Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology. The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed. These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function.

RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene silencing pathway found in eukaryotes, in which 21-nucleotide, small interfering RNAs (siRNAs) guide destruction of a corresponding target mRNA. RNAi is a natural mechanism for both genome surveillance and gene regulation. Moreover, siRNAs can be transfected into cultured mammalian cells, causing the sequence-specific ‘knock down’ of an mRNA. My work in the Zamore lab has centered around the Drosophilain vitro system and cultured mammalian cells to study the RNA interference (RNAi) pathway. small interfering RNAs (siRNAs) are incorporated into the RNA-induced silencing complex (RISC), which culminates in the cleavage of a complementary target mRNA. Previous work proved that certain structural features of siRNAs are essential for RNAi in flies, including the requirement for 5´ phosphates and 3´ hydroxyl groups. In cultured mammalian cells, the requirement for a 5´ phosphate also holds true, but we found no evidence to support the necessity for 3´ hydroxyls in either system. In addition, siRNAs can act as single strands entering the pathway downstream of double-stranded siRNAs, both of which are competent in directing the cleavage of its cognate mRNA at a single site. While these key features are a requirement for functional siRNAs, alone they do not determine the efficiency to which an siRNA can enter the RISC. In fact, both strands of an siRNA can enter RISC to a different degree as determined by the stabilities of the 5´ ends of the siRNA strand, a phenomenon termed ‘functional asymmetry’. This characteristic is also reflected in another class of small RNAs involved in gene silencing known as microRNAs (miRNAs), which are processed from long hairpin RNA structures into mature, single-stranded non-coding RNAs. The asymmetric loading of siRNAs suggests that miRNAs are initially generated from siRNA-like duplexes cleaved from the stem of the hairpins. The strand whose 5´ end is less tightly paired will be processed into the mature miRNA, while the other strand is destroyed. By applying the rules of siRNA asymmetry it is possible to predict which side of the stem will be processed into the mature miRNA, a finding verified experimentally by our lab and others. This discovery also has additional implications in designing highly effective siRNAs and in reducing siRNA off-target effects. We used these results to design siRNAs that target the single nucleotide polymorphism in superoxide dismutase that causes the familial form of amyotrophic lateral sclerosis (ALS), but leave the wild-type mRNA intact and functional. Our experiments have helped define the ‘rules’ for creating SNP-specific siRNAs. In particular, we found that only siRNAs with a purine:purine mismatch to the allele not intended for destruction show good discrimination. The placement of the mismatch in a tiled set of siRNAs shows that mismatches located in the 5´ region of the siRNA, a region shown to be responsible for siRNA binding, can not discriminate between alleles. In contrast, mismatches in the 3´ region of the siRNA, the region contributing to catalysis, discriminate between wild-type and mutant alleles. This work is an important step in creating allele-specific siRNAs as therapeutics for dominant negative genetic diseases. But how does RISC cleave its target? By isolating both the 5´ and 3´ cleavage products produced by RISC in the Drosophila in vitro system, we discovered that RISC acts as a Mg2+-dependent endonuclease that cleaves a single phosphodiester bond in the mRNA target, leaving 5´ phosphate and 3´ hydroxyl groups. These findings were a critical step in the demonstration that Argonaute, a protein known to be a component of RISC, is the RNAi endonuclease.

ATP plays a critical role in the regulation of many enzyme processes. In this work, I have focused on the ATP mediated regulation of the recombination processes catalyzed by the E. coliRecA and the human Rad51 proteins. The RecA protein is a multifunctional enzyme, which plays a central role in the processes of recombinational DNA repair, homologous genetic recombination and in the activation of the cellular SOS response to DNA damage. Each of these functions requires a common activating step, which is the formation of a RecA-ATP-ssDNA nucleoprotein filament. The binding of ATP results in the induction of a cooperative, high affinity ssDNA binding state within RecA (Menetski & Kowalczykowski, 1985b; Silver & Fersht, 1982). Data presented here identifies Gln194 as the NTP binding site "γ-phosphate sensor", in that mutations introduced at this residue disrupt all ATP induced RecA activities, while basal enzyme function is maintained. Additionally, we have dissected the parameters contributing to cooperative nucleoprotein filament assembly in the presence of cofactor. We show that the dramatic increase in the affinity of RecA for ssDNA in the presence of ATP is a result of a significant increase in the cooperative nature of filament assembly and not an increase in the intrinsic affinity of a RecA monomer for ssDNA. Previous work using both mutagenesis and engineered disulfides to study the subunit interface of the RecA protein has demonstrated the importance of Phe217 for the maintenance of both the structural and functional properties of the protein (Skiba & Knight, 1994; Logan et al., 1997; Skiba et al., 1999). A Phe217Tyr mutation results in a striking increase in cooperative filament assembly. In this work, we identify Phe217 as a key residue within the subunit interface and clearly show that Phe217 is required for the transmission of ATP mediated allosteric information throughout the RecA nucleoprotein filament. The human Rad51 (hRad51) protein, like its bacterial homolog RecA, catalyzes genetic recombination between homologous single and double stranded DNA substrates. This suggests that the overall process of homologous recombination may be conserved from bacteria to humans. Using IAsys biosensor technology, we examined the effect of ATP on the binding of hRad51 to ssDNA. Unlike RecA, we show that hRad51 binds cooperatively and with high affinity to ssDNA both in the presence and absence of nucleotide cofactor. These results show that ATP plays a fundamentally different role in hRad51 vs.RecA mediated processes. In summary, through the work presented in this dissertation, we have defined the critical molecular determinants for ATP mediated allosteric regulation within RecA. Furthermore, we have shown that ATP is not utilized by Rad51 in the same manner as shown for RecA, clearly defining a profound mechanistic difference between the two proteins. Future studies will define the requirement for ATP in hRad51 mediated processes.

RNA silencing is an evolutionary conserved sequence-specific mechanism of regulation of gene expression. RNA interference (RNAi), a type of RNA silencing in animals, is based on recognition and endonucleolytic cleavage of target mRNA complimentary in sequence to 21-nucleotide (nt) small RNA guides, called small interfering RNAs (siRNAs). Another class of 21-nt small RNAs, called micro RNAs (miRNAs), is endogenously encoded in eukaryotic genomes. Both production of siRNAs from long double-stranded RNA (dsRNA) and biogenesis of miRNAs from hairpin structures are governed by the ribonuclease III enzyme Dicer. Although produced as duplex molecules, siRNAs and miRNAs are assembled into effector complex, called the RNA-induced silencing complex (RISC), as single-strands. A member of the Argonaute family of small RNA-binding proteins lies at the core of all known RNA silencing effector complexes. Plants and animals contain multiple Argonaute paralogs. In addition to endonucleolytic cleavage, Argonaute proteins can direct translational repression/destabilization of mRNA or transcriptional silencing of DNA sequences by the siRNAdirected production of silent heterochromatin. The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. That only one Dicer, RdRP and Argonaute is expressed in S. pombe might reflect the extreme specialization of RNA silencing pathways regulating targets only at the transcriptional level in this organism. We decided to test if classical RNAi can be induced in S. pombe. We introduced a dsRNA hairpin corresponding to a GFP transgene. GFP silencing triggered by dsRNA reflected a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe required dcr1, rdp1, and ago1, but did not require chp1, tas3, or swi6, genes required for transcriptional silencing. We concluded that the RNAi machinery in S. pombecould direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that, in spite of specialization in distinct siRNA-directed silencing pathways, these three proteins fulfill a common biochemical function. In Drosophila, miRNA and RNAi pathways are both genetically and biochemically distinct. Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)/Loquacious complex produces miRNAs. Argonaute proteins can be divided by sequence similarity into two classes: in flies, the Ago subfamily includes Argonaute1 (Ago1) and Argonaute2 (Ago2), whereas the Piwi subfamily includes Aubergine, Piwi and Argonaute 3. siRNAs and miRNAs direct posttranscriptional gene silencing through effector complexes containing Ago1 or Ago2. The third class of small RNAs, called repeat-associated small interfering RNAs (rasiRNAs), is produced endogenously in the Drosophilagerm line. rasiRNAs mediate silencing of endogenous selfish genetic elements such as retrotransposons and repetitive sequences to ensure genomic stability. We examined the genetic requirements for biogenesis of rasiRNAs in both male and female germ line of Drosophilaand silencing of 8 different selfish elements, including tree LTR retrotransposons, two non-LTR retrotransposons, and three repetitive sequences. We find that biogenesis of rasiRNAs is different from that of miRNAs and siRNAs. rasiRNA production appears not to require Dicer-1 or Dicer-2. rasiRNAs lack the 2´,3´ hydroxy termini characteristic of animal siRNA and miRNA. While siRNAs derive from both the sense and antisense strands of their dsRNA precursors, rasiRNAs accumulate in antisense polarity to their corresponding target mRNAs. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. We find that rasiRNAs silence their target RNAs posttranscriptionally: mutations that abrogate rasiRNA function dramatically increase the steady-state mRNA level of rasiRNA targets, but do not alter their rate of transcription, measured by nuclear run-on assay. Our data suggest that rasiRNAs protect the fly germ line through a silencing mechanism distinct from both the miRNA and RNAi pathways.

Les dérivés phosphorylés de nucléosides naturels ou d'analogues (appelés nucléotides) sont des composés primordiaux largement utilisés comme outils biologiques. Certains d'entre eux présentent également un fort potentiel thérapeutique en particulier comme agents antiviraux ou antitumoraux. La préparation des dérivés polyphosphorylés de nucléosides demeure cependant un challenge du fait de rendements de synthèse parfois faibles et des multiples étapes de purification longues et fastidieuses. Ce manuscrit relève donc de la problématique globale de synthèse de ces composés. Le premier chapitre est consacré à l’étude non exhaustive des méthodes de synthèse décrites dans la littérature pour ces dérivés, en rappelant les avantages et inconvénients de ces approches. Le deuxième chapitre rapporte la voie de synthèse en milieux aqueux de nucléotides, et de quelques analogues, que nous avons développée. Des résultats préliminaires de mécanosynthèse basée sur cette même approche sont également présentés. Enfin, le troisième chapitre porte sur deux voies de synthèse supportée de nucléotides. L’une implique la cytidine et son ancrage sur polyéthylène glycol afin de fournir les dérivés 5’-di- et triphosphate correspondants. L’autre porte sur le développement d’un support tétrapode inédit en vue de son utilisation pour la synthèse de dérivés de l'adénosine. / Phosphorylated derivatives of endogenous nucleosides or analogues (called nucleotides) are crucial compounds widely used as biological tools. Some of them also have a high therapeutic potential, in particular as antiviral or antitumor agents. However, the synthesis of polyphosphorylated derivatives of nucleosides remains a challenge due to low yields as well as long and tedious multiple purification steps. Thus, this manuscript is dealing with the overall problem of synthesis of these compounds. The first chapter is devoted to the non-exhaustive study of the synthetic methods described in the literature for such compounds, recalling the advantages and disadvantages of these approaches. In the second chapter, we report our contribution to an original synthetic pathway in aqueous media of some nucleotides and analogues. Some preliminary results of mecanosynthesis based on the same approach are also presented. Finally, the third chapter refers to supported synthesis of nucleotides with two different approaches. One involves cytidine and its anchoring on polyethylene glycol in order to provide the corresponding 5'-di- and triphosphate derivatives. The other concerns the development of a novel tetrapod support that may be valuable for the synthesis of adenosine derivatives.

The overall objective of this study was to identify novel proteins of the nuclear matrix in order to contribute to a better understanding of nuclear structure and organization. To accomplish this, a monoclonal antibody specific for the nuclear matrix was used to screen a human λgt11 expression library. Several cDNAs were isolated, cloned, sequenced, and shown to represent NuMA, the nuclear mitotic spindle apparatus protein. Further characterization of the gene and RNA was undertaken in an effort to obtain information about NuMA. The NuMA gene was present at a single site on human chromosome 11q13. Northern and PCR analysis of NuMA mRNA showed a major 7.2 kb transcript and minor forms of 8.0 and 3.0 kb. The minor forms were shown to be alternatively spliced although their functional significance is not yet understood. Immunofluorescence microscopy demonstrated that NuMA oscillates between the nucleus and the microtubule spindle apparatus during the mitotic cell cycle. NuMA appeared as a 200-275 kDa protein detectable in all mammalian cells except human neutrophils. To determine whether NuMA's changes in intracellular distribution correlated with post-translational modifications, the protein's phosphorylation state was examined through the cell cycle using highly synchronized cells. NuMA was a phosphoprotein in interphase and underwent additional phosphorylation events in mitosis. The mitotic phosphorylation events occurred with similar timing to lamin B (G2/M transition) and were concomitant with NuMA's release from the nucleus and its association with the mitotic spindle. However, the mitotic phosphorylation occurred in the absence of spindle formation. Dephosphorylation of NuMA did not correlate with reassociation with the nuclear matrix but occurred in two distinct steps after nuclear reformation. Based on the timing of these events, phosphorylation may playa role in nuclear processes. In conclusion, the work in this dissertation identified NuMA, a nuclear matrix protein and showed that it is phosphorylated during the cell cycle and may be important for nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.

Human erythrocyte glucose sugar transport displays a complexity that is not explained by available models. Sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions (intracellular [sugar] = extracellular [sugar]). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Two models for biphasic sugar transport are presented in which 3MG must overcome a sugar-specific, physical (diffusional) or chemical (anomerization) barrier to equilibrate with cell water. The anomerization model was rejected through several lines of direct experimental investigation. 1) The sizes of the fast and slow phases of sugar transport do not correlate with the equilibrium anomer distributions of all GLUT1 sugar substrates. 2) Increasing the rate of anomerization by addition of exogenous intracellular mutarotase has no effect on biphasic transport kinetics. 3) Direct measurement of initial rates of sugar uptake or exchange demonstrates that GLUT1 shows no anomer preference. The physical barrier model was further refined by the use of the counterflow condition (intracellular [sugar] >> extracellular [sugar]). The presence of a physical barrier alone was unable to explain the complex counterflow time courses observed. As a result, the model was modified to include the action of a specific sugar export that is compartmentalized from rapidly equilibrating, GLUT1-mediated uptake and exit.

Metazoan genomes contain thousands of protein-coding and non-coding RNA genes, most of which are differentially expressed, i.e., at different locations, at different times during development, or in response to environmental signals. Differential gene expression is achieved through complex regulatory networks that are controlled in part by two types of trans-regulators: transcription factors (TFs) and microRNAs (miRNAs). TFs bind to cis-regulatory DNA elements that are often located in or near their target genes, while microRNAs hybridize to cis-regulatory RNA elements mostly located in the 3’ untranslated region (3’UTR) of their target mRNAs. My work in the Walhout lab has centered on understanding how these trans-regulators interact with each other in the context of gene regulatory networks to coordinate gene expression at the genome-scale level. Our model organism is the free-living nematode Caenorahbditis elegans, which possess approximately 950 predicted TFs and more than 100 miRNAs Whereas much attention has focused on finding the protein-coding target genes of both miRNAs and TFs, the transcriptional networks that regulate miRNA expression remain largely unexplored. To this end, we have embarked in the task of mapping the first genome-scale miRNA regulatory network. This network contains experimentally mapped transcriptional TF=>miRNA interactions, as well as computationally predicted post-transcriptional miRNA=>TF interactions. The work presented here, along with data reported by other groups, have revealed the existence of reciprocal regulation between these two types of regulators, as well as extensive coordination in the regulation of shared target genes. Our studies have also identified common mechanisms by which miRNAs and TFs function to control gene expression and have suggested an inherent difference in the network properties of both types of regulators. Reverse genetic approaches have been extensively used to delineate the biological function of protein-coding genes. For instance, genome-wide RNAi screens have revealed critical roles for TFs in C. elegans development and physiology. However, reverse genetic approaches have not been very insightful in the case of non-coding genes: A single null mutation does not result in an easily detectable phenotype for most C. elegans miRNA genes. To help delineate the biological function of miRNAs we sought to determine when and where they are expressed. Specifically, we generated a collection of transgenic C. elegans strains, each containing a miRNA promoter::gfp (Pmir::gfp) fusion construct. The particular pattern of expression of each miRNA gene should help to identify potential genetic interactors that exhibit similar expression patterns, and to design experiments to test the phenotypes of miRNA mutants.

During my thesis work I studied the expression and potential function of brain expressed microRNAs (miRNAs) in human prefrontal cortex (PFC). Initially, I used combinatorial computational analysis and microarray data to identify miRNAs that are predicted with high probability to target the human Brain Derived Neurotrophic Factor (BDNF) 3’ Untranslated Region (3’UTR) and are expressed in moderate to high levels in adult human prefrontal cortex. A subset of 10 miRNAs segregating into 5 different miRNA families (miR-30a-d, miR-103/107, miR-16/195, miR-191 and miR-495) met the above criteria. I then designed a protocol to detect these miRNAs with Locked Nucleic Acid (LNA) in situ hybridization in human prefrontal cortex and determine their layer and cellular expression patterns. LNA in situ revealed differential lamina and cellular enrichment of BDNF-related miRNAs. As an example, miR-30a-5p was found to be enriched in large pyramidal neurons of layer 3, which was verified using laser capture microdissection of layer 3 pyramidal neurons and quantitative Real Time Polymerase Chain Reaction (qRT-PCR) following dissection of upper and deeper layers of human PFC. Parallel to this, I used miRNA qRT-PCR to determine the developmental expression of miRNAs using postmortem PFC tissues ranging from embryonic age to old adulthood and compared miRNA to BDNF protein levels. My results revealed a robust inverse correlation between BDNF-related miRNAs and BDNF protein during late maturation and aging of human prefrontal cortex. In vitro luciferase assays and/or lentivirus mediated neuronal miRNA overexpression experiments validated that at least two miRNAs, miR-30a-5p and miR-195, target human BDNF 3’UTR and mediate its translational repression. In the second part of my thesis work I measured levels of miR-30a and miR-195 in the prefrontal cortex of patients with schizophrenia and compared them with levels of BDNF protein and BDNF-related GABAergic mRNAs. According to my results differences in miR-195 levels in a subset of subjects diagnosed with schizophrenia were found to be associated with disease related changes in BDNF protein levels and deficits in BDNF dependent GABAergic gene expression. In the last part of my work I focused on miR-30b, another member of the miR-30 family, which I found to be reduced in the prefrontal cortex of female but not male subjects with schizophrenia. More importantly, disease related changes in miR-30b levels were strongly associated with the age of onset of the disease. Additional experiments in mouse cortex and hippocampus revealed a gender dimorphic expression pattern of this miRNA with higher expression in female brain. Collectively, my results suggest that miRNAs could participate in novel molecular pathways that play an important role during cortical development and maturation and are potentially linked to the pathophysiology of neuropsychiatric disease.

Nutrientes específicos, denominados farmaconutrientes, demonstraram possuir a capacidade de modular a resposta imunológica e inflamatória de animais e seres humanos, em estudos clínicos e laboratoriais. Dentre os substratos conhecidos, os que têm maior relevância e ação imunomoduladora são a arginina, glutamina, ácido graxo n-3 e nucleotídeos. No entanto, revisões sistemáticas e meta-análises buscam consenso em relação aos vários e controversos resultados publicados sobre os possíveis benefícios da imunonutrição em pacientes críticos. Nossos objetivos foram avaliar a efetividade das dietas enriquecidas com Imunonutrientes na redução de complicações e mortalidade nos diferentes tipos de pacientes críticos. O presente estudo é uma revisão sistemática com metanálise onde foram inseridos ensaios clínicos randomizados avaliando o uso de nutrientes imunomoduladores em doente adulto de ambos os sexos, definido como crítico traumatizado, séptico, queimado ou cirúrgico; as dietas utilizadas deveriam conter um ou mais dos imunonutrientes, em qualquer dose, administradas por via enteral comparadas à dieta padrão pela mesma via em pelo menos um dos grupos de comparação. As bases de dados consultadas foram Pubmed e Cinhal, utilizando os termos: Immunonutrition, arginine, glutamine, n-3, nucleotides e criticall illness. De 206 artigos encontrados inicialmente, apenas 35 preencheram os critérios de elegibilidade estabelecidos. Destes 35 ensaios clínicos, 18 foram conduzidos em pacientes cirúrgicos, 6 em pacientes traumatizados, 5 em pacientes queimados, 5 em pacientes críticos em geral e 1 em pacientes sépticos. Para a população geral, não houve redução significativa do risco de morrer indicada pelo RR de 0,84 (IC de 0,68 a 1,05) e os queimados foram mais protegidos de morrer com RR de 0,25 (IC de 0,09 a 0,66); as complicações infecciosas foram reduzidas com RR de 0,56 (IC de 0,42 a 0,73); a incidência de sepse foi reduzida com RR de 0,45 (IC de 0,29 a 0,69), especialmente nos pacientes traumatizados com RR de 0,42 (IC de 0,26 a 0,68); a incidência de abscesso abdominal também foi reduzida com RR de 0,39 (IC de 0,21 a 0,72) e também de bacteremia com RR de 0,46 (IC de 0,31 a 0,66); o tempo de internação hospitalar diminuiu -3,9 dias (IC de -5,0 a -2,8). Para a população de pacientes cirúrgicos: o óbito não apresentou redução significativa do RR de 0,93 (IC de 0,44 a 1,95), as complicações infecciosas foram reduzidas com RR de 0,51 (IC de 0,41 a 0,63), o tempo de internação hospitalar foi reduzido - 3,41 dias (IC de -4,25 a -2,58) e o tempo de internação em UTI -1,72 dias (IC de -2,12 a -1,31). A utilização de dietas com nutrientes imunomoduladores não alterou a mortalidade em doentes críticos ou cirúrgicos. Indivíduos com mais de 60 anos são menos protegidos de morrer pela utilização de dietas com imunomoduladores. As complicações infecciosas são reduzidas em pacientes críticos com a utilização de imunonutrição, em especial a população de pacientes cirúrgicos. Dietas com mais de 10g/l de arginina protegem mais os pacientes críticos da incidência de complicações infecciosas. Pacientes traumatizados são protegidos da incidência de sepse pela utilização da imunonutrição. O tempo de internação hospitalar e o tempo de internação em UTI foram reduzidos em críticos e cirúrgicos com imunonutrição. A execução de ensaios clínicos explanatórios nos diferentes tipos de doentes críticos com um nutriente imunomodulador isolado, seguida de ensaios pragmáticos de acordo com os resultados dos anteriores, é um fato a ser considerado em futuras pesquisas. / Specific nutrients, the so-called pharmaconutrients, showed a capability to modulate human and animal immune answers in clinical and laboratory studies. Amongst the well-known substracts, arginine, glutamine, fatty accid n-3, and nucleotide showed the most relevant immunomodulator actions. However, systematic revisions and meta-analysis are still in search of agreement due to several controversial results published in the literature on the probable benefits of the immunonutrition of the critically ill. Evaluate the effectiveness of the immunonutrients enriched diets in the reduction of complications and mortality of different types of the critically ill patients. The present study is a meta-analysis systematic revision where randomized clinical trials were included, evaluating the immunomodulator nutrients use in ill adults of both genders, defined as critical traumatized, septicemic, burned, or surgical patients; diets administered should contain one or more immunonutrients, at any dose, ministered by enteral route and compared with a pattern diet offered by the same via at least to one of the comparison groups. Pubmed and Cinhal data banks were searched for the following terms: immunonutrition, arginine, glutamine, n-3, nucleotides and critical illness. In 206 articles were initially found, but only 35 agreed to the established electability criteria: 18 were conducted in the surgical patients, 6 in the traumatized patients, 5 in the burned patients, 5 in the general critical patients, and 1 in the septicemic patients. Considering the general population: obit did not show a significant RR reduction of 0.84 (IC from 0.68 to 1.05); burned patients were more protected from death with RR of 0.25 (IC from 0.09 to 0.66); infections complications were reduced with RR of 0.56 (IC from 0.42 to 0.73); sepsis incidence was reduced with RR of 0.45 (IC from 0.29 to 0.69); specially in the traumatized patients with RR of 0.42 (IC from 0.26 to 0.68); abdominal abscess incidence was also reduced with RR of 0.39 (IC from 0.21 to 0.72) and also bacteremia with RR of 0.46 (IC from 0.31 to 0.66); hospitalization time was also reduced in -3.9 days (IC from -5.0 to -2.8). Considering the surgical patients population: obit did not present a significant RR reduction of 0.93 (IC from 0.44 to 1.95); infections complications were reduced with RR of 0.51 (IC from 0.41 to 0.63); hospitalization period was reduced in -3.41 days (IC from -4.25 to -2.58), and in the ICU time was reduced in -1.72 days (IC from -2.12 to -1.31). Administration of immunomodulator nutrient diets did not change the mortality of the critically ill or surgical patients. Individuals over 60 years are less protected from death with the administration of immunomodulator diets. Infections complications showed a reduction in the critically ill patients with the utilization of immunonutrition, specially the surgical patients population. Diets containing more than 10g/l of arginine protect the critical patients from the incidence of infectious complications. Traumatized patients are protected from the incidence of sepsis by the administration of the immunonutrition. Hospitalization and ICU time were reduced for critical and surgical patients in use of immunonutrition. Explanatory clinical trials in different types of critically ill patients using an isolated immunomodulator nutrient followed by pragmatic trials according to previous results should be considered in future research.