O presente estudo tem como objetivo investigar a frequência dos diversos polimorfismos dos genes KIR (Killer Immunoglobulin-like Receptors) e HLA C1 e C2 em um grupo de pacientes com câncer de mama e comparar com um grupo controle de indivíduos sadios. As células natural killer (NK) são linfócitos que diferem das células T e B e que fazem parte da imunidade natural, reconhecendo as moléculas HLA (Antígenos Leucocitários Humano) de classe I em células infectadas por vírus ou em células tumorais, através de seus receptores de membrana. Os principais receptores das células NK são conhecidos como receptores KIR, sendo codificados por genes localizados no cromossomo 19q13.4 e classificados em grupos funcionais supressores e ativadores. Neste estudo, analisamos 15 genes KIR e alelos do sistema HLA de classe I em 230 pacientes caucasóides e em 278 controles, usando a técnica de PCR com primers específicos (PCR-SSO e PCR-SSP). Nossos resultados demonstraram uma frequência maior do genótipo supressor 2DL2 (P<0,001) em pacientes com câncer de mama, quando comparados ao grupo controle. Os genes HLA-C2 e HLA-BW4 não apresentaram diferenças significantes entre os grupos. Contudo, o gene HLA-C1 foi observado em maior frequência nos pacientes com câncer de mama. Considerando que estes achados sugerem uma potencial associação entre o sistema de genes KIR, HLA classe I e o câncer de mama, estudos adicionais sobre este tema são necessários. / We investigated the frequency of various KIR (Killer Immunoglobulin-like Receptors) and HLA C1 and C2 gene polymorphisms in a group of patients with breast cancer and healthy controls. Natural Killer (NK) cells are lymphocytes that differ from T and B cells and are part of the innate immune system, recognizing class I Human Leukocyte Antigens (HLA) molecules on target cells (virus-infected as well as cancer cells), through specific cell surface receptors. KIR comprises the main class of NK receptors, being encoded by genes located in chromosome 19q13.4. They possess both suppressor and activating functional groups. Fifteen KIR genes and class I HLA alleles obtained from 230 Caucasians patients, as well as 278 controls were studied, using PCR techniques with specific primers (PCR-SSO and PCR-SSP). Our results showed a higher frequency of suppressor genotype 2DL2 (P<0,001) in patients with breast cancer as compared to controls. No significant difference between HLA-C2 and HLA-BW4 alleles were observed between the study groups. Notably, a higher frequency of HLA-C1 gene was noted in patients with breast cancer. Our results suggest a potential association between KIR genes, HLA class I and breast cancer, deserving further investigation.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-11-11T20:37:41Z No. of bitstreams: 2 Dissertação - Leandro Prado Felício - 2013.pdf: 4839350 bytes, checksum: 07898140311917429ab55aa63ad5324e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2014-11-11T20:38:05Z (GMT) No. of bitstreams: 2 Dissertação - Leandro Prado Felício - 2013.pdf: 4839350 bytes, checksum: 07898140311917429ab55aa63ad5324e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-11T20:38:05Z (GMT). No. of bitstreams: 2 Dissertação - Leandro Prado Felício - 2013.pdf: 4839350 bytes, checksum: 07898140311917429ab55aa63ad5324e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-01-31 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The HLA-E locus is a Human Major Histocompatibility Complex (MHC) gene associated with immune-modulation and suppression of the immune response by the interaction with specific NK and T cell receptors. The HLA-E gene is considered the most conserved locus in the human HLA; however, this low variability might be a consequence of the scarce number of studies focusing this subject. In this mastering thesis we assessed the HLA-E coding and 3’ untranslated region variability in a group of individuals from Brazil and the results were evaluated together with data from the 1000Genomes Consortium. Altogether, only 28 variation sites were found in approximately 2724 bp evaluated. These variation sites were arranged into 33 haplotypes, most of them (98.2%) encoding one of the two HLA-E molecules found worldwide, i.e., the molecules associated with the allele groups E*01:01 and E*01:03. Interestingly, 85% of all haplotypes were represented by only three different sequences, each of them associated with one of the main known HLA-E coding alleles, E*01:01:01, E*01:03:01 and E*01:03:02, all of them found worldwide. This phenomenon, together with the comparisons with other primate sequences, reveals that these two main allele groups (and molecules) arose early before human speciation, and indicates that E*01:03:01 might be the oldest allele. In addition, the low nucleotide diversity found for the HLA-E coding and 3’UTR in worldwide populations suggests that the HLA-E gene is in fact a conserved gene, which might be a consequence of its key role in the modulation of the immune system. / O loco HLA-E é um gene do Complexo Principal de Histocompatibilidade Humano (MHC), cujo produto está relacionado com a modulação e supressão da resposta imunitária por meio da interação com receptores específicos das células NK e linfócitos T. O gene HLA-E é considerado o loco menos polimórfico dos genes do complexo HLA, no entanto, esta baixa variabilidade pode ser uma consequência do pequeno número de estudos realizados sobre esse tema. No presente trabalho, a variabilidade das regiões codificadoras e 3’ não traduzida do gene HLA-E foi analisada em amostras brasileiras e os resultados foram comparados com dados obtidos pelo projeto 1000Genomes. Considerando todas as populações avaliadas, apenas 28 pontos de variação foram encontrados em uma região de aproximadamente 2724-pb. Estes pontos de variação estão arranjados em 33 haplótipos diferentes, a maioria deles (98%) codificando uma das duas moléculas HLA-E frequentemente encontradas, E*01:01 e E*01:03. Ainda, 85% dos haplótipos encontrados foram representados por apenas três sequências diferentes, cada uma deles associada a um dos principais alelos da região codificadora do gene HLA-E, E*01:01:01, E*01:03:01 e E*01:03:02. Todas essas sequências foram encontradas em todas as populações avaliadas. Este fenômeno, em conjunto com as comparações envolvendo sequências de primatas, sugere que estes dois grupos de alelos principais (e moléculas) surgiram antes da especiação e dispersão humana, além de indicar que o alelo E*01:03:01 pode ser o mais antigo dentre os demais. Ainda, a baixa diversidade nucleotídica encontrada para a região codificadora e 3' NT do gene HLA-E em populações de todo o mundo sugere que este gene é, de fato, bastante conservado, provavelmente devido ao seu papel chave na modulação das respostas imunes.

The human leukocyte antigen (HLA) is the most polymorphic region in the human genome and accounts for more than 10% of human diversity. This region plays an important role in matching donors and recipients for transplantation. The South African Bone Marrow Registry (SABMR) does not reflect the demographics of the South African population. The large number of polymorphisms resulting from HLA diversity in the Black South African population and their limited representation in the SABMR reduce the chances of finding adequate matches between donors and recipients in this group. Umbilical cord blood is an alternative to bone marrow for the treatment of fatal diseases. Less strict HLA matching is required due to the naive nature of the T cells in cord blood. A public umbilical cord blood bank is a necessity in trying to cater for the diverse population in South Africa. However, the ethnic diversity of the South African population poses a great challenge in constituting a public umbilical cord blood bank that is representative of the entire population. The Roche designed next generation sequencing (NGS) high resolution (HR) HLA typing kit enables sequencing of additional HLA exons and could improve the degree of matching between individuals to ultimately decrease adverse reactions. An extensive study of the literature was performed to establish the demographics, linguistics, and HLA diversity of the South African population to determine how a public cord blood bank should be constituted. In addition, HLA genotyping was performed by 454 NGS on 20 samples that had previously been HLA typed by conventional methods. The 454 NGS technique made use of a Roche designed medium and high resolution HLA typing kit to genotype the samples. It was possible to assign accurate genotypes to 95.5% of the loci of interest for the total number of 20 samples using the MR kit, compared with 98.5% using the HR kit. In conclusion, the present study indicates the extreme HLA diversity in the South African population, and therefore, recommends constituting the first public umbilical cord blood bank in Gauteng on the basis of race or major ethnic groupings. A minimum number of 10 000 cord blood units is needed to initiate the bank. Furthermore, the 454 NGS platform together with the HR HLA typing kit display potential as an alternative method to be used in a public cord blood bank, as well as routine clinical and diagnostic laboratories, to ultimately improve HLA matching between donors and recipients. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Immunology / unrestricted

Data Distribution Management (DDM) deals with two basic problems: how to distribute data generated at the application layer among underlying nodes in a distributed system and how to retrieve data back whenever it is necessary. This thesis explores DDM in two different network environments: peer-to-peer (P2P) overlay networks and cluster-based network environments. DDM in P2P overlay networks is considered a more complete concept of building and maintaining a P2P overlay architecture than a simple data fetching scheme, and is closely related to the more commonly known associative searching or queries. DDM in the cluster-based network environment is one of the important services provided by the simulation middle-ware to support real-time distributed interactive simulations. The only common feature shared by DDM in both environments is that they are all built to provide data indexing service. Because of these fundamental differences, we have designed and developed a novel distributed data structure, Hierarchically Distributed Tree (HD Tree), to support range queries in P2P overlay networks. All the relevant problems of a distributed data structure, including the scalability, self-organizing, fault-tolerance, and load balancing have been studied. Both theoretical analysis and experimental results show that the HD Tree is able to give a complete view of system states when processing multi-dimensional range queries at different levels of selectivity and in various error-prone routing environments. On the other hand, a novel DDM scheme, Adaptive Grid-based DDM scheme, is proposed to improve the DDM performance in the cluster-based network environment. This new DDM scheme evaluates the input size of a simulation based on probability models. The optimum DDM performance is best approached by adapting the simulation running in a mode that is most appropriate to the size of the simulation.

O presente estudo tem como objetivo investigar a frequência dos diversos polimorfismos dos genes KIR (Killer Immunoglobulin-like Receptors) e HLA C1 e C2 em um grupo de pacientes com câncer de mama e comparar com um grupo controle de indivíduos sadios. As células natural killer (NK) são linfócitos que diferem das células T e B e que fazem parte da imunidade natural, reconhecendo as moléculas HLA (Antígenos Leucocitários Humano) de classe I em células infectadas por vírus ou em células tumorais, através de seus receptores de membrana. Os principais receptores das células NK são conhecidos como receptores KIR, sendo codificados por genes localizados no cromossomo 19q13.4 e classificados em grupos funcionais supressores e ativadores. Neste estudo, analisamos 15 genes KIR e alelos do sistema HLA de classe I em 230 pacientes caucasóides e em 278 controles, usando a técnica de PCR com primers específicos (PCR-SSO e PCR-SSP). Nossos resultados demonstraram uma frequência maior do genótipo supressor 2DL2 (P<0,001) em pacientes com câncer de mama, quando comparados ao grupo controle. Os genes HLA-C2 e HLA-BW4 não apresentaram diferenças significantes entre os grupos. Contudo, o gene HLA-C1 foi observado em maior frequência nos pacientes com câncer de mama. Considerando que estes achados sugerem uma potencial associação entre o sistema de genes KIR, HLA classe I e o câncer de mama, estudos adicionais sobre este tema são necessários. / We investigated the frequency of various KIR (Killer Immunoglobulin-like Receptors) and HLA C1 and C2 gene polymorphisms in a group of patients with breast cancer and healthy controls. Natural Killer (NK) cells are lymphocytes that differ from T and B cells and are part of the innate immune system, recognizing class I Human Leukocyte Antigens (HLA) molecules on target cells (virus-infected as well as cancer cells), through specific cell surface receptors. KIR comprises the main class of NK receptors, being encoded by genes located in chromosome 19q13.4. They possess both suppressor and activating functional groups. Fifteen KIR genes and class I HLA alleles obtained from 230 Caucasians patients, as well as 278 controls were studied, using PCR techniques with specific primers (PCR-SSO and PCR-SSP). Our results showed a higher frequency of suppressor genotype 2DL2 (P<0,001) in patients with breast cancer as compared to controls. No significant difference between HLA-C2 and HLA-BW4 alleles were observed between the study groups. Notably, a higher frequency of HLA-C1 gene was noted in patients with breast cancer. Our results suggest a potential association between KIR genes, HLA class I and breast cancer, deserving further investigation.

Typning av hla-b*27:En jämförelsestudie mellan två analysmetoder för att påvisa HLA-B*27 molekylen i ankyloserande spondylitCarolina BermudezBermudez, C. Typning av HLA-B*27. En jämförelsestudie mellan två analysmetoder för att påvisa HLA-B*27 molekylen i Ankyloserande spondylit. Examensarbete i Biomedicinsk vetenskap, 15 högskolepoäng. Malmö universitet: Fakulteten för hälsa och samhälle, institutionen för Biomedicinsk vetenskap, 2018.Human leukocyt antigen (HLA) är vävnadsantigener, belägna på våra vita blodkroppar. HLA-B*27 allelen är starkt kopplat till Ankyloserande spondylit (AS). Det är en kronisk inflammatorisk ledsjukdom, som främst attackerar ryggraden, bäckenet och bröstkorgen. Det finns idag ingen enskild laborativ metod som med full säkerhet kan fastställa diagnos av denna sjukdom, innan de kliniska symtomen uppträder. Typning av HLA-B*27 ger endast information om närvaro eller frånvaro av antigenet, vid utredning av AS. Vidare är HLA-B*27 en polymorf och de olika alleltyperna varierar kraftigt, bland skilda etniska grupper samt mellan geografiska områden. Genetiska- och miljöfaktorer påverkar också. Sjukdomsutveckling i samband med närvaro av HLA-B*27 allelen, varierar därför från individ till individ. Därmed fungerar metoden endast som ett komplement-verktyg, för att ytterligare bekräfta diagnos. Syftet med denna studie var att med realtids-polymerase chain reaction (PCR), utföra typning av HLA-B*27 med Linkseq kit samt jämföra analysresultaten med uthämtade resultat från intern sjukhusdatabas, där typning av HLA-B*27 hade utförts med PCR-SSP (sekvens-specifika primers). Samtliga resultat stämde överens till 100%, vilket indikerar att metoden fungerar bra. Det finns studier som visat att HLA-B*27 molekylens fria tunga kedjor (HLA-B*272) har en starkare benägenhet än andra HLA-molekyler att binda in till killer immunoglobine-like receptorer (KIRs). Inbindning till KIRs med efterföljande ökad stimulering av interleukiner (IL) främst IL-17 och IL-23 bidrar till sjukdomsutvecklingen av AS. Dock finns ingen HLA-B*272 specifik antikropp som kan bevisa detta och det behövs därför ytterligare undersökning för att hitta en sådan. Därefter skulle en ny laborativ metod kunna utvecklas för att fastställa diagnos av AS i ett tidigt skede, innan de kliniska symtomen uppvisas. Nyckelord: Allelvarianter, Ankyloserande spondylit, HLA-B*27, KIR, PCR-SSP, Realtids-PCR. / typing of hla-b*27:a comparison study between two analysing methods for the detection of the HLA-B*27 molecule in ankylosing spondylitisCarolina BermudezBermudez, C. Typing of HLA-B*27. A comparison study between two analysing methods for the detection of the HLA-B*27 molecule in Ankylosing spondylitis. Degree project in Biomedical Laboratory Science, 15 credit points. Malmö University: Faculty of Health and Society, Department of Biomedical science, 2018.Human leukocyte antigen (HLA) are tissue antigens located on our white blood cells. The HLA-B*27 allele is strongly related to Ankylosing spondylitis (AS). It is a chronical inflammatory rheumatic disease that primarily affects the spine, the pelvis and the chest. At present, there is no single laboratory method that with all certainty may determine diagnosis of this disease, before the clinical symptoms appear. Typing of HLA-B*27 only gives information about the presence or absence of the antigen, upon the investigation of AS. Furthermore, HLA-B*27 is a polymorph and the different types of alleles, strongly vary among different ethnic groups and also between geographic regions. Genetic- and environmental factors also affect. Development of disease in conjunction with the presence of the HLA-B*27 allele, therefore varies from one individual to another. So, the method only functions as a complementary tool, to further confirm diagnosis. The aim of this study was to perform HLA-B*27 typing with realtime-polymerase chain reaction (PCR) using Linkseq kit and compare the analysed results with those results that were retrieved from the internal database of the hospital, where typing of HLA-B*27 had been performed with PCR-SSP (sequence specific primers). All results agreed with 100%, which indicates that the method functions well. There are studies that show that the heavy chains (HLA-B*272) of the HLA-B*27 molecule have a stronger affinity than other HLA-molecules of binding in to killer immunoglobulin-like receptors (KIRs). Increased stimulation of interleukins (IL) primarily IL17 and IL23, following binding to KIRs, contributes to the pathogenesis of ankylosing spondylitis. However, there is no HLA-B*272 specific antibody that may prove this and therefore more investigation is needed, in order to find one. A new laboratory method could then be developed to determine diagnosis of AS at an early stage, before the clinical symptoms emerge. Keyword: Allelvariants, Ankylosing spondylitis, HLA-B*27, KIR, PCR-SSP, Realtime-PCR.

Malgré le succès de l’utilisation des lymphocytes T modifiées par des récepteur antigéniques chimériques (CAR) contre les leucémies, celles-ci présentent des limites comme leur risque de CRS et leur inefficacité dans les tumeurs solides. Plusieurs autres immunothérapies cellulaires ont été proposées pour pallier à ces inconvénients. Les cellules natural killer (NK) ont plusieurs propriétés qui en font une alternative avantageuse aux cellules T dans les immunothérapies. Cependant, les cellules NK restent difficiles à modifier avec les outils actuels et leur efficacité reste limitée par les mécanismes immunosuppresseurs des tumeurs. Nous avons réussi à augmenter l’efficacité de transduction avec une nouvelle glycoprotéine, le BaEVRless. Nous avons aussi démontré que cette enveloppe ne provoque pas de modification du phénotype ou de l’activité intrinsèque des cellules NK. Dans un modèle de leucémie, nous avons déterminé que l’utilisation du BaEVRless permet la production de cellules CAR-NK fonctionnelles. Les cellules NK peuvent aussi être transduites efficacement par des constructions lentivirales portant les séquences codant pour deux constructions CAR simultanément. Nous avons aussi démontré que l’édition génomique des NK par la technologie Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) est possible en utilisant une livraison non-virale. Avec cette méthode, nous avons pu réduire l’expression de NKG2A. Les cellules NK avec une expresssion réduite de NKG2A étaient résistantes à l’inhibition par HLA-E, exprimé sur des lignées de cancer du sein et du colon. Cet effet a été confirmé in vivo dans un modèle préclinique xenogénique. Ces résultats montrent deux stratégies qui pourraient permettre d’améliorer les immunothérapies à base de cellules NK. / Despite the overwhelming success of chimeric antigen receptor (CAR)-modified T lymphocytes against leukemias, some limitations have been observed, such as the risk of developing CRS and the lack of efficiency in solid tumor settings. Many other cell-based immunotherapies have been explored to circumvent those caveats. Natural killer (NK) cells present many advantageous properties that could make them a very promising alternative to T cells in immunotherapies. However, NK cells have some caveats, mainly they are hard to modify using conventional tools and they are sensitive to many inhibitory signals expressed by cancer cells. We managed to greatly improve the efficiency of transduction using a novel viral glycoprotein, BaEVRless. In the process, we determined that this novel enveloppe glycoprotein did not modify the phenotype or intrinsic activity of the transduced NK cells. In a leukemia model, we also showed that the BaEVRless can be used to generate functionnal CAR-NK cells. Moreover, the NK cells can be transduced with larger lentiviral constructions bearing two simultaneous CAR-coding sequences. We also demonstrated that Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) modification of NK cells using a non-viral approach was possible. Using this approach, we generated NK cells with lower NKG2A expression, that were resistant to the inhibitory effects of HLA-E. This affect was seen in a breast cancer model and a colon cancer model. The in vitro results were confirmed in an in vivo preclinical xenogeneic model. Together, those results represent two improvements applicable to NK cell-based immunotherapies.

Antigen presentation and recognition are key processes of the immune system necessary to initiate the adaptive immune response. Longstanding goals of these fields have been to understand the molecular mechanism of MHC II-peptide binding, the way in which dysregulation of this process can lead to disease, and determining how γδTCRs recognize their ligands. To examine some of these outstanding questions, I designed photocleavable peptides that could bind HLA-DR1 and could be used to facilitate peptide exchange. I also performed studies to understand whether peptide exchange on HLA-DR1 can be affected by glycation modifications, which occur in hyperglycemic conditions such as diabetes. I observed that while glycation modifications on HLA-DR1 did not affect peptide exchange, these modifications decreased the catalytic effect of HLA-DM on this reaction, which could affect antigen presentation in diabetic patients. For studies on antigen recognition by γδTCRs, I focused on γδNKT cells, a subset of γδT cells known to play a role during Listeria infection. I used four different variants of the γδNKT TCR to study the restrictions on Vγ junctional region usage by this TCR for ligand recognition. I found that all the TCR variants I examined could recognize cells infected with Listeria, indicating that this TCR is not restricted by γ-chain usage in order to recognize ligand. My research generated reagents that could serve in future studies of HLA-DR1 peptide binding and contributed to understanding the effect of hyperglycemic conditions on antigen presentation, as well as provided greater understanding of γδTCR restriction for ligand recognition.

MHC II proteins are heterodimeric glycoproteins that form complexes with antigenic peptides in order to elicit a CD4+ adaptive immune response. Even though there have been numerous MHC II-peptide crystal structures solved, there is little insight into the dynamic process of peptide loading. Through biochemical and biophysical studies, it has been shown that MHC II adopt multiple conformations throughout the peptide loading process. At least one of these conformations is stabilized by the MHC II-like homologue, HLA-DM. The main focus of this thesis is to elucidate alternate conformers of MHC II in an effort to better understand the structural features that enable HLA-DM catalyzed peptide loading. In this thesis, two altered conformations of HLA-DR were investigated, one modeled in the absence of peptide using molecular dynamics, and one stabilized by the mutation αF54C. The model for the peptide-free form of HLA-DR1 was derived from a molecular dynamics simulation. In this model, part of the alpha-subunit extended-strand region proximal to the peptide binding groove is folded into the peptide-binding groove such that the architecture of the critical peptide binding pocket, P1, as well as the invariant hydrogen bonding network were maintained. Biochemical studies aimed at validating the predicted structural changes were consistent with the model generated from the simulations. Next, structural studies were carried out on an MHC II mutant, αF54C, which was shown to have unique peptide binding characteristics as well as enhanced susceptibility to HLA-DM. Although this mutation did not affect the affinity for peptide, there was a striking increase in the rate of intrinsic peptide release. Both αF54C and αF54A were over 100-fold more susceptible to HLADM catalyzed peptide release than wild type as well as other mutants introduced along the peptide binding groove. In addition, mutation of the αF54 position results in a higher affinity for HLA-DM, which, unlike wild type, is detectable by surface plasmon resonance. Crystallographic studies resulted in a 2.3 Å resolution structure for the αF54C-Clip complex. There were two molecules in the asymmetric unit, one of which had no obvious deviations from other MHC II-pep complexes and one which had a conformational change as a result of a crystal contact on the αF51 residue, a residue which has been shown to be involved in the HLA-DM/HLA-DR binding interface. The crystal structure of wild type HLA-DR1- Clip was also solved, but did not have the altered conformation even though there was a similar crystal contact at the αF51. These data suggest the altered conformation seen in the mutant structure, results from increased lability in the extended stand region due to the αF54C mutation. As a result of this work, we have developed a new mechanistic model for how structural features of MHC II influence DM mediated peptide release.

Computer modeling and simulation is a practical way to design and test a system without actually having to build it. Simulation has many benefits which apply to many different domains: it reduces costs creating different prototypes for mechanical engineers, increases the safety of chemical engineers exposed to dangerous chemicals, speeds up the time to model physical reactions, and trains soldiers to prepare for battle. The motivation behind this work is to build a common software framework that can be used to create new networking simulators on top of an HLA-based federation for distributed simulation. The goals are to model and simulate networking architectures and protocols by developing a common underlying simulation infrastructure and to reduce the time a developer has to learn the semantics of message passing and time management to free more time for experimentation and data collection and reporting. This is accomplished by evolving the simulation engine through three different applications that model three different types of network protocols. Computer networking is a good candidate for simulation because of the Internet's rapid growth that has spawned off the need for new protocols and algorithms and the desire for a common infrastructure to model these protocols and algorithms. One simulation, the 3DInterconnect simulator, simulates data transmitting through a hardware k-array n-cube network interconnect. Performance results show that k-array n-cube topologies can sustain higher traffic load than the currently used interconnects. The second simulator, Cluster Leader Logic Algorithm Simulator, simulates an ad-hoc wireless routing protocol that uses a data distribution methodology based on the GPS-QHRA routing protocol. CLL algorithm can realize a maximum of 45% power savings and maximum 25% reduced queuing delay compared to GPS-QHRA. The third simulator simulates a grid resource discovery protocol for helping Virtual Organizations to find resource on a grid network to compute or store data on. Results show that worst-case 99.43% of the discovery messages are able to find a resource provider to use for computation. The simulation engine was then built to perform basic HLA operations. Results show successful HLA functions including creating, joining, and resigning from a federation, time management, and event publication and subscription.