A study of protein targeting reveals insights into mitigating protein aggregation

A unique chloroplast Signal Recognition Particle (SRP) in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll-a/b binding (LHC) proteins. Our study of the thermodynamics and kinetics of the GTPases of the system demonstrates that GTPase complex assembly and activation are highly coupled in the chloroplast GTPases, suggesting they may forego the GTPase activation step as a key regulatory point. This reflects adaptations of the chloroplast SRP to the delivery of their unique substrate protein. Devotion to one highly hydrophobic family of proteins also may have allowed the chloroplast SRP system to evolve an efficient chaperone in the cpSRP43 subunit. To understand the mechanism of disaggregation, we showed that LHC proteins form micellar, disc-shaped aggregates that present a recognition motif (L18) on the aggregate surface. Further molecular genetic and structure-activity analyses reveal that the action of cpSRP43 can be dissected into two steps: (i) initial recognition of L18 on the aggregate surface; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. We also tested the adaptability of cpSRP43 for alternative substrates, specifically in attempts to improve membrane protein expression and inhibition of amyloid beta fibrillization. These preliminary results attest to cpSRP43’s potential as a molecular chaperone and provides the impetus for further engineering endeavors to address problems that stem from protein aggregation.

Chemistry of low-valent platinum dimers

Physical and chemical properties of low-valent platinum dimers, namely [Pt_2(P_2O_5H_2)4]^(4-) and Pt_2(µ-dppm)_2Cl_2, have been investigated using a variety of structural and spectroscopic techniques.

Platinum(II) d^8-d^8 dimers have been shown to exhibit much thermal and photochemical reactivity. Chapter 2 describes studies aimed at elucidating the excited state reduction potenetial of [Pt_2(P_2O_5H_2)4]^(4-), Pt_2, in organic media. By conducting excited state electron transfer studies using derivatized pyridiniums and benzophenones, the excited state reduction potential has been estimated to be ~2 V. The Pt_2 complex undergoes partial oxidation to form Pt(II,III) linear chains. Chapter 3 describes the structural and spectroscopic techniques used to determine the translational symmetries of these [Pt_2(P_2O_5H_2)4]^(4-) (X = Cl, Br), Pt_2X, chains. Pt_2Br has been found to be intermediate between (AAB)_n and (AABCCB)_n, while, Pt_2Cl is of (AABCCB)_n translational symmetry. Investigations into the electronic transitions of Pt_2Cl and Pt_2Br were conducted using high pressure techniques and are presented in Chapter 4. The Pt_2X electronic spectrum exhibits bands attributable to the reduced Pt2 complex and the oxidized Pt_2X_2 complex [Pt_2(P_2O_5H_2)4]^(4-) along with an intervalence charge-tranfer band characteristic of a mixed-valence solid.

Photophysical investigations of a new luminescent chromophore, Pt_2(µ-dppm)_2Cl_2, a d^9-d^9 dimer, and its analogs are described in Chapter 5. The absorption band directly responsible for the observed emission is believed to be very weak and, as of yet, unobserved. Attempts to determine the spin multiplicty and approximate energy of this unobserved transition are described in Chapter 6. Excited-state energy transfer studies indicate that this absorption band is a triplet transition at -13,000 cm^(-1). Although, the Pt_2(µ-dppm)_2Cl_2 excited state is non-luminescent in fluid solution, it has been shown to undergo thermal electron transfer to tetracyanoethylene and photoinduced electron transfer to methylviologen. These experiments are presented in Chapter 7. Preliminary studies, described in Chapter 8, of non-bridged d^9-d^9 platinum(I) dimers have shown that [Pt_2(CNCH_3)_6]^(2+) serves as a versatile precursor in the synthesis of new d^8-d^8 A-frame complexes.

A retrofitting framework for pre-Northridge steel moment-frame buildings

In the 1994 M_w 6.7 Northridge and 1995 M_w 6.9 Kobe earthquakes, steel moment-frame buildings were exposed to an unexpected flaw. The commonly utilized welded unreinforced flange, bolted web connections were observed to experience brittle fractures in a number of buildings, even at low levels of seismic demand. A majority of these buildings have not been retrofitted and may be susceptible to structural collapse in a major earthquake.

This dissertation presents a case study of retrofitting a 20-story pre-Northridge steel moment-frame building. Twelve retrofit schemes are developed that present some range in degree of intervention. Three retrofitting techniques are considered: upgrading the brittle beam-to-column moment resisting connections, and implementing either conventional or buckling-restrained brace elements within the existing moment-frame bays. The retrofit schemes include some that are designed to the basic safety objective of ASCE-41 Seismic Rehabilitation of Existing Buildings.

Detailed finite element models of the base line building and the retrofit schemes are constructed. The models include considerations of brittle beam-to-column moment resisting connection fractures, column splice fractures, column baseplate fractures, accidental contributions from ``simple'' non-moment resisting beam-to-column connections to the lateral force-resisting system, and composite actions of beams with the overlying floor system. In addition, foundation interaction is included through nonlinear translational springs underneath basement columns.

To investigate the effectiveness of the retrofit schemes, the building models are analyzed under ground motions from three large magnitude simulated earthquakes that cause intense shaking in the greater Los Angeles metropolitan area, and under recorded ground motions from actual earthquakes. It is found that retrofit schemes that convert the existing moment-frames into braced-frames by implementing either conventional or buckling-restrained braces are effective in limiting structural damage and mitigating structural collapse. In the three simulated earthquakes, a 20% chance of simulated collapse is realized at PGV of around 0.6 m/s for the base line model, but at PGV of around 1.8 m/s for some of the retrofit schemes. However, conventional braces are observed to deteriorate rapidly. Hence, if a braced-frame that employs conventional braces survives a large earthquake, it is questionable how much service the braces provide in potential aftershocks.

Mechanisms of substrate selection by the signal recognition particle

The signal recognition particle (SRP) targets membrane and secretory proteins to their correct cellular destination with remarkably high fidelity. Previous studies have shown that multiple checkpoints exist within this targeting pathway that allows ‘correct cargo’ to be quickly and efficiently targeted and for ‘incorrect cargo’ to be promptly rejected. In this work, we delved further into understanding the mechanisms of how substrates are selected or discarded by the SRP. First, we discovered the role of the SRP fingerloop and how it activates the SRP and SRP receptor (SR) GTPases to target and unload cargo in response to signal sequence binding. Second, we learned how an ‘avoidance signal’ found in the bacterial autotransporter, EspP, allows this protein to escape the SRP pathway by causing the SRP and SR to form a ‘distorted’ complex that is inefficient in delivering the cargo to the membrane. Lastly, we determined how Trigger Factor, a co-translational chaperone, helps SRP discriminate against ‘incorrect cargo’ at three distinct stages: SRP binding to RNC; targeting of RNC to the membrane via SRP-FtsY assembly; and stronger antagonism of SRP targeting of ribosomes bearing nascent polypeptides that exceed a critical length. Overall, results delineate the rich underlying mechanisms by which SRP recognizes its substrates, which in turn activates the targeting pathway and provides a conceptual foundation to understand how timely and accurate selection of substrates is achieved by this protein targeting machinery.

Suspension mechanics: I. Inertial and non-Newtonian migration of neutrally buoyant rigid spheres in two-dimensional unidirectional flows; II. The effect of viscoelasticity on the creeping motion of a train of neutrally buoyant Newtonian drops through a circular tube

The lateral migration of neutrally buoyant rigid spheres in two-dimensional unidirectional flows was studied theoretically. The cases of both inertia-induced migration in a Newtonian fluid and normal stress-induced migration in a second-order fluid were considered. Analytical results for the lateral velocities were obtained, and the equilibrium positions and trajectories of the spheres compared favorably with the experimental data available in the literature. The effective viscosity was obtained for a dilute suspension of spheres which were simultaneously undergoing inertia-induced migration and translational Brownian motion in a plane Poiseuille flow. The migration of spheres suspended in a second-order fluid inside a screw extruder was also considered.

The creeping motion of neutrally buoyant concentrically located Newtonian drops through a circular tube was studied experimentally for drops which have an undeformed radius comparable to that of the tube. Both a Newtonian and a viscoelastic suspending fluid were used in order to determine the influence of viscoelasticity. The extra pressure drop due to the presence of the suspended drops, the shape and velocity of the drops, and the streamlines of the flow were obtained for various viscosity ratios, total flow rates, and drop sizes. The results were compared with existing theoretical and experimental data.

Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Energy transfer in molecular collisions

Two general, numerically exact, quantum mechanical methods have been developed for the calculation of energy transfer in molecular collisions. The methods do not treat electronic transitions because of the exchange symmetry of the electrons. All interactions between the atoms in the system are written as potential energies.

The first method is a matrix generalization of the invariant imbedding procedure, ^{17, 20} adapted for multi-channel collision processes. The second method is based on a direct integration of the matrix Schrödinger equation, with a re-orthogonalization transform applied during the integration.

Both methods have been applied to a collinear collision model for two diatoms, interacting via a repulsive exponential potential. Two major studies were performed. The first was to determine the energy dependence of the transition probabilities for an H₂ on the H₂ model system. Transitions are possible between translational energy and vibrational energy, and from vibrational modes of one H₂ to the other H₂. The second study was to determine the variation of vibrational energy transfer probability with differences in natural frequency of two diatoms similar to N₂.

Comparisons were made to previous approximate analytical solutions of this same problem. For translational to vibrational energy transfer, the previous approximations were not adequate. For vibrational to vibrational energy transfer of one vibrational quantum, the approximations were quite good.

Normas Tradutórias: o caso dos Artigos Científicos e suas Condicionantes Culturais

Esta dissertação investiga o gênero artigo científico e suas condicionantes culturais, ou seja, as marcas textuais que podem apresentar determinada dificuldade para o tradutor por problemas de interculturalidade, a partir da perspectiva do conceito de normas nos estudos da tradução e da descrição do gênero artigo científico. O objetivo deste trabalho é identificar estas condicionantes em artigos científicos da área de Geriatria e Gerontologia, exemplificando partes deste universo de condicionantes através do levantamento das características desse gênero, assim como da comparação das traduções. Demonstrar através da reflexão teórica, de exemplos práticos e de análises comparativas, como a tradução se beneficia do estudo de gêneros, das normas e do levantamento das condicionantes culturais para auxiliar a tarefa tradutória de artigos científicos. Os procedimentos de análise dos corpora foram baseados no modelo de Lambert e Van Gorp (1985) para a análise da tradução literária, adaptado aqui à tradução técnica. Finalmente, analisando as condicionantes culturais levantadas nesta pesquisa, assim como as características do gênero, o estudo culmina com reflexões a respeito da tradução de artigos científicos

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding beta-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic beta-amylase encoding genes in pgi1 leaves, which was accompanied by increased beta-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P) H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

Análise do modo de apropriação do marco regulatório do uso de animais na pesquisa científica no Brasil: estudo de caso da Fundação Oswaldo Cruz

Após 13 anos de debate parlamentar, o governo federal instituiu em 8 de outubro de 2008 uma nova ordem jurídico-administrativa através da Lei n.11.794 que foi regulamentada em 15 de julho de 2009 com o Decreto 6.899. A nova legislação introduziu modelos de condutas que antes não existiam, criou o Conselho Nacional de Controle de Experimentação Animal (CONCEA) e tornou obrigatória a implantação de Comissões de Ética no Uso de Animais (CEUAs) pelas instituições que criam ou utilizam animais para ensino e pesquisa. Partindo do pressuposto de que a Lei n. 11794/2008 é uma política pública regulatória de ciência, tecnologia e inovação em saúde (CT&I/S), o objetivo deste trabalho é demonstrar que a implementação da política regulatória em uma instituição pública federal de pesquisa em saúde é um processo atravessado por relações transnacionais, pelas interpretações que os atores da pesquisa fazem da Lei com base em suas práticas de trabalho, e pelas ações institucionais de tradução local de outras políticas governamentais. O estudo contempla dois níveis de descrição e análise complementares: o da formulação e o da implementação da política regulatória na Fiocruz. O primeiro descreve e analisa o processo de tramitação do Projeto de Lei que originou a Lei e sua regulamentação pelo CONCEA, até dezembro de 2011. O segundo é um estudo de caso inspirado pela abordagem interpretativa, centrado na tradução da Lei pelos pesquisadores, especialistas e gestores da Fiocruz em relação às suas práticas de trabalho. A tese formulada resulta do seguinte percurso metodológico: leitura do movimento de tradução dos interesses dos atores envolvidos no jogo parlamentar do Congresso Nacional de formulação, discussão e aprovação da Lei; delineamento do espaço institucional em que as interpretações dos pesquisadores estão embebidas e que as políticas de CT&I/S são implementadas; leitura das ações e dos programas em CT&I/S do MS para a Fiocruz entre 2004 e 2010; um ano de observação participante nas reuniões da Comissão de Ética no Uso de Animais da Fiocruz; entrevistas semi-estruturadas com 22 pesquisadores, gestores e especialistas em animais de laboratório para compreender o enquadramento da Lei pelos atores nas suas práticas de trabalho. Concluimos afirmando que a aproximação entre a pesquisa pré-clinica e clínica é uma estratégia de contorno às restrições vivenciadas pelos pesquisadores nos processos de trabalho com animais de pesquisa que se desenvolve no cenário da pesquisa translacional e transnacional. O modelo animal é um dispositivo que divide as comunidades instrumentais de pesquisadores biomédicos entre os que utilizam e os que não utilizam animais. A governança da pesquisa biomédica com animais de laboratório não está estabilizada no espaço institucional: as diferentes comunidades instrumentais de usuários de animais, produtores de animais, gestores e membros da CEUA estão em disputa.

Da literatura para a dança: a prosa-poética de Gertrude Stein em tradução intersemiótica

O escopo desta tese é a relação entre a prosa-poética da escritora norte-americana Gertrude Stein, através de seus retratos e peças, e traduções intersemióticas para dança contemporânea. O corpus analítico articula os retratos Orta or One Dancing, If I Told Him: A Completed Portrait of Picasso, A Valentine to Sherwood Anderson, e as peças Four Saints in Three Acts, Listen to Me e Three Sisters Who Are Not Sisters de Gertrude Stein e os espetáculos de dança [5.sobre.o.mesmo], Shutters Shut, Always Now Slowly, ,e[dez episódios sobre a prosa topovisual de gertrude stein]. A natureza dos campos colocados em comparação literatura & dança demandou a conjugação de duas vertentes de estudo ligadas às especificidades performática e tradutória dos objetos selecionados: de um lado, seguimos encaminhamentos surgidos de uma derivação específica da Comparatística tradicional, os Estudos Interartes ou Artes Comparativas; de outro, os Estudos de Intermidialidade, relacionados aos Estudos das Mídias. A abordagem dos exemplos analisados sob a perspectiva comparativa baseia-se em Estudos de Tradução, com especial referência à noção de transcriação de Haroldo de Campos, e na semiótica de Charles S.Peirce. No primeiro capítulo, definimos nossa abordagem teórica; a seguir, apresentamos a obra de Gertrude Stein e as principais propriedades que transformaram sua obra em uma das principais referências literárias e estéticas do século XX; e, para finalizar, analisamos as traduções, com especial atenção para a transcriação da percepção do tempo e da construção sintática steineanas. Concluímos sugerindo que as traduções para dança são modos de interpretação e leitura dos textos literários, bem como formas radicais de crítica de arte ou literária

Análise estática e dinâmica de lajes nervuradas de concreto armado.

Nesta dissertação desenvolve-se um estudo sobre o comportamento estrutural de lajes nervuradas de concreto armado, tendo em mente o emprego destas em sistemas estruturais na engenharia civil, de forma viável sob o ponto de vista técnico e econômico. No cenário atual, diversos tipos de sistemas estruturais têm sido desenvolvidos, dentre estes as lajes nervuradas de concreto armado com base no sistema Reduzcon. Essas lajes nervuradas têm como principais características o baixo consumo de material e o peso próprio bastante reduzido. Este sistema utiliza cubas cilíndricas invertidas metálicas denominadas BRC (Barrote Redutor de Concreto), que suportam o concreto da laje. Assim sendo, este trabalho de pesquisa apresenta um estudo acerca da resposta estática e dinâmica do sistema estrutural em questão, em relação aos deslocamentos translacionais verticais e tensões; e, bem como, da resposta dinâmica do sistema sob o ponto de vista de conforto humano. Ênfase especial é dada às atividades humanas rítmicas, com base no emprego de modelos de carregamento que representem as ações inerentes à ginástica aeróbica. A definição das ações dinâmicas atuantes sobre os modelos estruturais foi feita com base em resultados de testes experimentais que levam em conta os efeitos de multidão. A análise numérica fundamenta-se na modelagem computacional do sistema, através do Método dos Elementos Finitos (MEF), de forma a representar apropriadamente o comportamento estrutural das lajes Reduzcon. São empregadas técnicas usuais de discretização, por meio do programa ANSYS. As vigas de bordo, nervuras e a laje de concreto são simuladas por elementos finitos sólidos tridimensionais.

Mecanismos celulares e moleculares envolvidos com alterações na expressão de glicanos durante a progressão do câncer colorretal

O câncer colorretal representa uma das maiores causas de morbidade e mortalidade relacionadas ao câncer. No Brasil, é o terceiro tipo de câncer mais frequente em homens e mulheres. Muitos estudos estão sendo desenvolvidos no sentido de esclarecer os diversos aspectos moleculares que regulam as alterações fenotípicas exibidas pelas células que constituem o câncer colorretal, no entanto, comparativamente, ainda são poucos os que são dedicados a investigar o papel de modificações co- e pós-traducionais neste processo. Entre os vários tipos destas modificações que ocorrem em proteínas, a glicosilação é a mais comum. Cogita-se que aproximadamente cinquenta por cento de todas as proteínas são glicosiladas. Durante a transformação maligna, mudanças no perfil de expressão de glicanos (carboidratos covalentemente ligados a proteínas ou lipídios) estão envolvidas em uma variedade de mecanismos celulares, tais como: perda da adesão célula-célula e célula matriz, migração, invasão e evasão da apoptose. Neste estudo, foi investigada a atividade antitumoral de inibidores da biossíntese de N-glicanos, swainsonina e tunicamicina, em células derivadas de câncer colorretal (Caco-2, HCT-116 e HT-29). Os resultados obtidos mostram que o tratamento das células HCT-116 com tunicamicina inibe mecanismos celulares relacionados ao fenótipo maligno, como formação de colônia dependente e independente de ancoragem, migração e invasão. Estes resultados sugerem que modulação da biossíntese de N-glicanos parece ser uma potencial ferramenta terapêutica para o tratamento do câncer colorretal. Em outra etapa do trabalho, foram avaliados também o impacto da estimulação com insulina e IGF-1 na expressão N-glicanos bissectados em células tumorais MDA-MB-435. Os resultados obtidos confirmaram também a existência de uma relação entre a estimulação dos receptores de insulina e IGF-1 e a regulação da expressão de N-glicanos bissectados em células tumorais MDA-MB-435, fornecendo assim informações relevantes sobre o papel desempenhado pela sinalização de insulina e IGF-1 durante a progressão de carcinomas.

A modular planar robotic manipulandum with end-point torque control.

Robotic manipulanda are extensively used in investigation of the motor control of human arm movements. They permit the application of translational forces to the arm based on its state and can be used to probe issues ranging from mechanisms of neural control to biomechanics. However, most current designs are optimized for studying either motor learning or stiffness. Even fewer include end-point torque control which is important for the simulation of objects and the study of tool use. Here we describe a modular, general purpose, two-dimensional planar manipulandum (vBOT) primarily optimized for dynamic learning paradigms. It employs a carbon fibre arm arranged as a parallelogram which is driven by motors via timing pulleys. The design minimizes the intrinsic dynamics of the manipulandum without active compensation. A novel variant of the design (WristBOT) can apply torques at the handle using an add-on cable drive mechanism. In a second variant (StiffBOT) a more rigid arm can be substituted and zero backlash belts can be used, making the StiffBOT more suitable for the study of stiffness. The three variants can be used with custom built display rigs, mounting, and air tables. We investigated the performance of the vBOT and its variants in terms of effective end-point mass, viscosity and stiffness. Finally we present an object manipulation task using the WristBOT. This demonstrates that subjects can perceive the orientation of the principal axis of an object based on haptic feedback arising from its rotational dynamics.

Rapid evolution of an X-linked microRNA cluster in primates

MicroRNAs (miRNAs) are a growing class of small RNAs ( about 22 nt) that play crucial regulatory roles in the genome by targeting mRNAs for cleavage or translational repression. Most of the identified miRNAs are highly conserved among species, indicating