Produção de um modelo animal para o estudo de doenças causadoras de desmielinização do sistema nervoso central

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Síntese, caracterização e avaliação biológica de quelatos metálicos contendo ligantes de origem natural

O trabalho que será aqui apresentado teve como finalidade reunir os resultados obtidos dentro do Laboratório de Organometálicos II em relação aos estudos realizados acerca da interação de íons metálicos com ligantes orgânicos naturais. Os dados apresentados foram obtidos pela autora deste trabalho, e também pelos professores Francisco Otávio Cintra Ferrarini, José Roberto Stefanelli Junior e pela aluna de Iniciação Científica Angélica Romão Carvalho. Hidrolisado proteico de soro de leite bovino e torta de linhaça foram os materiais obtidos, após uma série de operações que envolveram filtração com caulim, diálise, hidrólise enzimática, liofilização, etc., após as quais foram utilizados neste trabalho como ligantes de origem natural. Os metais de transição cobre, ferro, cobalto, zinco, paládio, platina e prata foram utilizados nas reações de complexação com os hidrolisados para obtenção de complexos metálicos, através do uso de diferentes rotas sintéticas (convencional, micro-ondas, hidrotérmica, difusão) e parâmetros experimentais (pH, solvente, natureza do precursor, etc.). A escolha dos íons metálicos baseou-se no interesse em preparar compostos que apresentassem ao mesmo tempo alta biodisponibilidade, baixa citotoxicidade e diversas aplicações biológicas. O uso do cobalto deveu-se à reconhecida propriedade que os micro-organismos do sistema digestório dos ruminantes apresenta de sintetizar a vitamina B12 e, portanto, os compostos desse metal podem ser usados em rações visando um melhor desenvolvimento do animal. O ferro, conhecidamente, é um importante metal para os seres humanos, estando presente na hemoglobina, sendo assim, também consiste em uma substância nutracêutica de relevado interesse. À propósito, cabe salientar que os compostos de ferro (III) obtidos nesse trabalho serão objeto de avaliação do uso como suplemento alimentar, em colaboração com o Departamento de...

SCWDS BRIEFS: Volume 20, Number 2 (July 2004)

National Veterinary Services Laboratories in Ames, Iowa, confirmed vesicular stomatitis (VS) in horses at one premises in Texas. As of July 21, 2004, infected animals were identified on a total of 45 premises in Colorado (11), New Mexico (21), and Texas (13). These are the first reports of VS in livestock in the United States since the 1998 epizootic. SCWDS VS Studies Chronic Wasting Disease Developments: nearly 118,000 wild white-tailed deer, mule deer, and elk were tested in the United States from October 2002 to September 2003, with 592 animals testing positive for the CWD prion. More than $38,000,000 was spent by federal and state wildlife and animal health agencies on CWD-related activities during this same period. The Second International Chronic Wasting Disease Symposium, hosted by the Wisconsin Department of Natural Resources, will be held in Madison, Wisconsin, July 12-14, 2005. Crow Decoys Used in West Nile Virus Study Although Lyme disease caused by a spirochete bacterium, Borrelia burgdorferi, is relatively rare in the southeastern United States, a Lyme disease-like infection referred to as Southern Tick-Associated Rash Illness (STARI) and thought to be caused by Borrelia lonestari, has been recognized in people in this region. The Ohio Division of Wildlife joined SCWDS as an associate member beginning July 1, 2004. The Final Report of the 2003 Hemorrhagic Disease (HD) Surveillance project has been completed and distributed to all cooperators. New of Caroline Duffie, Robbie Edalgo and wife Jen, Clay George, Darrell Kavanaugh, Lynn Lewis-Weiss’s husband, Dr. Kevin Weiss, and Nate Mechlin. New SCWDS staff include Brian Chandler, Jay Cumbee, Ginger Goekjian, Bill Hamrick, Sabrina McGraw, Kerri Pedersen, and Ben Wilcox. Recent SCWDS Publications Available

Produção de um modelo animal para o estudo de doenças causadoras de desmielinização do sistema nervoso central

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Síntese, caracterização e avaliação biológica de quelatos metálicos contendo ligantes de origem natural

O trabalho que será aqui apresentado teve como finalidade reunir os resultados obtidos dentro do Laboratório de Organometálicos II em relação aos estudos realizados acerca da interação de íons metálicos com ligantes orgânicos naturais. Os dados apresentados foram obtidos pela autora deste trabalho, e também pelos professores Francisco Otávio Cintra Ferrarini, José Roberto Stefanelli Junior e pela aluna de Iniciação Científica Angélica Romão Carvalho. Hidrolisado proteico de soro de leite bovino e torta de linhaça foram os materiais obtidos, após uma série de operações que envolveram filtração com caulim, diálise, hidrólise enzimática, liofilização, etc., após as quais foram utilizados neste trabalho como ligantes de origem natural. Os metais de transição cobre, ferro, cobalto, zinco, paládio, platina e prata foram utilizados nas reações de complexação com os hidrolisados para obtenção de complexos metálicos, através do uso de diferentes rotas sintéticas (convencional, micro-ondas, hidrotérmica, difusão) e parâmetros experimentais (pH, solvente, natureza do precursor, etc.). A escolha dos íons metálicos baseou-se no interesse em preparar compostos que apresentassem ao mesmo tempo alta biodisponibilidade, baixa citotoxicidade e diversas aplicações biológicas. O uso do cobalto deveu-se à reconhecida propriedade que os micro-organismos do sistema digestório dos ruminantes apresenta de sintetizar a vitamina B12 e, portanto, os compostos desse metal podem ser usados em rações visando um melhor desenvolvimento do animal. O ferro, conhecidamente, é um importante metal para os seres humanos, estando presente na hemoglobina, sendo assim, também consiste em uma substância nutracêutica de relevado interesse. À propósito, cabe salientar que os compostos de ferro (III) obtidos nesse trabalho serão objeto de avaliação do uso como suplemento alimentar, em colaboração com o Departamento de...

Interaction of dietary vitamin D-3 and sunlight exposure on B. indicus cattle: Animal performance, carcass traits, and meat quality

Attempts to improve beef tenderness through supplementation with dietary vitamin D-3 have been challenged by null results and negative impacts on animal performance and carcass traits. Because vitamin D-3 is also synthesised by the animal via ultraviolet radiation from sunlight, the effectiveness of supplementation with dietary vitamin D-3 may be modulated by the degree of exposure of the animal to sunlight. Hence, this work aimed to verify whether dietary vitamin D-3 modifies meat quality without negatively affecting animal performance and carcass traits in B. indicus beef cattle that were either exposed to or protected from natural sunlight. Forty-two (411 +/- 38 kg) Nellore-type castrated males were fed a high-concentrate diet for 45 days after assignment to a treatment group. The treatments comprised combinations of three levels of vitamin D3 [ViTD - none (V0) or 2 x 10(6) IU of vitamin D-3 administered for either 2 (V2) or 8 (V8) consecutive days pre-slaughter] and two shading conditions (SHADE - unshaded or shaded). The post-mortem (pm) measurements were taken in the Longissimus thoracis et lumborum muscle. The animal performance and carcass traits were unaffected by ViTD or SHADE The V2 treatment increased the Myofibrillar Fragmentation Index in shaded animals compared to unshaded ones. Animals under shade had higher muscle calcium concentration. There was no effect of either ViTD or SHADE on the shear force. The L* values were higher at 24 h pm than at 0 and 1 h pm, with no differences among the animals in the ViTD or SHADE groups. Higher a* values were observed among animals in the V8 group than in the V0 group, and higher b* values were observed among animals in the V8 group than in the V2 or V0 groups, which were not different. In conclusion, ViTD and SHADE did not affect animal performance, carcass traits or shear force, whereas animals receiving a lower ViTD dosage and SHADE exhibited altered myofibrillar fragmentation. ViTD affected the colour parameters, and changes in the lightness of the beef related to the time pm were found in meat from animals under SHADE. (c) 2012 Elsevier B.V. All rights reserved.

Taxonomy, Hemipenial Morphology, and Natural History of Two Poorly Known Species of Anadia (Gymnophthalmidae) from Northern South America

Anadia pariaensis Rivas, La Marca, and Oliveros, 1999, and Anadia steyeri Nieden, 1914, are two particularly rare and poorly known lizards described from single specimens. In the case of A. pariaensis, it remains known from the holotype, whereas A. steyeri is known from three additional specimens reported in the literature after the original description of the species. A single new specimen of A. pariaensis and five of A. steyeri, including the first adult males recorded for both species, make possible a more representative description of both species, including descriptions of the hemipenes. Despite both species presenting some similar morphological characteristics, the examination of the hemipenial morphology revealed very different organs. The hemipenis of A. steyeri presents some characteristics that resemble the organs of two species from the Santa Marta Mountain Range in the "bitaeniata-group" (Anadia pulchella and Anadia altaserrania). On the other hand, the hemipenes of A. pariaensis are unique morphologically and cannot be associated with the hemipenes known from other species in the genus. We describe variation within both species, and we comment on possible sexual dimorphism (number and arrangement of the femoral pores), natural history, and the known geographic distribution of the species. We also comment on Anadia bumanguesa Rueda-Almonacid and Caicedo 2004 based on a new specimen, the second known. This species may be a synonym of A. steyeri.

Stem Cells as a therapy for myocardial infarction in animal models

Advances in stem cell biology have challenged the notion that infarcted myocardium is irreparable. The pluripotent ability of stem cells to differentiate into specialized cell lines began to garner intense interest within cardiology when it was shown in animal models that intramyocardial injection of bone marrow stem cells (MSCs), or the mobilization of bone marrow stem cells with spontaneous homing to myocardium, could improve cardiac function and survival after induced myocardial infarction (MI) [1, 2]. Furthermore, the existence of stem cells in myocardium has been identified in animal heart [3, 4], and intense research is under way in an attempt to clarify their potential clinical application for patients with myocardial infarction. To date, in order to identify the best one, different kinds of stem cells have been studied; these have been derived from embryo or adult tissues (i.e. bone marrow, heart, peripheral blood etc.). Currently, three different biologic therapies for cardiovascular diseases are under investigation: cell therapy, gene therapy and the more recent “tissue-engineering” therapy . During my Ph.D. course, first I focalised my study on the isolation and characterization of Cardiac Stem Cells (CSCs) in wild-type and transgenic mice and for this purpose I attended, for more than one year, the Cardiovascular Research Institute of the New York Medical College, in Valhalla (NY, USA) under the direction of Doctor Piero Anversa. During this period I learnt different Immunohistochemical and Biomolecular techniques, useful for investigating the regenerative potential of stem cells. Then, during the next two years, I studied the new approach of cardiac regenerative medicine based on “tissue-engineering” in order to investigate a new strategy to regenerate the infracted myocardium. Tissue-engineering is a promising approach that makes possible the creation of new functional tissue to replace lost or failing tissue. This new discipline combines isolated functioning cells and biodegradable 3-dimensional (3D) polymeric scaffolds. The scaffold temporarily provides the biomechanical support for the cells until they produce their own extracellular matrix. Because tissue-engineering constructs contain living cells, they may have the potential for growth and cellular self-repair and remodeling. In the present study, I examined whether the tissue-engineering strategy within hyaluron-based scaffolds would result in the formation of alternative cardiac tissue that could replace the scar and improve cardiac function after MI in syngeneic heterotopic rat hearts. Rat hearts were explanted, subjected to left coronary descending artery occlusion, and then grafted into the abdomen (aorta-aorta anastomosis) of receiving syngeneic rat. After 2 weeks, a pouch of 3 mm2 was made in the thickness of the ventricular wall at the level of the post-infarction scar. The hyaluronic scaffold, previously engineered for 3 weeks with rat MSCs, was introduced into the pouch and the myocardial edges sutured with few stitches. Two weeks later we evaluated the cardiac function by M-Mode echocardiography and the myocardial morphology by microscope analysis. We chose bone marrow-derived mensenchymal stem cells (MSCs) because they have shown great signaling and regenerative properties when delivered to heart tissue following a myocardial infarction (MI). However, while the object of cell transplantation is to improve ventricular function, cardiac cell transplantation has had limited success because of poor graft viability and low cell retention, that’s why we decided to combine MSCs with a biopolimeric scaffold. At the end of the experiments we observed that the hyaluronan fibres had not been substantially degraded 2 weeks after heart-transplantation. Most MSCs had migrated to the surrounding infarcted area where they were especially found close to small-sized vessels. Scar tissue was moderated in the engrafted region and the thickness of the corresponding ventricular wall was comparable to that of the non-infarcted remote area. Also, the left ventricular shortening fraction, evaluated by M-Mode echocardiography, was found a little bit increased when compared to that measured just before construct transplantation. Therefore, this study suggests that post-infarction myocardial remodelling can be favourably affected by the grafting of MSCs delivered through a hyaluron-based scaffold

Estudio etiopatogénico del circovirus porcino tipo 2 asociado a virus y bacterias en una infección natural

Programa de doctorado: Sanidad y patología animal

Natural patterns and anthropogenic disturbance in north Adriatic marine benthic assemblages: descriptive and methodological studies

Marine soft bottom systems show a high variability across multiple spatial and temporal scales. Both natural and anthropogenic sources of disturbance act together in affecting benthic sedimentary characteristics and species distribution. The description of such spatial variability is required to understand the ecological processes behind them. However, in order to have a better estimate of spatial patterns, methods that take into account the complexity of the sedimentary system are required. This PhD thesis aims to give a significant contribution both in improving the methodological approaches to the study of biological variability in soft bottom habitats and in increasing the knowledge of the effect that different process (both natural and anthropogenic) could have on the benthic communities of a large area in the North Adriatic Sea. Beta diversity is a measure of the variability in species composition, and Whittaker’s index has become the most widely used measure of beta-diversity. However, application of the Whittaker index to soft bottom assemblages of the Adriatic Sea highlighted its sensitivity to rare species (species recorded in a single sample). This over-weighting of rare species induces biased estimates of the heterogeneity, thus it becomes difficult to compare assemblages containing a high proportion of rare species. In benthic communities, the unusual large number of rare species is frequently attributed to a combination of sampling errors and insufficient sampling effort. In order to reduce the influence of rare species on the measure of beta diversity, I have developed an alternative index based on simple probabilistic considerations. It turns out that this probability index is an ordinary Michaelis-Menten transformation of Whittaker's index but behaves more favourably when species heterogeneity increases. The suggested index therefore seems appropriate when comparing patterns of complexity in marine benthic assemblages. Although the new index makes an important contribution to the study of biodiversity in sedimentary environment, it remains to be seen which processes, and at what scales, influence benthic patterns. The ability to predict the effects of ecological phenomena on benthic fauna highly depends on both spatial and temporal scales of variation. Once defined, implicitly or explicitly, these scales influence the questions asked, the methodological approaches and the interpretation of results. Problem often arise when representative samples are not taken and results are over-generalized, as can happen when results from small-scale experiments are used for resource planning and management. Such issues, although globally recognized, are far from been resolved in the North Adriatic Sea. This area is potentially affected by both natural (e.g. river inflow, eutrophication) and anthropogenic (e.g. gas extraction, fish-trawling) sources of disturbance. Although few studies in this area aimed at understanding which of these processes mainly affect macrobenthos, these have been conducted at a small spatial scale, as they were designated to examine local changes in benthic communities or particular species. However, in order to better describe all the putative processes occurring in the entire area, a high sampling effort performed at a large spatial scale is required. The sedimentary environment of the western part of the Adriatic Sea was extensively studied in this thesis. I have described, in detail, spatial patterns both in terms of sedimentary characteristics and macrobenthic organisms and have suggested putative processes (natural or of human origin) that might affect the benthic environment of the entire area. In particular I have examined the effect of off shore gas platforms on benthic diversity and tested their effect over a background of natural spatial variability. The results obtained suggest that natural processes in the North Adriatic such as river outflow and euthrophication show an inter-annual variability that might have important consequences on benthic assemblages, affecting for example their spatial pattern moving away from the coast and along a North to South gradient. Depth-related factors, such as food supply, light, temperature and salinity play an important role in explaining large scale benthic spatial variability (i.e., affecting both the abundance patterns and beta diversity). Nonetheless, more locally, effects probably related to an organic enrichment or pollution from Po river input has been observed. All these processes, together with few human-induced sources of variability (e.g. fishing disturbance), have a higher effect on macrofauna distribution than any effect related to the presence of gas platforms. The main effect of gas platforms is restricted mainly to small spatial scales and related to a change in habitat complexity due to a natural dislodgement or structure cleaning of mussels that colonize their legs. The accumulation of mussels on the sediment reasonably affects benthic infauna composition. All the components of the study presented in this thesis highlight the need to carefully consider methodological aspects related to the study of sedimentary habitats. With particular regards to the North Adriatic Sea, a multi-scale analysis along natural and anthopogenic gradients was useful for detecting the influence of all the processes affecting the sedimentary environment. In the future, applying a similar approach may lead to an unambiguous assessment of the state of the benthic community in the North Adriatic Sea. Such assessment may be useful in understanding if any anthropogenic source of disturbance has a negative effect on the marine environment, and if so, planning sustainable strategies for a proper management of the affected area.

Rare and complicated forms of infantile hemangiomas: new therapeutic outlooks

Infantile hemangiomas (IHs) are the most common benign neoplastic pathology of childhood; their natural history generally involves three phases: after the onset, which usually occurs in the first weeks of life, there is the proliferation phase where the IH reaches its maximum development and it is followed by the spontaneous involution which leads to the IH regression. The duration and the extent of these phases may vary widely even though in most of the cases the involution process begins around twelve months of life and the regression, complete or partial, is completed around seventh-ninth year of life. The majority of the IHs does not require any treatment. However, 10%-20% is likely to develop serious complications, functional impairments or aesthetic alterations and entail a timely treatment. Although there is no treatment protocol currently shared, therapies usually used in cases with a complication risk consist in: systemic or intralesional steroids as a first choice; interferon α, vincristine and/or bleomicin as second or third choice and/or surgical treatment. Propranolol, a non-selective beta-blocker, has been used for cardiovascular diseases even in childhood for decades. Since 2008 it has been widely used in the IHs treatment, although it is still "off-label". In literature there are hundreds of cases and some clinical studies that show the effectiveness and safety of this drug for this indication. Thanks to a multidisciplinary team (Dermatologists, Cardiologists, Paediatricians, and Radiologists) of S. Orsola-Malpighi Hospital, a clinical study, which has been previously approved by the ethics committee, is carried out in order to evaluate the efficacy and safety of systemic propranolol in the treatment of IHs in paediatric age. At the end of 2012, 78 patients underwent this treatment: the results we have obtained so far show a good efficacy and safety profile in agreement with the data provided by the literature.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.

The geometry of Lagrangian fibres

If the generic fibre f−1(c) of a Lagrangian fibration f : X → B on a complex Poisson– variety X is smooth, compact, and connected, it is isomorphic to the compactification of a complex abelian Lie–group. For affine Lagrangian fibres it is not clear what the structure of the fibre is. Adler and van Moerbeke developed a strategy to prove that the generic fibre of a Lagrangian fibration is isomorphic to the affine part of an abelian variety.rnWe extend their strategy to verify that the generic fibre of a given Lagrangian fibration is the affine part of a (C∗)r–extension of an abelian variety. This strategy turned out to be successful for all examples we studied. Additionally we studied examples of Lagrangian fibrations that have the affine part of a ramified cyclic cover of an abelian variety as generic fibre. We obtained an embedding in a Lagrangian fibration that has the affine part of a C∗–extension of an abelian variety as generic fibre. This embedding is not an embedding in the category of Lagrangian fibrations. The C∗–quotient of the new Lagrangian fibration defines in a natural way a deformation of the cyclic quotient of the original Lagrangian fibration.

Gene therapy for a novel mouse model of Canavan disease

Canavan disease (CD) is a rare leukodystrophy caused by loss-of-function mutations in the gene encoding aspartoacylase (ASPA), an oligodendrocyte-enriched enzyme. It is characterised by the accumulation of the ASPA substrate N-acetylaspartate (NAA) in brain, blood and urine, leading to a spongiform vacuolisation of the brain, severe motoric and cognitive impairments and premature death. To date, no therapy is available due to the lack of a gene-transfer system allowing transgene expression in oligodendrocytes (OLs) and the restoration of the missing enzyme. Hence, the aim of this study was to establish a novel gene-transfer system and its preclinical evaluation in a CD animal model.rnIn the first part of this thesis, a novel ASPA mouse mutant was generated. A βgeo cassette (including the genes encoding β-galactosidase and neomycin) flanked by frt sites was inserted into intron 1 of the intact aspa gene. Additionally, exon 2 was flanked by loxP sites for optional conditional deletion of the targeted locus. The resulting ASPA-deficient aspalacZ/lacZ-mouse was found to be an accurate model of CD and an important tool to identify novel aspects of its complex pathology. Homozygous mutants showed a CD-like histopathology, neurological impairment, behavioural deficits as well as a reduced body weight. Additionally, MRI data revealed changes in brain metabolite composition. rnRecombinant adeno-associated viral (rAAV) vectors have become a versatile tool for gene transfer to the central nervous system because they are efficient, non-toxic and replication-deficient. Based on the natural neurotropism of AAV vectors, AAV-based gene delivery has entered the clinics for the treatment of neurodegenerative diseases. However, the lack of AAV vectors with oligodendroglial tropism has precluded gene therapy for leukodystrophies. In the second part of this work, it was shown that the transduction profile of established AAV serotypes can be targeted towards OLs in a transcriptional approach, using the oligodendrocyte-specific myelin basic protein (MBP) promoter to drive transgene expression in OLs.rnIn the last part of this work, the therapeutic efficacy of AAV-mediated aspa gene transfer to OLs of juvenile aspalacZ/lacZ mice was evaluated. AAV-aspa injections into multiple sites of the brain parenchyma resulted in transduction of OLs in the grey and white matter throughout the brain. Histological abnormalities in the brain of ASPA-deficient mice were ameliorated and accompanied by a reduction of NAA levels. Furthermore, the treatment resulted in normalisation of body weight, motor function and nest-building behaviour. These data provide a proof-of-concept for a successful gene therapy of Canavan disease. This might pave the way towards translation into clinical application and serve as the basis for the genetic treatment of other leukodystrophies.

Early bursts of body size and shape evolution are rare in comparative data

George Gaylord Simpson famously postulated that much of life's diversity originated as adaptive radiations-more or less simultaneous divergences of numerous lines from a single ancestral adaptive type. However, identifying adaptive radiations has proven difficult due to a lack of broad-scale comparative datasets. Here, we use phylogenetic comparative data on body size and shape in a diversity of animal clades to test a key model of adaptive radiation, in which initially rapid morphological evolution is followed by relative stasis. We compared the fit of this model to both single selective peak and random walk models. We found little support for the early-burst model of adaptive radiation, whereas both other models, particularly that of selective peaks, were commonly supported. In addition, we found that the net rate of morphological evolution varied inversely with clade age. The youngest clades appear to evolve most rapidly because long-term change typically does not attain the amount of divergence predicted from rates measured over short time scales. Across our entire analysis, the dominant pattern was one of constraints shaping evolution continually through time rather than rapid evolution followed by stasis. We suggest that the classical model of adaptive radiation, where morphological evolution is initially rapid and slows through time, may be rare in comparative data.