Aspasing Out Metacaspases and Caspases: Proteases of Many Trades

Execution of programmed cell death (PCD) in nonmetazoan organisms is morphologically different from apoptotic PCD in animals and lacks a number of key molecular components of apoptotic machinery, including caspases. Yet protozoan, fungal, and plant cells exhibit caspase-like proteolytic activities, which increase in a PCD-dependent manner. This poses a question whether nonmetazoan organisms contain structurally dissimilar proteases that functionally substitute for caspases. Putative ancestors of caspases, metacaspases, are candidates for this role; however, their distinct substrate specificity raises doubts. The identification of a common biological target of caspases and metacaspases and previously unknown functions unrelated to cell death of metacaspases provide new food for thought.

Impact of vitamin D3 on cutaneous immunity and antimicrobial peptide expression

Antimicrobial peptides (AMPs) are effectors of cutaneous innate immunity and protect primarily against microbial infections. An array of AMPs can be found in and on the skin. Those include peptides that were first discovered for their antimicrobial properties but also proteins with antimicrobial activity first characterized for their activity as chemokines, enzymes, enzyme inhibitors and neuropeptides. Cathelicidins were among the first families of AMPs discovered in skin. They are now known to exert a dual role in innate immune defense: they have direct antimicrobial activity and will also initiate a host cellular response resulting in cytokine release, inflammation and angiogenesis. Altered cathelicidin expression and function was observed in several common inflammatory skin diseases such as atopic dermatitis, rosacea and psoriasis. Until recently the molecular mechanisms underlying cathelicidin regulation were not known. Lately, vitamin D3 was identified as the major regulator of cathelicidin expression and entered the spotlight as an immune modulator with impact on both, innate and adaptive immunity. Therapies targeting vitamin D3 signalling may provide novel approaches for the treatment of infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions through AMP regulation.

Functional and pharmacological characterization of volume-regulated anion channels in human normal and cystic fibrosis bronchial and nasal epithelial cells

Volume-regulated anion channels (VRACs) are widely present in various cell types and have important functions ranging from regulatory volume decrease to control of cell proliferation and apoptosis. Here we aimed to compare the biophysical features and pharmacological profiles of VRAC currents in healthy and cystic fibrosis (CF) respiratory epithelial cells in order to characterize these currents both functionally and pharmacologically. Whole-cell electrophysiology was used to characterize the VRAC current in normal (16HBE14o-; HBE) and CF cell lines (CFBE14o-; CFBE), as well as in native human nasal epithelial cells. Application of hypotonic solution produced current responses of similar sizes in both HBE and CFBE cells. Biophysical properties of VRACs, such as instantaneous activation and deactivation upon voltage step, some inactivation at potentials positive to 40 mV and outwardly-rectifying I-V curves, were indistinguishable in both cell types. Extensive pharmacological analysis of the currents revealed a similar pharmacological profile in response to three blockers--NPPB, DCPIB and DIDS. Native primary human nasal epithelial cells from both healthy and CF volunteers also showed typical VRAC responses of comparable sizes. VRACs in these cells were more sensitive to external solution hypotonicity compared to HBE and CFBE cells. In all cell types studied robust VRAC currents could be induced at constant cell volume by G-protein activation with GTPγS infusion. This study provides the first extensive comparative functional and pharmacological analysis of VRAC currents in normal and CF airway epithelial cells and shows that VRACs are unimpaired molecularly or functionally in CF.

Aging in personal and social immunity: do immune traits senesce at the same rate?

How much should an individual invest in immunity as it grows older? Immunity is costly and its value is likely to change across an organism's lifespan. A limited number of studies have focused on how personal immune investment changes with age in insects, but we do not know how social immunity, immune responses that protect kin, changes across lifespan, or how resources are divided between these two arms of the immune response. In this study, both personal and social immune functions are considered in the burying beetle, Nicrophorus vespilloides. We show that personal immune function declines (phenoloxidase levels) or is maintained (defensin expression) across lifespan in nonbreeding beetles but is maintained (phenoloxidase levels) or even upregulated (defensin expression) in breeding individuals. In contrast, social immunity increases in breeding burying beetles up to middle age, before decreasing in old age. Social immunity is not affected by a wounding challenge across lifespan, whereas personal immunity, through PO, is upregulated following wounding to a similar extent across lifespan. Personal immune function may be prioritized in younger individuals in order to ensure survival until reproductive maturity. If not breeding, this may then drop off in later life as state declines. As burying beetles are ephemeral breeders, breeding opportunities in later life may be rare. When allowed to breed, beetles may therefore invest heavily in "staying alive" in order to complete what could potentially be their final reproductive opportunity. As parental care is important for the survival and growth of offspring in this genus, staying alive to provide care behaviors will clearly have fitness payoffs. This study shows that all immune traits do not senesce at the same rate. In fact, the patterns observed depend upon the immune traits measured and the breeding status of the individual.

Oxidation and glycoxidation of phosphatidylserine and their effect in immune response : a lipidomic approach

Phosphatidylserine (PS) is a member of the class of phospholipids, and is distributed among all cells of mammalians, playing important roles in diverse biological processes, including blood clotting and apoptosis. When externalized, PS is a ligand that is recognized on apoptotic cells. It has been considered that before externalization PS is oxidized and oxPS enhance the recognition by macrophages receptors, however the knowledge about oxidation of PS is still limited. PS, like others phospholipids, has two fatty acyl chains and one polar head group, in this case is the amino acid serine. The modifications in PS structure can occur by oxidation of the unsaturated fatty acyl chains and by glycation of the polar head group, due to free amine group, thus increasing the susceptibility to oxidative events. The main goal of this work was to characterize and identify oxidized and glycoxidized PS, contributing to the knowledge of the biological role of oxidation products of PS, as well as of glycated PS, in immune and inflammatory processes. To achieve this goal, PS standards (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- L-serine (POPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1- palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS) and 1-palmitoyl-2- arachidonoyl-sn-glycero-3-phospho-L-serine (PAPS)) and glycated PS (PAPS and POPS) were induced to oxidize in model systems, using different oxidant reagents: HO• and 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) . The detailed structural characterization of the oxidative products was performed by ESI-MS and MS/MS coupled to separation techniques such as off line TLC-MS and on line LC-MS, in order to obtained better characterization of the larger number of PS and glycated PS oxidation products. The results obtained in this work allowed to identify several oxidation products of PS and glycated PS with modifications in unsaturated fatty acyl chain. Also, oxidation products formed due to structural changes in the serine polar head with formation of terminal acetamide, terminal hydroperoxyacetaldehyde.and terminal acetic acid (glycerophosphacetic acid, GPAA) were identified. The mass spectrometric specific fragmentation pathway of each type of oxidation product was determined using different mass spectrometry approaches. Based on the identified fragmentation pathways, targeted lipidomic analysis was performed to detect oxidation products modified in serine polar head in HaCaT cell line treated with AAPH. The GPAA was detected in HaCaT cells treated with AAPH to induce oxidative stress, thus confirming that modifications in PS polar head is possible to occur in biological systems. Furthermore, it was found that glycated PS species are more prone to oxidative modifications when compared with non glycated PS. During oxidation of glycated PS, besides the oxidation in acyl chains, new oxidation products due to oxidation of the glucose moiety were identified, including PS advanced glycation end products (PSAGES). To investigate if UVA oxidative stress exerted changes in the lipidome of melanoma cell lines, particularly in PS profile, a lipidomic analysis was performed. The lipid profile was obtained using HILIC-LC-MS and GC-MS analysis of the total lipid extracts obtained from human melanoma cell line (SKMEL- 28) after UVA irradiation at 0, 2 and 24 hours. The results did not showed significant differences in PS content. At molecular level, only PS (18:0:18:1) decreased at the moment of irradiation. The most significant changes in phospholipids content occurred in phosphatidylcholines (PC) and phosphatidylinositol (PI) classes, with an increase of mono-unsaturated fatty acid (MUFA), similarly as observed for the fatty acid analysis. Overall, these data indicate that the observed membrane lipid changes associated with lipogenesis after UVA exposure may be correlated with malignant transformations associated with cancer development and progression. Despite of UVA radiation is associated with oxidative damage, in this work was not possible observe oxidation phospholipids. The anti/pro-inflammatory properties of the oxidized PLPS (oxPLPS) versus non-oxidized PLPS were tested on LPS stimulated RAW 264.7 macrophages. The modulation of intracellular signaling pathways such as NF-kB and MAPK cascades by oxPLPS and PS was also examined in this study. The results obtained from evaluation of anti/pro-inflammatory properties showed that neither PLPS or oxPLPS species activated the macrophages. Moreover only oxidized PLS were found to significantly inhibit NO production and iNOS and il1β gene transcription induced by LPS. The analysis at molecular level showed that this was the result of the attenuation of LPS-induced c-Jun-N-terminal kinase (JNK) and p65 NF-kB nuclear translocation. Overall these data suggest that oxPLPS, but not native PLPS, mitigates pro-inflammatory signaling in macrophages, contributing to containment of inflammation during apoptotic cell engulfment. The results obtained in this work provides new information on the modifications of PS, facilitating the identification of oxidized species in complex samples, namely under physiopathologic conditions and also contributes to a better understanding of the role of oxPS and PS in the inflammatory response, in the apoptotic process and other biological functions.

Mathematica in the classroom: new tools for exploring precalculus and differential calculus

Prémio de Melhor Artigo de Jovem Investigador atribuído pela empresa Timberlake, apresentado na 1ª Conferência Nacional sobre Computação Simbólica no Ensino e na Investigação - CSEI2012, que decorreu no IST nos dias 2 e 3 de Abril.

Joint inversion of geophysical and hydrologic data during water infiltration

Tese de doutoramento, Ciências Geofísicas e da Geoinformação (Geofísica), Universidade de Lisboa, Faculdade de Ciências, 2014

Program slice metrics and their potential role in DSL design, position paper

The advantages a DSL and the benefits its use potentially brings imply that informed decisions on the design of a domain specific language are of paramount importance for its use. We believe that the foundations of such decisions should be informed by analysis of data empirically collected from systems to highlight salient features that should then form the basis of a DSL. To support this theory, we describe an empirical study of a large OSS called Barcode, written in C, and from which we collected two well-known 'slice' based metrics. We analyzed multiple versions of the system and sliced its functions in three separate ways (i.e., input, output and global variables). The purpose of the study was to try and identify sensitivities and traits in those metrics that might inform features of a potential slice-based DSL. Results indicated that cohesion was adversely affected through the use of global variables and that appreciation of the role of function inputs and outputs can be revealed through slicing. The study presented is motivated primarily by the problems with current tools and interfaces experienced directly by the authors in extracting slicing data and the need to promote the benefits that analysis of slice data and slicing in general can offer.

What is universal and what differs in language development?

Goldin-Meadow (2015) presents an exceptional synthesis of work from studies of children acquiring language under variable circumstances of input or processing abilities. Deaf children who acquire homesign without any well- formed model from which to learn language represent a powerful example. Goldin-Meadow argues that the resilient properties of language that nevertheless emerge include simple syntactic structures, hierarchical organisa- tion, markers modulating the meaning of sentences, and social-communicative functions. Among the fragile or input-dependent properties are the orders that the language follows, the parts into which words are decomposed, and the features that distinguish nominals from predicates. Separation of these two types of properties poses questions concerning the innate constraints on language acquisition (perhaps these equate to the resilient properties) and con‐ cerning the specificity of processes to language (e.g., whether properties such as hierarchical organisation are specific to language or originate in the structure of thought). The study of the resilient properties of human language in the face of adversity and the relation of these properties to the information that is encoded in the human genome represent a research strategy that draws inferences about species universals (properties that all humans share) from data about individual differences (IDs; factors that make humans different from one another). In the following, we suggest three reasons to be cautious about this approach.

Crowdsourcing innovation intermediaries functions

Open innovation is a hot topic in innovation management. Its basic premise is open up the innovation process. The innovation process, in general sense, may be seen as the process of designing, developing and commercializing a novel product or service to improve the value added of a company. The development of Web 2.0 tools facilitates this kind of contributions, opening space to the emergence of crowdsourcing innovation initiatives. Crowdsourcing is a form of outsourcing not directed to other companies but to the crowd by means of an open call mostly through an Internet platform. Innovation intermediaries, in general sense, are organizations that work to enable innovation, that just act as brokers or agents between two or more parties. Usually, they are also engaged in other activities like inter-organizational networking and technology development and related activities. A crowdsourcing innovation intermediary is an organization that mediates the communication and relationship between the seekers – companies that aspire to solve some problem or to take advantage of any business opportunity – with a crowd that is prone to give ideas based on their knowledge, experience and wisdom. This paper identifies and analyses the functions to be performed by an intermediary of crowdsourcing innovation through grounded theory analyses from literature. The resulting model is presented and explained. The resulting model summarizes eight main functions that can be performed by a crowdsourcing process, namely, diagnoses, mediation, linking knowledge, community, evaluation, project management, intellectual property governance and marketing and support. These functions are associated with a learning cycle process which covers all the crowdsourcing activities that can be realized by the broker.

Sexual dimorphism in resource acquisition and deployment: both size and timing matter.

Background and Aims The males and females of many dioecious plant species differ from one another in important life-history traits, such as their size. If male and female reproductive functions draw on different resources, for example, one should expect males and females to display different allocation strategies as they grow. Importantly, these strategies may differ not only between the two sexes, but also between plants of different age and therefore size. Results are presented from an experiment that asks whether males and females of Mercurialis annua, an annual plant with indeterminate growth, differ over time in their allocation of two potentially limiting resources (carbon and nitrogen) to vegetative (below-and above-ground) and reproductive tissues.Methods Comparisons were made of the temporal patterns of biomass allocation to shoots, roots and reproduction and the nitrogen content in the leaves between the sexes of M. annua by harvesting plants of each sex after growth over different periods of time.Key Results and Conclusions Males and females differed in their temporal patterns of allocation. Males allocated more to reproduction than females at early stages, but this trend was reversed at later stages. Importantly, males allocated proportionally more of their biomass towards roots at later stages, but the roots of females were larger in absolute terms. The study points to the important role played by both the timing of resource deployment and the relative versus absolute sizes of the sinks and sources in sexual dimorphism of an annual plant.

Effects of intracerebroventricular bombesin administration on local cerebral glucose utilization in the restrained and unrestrained rat

Intracerebroventricular (ICV) administration of bombesin (BN) induces a syndrome characterized by stereotypic locomotion and grooming, hyperactivity and sleep elimination, hyperglycemia and hypothermia, hyperhemodynamics, feeding inhibition, and gastrointestinal function changes. Mammalian BN-like peptides (MBNs), e.g. gastrin-releasing peptide (GRP), Neuromedin C (NMC), and Neuromedin B (NMB), have been detected in the central nervous system. Radio-labeled BN binds to specific sites in discrete cerebral regions. Two specific BN receptor subtypes (GRP receptor and NMB receptor) have been identified in numerous brain regions. The quantitative 2-[14C]deoxyglucose ([14C]20G) autoradiographic method was used to map local cerebral glucose utilization (LCGU) in the rat brain following ICV injection of BN (vehicle, BN O.1Jlg, O.5Jlg). At each dose, experiments were conducted in freely moving or restrained conditions to determine whether alterations in cerebral function were the result of BN central administration, or were the result of BN-induced motor stereotypy. The anteroventral thalamic nucleus (AV) (p=O.029), especially its ventrolateral portion (AVVL) (pand the suprachiasmatic nucleus (SCh) (p=O.003) exhibited BN treatment effects. BN effects on LCGU were also observed in the median eminence (ME) (p=O.011). Restraint, however, decreased LCGU in the lateral dorsal thalamic nucleus, ventrolateral and dorsomedial parts (LOVL and LOOM) (p=O.044, p=O.009), and the lateral geniculate (LG) (p=O.027). In sum, BN induced a marked and highly localized alteration in cerebral metabolism within parts of the anterior thalamus, which is the principle relay in the limbic circuitry. BN effects were also observed in IGr, Mi, SCh, and ME. Effects of restraint were found in LOVL, LOOM, and LG. It is suggested that increased LCGU in AV and AVVL may be the result of functional change in the limbic circuitry and the hypothalamus caused by BN receptor functional modification. In IGr, increased LCGU following BN administration is considered to be mainly the result the activation of NMB receptor, a subtype of BN receptors. In SCh, increased LCGU is believed to be caused both by BN effects on the thalamic, the hypothalamic, and the limbic functions and by activation of GRP receptor, another BN receptors subtype found in SCh. In ME, increased LCGU is suggested to be caused by BN effects on the hypothalamic functions, especially those related to the neuroendocrine functions. None of the alterations seen in these regions reflects the emission of stereotyped motor behaviors. Rather, they reflect a direct influence of BN central administration upon functioning of the cerebral regions influenced by BN administration. The restraint effects seen in LO, including LOOM and LOVL, are suggested to be the result of altered behavioral expression. The restraint effects seen in LG is suggested to be the result of reduced locomotion.

The BTB/POZ Transcription Factor Miz-1 Is Required To Regulate The Commitment, Survival And Differentiation Of Early B And T Cell Lineages

Les lymphocytes B et T sont issus de cellules progénitrices lymphoïdes de la moelle osseuse qui se différencient grâce à l’action de facteurs de transcription, cytokines et voies de signalisation, dont l’interleukine-7 (IL-7)/IL-7 récepteur (IL-7R). Le facteur de transcription c-Myc est exprimé par les cellules lymphoïdes et contrôle leur croissance et leur différenciation. Cette régulation transcriptionnelle peut être coordonnée par le complexe c-Myc/Myc-Interacting Zinc finger protein-1 (Miz-1). Le but de ce projet était de comprendre les mécanismes qui impliquent Miz-1 et le complexe c-Myc/Miz-1 dans le développement des lymphocytes B et T. Pour réaliser ce projet, des souris déficientes pour le domaine de transactivation de Miz-1 (Miz-1POZ) et des souris à allèles mutantes pour c-MycV394D, mutation qui empêche l’interaction avec Miz-1, ont été générées. La caractérisation des souris Miz 1POZ a démontré que l’inactivation de Miz-1 perturbe le développement des lymphocytes B et T aux stades précoces de leur différenciation qui dépend de l’IL-7. L’analyse de la cascade de signalisation IL-7/IL-7R a montré que ces cellules surexpriment la protéine inhibitrice SOCS1 qui empêche la phosphorylation de STAT5 et perturbe la régulation à la hausse de la protéine de survie Bcl-2. De plus, Miz-1 se lie directement au promoteur de SOCS1 et contrôle son activité. En plus de contrôler l’axe IL-7/IL-7R/STAT5/Bcl-2 spécifiquement aux stades précoces du développement afin d’assurer la survie des progéniteurs B et T, Miz-1 régule l’axe EBF/Pax-5/Rag-1/2 dans les cellules B afin de coordonner les signaux nécessaires pour la différenciation des cellules immatures. La caractérisation des souris c-MycV394D a montré, quant à elle, que les fonctions de Miz-1 dans les cellules B et T semblent indépendantes de c-Myc. Les cellules T des souris Miz-1POZ ont un défaut de différenciation additionnel au niveau de la -sélection, étape où les signaux initiés par le TCR remplacent ceux induits par IL-7 pour assurer la prolifération et la différenciation des thymocytes en stades plus matures. À cette étape du développement, une forme fonctionnelle de Miz-1 semble être requise pour contrôler le niveau d’activation de la voie p53, induite lors du processus de réarrangement V(D)J du TCR. L’expression de gènes pro-apoptotiques PUMA, NOXA, Bax et du régulateur de cycle cellulaire p21CIP1 est régulée à la hausse dans les cellules des souris Miz-1POZ. Ceci provoque un débalancement pro-apoptotique qui empêche la progression du cycle cellulaire des cellules TCR-positives. La survie des cellules peut être rétablie à ce stade de différenciation en assurant une coordination adéquate entre les signaux initiés par l’introduction d’un TCR transgénique et d’un transgène codant pour la protéine Bcl-2. En conclusion, ces études ont montré que Miz-1 intervient à deux niveaux du développement lymphoïde: l’un précoce en contrôlant la signalisation induite par l’IL-7 dans les cellules B et T, en plus de l’axe EBF/Pax-5/Rag-1/2 dans les cellules B; et l’autre tardif, en coordonnant les signaux de survie issus par le TCR et p53 dans les cellules T. Étant donné que les thymocytes et lymphocytes B immatures sont sujets à plusieurs rondes de prolifération, ces études serviront à mieux comprendre l’implication des régulateurs du cycle cellulaire comme c-Myc et Miz-1 dans la génération des signaux nécessaires à la différenciation non aberrante et à la survie des ces cellules. Enfin, les modèles expérimentaux, souris déficientes ou à allèles mutantes, utilisés pour ce travail permettront de mieux définir les bases moléculaires de la transformation maligne des lymphocytes B et T et de révéler les mécanismes conduisant au lymphome.

Muscarinic M1 and M3 Receptors,IP3 and cGMP Functional Regulation in Streptozotocin Induced Diabetic Rats: Insulin and Somatotropin Induced Rejuvenation as a Function of Age

The present study describes that acetylcholine through muscarinic Ml and M3 receptors play an important role in the brain function during diabetes as a function of age. Cholinergic activity as indicated by acetylcholine esterase, a marker for cholinergic function, decreased in the brain regions - the cerebral cortex, brainstem and corpus striatum of old rats compared to young rats. in diabetic condition, it was increased in both young and old rats in cerebral cortex, and corpus striatum while in brainstem it was decreased. The functional changes in the muscarinic receptors were studied in the brain regions and it showed that muscarinic M I receptors of old rats were down regulated in cerebral cortex while in corpus striatum and brainstem it was up regulated. Muscarinic M3 receptors of old rats showed no significant change in cerebral cortex while in corpus striatum and brainstem muscarinic receptors were down regulated. During diabetes, muscarinic M I receptors were down regulated in cerebral cortex and brainstem of young rats while in corpus striatum they were up regulated. In old rats, M I receptors were up regulated in cerebral cortex, corpus striatum and in brainstem they were down regulated. Muscarinic M3 receptors were up regulated in cerebral cortex and brainstem of young rats while in corpus striatum they were down regulated. In old rats, muscarinic M l receptors were up regulated in cerebral cortex, corpus striatum and brainstem. In insulin treated diabetic rats the activity of the receptors were reversed to near control. Pancreatic muscarinic M3 receptor activity increased in the pancreas of both young and old rats during diabetes. In vitro studies using carbachol and antagonists for muscarinic Ml and M3 receptor subtypes confirmed the specific receptor mediated neurotransmitter changes during diabetes. Calcium imaging studies revealed muscarinic M I mediated Ca2 + release from the pancreatic islet cells of young and old rats. Electrophysiological studies using EEG recording in young and old rats showed a brain activity difference during diabetes. Long term low dose STH and INS treated rat brain tissues were used for gene expression of muscarinic Ml, M3, glutamate NMDARl, mGlu-5,alpha2A, beta2, GABAAa1 and GABAB, DAD2 and 5-HT 2C receptors to observe the neurotransmitter receptor functional interrelationship for integrating memory, cognition and rejuvenating brain functions in young and old. Studies on neurotransmitter receptor interaction pathways and gene expression regulation by second messengers like IP3 and cGMP in turn will lead to the development of therapeutic agents to manage diabetes and brain activity.From this study it is suggested that functional improvement of muscarinic Ml, M3, glutamate NMDAR1, mGlu-5, alpha2A, beta2, GABAAa1 and GABAB, DAD2 and 5-HT 2C receptors mediated through IP3 and cGMP will lead to therapeutic applications in the management of diabetes. Also, our results from long term low dose STH and INS treatment showed rejuvenation of the brain function which has clinical significance in maintaining healthy period of life as a function of age.

An Error-Localization, Validation and Optimization Tool for Embedded Code Augmentation: an Architecture Oriented Approach

Embedded systems are usually designed for a single or a specified set of tasks. This specificity means the system design as well as its hardware/software development can be highly optimized. Embedded software must meet the requirements such as high reliability operation on resource-constrained platforms, real time constraints and rapid development. This necessitates the adoption of static machine codes analysis tools running on a host machine for the validation and optimization of embedded system codes, which can help meet all of these goals. This could significantly augment the software quality and is still a challenging field.Embedded systems are usually designed for a single or a specified set of tasks. This specificity means the system design as well as its hardware/software development can be highly optimized. Embedded software must meet the requirements such as high reliability operation on resource-constrained platforms, real time constraints and rapid development. This necessitates the adoption of static machine codes analysis tools running on a host machine for the validation and optimization of embedded system codes, which can help meet all of these goals. This could significantly augment the software quality and is still a challenging field.Embedded systems are usually designed for a single or a specified set of tasks. This specificity means the system design as well as its hardware/software development can be highly optimized. Embedded software must meet the requirements such as high reliability operation on resource-constrained platforms, real time constraints and rapid development. This necessitates the adoption of static machine codes analysis tools running on a host machine for the validation and optimization of embedded system codes, which can help meet all of these goals. This could significantly augment the software quality and is still a challenging field.Embedded systems are usually designed for a single or a specified set of tasks. This specificity means the system design as well as its hardware/software development can be highly optimized. Embedded software must meet the requirements such as high reliability operation on resource-constrained platforms, real time constraints and rapid development. This necessitates the adoption of static machine codes analysis tools running on a host machine for the validation and optimization of embedded system codes, which can help meet all of these goals. This could significantly augment the software quality and is still a challenging field.This dissertation contributes to an architecture oriented code validation, error localization and optimization technique assisting the embedded system designer in software debugging, to make it more effective at early detection of software bugs that are otherwise hard to detect, using the static analysis of machine codes. The focus of this work is to develop methods that automatically localize faults as well as optimize the code and thus improve the debugging process as well as quality of the code.Validation is done with the help of rules of inferences formulated for the target processor. The rules govern the occurrence of illegitimate/out of place instructions and code sequences for executing the computational and integrated peripheral functions. The stipulated rules are encoded in propositional logic formulae and their compliance is tested individually in all possible execution paths of the application programs. An incorrect sequence of machine code pattern is identified using slicing techniques on the control flow graph generated from the machine code.An algorithm to assist the compiler to eliminate the redundant bank switching codes and decide on optimum data allocation to banked memory resulting in minimum number of bank switching codes in embedded system software is proposed. A relation matrix and a state transition diagram formed for the active memory bank state transition corresponding to each bank selection instruction is used for the detection of redundant codes. Instances of code redundancy based on the stipulated rules for the target processor are identified.This validation and optimization tool can be integrated to the system development environment. It is a novel approach independent of compiler/assembler, applicable to a wide range of processors once appropriate rules are formulated. Program states are identified mainly with machine code pattern, which drastically reduces the state space creation contributing to an improved state-of-the-art model checking. Though the technique described is general, the implementation is architecture oriented, and hence the feasibility study is conducted on PIC16F87X microcontrollers. The proposed tool will be very useful in steering novices towards correct use of difficult microcontroller features in developing embedded systems.