humans; humans are superior to animals) or to a neutral control condition. Encouragingly, when animals were described as similar to humans, prejudice towards non-human animals and immigrants was significantly lower, and to some extent this finding was also true for people naturally high in prejudice (i.e., high in Social Dominance Orientation or Right-Wing Authoritarianism). Inducing perceptions that nonhuman animals are similar to humans was particularly effective at reducing the tendency to dehumanize immigrants ("re-humanization"), lowering feelings of personal threat regarding one's animal-nature, and at increasing inclusive intergroup representations and empathy, all of which uniquely accounted for the significant decreases in prejudiced attitudes. Implications for research, theory and prejudice interventions are considered.