**8. References**

Bäck, T. (1996). *Evolutionary Algorithms in Theory and Practice*, Oxford University Press, 1996

Bouchhida, O. (2011). *Etude et Optimisation des Performances d'Onduleur Monophasé et Triphasé à Commutation Pré-Calculée*. Habilitation Universitaire, Université Saad D'hleb Blida, Algérie

**Chapter 20**

© 2012 Bonaldi et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Bonaldi et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Predictive Maintenance by Electrical**

Erik Leandro Bonaldi, Levy Ely de Lacerda de Oliveira, Jonas Guedes Borges da Silva, Germano Lambert-Torresm

expensive, forcing the company to operate without a stand-by.

and Luiz Eduardo Borges da Silva

http://dx.doi.org/10.5772/48045

determined (Bonaldi et al, 2007).

**1. Introduction**

Additional information is available at the end of the chapter

**Signature Analysis to Induction Motors**

Industries always try to increase the reliability of their productive process. In this context, predictive maintenance performs a fundamental role in order to reach high availability and reliability concerning their pieces of equipment. Predictive maintenance can be understood as the action on the equipment, system or installations based on the previous knowledge about the operation condition or performance, obtained by means of parameters previously

Since the induction motors are the center of the vast majority of the industrial processes, this chapter gives total emphasis to the failure analysis and identification of this kind of electrical machine. Like all the rotating machines, the induction motors are exposed to many different adversities such as thermal and environmental stresses and mechanical damages, which demand maximum attention (Lambert-Torres et al., 2003). Usually, in industries, attention must be even larger since the downtime costs are very high. High and medium voltage induction motors are highly used in industrial processes. Many of them are strategic to the productive process and, because of that, looking for solutions that minimize the failure statistics is mandatory. In most cases, these motors are highly reliable and extremely

Many predictive techniques are applied to these motors to reduce the number of unplanned outage. The most common techniques applied to fault detection in induction motors are: vibration analysis, acoustical analysis, speed oscillations, partial discharges, circuit analysis, etc. The analyses based on mechanical concepts are established, but the techniques based on electrical signature analysis are being introduced only now. Because of that status, the application of Electrical Signature Analysis (ESA) to industries is the concern of this chapter.

Czarkowski, D.; Chudnovsky, D.; Chudnovsky, G. & Selesnick, I. (2002). Solving the optimal PWM problem for Single-phase inverters. *IEEE Transactions Circuits Syst*. I, Vol. 49, (April 2002), 465-475

Davis, L. (1991). *The Handbook of Genetic Algorithms*, Van Nostrand & Reinhold, 1991

**Chapter 20**


*2.3. Epithelial Surface*

The apical AE2 cell surface exhibits smooth areas surrounded by microvilli as described previously [1]. The pink cell has two larger plain areas and one of them shows an open surfactant secretion pore (Figure 2). (At this stage of exocytosis) the pore has a smaller diameter than the vesicle under secretion, cf. [1]. The green AE2 cell shows a larger plain surface area in the recess between the adjacent AE2 cells (Figure 1). Also the basolateral AE2 cell surface shows abundant microvilli. In contrast to the luminal microvilli, they may appear clustered in small groups, located in small niches of the cell surface. These microvilli are found both between adjacent epithelial cells (AE2 and AE2 or AE2 and AE1), but also at gaps in the basal lamina, where they may reach interstitial cells (Figure 3). They are even found above a continuous basal lamina (not shown).

The AE1 cell domains also exhibit a combination of smooth surface areas with surface enlargements (Figures 1 and 4). In contrast to the AE2 cells, this enlargement is realized primarily by protrusions of the plasma membrane filled with cytoplasm. However, some microvilli are found here as well, which can be identified by their regular structure and inner architecture of the cytoskeleton. Some microvilli may share a common basis and some seem to rest on surface protrusions. Both AE1 cell domains show grooves bordered by cellular folds on their basal surface. These grooves stretch across the cells in a relatively orientated fashion along space. Beneath the groove of the yellow AE1 cell domain primarily extracellular matrix is found while it is primarily capillary endothelium beneath the blue cell domain (Figure 5). Additionally, the AE1 cells show abundant caveolae and it is the basal compartment that seems to house most of them (Figure 5B).

**Figure 2. Luminal AE2 cell surface and surfactant secretion pore.** (**A**) Detail from image 884 which shows an open surfactant secretion pore (arrowhead) and remnants of a lamellar body inside (asterisk). In this image the opening is much smaller than the profile diameter of the vesicle under secretion. (**B**) 3D model of the AE2 cell with the secretion pore (arrowhead) and the remnants of the lamellar body inside (yellow, asterisk). Note the plain cell surface in this area. (**C**) Same image content as in B but with a transparent cell body, which reveals the entire remnant of the lamellar body and indicates the size of the secretory vesicle (circumference marked by arrowheads), the projection of which is much larger than the secretion pore. Scale bars: 1 μm. The black lines in B and C indicate the position of the section plane of A.

**Figure 3. Basolateral surface of AE2 cells.** (**A**) Segmented electron micrograph (image 988 of the data set). The micrograph shows the segmentations of the pink and green AE2 cell as well as of the yellow AE1 cell domain with opaque outlines and transparent filling. Microvilli can be found at different sites of the AE2 cell, three of them are indicated on the micrograph: towards the adjacent green AE2 cell, which also shows microvilli at this site (filled arrowheads 2 and 3), towards the yellow AE1 cell domain (filled arrowhead 4), towards an interstitial cell (hash key) through a hole in the basal lamina (filled arrowhead 5). Turquoise circles indicate the lateral luminal cell border of the pink AE2 cell (empty arrowheads). The position at empty arrowhead 1 is found in the 3D model in C. The dotted line indicates the profile of the groove depicted in B. (**B**) 3D Model of the pink and green AE2 cells. The position of the segmented electron microscopic (EM) plane in A is indicated in black and the lateral luminal cell borders of the pink and green AE2 cells are indicated by turquoise and blue delineations, respectively (empty arrowheads; note that the positions do not correspond to the empty arrowheads in A). The pink AE2 cell has only one luminal surface. Note the abrupt appearance of a dense microvilli lawn on the luminal surface in some areas (asterisk). Filled arrowhead 5 indicates the region of the corresponding arrowhead 5 in A. Note also the surrounding basolateral microvilli, which may appear in larger groups. Two sites of basolateral groups of microvilli on the green cell are indicated (unnumbered filled arrowheads). Note the long and mostly smooth groove of the pink AE2 cell surface (dotted line). (**C**) Opaque 3D model of the pink AE2 cell and transparent models of the green AE2 cell and the yellow AE1 cell domain. The position of the segmented EM plane in A is indicated in black. The transparency of the yellow AE1 cell domain and the green AE2 cell enable visualization of intercellular microvilli. Numbered arrowheads correspond to the arrowheads in A: Filled arrowhead 2 and 3: Microvilli between the pink and green AE2 cells. Filled arrowhead 4: AE2 microvilli towards the yellow AE1 cell domain. The unnumbered filled arrowhead indicates another region with AE2 microvilli under the yellow AE1 cell domain. The lateral luminal AE2 cell borders are indicated by turquoise and blue delineations (cf. B). Empty arrowhead 1 refers to the corresponding empty arrowhead in A. Black arrows indicate the small parts of the cell where the cell extends beyond the available dataset. Scale bars: 1 μm.

**Figure 4. Luminal surface of AE1 cells.** (**A–C**) Images 1172, 1348 and 1520 of the dataset. Filled arrowheads 1 and 2 (**A**) indicate profiles of microvilli. Filled arrowheads 3 and 4 (**B**) indicate microvilli with a shared basis. Empty arrowheads 5 and 6 (**A**) and 7–10 (**C**) indicate profiles of plasma membrane protrusions filled with cytoplasm. The hash key indicates a region with smooth AE1 luminal surface. (**D**) Detail of the 3D model. Arrowheads and hash key refer to the particular profiles in A–C. The particular section planes 1172, 1348 and 1520 are labeled and emphasized by black color in the model. The different thickness of these black markings is caused by different segmentation intervals in these regions. Note the smooth surface of the cell in the center of the image and the surface enlargements around. Some of them seem to be concentrated in a surface cavity (asterisk). Scale bars: μm.

**Figure 5. Basolateral surface of AE1 cells.** (**A**) Detail of the segmented EM plane 1384 including parts of the yellow AE1 cell domain. At the basal side, small folds may appear which create a groove-like profile with the concavity pointing towards the interstitial space. The groove beyond the epithelium is filled with extracellular matrix (arrowhead 1). (**B**) A similar situation is found at the basal side of the blue AE1 cell domain, but here the groove is filled with capillary endothelium (arrowhead 2). Note the densely packed caveolae, which appear to be predominantly located in the basal compartment of the cell (asterisk). (**C**) View on the basal side of the 3D model. The model reveals that indeed the AE1 cell domains form grooves on their basal side, which are bordered by cellular folds. The groove in the AE2 cell surface, which has been already described in Figure 3, is also indicated by a dotted line. The EM plane 1384 is indicated by black color. Scale bars: 1 μm.

#### *2.4. Sites of Cell Contacts*

The AE1 cell domains contact each other with or without overlap. At a certain point, they may simply prod against each other (edge to edge), while a few micrometers further one cell domain slips under the other. Directly at the site of contact they may bulge out into the alveolus (Figures 6–8). On the basolateral side, cellular processes may interdigitate with the neighbor cell domain, while the luminal edges appear more regular (Figure 9).

At the contact site to AE2 cells, AE1 cells may simultaneously both crawl up and beneath AE2 cells. This is achieved by a y-shaped branching pattern with one branch crawling up and the other branch crawling beneath the AE2 cell. In 3D, this behavior reminds of a bowl partially enwrapping the AE2 cell (Figure 10). Additionally, the AE1 cells may send out thin, finger-like processes into the space between the AE2 cells and the underlying basal lamina. Such processes can be found in close proximity to AE2 microvilli (Figure 11).

The two AE2 cells contact each other edge to edge, with niches of microvilli facing each other (Figure 12).

**Figure 6. AE1/AE1 contact.** (**A**–**D**) show the x-plane reconstructions 1603, 1987, 2179 and 2251 of the data set (lateral view on the dataset) on the left side of the panel and a clipped model of the yellow and blue AE1 cell domains on the right side of the panel. The transect plane in the foreground corresponds to the EM plane on the left side. The site of cell contact is indicated by arrowheads. The distance between the different transect planes is indicated in micrometers between the models. Note how the yellow AE1 cell domain slips under the blue cell domain in A and C while the cell domains in B and D meet each other just edge to edge. Note also the bulging of the cell domains into the alveolar lumen at the contact site (asterisks on the EM images). Scale bars: 1 μm.

**Figure 7. AE1/AE1 contact.** (**A**,**B**) show the yellow and blue AE1 cell domain with their alveolar surfaces. On each image one of both cell domains is shown transparently (A: yellow, B: blue) to visualize overlap. Images (**C**,**D**) follow the same principle but show the basolateral surfaces. Scale bars: 1 μm.

**Figure 8. AE1/AE1 contact.** The image shows the contact of parts of the yellow and blue AE1 cell domains. The blue cell domain in the foreground is displayed transparently to enable visualization of the yellow AE1 cell domain. Note how the yellow AE1 cell domain slips beneath the blue cell domain. Scale bar: 1 μm.

**Figure 9. AE1/AE1 contact.** (**A**,**B**) again show the yellow and blue AE1 cell domain from a luminal (**A**) and a basolateral (**B**) perspective but without transparency. Note the irregular contact site on the abluminal side compared to the luminal side caused by interdigitating cell processes. Compare in particular the region emphasized by the ellipse. Scale bars: 1 μm.

**Figure 10. AE1/AE2 contact.** (**A**) 3D model of the two AE1 cell domains and AE2 cells. AE2 cell models are transparent to enable visualization of the yellow AE1 cell domain behind the AE2 cells. AE1 cells may branch at the edge of an AE2 cell and extend their branches beneath and over the AE2 cell to form a bowl-like structure that enwraps the AE2 cell partially. The yellow AE1 cell domain forms a bowl at the edge of the pink AE2 cell (behind the transparent AE2 cell model). Note also the y-shaped branching of the blue AE1 cell domain and its extension beneath the pink AE2 cell (arrow). The black line indicates the position of the transect image in B. (**B**) Reconstruction of the y-plane (lateral view on the dataset) at the position indicated by the black line in A. The profiles of the yellow AE1 cell domain and the pink AE2 cell are indicated by yellow and pink outlines. Note the y-shaped profile of the yellow AE1 cell domain. The surface of the yellow AE1 cell domain ("inner surface" of the bowl) looked at in A and C is indicated by arrowheads. (**C**) 3D model of the yellow and blue AE1 cell domain transected by the y-plane shown in B. The different angle of view compared to A facilitates the imagination of a bowl formed by the yellow AE1 cell domain. The arrowhead indicates where the image plane transects the bowl of the yellow AE1 cell domain. Scale bars: 1 μm.

**Figure 11. AE1/AE2 contact.** (**A**) Detail of segmented image 1208. The image shows the segmentation of the pink and green AE2 cells as well as of the yellow and blue AE1 cell domain. Note the very small profiles of the yellow AE1 cell domain (arrowheads 1–5) and the small profile of the blue AE1 cell domain (arrowhead 6). These will probably be overseen if only single images are investigated. If they are recognized, it will almost be impossible to assign them to different cells (compare B). Only the sequence of images reveals their belonging. (**B**) The same EM image as in A but without segmentations. (**C**) 3D Modell of the yellow and blue AE1 cell domain as well as the pink and green AE2 cell with a view on the basolateral cell surfaces. The 3D model reveals that the labeled profiles in A belong to small and thin AE1 processes that crawl along the surface of the pink AE2 cell. The numbered arrowheads correspond to the arrowheads in A. Note also the processes of the yellow cell domain along the green AE2 cell. Some of the processes are found in close proximity to AE2 cell microvilli (asterisks). Scale bars: 1 μm.

**Figure 12. AE2/AE2 contact.** (**A**) EM image 1112. The image shows the green (top) and pink (bottom) AE2 cell contacting each other. Both cells show niches with microvilli on their basolateral surface facing each other (filled arrowheads). Between both cells also parts of the yellow AE1 cell domain are found (asterisk). The alveolar lumen is indicated by the hash key and the basal lamina by an empty arrowhead. (**B**) 3D model of the pink and green AE2 cell as well as the yellow and blue AE1 cell domains. The image plane of A is indicated in black. Arrowheads correspond to the arrowheads in A. The borders of the luminal/abluminal surface of the AE2 cells are indicated by turquoise (pink cell) and blue lines (green cell) (cf. Figure 3). The pink AE2 cell and its luminal/abluminal border are displayed transparently to visualize the green AE2 cell and yellow AE1 cell domain behind as well as the luminal/abluminal border of the green AE2 cell. Note the well defined niches with microvilli on the basolateral surface of the green AE2 cell (black arrowheads). Scale bars: 1 μm.

#### **3. Discussion**

Volume electron microscopic (EM) techniques for 3D modeling of biological structures, including "conventional" techniques like single sectioning transmission electron microscopy (ssTEM) and "new" techniques like SBF SEM and FIB SEM, have been reviewed extensively recently, e.g., [9–13] and some of them have already been used for 3D reconstructions of lung structure: ssTEM [5,7,8,14]; electron tomography [15]; array tomography [16] and SBF SEM [6,11]. Even FIB SEM has been applied [17–19], but the study of Købler et al. was rather focused on testing an alternative method to diamond knife ultramicrotomy and transmission electron microscopic imaging of nanotubes; the review of Ochs et al. gave an outlook on the potential of this technique in lung research; and Hegermann et al. demonstrated a method for correlative light and electron microscopy. Kremer et al. [11] showed a FIB SEM-based reconstruction of a small part of a *human* A549 cell. Here, to our knowledge, we provide the first extensive study of the alveolar epithelial 3D ultrastructure based on FIB SEM in a *human* lung sample. We were able to reconstruct an almost complete AE2 cell with two adjacent AE1 cell domains as well as a part of a neighboring AE2 cell. The model shows detailed reconstructions of the epithelial surface, including a surfactant secretion pore on an AE2 cell, enlargements of the apical AE1 cell surface, long folds bordering grooves on the basal AE1 cell surface, AE1/AE1, AE1/AE2 and AE2/AE2 contact sites, basolateral microvilli pits at AE2 cells and small AE1 processes beneath AE2 cells. The functional relevance of these findings will be discussed below.

AE1 cells seem to be responsible for the majority of fluid transport across the alveolar epithelium [20–22]. According to Dobbs et al. [20] different sodium channels (highly selective Na+ channel (HSC), non-selective Na+ channel (NSC) and cyclic nucleotide-gated channel (CNG)), the cystic fibrosis transmembrane regulator (chloride channel), potassium and water (Aquaporin 5) channels and Na+/K+ATPases are involved in that. The surface enlargements of the AE1 cell domains shown here may serve as a membrane compartment housing the particular transport proteins and, thus, be the ultrastructural correlate of liquid absorbtion.

Interestingly, when looking at Figure 4, one gets the idea, that the surface in a septal concavity is rich in these surface enlargements, while they seem to be reduced or even absent on convexities. Concave niches are filled with the (liquid) hypophase of surfactant [1,7] and these "puddles" may be the sites where most of the fluid absorption takes place. Microvilli on AE1 cells have been described previously [20] and were also found during *cat* lung development [8,14].

Fixation (cf. the studies of Gil et al. [23], Oldmixon et al. [24] and Oldmixon and Hoppin Jr. [25] or long-term storage can lead to microscopic artefacts. The following arguments, however, indicate that the non-microvillus surface enlargements are a real biological phenomenon: (1) The cytoplasm within these excrescences is quite electron dense, while lytic cell parts regularly appear electron light. (2) The surface enlargements seem to be concentrated at certain spots, while other areas remain smooth. (3) Microvillus-like surface irregularities without obvious microfilaments have also been described by Mercurio and Rhodin [7] in the *cat*. (4) Surface irregularities on AE1 cells can also be seen on some scanning electron micrographs in textbooks or other articles [1,3,5,26,27].

Another explanation for the surface irregularities was given by Mercurio and Rhodin [7], who suggested that they are incorporated into the cell membrane during inflation. This was concluded, because they also found a cell with a rather smooth surface, where they thought that the flatness was caused by trapped air in the airspaces. If this interpretation is true, however, then also the basolateral cell membrane needs membrane reserves, since the apical and basolateral membranes are separated by tight junctions [28]; Sirianni et al. [5] also mentioned the existence of discontinuous tight junctions. Such reserves could be provided by the cytoplasmic folds of the basolateral cell membrane or by the AE1 cell branches beneath the AE2 cells shown by our models.

The basal AE1 cell membrane folds, however, could also serve as mechanical stabilization, i. e., anchoring the epithelium in the septal wall by interdigitation with the interstitium to resist shearing forces. This principle is well established for the skin, where connective tissue papillae or ridges of the dermal papillary layer interdigitate with the epidermis at the dermal-epidermal junction [29] (p. 147).

With respect to AE1/AE1 cell contacts in the *cat* lung, Mercurio and Rhodin [7,8] described the possibility that adjacent cells may show alternating overlap or meet edge to edge. Our data suggest that this is also true for the *human* lung, even if the images and models shown here only demonstrate a clear overlap with the blue cell domain on top of the yellow cell or edge to edge contacts (Figures 6 and 7). What is the functional relevance of this overlap? Overlap per se enhances mechanical stability but additionally enlarges the contact area; in particular it may enlarge the available "contact area" for important cell junctions like the relatively wide (see [1,30]) tight junctions and/or gap junctions, for review, see [31]. The latter have been described to be in close topographic relation to the occluding junctions [30]. Mercurio and Rhodin [7] suggested that the overlap may change during breathing.

With respect to the AE1/AE2 contacts, we could confirm and extend our SBF-based presumption and Sirianni et al's [5] data that AE1 cells may extend both under and on the AE2 cells. Our data set and model revealed a y-shaped branching pattern in 3D (cf. Figure 2 of [5]) and additionally added that AE1 cells may extend various thin foot processes under AE2 cells.

Branches beneath AE2 cells may serve as membrane reserve during inspiration (see above). This may also be an explanation for the foot processes, but these may also be structural correlate of cellular interaction: Sirianni et al. [5] demonstrated linkage of AE1 and AE2 cells with interstitial fibroblasts via holes in the basal lamina, which seem to be primarily located beneath AE2 cells and Nabhan et al. [32] demonstrated that Wnt signaling between fibroblasts and alveolar epithelial stem cells (a small fraction of distinct AE2 cells) is necessary to maintain their stem cell status. Eventually, the small AE1 processes "look for" those holes for linkage to interstitial fibroblasts. Alternatively, the close proximity of these processes to AE2 microvilli pits in some cases (see Figure 11) could indicate exchange of material (too large for gap junctional transport) from AE1 to AE2 cells or vice versa (or fibroblasts).

These microvilli pits of AE2 cells, however, were also found towards other cell types (i.e. the other AE2 cell or interstitial cells (see Figure 3). Interestingly, they were also found above an intact basal lamina (not shown). Proximity to cells and holes in the basal lamina suggests a role in intercellular communication, material exchange or sensory functions. Localization above a closed basal lamina may be the structural correlate of basal lamina turnover or the initiation of creating a hole for consecutive subepithelial communication.

Summing up, the current FIB SEM study of a *human* lung sample provided new insights into the *human* alveolar epithelial cell morphology and topography. Our model reveals detailed reconstructions of the alveolar epithelial surface, including a surfactant secretion pore on an AE2 cell, enlargements of the apical AE1 cell surface, long folds bordering grooves on the basal AE1 cell surface, AE1/AE1, AE1/AE2 and AE2/AE2 contact sites, basolateral microvilli pits at AE2 cells and small AE1 processes beneath AE2 cells. These data may serve as morphological blueprint for molecular investigations of alveolar epithelial biology.

### **4. Materials and Methods**


**4. Activity profiling of extracts**

An alternative technique to the time-consuming and expensive methods previously used for creating extensive collections of isolated and structurally characterized natural products [29] is screening the mixtures of compounds obtained from extracts of cyanobacteria strains [11]. Yet, obtaining extracts with potential biologically active novel compounds is not always simple from primary screenings. This probability can be predicted by comparing the ratio of the binding potencies at two receptor sites for a known selective ligand and for an extract by the "differential smart screens" method [30]. Furthermore, by means of a database of the usefulness of an extensive variety of identified bioactive compounds the analysis of drugs with the unknown process is imaginable. Therefore, information about previously unidentified compounds can be gained, which is precious for the antibiotic applications stated below [31]:

These techniques could lead to a novel understanding of the potential effects of untested compounds (or exposure to compounds not structurally analogous and, thus, not expected to act via the same biological target) [2].

Bioinformatics and proteomics experiments are used in studies at the mRNA (transcriptome) or protein (proteome) levels, which help with the identification *Cyanobacteria Natural Products as Sources for Future Directions in* Antibiotic *Drug Discovery DOI: http://dx.doi.org/10.5772/intechopen.106364*

of DNA binding sites of transcription factors [32] and the adjustment of biological functions, respectively, in order to characterize the complex organism responses to environmental stimulates [2]. Microarrays have been used for the identification of regulon members and stimulons by many groups in the transcriptome measurement level [33, 34].

Two-dimensional gel electrophoresis in which proteins are separated according to their molecular weight and isoelectric point, is useful in most cases, but intricate protein samples can also be analyzed using the liquid chromatography-tandem MS (LC-MS/MS) in which protein and peptide combinations are supplied to a mass spectrometer (MS) from a HPLC system. Isotopic dilution strategies on a MS instrument (e.g., isotope-coded affinity tags or ICAT) can be used for a comparative quantification of protein expression. ICAT approaches were advantageous when first released but are limited by their inability to analyze more than two conditions without a large amount of multiplexing [2, 35]. Currently, a developed version of the iTRAQ approach can analyze eight different conditions simultaneously. Despite all these tools, the most useful method would involve a concurrent quantification of the expression of all the genes and proteins of interest from a biological sample.

### **5. Natural products as pharmacological instruments**

Aside from their curative activity, natural bioactive products can operate as pharmacological instruments demonstrating novel physiological features [14]. Cyanobacteria are stubbornly obstinate to genetic manipulation, which is accessible only for a small number of strains [3]. The modularity in cyanobacterial PKS-NRPS gene clusters authorizes the heterologous expression of natural bioactive products and, thus, genetic manipulation for combinatorial biosynthesis of innovative hybrid chemical bioactive products [4]. The prosperous production of nonribosomal and ribosomal peptides in heterologous hosts permits the usage of other cyanobacterial natural bioactive products [3]. Cyanobacteria usually synthesize multiple variants of the identical natural bioactive product; this can be ascribed to a deficiency of the inactivity of the NRPS tailoring enzymes or NRPS biosynthetic pathways. The genetic basis for this modification of secondary metabolite gene clusters is probably controlled by gene duplications, gene deletions, recombination, sequential mutation followed by natural selection, and loss and gain of tailoring enzymes [36]. However, the evolutionary and adaptive importance of these processes is deficiently understood.

### **6. Which cyanobacteria phyla produce therapeutics?**

Throughout the prior decade, several natural bacterial compounds have been described, all of which originated from five bacterial phyla: Bacteroidetes (34 compounds), cyanobacteria (220), actinobacteria (256), proteobacteria (78), firmicutes (35), and four bioactive compounds from taxonomically unknown sources [37]. The variety of cyanobacterial natural bioactive products gathers > 1100 secondary compounds recognized with composite chemical structures, stated from different genera [3]. These metabolites represent a broad range of bioactivities including some that may be related to their natural environment (antibacterial, antifungal, antiviral, and cytotoxic) [29], but others demonstrate a clear pharmaceutical interest, for example, they can be used as anticancer agents, immunomodulators, or protease inhibitors [38]. Cyanobacteria exhibit different growth forms, from unicellular to filamentous or colonial forms, and depending on their environmental conditions they may be surrounded by a mucilaginous or gelatinous sheath [29]. The PKS and NRPS genes seem to be more widespread in undifferentiated filamentous and heterocystous cyanobacterial strains. Despite the current taxonomic instability within cyanobacteria, which makes assessing the actual occurrence of natural products difficult, cyanobacterial compounds are mainly obtained from the lyngbya, symploca, microcystis, nostac, and hapalosiphon (**Table 1**) [3, 37].

*Cyanobacteria Natural Products as Sources for Future Directions in* Antibiotic *Drug Discovery DOI: http://dx.doi.org/10.5772/intechopen.106364*

**Table 1.**

*Current status of potential cyanobacteria therapeutics.*


**2. State of the Art and Literature**

It hardly needs stating that our approach constructively engages with a host of literature in the fields of religious studies, ritual studies and death studies. In all three fields, the concept of space is either foundational or has become important in recent decades. The study of ritual, in particular, has had space and place at heart. Van Gennep [1909] (1960) notes that all rites of passage are accompanied by a territorial passage (e.g., carrying the bride over the threshold or a procession with the newly dead to the cemetery), whereas Smith (1987) even entitled his major study of ritual *To take place*. As we have indicated, in spite of all the interest in ritual space and place, the concept of ritual space needs further refinement. A good illustration of this is, for instance, Grimes (2014, pp. 256–62) long list of (alleged) "misconceptualizations" of ritual space in binary oppositions or Alles' (2017) rejection of too rigid understandings of borders and boundaries that define ritual space. This has become even more pressing in view of the pluralization of societies in Northwestern Europe (e.g., Baumann 1992). Post and Molendijk (2007, p. 279) point at "the rise of increasingly multireligious urban ritual spaces"; an example *par excellence* of these ritual spaces is public cemeteries. In this article, we look at the "pluralistic communities of the dead" (Laqueur 2015, p. 294) in Maastricht's municipal cemetery Tongerseweg from its establishment in 1812 until today. What have been the dynamics of this ritual space in a period of over two centuries?

In the fields of religious studies and death studies, the "spatial turn" (Knott 2010) has become something of an orthodoxy. Academic scholars of death too have taken note of the concept of deathscapes (Kong 1999; Maddrell and Sidaway 2010) and understand cemeteries as sequestered places (Hüppi 1968; Rugg 2000; Sörries 2009). Much of the current literature refers to Foucault's 1967

1 Regarding the historical material, we maintain, against some skeptical currents, the possibility of reconstructing the meanings of, and authorial intentions behind, historical sources, but we cannot embark on this discussion in the present context (see, e.g., Bevir 1999, ch. 2 for a robust defense).

talk (published much later as Foucault 1986) "Des espaces autres", in which he invents the category of "heterotopia". Heterotopias are "real places ... which are something like counter-sites, a kind of effectively enacted utopia in which the real sites, all the other real sites that can be found within the culture are simultaneously represented, contested, and inverted" (Foucault 1986, p. 24).

Foucault reflects in the short text twice explicitly on cemeteries as instances of heterotopias. In the present context, we cannot review or engage the rich literature that comments on Foucault's sometimes cryptic remarks (but see, e.g., Dehaene and Cauter 2008; Spanu 2020). Suffice it to say that we see our effort here as a constructive engagement with Foucault's theory: our attempt to combine symbolic anthropological and historical research strategies dovetails with Foucault's attempt to see the category of the spatial as the sedimentation of the historical. Our effort here tries to mine the potential of Foucault's approach even where he (in our view) underutilizes the potential of the new category: most importantly, Foucault still talks of the cemetery as a unitary space, "where each family possesses its dark resting place" (Foucault 1986, p. 25). Such depictions of the cemetery as a depot of (bourgeois) family history may reflect iconic French cemeteries such as Père Lachaise. Against this background, it is, we tend to think, doubly interesting to analyze with the case study of Tongerseweg Maastricht, a cemetery that followed at its inception French regulations, and has over the course of its 200-year history reached a point where it has to grapple with cultural and legal changes in Dutch society that have ended cemeteries' monopoly as mandatory resting places, with all the reverberations of that change.

Foucault himself allows for heterotopias to juxtapose "several sites that are in themselves incompatible" (Foucault 1986, p. 25), but does not relate this in any way to cemeteries (his examples are the theatre, the cinema and the garden) and curiously he does not, for all his usual interest in power constellations, present cemeteries or, for that matter, other heterotopias as historically contingent ritual spaces that are coproduced by different actors.

For all the heuristic value of Foucault's concept of heterotopia, our approach highlights that the cemetery is less of a "counter site" but very much a continuation of the struggles, identity formations, appeals and positionings that characterize our society. Moreover, our approach highlights the internal divisions in the cemetery and the complex relationship with communities and groups.

Cemeteries are, in the words of Tarrés et al. (2018, p. 12), "spaces where communities can express their identities and their construction of otherness." The modern cemetery is a social and public arena in which identities can be claimed in a more or less ostensive way, subsumed, suppressed, denied or contested (see also Francis et al. 2005). The "new model of the cemetery" emerged at the end of the eighteenth and even more markedly in the beginning of the nineteenth century (Ariès 1985, p. 216; cf. Etlin 1984; Worpole 2003). Under the influence of the Enlightenment, mainly for hygienic reasons, the graveyards were to be located outside of the towns. The new cemeteries had a predetermined layout of grave plots. Some were even parklike with a geometrical ordering of space, dividing it up into distinctive grave fields (Kolnberger 2018; Heemels 2010, pp. 127–28). The future cemeteries envisioned in the eighteenth century took terribly long to materialize (Cappers 2012, p. 241). In the years just before the French Revolution, it followed from the plans that "the topography of the cemetery reproduces society as a whole," according to Ariès (Ariès [1977] 1981, p. 503). In his view, the "primary purpose of the cemetery is to present a microcosm of society" (Ariès [1977] 1981, p. 503).2

This idea of the modern cemetery as a "mirror image" of society has been taken up in the literature on cemeteries (e.g., Kamphuis 1999, passim; Sörries 2009, p. 177). Heemels (2006) examined this with regard to the main cemetery of Roermond, another town in the province of Limburg (Netherlands), for the period between 1870 and 1940. He found that in the predominantly Catholic town, the social stratification was clearly reflected in the locations of burials around 1880, but less so around 1930,

2 Other phrases used by Ariès are that "the cemetery is the image of public society" (Ariès [1977] 1981, p. 503) and that "[t]he cemetery is a schematic image of the society" (Ariès [1977] 1981, p. 553).

partly because this was affected by the earlier purchase of family graves (Heemels 2006, pp. 154–58). Maastricht, with twice the number of inhabitants compared to Roermond, knew and knows greater diversity. As Francis et al. (2005, p. 195) note, "for members of minority and immigrant ethnic groups, cemeteries can play a significant role in creating a sense of community beyond the strictly familial.". We are interested in the question of how the cemetery inscribes the identities of various communities in spatial terms.

At the same time, we argue that the spatial order of a cemetery as a ritual space should not be considered as a mere reflection of society. Clifford Geertz (1973, pp. 93–5) distinction between a model *of* reality, in contrast to a model *for* reality is still helpful here. In other words, we need to distinguish what is from normative ideals, from what actors believe ought to be. How space in the cemetery is divided up in bounded areas can also speak to aspirations or to "an idealized conception of the social order" (Rugg 2013, p. 5) and feed back to the social relations of the living.

We want to make headway with an analysis of the complexities of such a process of "cocreation" of ritual space that gives full weight to the recognition of the cemetery being home to plural communities, whose diversity is reflected or enhanced, but also downplayed, mitigated or denied in the complex interactions that create the cemetery as a plural ritual space.

Those complexities have not been fully understood, even though helpful, partial observations have been noted in the literature. For instance, Sutherland (1978, p. 47) highlighted the interactive nature of the nexus between social organization and physical environment:

When we try to understand how societies use space, how they divide it up both conceptually and physically, we are looking at a combination of their form of social organization and their perception of the physical environment. Both of these elements, the social as well as the physical, will form a single interacting whole in which no one element can be said to determine the other.

Social identities or categories are marked by boundaries in physical space. They have to be understood in relative terms. The boundaries distinguishing one from the other are betwixt and between. They separate and connect at the same time. Because the boundaries need to stand out, being neither the one nor the other and both at once, and, therefore, are ambiguous, they happen to be addressed with ritual (Leach [1976] 1978, pp. 41–4). Identities are always relational: "the identity *is* in important senses the boundary" (Jenkins 2008, p. 142). Cemeteries do not passively "track" societal developments; they are constructs that have been and are continuously shaped, actively, on the basis of social, political and economic ideas and ideals at different levels of government and on the basis of the aspirations and ideals of their users. As a result of explicit or implicit contestation, the cemetery can be conceived of as an arena (Turner 1974, pp. 129–36). Cemeteries should be considered "temporal topographies" in which space and time coalesce (Nielsen 2017). "As liminal, betwixt-and-between sites where geography and chronology are reshaped and history is made spatial, cemeteries are places of social, religious and ethnic continuity and belonging" (Francis et al. 2005, p. 195). In order to tease out how cemeteries as ritual spaces produce communal identities, special attention should be given to processes of territorial segmentation.

The cemetery is often seen as the epitome of modernity. The churchyard, in contrast, has become associated with tradition. Rugg (2013, p. 9ff), however, argues that the binary opposition rests on stereotypes. She speaks of "false dichotomies" and relates that a churchyard can also be profitable and express social distinction, whereas a cemetery is not necessarily secular but can, in part or whole, be consecrated (ibidem). Although Rugg does not mention them, the stereotypical contrast fits well with Tönnies' notions of civil society (*Gesellschaft*) and community (*Gemeinschaft*), respectively (see Tönnies [1887] 2001). Cemetery Tongerseweg, as we will see, was also for the largest part consecrated.3

The present article demonstrates the need for and usefulness of a fresh look at the nexus of ritual space and cemeteries with a case study, the monumental Cemetery Tongerseweg in Maastricht, The Netherlands. Tongerseweg is a particularly rich case study due to it being a municipal, public cemetery in a culture defined by Roman Catholicism: the cemetery has its roots in the burial reforms ushered in under French rule during the Napoleonic era, is still in active use after two hundred years and currently undergoes a new wave of redevelopment. After a short overview of the history of the cemetery (3), we will analyze Cemetery Tongerseweg in four episodes (nineteenth century (4), early twentieth century (5), late twentieth century (6), future planning (7)) as a pluralistic ritual space that is continuously (re)constituted and (re)negotiated by administrators, users and their allies. We continue (8) with a discussion of the relationship between ritual spaces and communities in which we propose a new "Arena Model" of this dynamic relationship. We end with a brief reflection on the applicability of the Arena Model beyond the case of Maastricht (9).

## **3. Cemetery Tongerseweg, Maastricht**

Cemetery Tongerseweg was planned and realized in the early nineteenth century under French rule, in the name of the hygiene and societal reform. The decision to build a general cemetery to the west of the crowded medieval town center of Maastricht was made in 1805. The cemetery was to replace several cemeteries run by religious communities (Catholic, Lutheran, Calvinist, Jewish) in the old center and was to cater for the dead of all religions and social echelons (Minis 1994, 2012; Noten 1998). The French legal situation at the time was defined by a law from 1804, leaving municipalities the choice between separate cemeteries for the different religions or indeed a single communal one, with provisions for the different faiths. In that case, walls, hedges or canals should separate the different sections, which each should be given separate entrances.4 In Maastricht, this was seen as an opportunity to fuse the different cemeteries into a single one, following the French example (cf. Ariès [1977] 1981; Van Helsdingen 1987; for further details, see Cappers 2012, pp. 240, 253). In 1809, the site at the road towards Tongeren (in Belgium) was selected, then located in the village of Oud-Vroenhoven (Bruijnesteijn van Coppenraet and Denessen 2002, p. 21). The main, Catholic part of the cemetery was consecrated on the 27th of January 1812 by Hendrik Partouns, vicar of the Roman Catholic Saint-Nicholas Church (Minis 2012).

With growing needs, the cemetery was extended three times in the north-eastern direction, towards the city center of Maastricht (in 1857, 1910 and 1956; see Figure 1) and is today part of Maastricht's western perimeter. Each extension came with significant cemetery developments which related to changing religious, political, legal and economic circumstances. For instance, after the first extension, the Catholic dead could be buried in grave plots of three distinctive classes: the most expensive were alongside the pathways, whereas the cheapest plots were out of view and practically out of reach (Heemels 2010, p. 130). Furthermore, a Catholic chapel was added to the cemetery in 1885, founded by the Clarenboets family and representing the four Catholic parishes of Maastricht of that time (Van Raak 1995, p. 95). With the second extension, new grave fields were opened for Socialists and other "dissenters". The third extension drastically altered the plan of the cemetery; a new main entrance was created at its northern perimeter, next to parking lots along the street (Figure 1, C).

3 Cappers (2012, p. 247) explains that due to the Napoleonic Concordat of 1801, his agreement with the pope, the Roman Catholic Church regained its status as a State Church. This was also relevant for Maastricht, which was incorporated into the French department Meuse-Inférieure between 1795 and 1814.

4 *Décret impérial sur les sépultures* du 23 prairial an XII (12 June 1804), article 15: "Dans les communes où l'on professe plusieurs cultes, chaque culte doit avoir un lieu d'inhumation particulier, et dans le cas où il n'y aurait qu'un seul cimetière, on le partagera par des murs, des haies ou fossés, en autant de parties qu'il y a de cultes différents, avec une entrée particulière pour chacune, et en proportionnant cet espace au nombre habitants de chaque culte" (Fortuijn 1840, p. 309).

At present, over 125,000 people are buried in the over twelve hectares of consecrated and unconsecrated ground. As the numbers of traditional burials are decreasing, and the amount of people who opt for cremation is increasing, the story of the cemetery's development does not end here. A new business plan (Gemeente Maastricht 2018) prepares the cemetery for the future by making possible a fourth extension towards the Northwest. In the main section of this article, we will delve into these historical and future developments in more detail.

**Figure 1.** Schematic map of the development of Cemetery Tongerseweg (T.K.).


*Article* **High Hydrostatic Pressure-Assisted Enzymatic Hydrolysis A**ff**ect Mealworm Allergenic Proteins**

**Abir Boukil 1 , Véronique Perreault 1 , Julien Chamberland 1 , Samir Mezdour 2 , Yves Pouliot 1 and Alain Doyen 1,\***

Academic Editors: Przemyslaw Lukasz Kowalczewski and Antonio-José Trujillo Received: 12 May 2020; Accepted: 4 June 2020; Published: 9 June 2020

**Abstract:** Edible insects have garnered increased interest as alternative protein sources due to the world's growing population. However, the allergenicity of specific insect proteins is a major concern for both industry and consumers. This preliminary study investigated the capacity of high hydrostatic pressure (HHP) coupled to enzymatic hydrolysis by Alcalase® or pepsin in order to improve the in vitro digestion of mealworm proteins, specifically allergenic proteins. Pressurization was applied as pretreatment before in vitro digestion or, simultaneously, during hydrolysis. The degree of hydrolysis was compared between the different treatments and a mass spectrometry-based proteomic method was used to determine the efficiency of allergenic protein hydrolysis. Only the Alcalase® hydrolysis under pressure improved the degree of hydrolysis of mealworm proteins. Moreover, the in vitro digestion of the main allergenic proteins was increased by pressurization conditions that were specifically coupled to pepsin hydrolysis. Consequently, HHP-assisted enzymatic hydrolysis represents an alternative strategy to conventional hydrolysis for generating a large amount of peptide originating from allergenic mealworm proteins, and for lowering their immunoreactivity, for food, nutraceutical, and pharmaceutical applications.

**Keywords:** high hydrostatic pressure; mealworm proteins; enzymatic hydrolysis; allergenic proteins; proteomics analysis


**Acknowledgements**

I would like to thank National Research Foundation (NRF, grant no. 99270) and Nanotechnology Innovation Centre (UJ Water Node) for providing financial support and the University of Johannesburg for making this study possible by making laboratory facilities available.


The Toxicity of Environmental Pollutants

*Edited by Daniel Junqueira Dorta and Danielle Palma de Oliveira*

Published in London, United Kingdom

The Toxicity of Environmental Pollutants http://dx.doi.org/10.5772/intechopen.98127 Edited by Daniel Junqueira Dorta and Danielle Palma de Oliveira

#### Contributors

Benjamin Ampomah Asiedu, Ahmed Nasri, Mohamed Allouche, Amel Hannachi, Abdelwaheb Aydi, Patricia Aïssa, Hamouda Beyrem, Ezzeddine Mahmoudi, Bamba Massa Ismaë, Sorho Siaka, Saganuwan Alhaji Saganuwan, Çiğdem Sevim, Mehtap Kara, Selma Yazar, Peter Ostoich, Michaela Beltcheva, Roumiana Metcheva, Jose Antonio Heredia Rojas, Luciana Maria Saran, Ane Gabriele Vaz Souza, Tainá Carolini Maria, Lucia Maria Carareto Alves, Deepak S. Khobragade, Raymundo Erazo, Benito Buendía Quispe, Mahipal S. Sankhla, Pritam P. Pandit, Anubhav Singh, Anuj Sharma, Varad Nagar, Rushikesh L. Chopde, Kumud K. Awasthi, Garima Awasthi, Vinay Aseri, Rohit K. Verma, Sumit K. Choudhary, Daniel Junqueira Dorta, Raul Ghiraldelli Miranda, Carolina Ferreira Sampaio, Fernanda Gomes Leite, Flavia Duarte Maia

#### © The Editor(s) and the Author(s) 2022

The rights of the editor(s) and the author(s) have been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights to the book as a whole are reserved by INTECHOPEN LIMITED. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECHOPEN LIMITED's written permission. Enquiries concerning the use of the book should be directed to INTECHOPEN LIMITED rights and permissions department (permissions@intechopen.com).

Violations are liable to prosecution under the governing Copyright Law.

Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be found at http://www.intechopen.com/copyright-policy.html.

#### Notice

Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

First published in London, United Kingdom, 2022 by IntechOpen IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, 5 Princes Gate Court, London, SW7 2QJ, United Kingdom

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library

Additional hard and PDF copies can be obtained from<EMAIL_ADDRESS>
The Toxicity of Environmental Pollutants Edited by Daniel Junqueira Dorta and Danielle Palma de Oliveira p. cm. Print ISBN 978-1-80355-579-9 Online ISBN 978-1-80355-580-5 eBook (PDF) ISBN 978-1-80355-581-2


**5. Conclusions**

This study investigated the change in the optimum angle of knee extension force production due to growth. The results showed that the optimal angle is temporarily changed to the loose position of the quadriceps femoris muscle at approximately the same time as the increase in femur length in a previous study, and returned to the original within a few years. This suggests that the force–angle relationship temporarily changes with muscle elongation due to skeletal growth.

**Author Contributions:** Conceptualization, J.S. and T.M.; methodology, J.S.; formal analysis, J.S.; data curation, J.S. and A.S.; writing—original draft preparation, J.S.; writing—review and editing, S.T.; supervision, S.T.; project administration, S.I., H.S. and S.T.; funding acquisition, J.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a Grant-in-Aid from the Japan Society for the Promotion of Science Fellows (18J01881).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Review Committee on Human Research of Waseda University (2013-167).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We appreciate Shinoda for helping in recruitment.

**Conflicts of Interest:** The authors declare no conflict of interest.


**6.2** *Aerva lanata*

*A. lanata* (L.) A. L. Juss. ex Schult (Amaranthaceae) is a perennial herb, frequently becoming more or less woody at the base. The stems can be erect to prostrate, sometimes scrambling or climbing into other plants for support. It is widespread in the tropics and subtropics of Africa through Asia to the Philippines and New Guinea. It is used traditionally for treating cough, sore throat, indigestion, wounds, and diabetics and as a vermifuge for children [75]. The ethanol extract of aerial parts of *A. lanata* at 100 μg/mL significantly (\*\*\*p < 0.01) exhibited percentage decreased contraction in the isolated goat tracheal chain preparation model. Also in clonidine induced mast cell degranulation, the extract at 30 and 60 mg/kg administered orally, showed percentage protection of 64.2 and 68.9%, respectively [76].


**3. Results**

The distribution of ESA applications in the three observed dialysis centers can be seen in table 1. Totally 185 of 233 patients received at least one ESA dosage. The mean value of ferritin was 538 mg/L. The transferrin saturation (TSAT) did not differ significantly in the observed dialysis units.

Table 1. Application of ESA per dialysis unit and overall.

The mean weekly ESA dosage can be seen in table 2:

Table 2. Mean weekly ESA dosage

162 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

233 chronic hemodialysis patients were included in this clinical evaluation (dialysis center 1 (D1) n= 94, D2 n= 35, D3 n= 104 patients). 54.9% were male; the mean age was 63.8 years (range 22 - 89). The patients in all three centers were comparable with regard to gender distribution, mean age, mean time on dialysis, and distribution of underlying kidney disease. The clinical evaluation was carried out for 12 months retrospectively. Laborchemical parameters were estimated for hemoglobin (Hb) every 2 weeks (labanalyzer), CRP (turbidometry), albumin (alb; nephelometry) and ferritin (chemiluminescence technique) every three months. Serum iron (photometry) and transferrin (turbidometry) were necessary to calculate the transferrin saturation (every 4 weeks). Single pool kt/V was evaluated every 3 months with the Daugirdas technique (Daugirdas, 1993). Intraindividual variability of hemoglobin (Hbvar) was defined as the difference between minimal and maximal concentration (range) and by time to reach the target between Hb 6.8 mmol/l and 8.0 mmol/l within 9 months. Relevant changes in ESA dosages were defined as an elevation greater than two fold and lowering of a half of the

ESA dosage, the end of ESA application or the start with more than 4200 U/week.

Hemodialysis (HD) was performed by MTS 5008 (Fresenius Medical Care), low flux dialyser FX8, FX10 (helixone, Fresenius Medical Care), QB 300 ml/min, QD 500 ml/min, ultrapure dialysis fluid, online hemodiafiltration (olHDF) by MTS 5008 (Fresenius Medical Care; automatic procedure with factor 1.2), high flux dialyser FX 60, FX80 (helixone, Fresenius Medical Care), QB 300 ml/min, QD 350…..360 ml/min, QS 51….60 ml/min, ultrapure dialysis fluid and Nikkiso DBB 05 (Nikkiso Medical Ltd.), high flux dialyser FDY 15 G (PEPA, Nikkiso Medical Ltd.), QB 300 ml/min, QD 700 ml/min, QS 60 ml/min, ultrapure dialysis fluid (QB… blood flow; QD…dialysate flow; Qs…substitution flow). The group "mixed" contained patients started with HD and switched to olHDF (at least 6 months olHDF). We compared the mean values of collected serum parameters three times a month. Descriptive statistical evaluation was calculated by mean, standard deviation and significance by Wilcoxon test, correlation by Spearman rang correlation. The level of

The distribution of ESA applications in the three observed dialysis centers can be seen in table 1. Totally 185 of 233 patients received at least one ESA dosage. The mean value of ferritin was 538 mg/L. The transferrin saturation (TSAT) did not differ significantly in the

> **Application of ESA** 37 39.4 4 11.4 7 6.7 48 **20.6 without ESA At least 1 ESA dosage** 57 60.6 31 88.6 97 93.3 185 **79.4**

Table 1. Application of ESA per dialysis unit and overall.

The mean weekly ESA dosage can be seen in table 2:

**Dialysis unit Total 1 2 3 N % N % N % N %**

**2. Materials and methods**

significance was defined as p< 0.05.

**3. Results**

observed dialysis units.

In nearly all patients (98.9%) an adjustment of ESA dosage was essential. Relevant changes in ESA dosages were defined as an elevation greater than two fold and lowering of a half of the ESA dosage, the end of ESA application or the start with more than 4200 U/week.

The mean value of Hb (measured per patient over the whole study time) was 7.35 mmol/l (Table 3). In patients without ESA application during the 12 months study the mean value of Hb was larger (7.66 mmol/l) in comparison to patients with ESA dosage (7.27 mmol/l).

Table 3. Mean Hb concentrations

Influence of Online Hemodiafiltration on Hemoglobin Level,

Table 6. Relationship between treatment mode and Hb value

**the Hb-value**

**the Hb-value**

**HD Mean treatment efficacy (spkt/V) N**

Table 8. Single Pool Kt/V as a function of the treatment mode

0.66±0.28 mmol/l vs olHDF 0.53±0.16 mmol/l, p<= 0.05): Table 7.

**Intra-individual standard deviation of**

**Intra-individual standard deviation of**

**Treatment mode**

**HD**

**All patients**

**All patients**

**HDF**

mode

ESA-Dosage and Serum Albumin – A Retrospective, Multicenter Analysis 165

**HD Mean Hb [mmol/L] N** 40 32 74 146

**HDF Mean Hb [mmol/L] N** 15 . 4 19

**Mixed Mean Hb [mmol/L] N** 39 3 26 68

**Total Mean Hb [mmol/L] N** 94 35 104 233

In the olHDF group the intraindividual Hbvar was significantly lower than in HD (HD

Table 7. Intra-individual standard deviation of the Hb-values as a function of the treatment

In the subanalysis the single pool Kt/V (spkt/V) was >1.2 on average in all centers. But there is a significant improvement of spKt/V for olHDF compared to HD (p = 0.04): Table 8:

**HDF Mean treatment efficacy (spkt/V) N** 15 . 4 19

**Mixed Mean treatment efficacy (spkt/V) N** 38 3 26 67

**Dialysis unit**

**Mean** 7.37 7.16 7.23 **7.25 SD** 0.43 0.59 0.53 0.52

**Mean** 7.58 . 7.50 **7.56 SD** 0.38 . 0.25 0.35

**Mean** 7.56 7.22 7.41 **7.49 SD** 0.46 0.11 0.61 0.52

**Mean** 7.48 7.17 7.29 **7.35 SD** 0.44 0.57 0.55 0.52

**Dialysis unit**

**N** 40 32 74 146 **Mean** 0.69 0.51 0.71 **0.66 SD** 0.29 0.24 0.26 0.28

**N** 15 . 4 19 **Mean** 0.52 . 0.57 **0.53 SD** 0.11 . 0.30 0.16

**Dialysis unit**

**1 2 3**

**Mean** 1.46 1.32 1.55 **1.48 SD** 0.60 0.29 0.38 0.44

**Mean** 1.57 . 1.82 **1.62 SD** 0.17 . 0.52 0.28

**Mean** 1.48 1.16 1.45 **1.45 SD** 0.24 0.04 0.31 0.27

**Total**

**1 2 3 Total**

**1 2 3 Total**

Table 4 shows the intra-individual variability of hemoglobin (Hbvar):

Table 4. Means of haemoglobin variability

The relation between the treatment mode (HD, olHDF) and ESA dosage as well as Hb is shown in the tables 5 and 6:

Table 5. Relationship between treatment mode and required weekly ESA dosage

Hb was larger in the olHDF group and the required ESA dosage to reach the Hb concentration lower (Hb olHDF 7.56± 0.35 mmol/l, HD 7.25± 0.52 mmol/l, p= 0.01; ESA/week olHDF 4407± 4660 U/l, HD 6809±7293 U/l, p= 0.1): Table 6.

**ESA** 37 4 7 48 **without ESA Hb range (min-max) N**

**at least one ESA dosage Hb range (min-max) N** 57 31 97 185

**Total Hb range (min-max) N** 94 35 104 233

The relation between the treatment mode (HD, olHDF) and ESA dosage as well as Hb is

**HD All patients N** 40 32 74 146

**HDF All patients N** 15 . 4 19

**Mixed All patients N** 39 3 26 68

Hb was larger in the olHDF group and the required ESA dosage to reach the Hb concentration lower (Hb olHDF 7.56± 0.35 mmol/l, HD 7.25± 0.52 mmol/l, p= 0.01;

Table 5. Relationship between treatment mode and required weekly ESA dosage

ESA/week olHDF 4407± 4660 U/l, HD 6809±7293 U/l, p= 0.1): Table 6.

**Patients with at least one ESA dosage N** 22 28 69 119

**Patients with at least one ESA dosage N** 9 . 4 13

**Patients with at least one ESA dosage N** 26 3 24 53

**Dialysis unit**

**Mean** 1.98 1.35 1.93 **1.92 SD** 0.92 0.68 1.11 0.93

**Mean** 2.20 1.74 2.41 **2.23 SD** 0.73 0.85 0.88 0.86

**Mean** 2.11 1.70 2.37 **2.17 SD** 0.81 0.83 0.90 0.88

**Dialysis unit**

**Mean** 3608 6132 8833 **6809 SD** 5058 5511 8313 7293

**Mean** 6560 7009 9473 **8354 SD** 5217 5339 8248 7234

**Mean** 3515 . 7750 **4407 SD** 4160 . 5535 4660

**Mean** 5859 . 7750 **6441 SD** 3853 . 5535 4287

**Mean** 3505 3821 10378 **6147 SD** 3960 1500 9441 7313

**Mean** 5257 3821 11243 **7887 SD** 3775 1500 9314 7410

**1 2 3 Total**

**1 2 3 Total**

Table 4 shows the intra-individual variability of hemoglobin (Hbvar):

Table 4. Means of haemoglobin variability

shown in the tables 5 and 6:

Table 6. Relationship between treatment mode and Hb value

In the olHDF group the intraindividual Hbvar was significantly lower than in HD (HD 0.66±0.28 mmol/l vs olHDF 0.53±0.16 mmol/l, p<= 0.05): Table 7.

Table 7. Intra-individual standard deviation of the Hb-values as a function of the treatment mode

In the subanalysis the single pool Kt/V (spkt/V) was >1.2 on average in all centers. But there is a significant improvement of spKt/V for olHDF compared to HD (p = 0.04): Table 8:

Table 8. Single Pool Kt/V as a function of the treatment mode

Influence of Online Hemodiafiltration on Hemoglobin Level,

 **[g/l]**

**HDF albumin [g/l]**

**Mixed albumin [g/l]**

Table 12. Relationship between albumin levels and treatment mode

 **[mg/l]**

**[mg/l]**

 **[mg/l]**

Table 13. Relationship between CRP levels and treatment mode

dosage and Hbvar were reduced in comparison to the other centers.

**Mode**

**Mode**

**4. Discussion**

reduced ESA needs.

**HDF CRP**

**Mixed CRP**

ESA-Dosage and Serum Albumin – A Retrospective, Multicenter Analysis 167

**HD albumin** 40 32 74 146

**HD CRP** 39 30 71 140

Our retrospective analysis was performed in three different dialysis centers for 12 months. The D1 center had the largest percentage of patients treated with olHDF (olHDF+"mixed") (57 % in D1 vs. 9 % in D2, 29 % in D3). In D1 the lowest dosage of ESA to reach the Hb target was used (Table 2; D1 vs D2 p= 0.003; D1 vs D3 p< 0.0001), the smallest number of D1 patients were treated with ESA and the time in target was longer than in D2 and D3. In addition, it could be demonstrated that in D1 patients the frequency of adaptation of ESA

Concerning the ferritin values and the transferrrin saturation (TSAT) there were no noticeable differences between the observed centers. But the subanalysis shows a positive correlation of the overall TSAT values with the Hb values (p = 0.002, see Table 11) and a negative one with the mean ESA consumption (p = 0.05). These results comply with the expectation because an improved Hb value is connected with a larger TSAT level and

The treatment efficacy (single pool and equilibrated Kt/V; spKt/V, eKt/V), which was measured periodically in the 3 dialysis units, did not show any significant influence on ESA

**N**

**N**

**Dialysis unit**

**Mean** 39.89 39.35 38.58 **39.11 SD** 3.10 2.11 2.72 2.76

**N** 14 . 4 18 **Mean** 40.74 . 40.24 **40.63 SD** 2.32 . 2.17 2.23

**N** 39 3 26 68 **Mean** 40.51 40.35 39.24 **40.02 SD** 3.35 3.06 3.21 3.30

**Dialysis unit**

**Mean** 18.56 15.52 14.94 **16.07 SD** 26.70 8.82 10.05 16.26

**N** 12 . 2 14 **Mean** 10.81 . 4.81 **9.96 SD** 8.68 . 0.22 8.28

**N** 36 3 25 64 **Mean** 14.52 7.36 10.43 **12.59 SD** 10.79 2.55 7.81 9.66

**1 2 3 Total**

**1 2 3 Total**

Further analyses regarded the relationship between CRP and albumin. The tables 9 and 10 show the mean levels of CRP and albumin:

Table 9. Mean CRP level per dialysis unit and overall

Table 10. Mean albumin level per dialysis unit and overall

For all patients the Hb level was negatively correlated to CRP (r= - 0.24, p< 0.0005) and positively to Albumin (r= 0.30, p< 0.0001) and TSAT (r= 0.20, p< 0.005): see table 11:

Table 11. Correlation of the total values for CRP, Albumin and TSAT with Hemoglobin

In a subanalysis we found significantly larger albumin levels and lower CRP concentrations in olHDF vs HD (albumin olHDF 40.63+/-2.23 g/l, HD 39.11± 2.76 g/l, p< 0.05; CRP olHDF 9.96± 8.28 mg/l, HD 16.07 ± 16.26 mg/l, p< 0.05): Tables 12 and 13:

Table 12. Relationship between albumin levels and treatment mode

Table 13. Relationship between CRP levels and treatment mode


**Acronyms**

the cathodic one. On the other hand, a failure of the identification procedure, that is, a poor fit of the polarization curves with varying operating conditions, typically

*Identification by means of a hybrid PSO-DE algorithm run simultaneously on all polarization curves. Dashed*

*lines: experimental, continuous lines: optimized models (courtesy of Journal of Power Sources).*

The decarbonization of power grids and the development of electric vehicles and renewable resources are promoting researches on advanced fuel cells for both stationary and mobile applications. Several programs are running that aim at reducing costs and improve performance and duration. These researches can be strongly supported by numerical models which are the core of a computer-aided engineering approach capable of reducing tentative experimentation by selecting those solutions which result more competitive on the basis of analytical/numerical computations, both in terms of device design and its operation. Effective models must take into account all relevant chemical-physical-electrical quantities, their interdependence, and their evolutions. These models play an interactive game with diagnostic and measurement issues, because their reliability depends on their fitting to the cases under investigation and this occurrence involves the determination of the many parameters used in nonlinear equations. Design optimizations based on numerical procedures are strong tools not only in identifying device materials and geometries capable of competitive performance, but also in determining the whole FC system,

Fuel cell computational modeling confirms to be an important topic of applied research, involving multiphysics, multiscale problems which are still challenging for the researchers working on this subject, both at the scientific and industrial level.

This work was supported by MAESTRA 2011—"From Materials for Membrane-Electrode Assemblies to Electric Energy Conversion and Storage Devices," the 2011

hints at weaknesses in the model.

*Thermodynamics and Energy Engineering*

including static converters and system supervisors.

University of Padua Strategic Project cod. STPD11XNRY\_002.

**7. Conclusion**

**Figure 7.**

**Acknowledgements**

**44**

*Thermodynamics and Energy Engineering*

**References**

cr020718s

[1] Wang CY. Fundamental models for fuel cell engineering. Chemical Reviews. 2004;**104**:4727-4766. DOI: 10.1021/

*DOI: http://dx.doi.org/10.5772/intechopen.89048*

*Distributed and Lumped Parameter Models for Fuel Cells*

Science. 2011;**37**:204-220. DOI: 10.1016/j.

[10] Vilekar SA, Datta R. The effect of hydrogen crossover on open-circuit voltage in polymer electrolyte

membrane fuel cells. Journal of Power Sources. 2010;**195**(8):2241-2247. DOI: 10.1016/j.jpowsour.2009.10.023

[11] Mench MM. Fuel Cell Engines.

[13] Sundmacher K, Scott K. Direct methanol polymer electrolyte fuel cell: Analysis of charge and mass transfer in the vapor-liquid-solid system. Chemical Engineering Science. 1999;**54**:2927-2936. DOI: 10.1016/S0009-2509(98)00344-3

[14] Guo H, Ma C. 2-D analytical model

[15] Alotto P, Guarnieri M, Moro F. Optimal design of micro direct methanol fuel cells for low-power applications. IEEE Transactions on Magnetics. 2009a;**45**(3):1570-1573. DOI:

10.1109/TMAG.2009.2012745

methanol fuel cell for portable

Industrial Electronics. 2010;**57**: 1865-1873. DOI: 10.1109/ TIE.2009.2027916

[16] Guarnieri M, Di Noto V, Moro F. A dynamic circuit model of a small direct

electronic devices. IEEE Transactions on

[17] Kulikovsky AA. The voltage-current curve of a direct methanol fuel cell:

of a direct methanol fuel cell. Electrochemistry Communications. 2004;**6**:306-312. DOI: 10.1016/j.

elecom.2004.01.005

[12] Scott K, Taama W, Cruickshank J. Performance and modeling of a direct methanol solid polymer electrolyte fuel cell. Journal of Power Sources. 1997;**65**: 159-171. DOI: 10.1016/S0378-7753(97)

Hoboken: Wiley; 2008

02485-3

pecs.2010.06.001

[2] Carnes B, Djilali N. Systematic parameter estimation for PEM fuel cell models. Journal of Power Sources. 2005;

[3] O'Hayre R, Cha SW, Coltella W, Prinz FB. Fuel Cell Fundamentals. New

York: John Wiley & Sons; 2009

[5] Parthasarathy A, Srinivasan S, Appleby AJ, Martin CR. Temperature dependence of the electrode kinetics of oxygen reduction at the platinum/ Nafion® interface – A microelectrode

investigation. Journal of the

(01)00322-2

**47**

Electrochemical Society. 1992;**139**(9): 2530-2537. DOI: 10.1149/1.2221258

[6] You L, Liu H. A two-phase flow and transport model for the cathode of PEM fuel cells. International Journal of Heat and Mass Transfer. 2002;**45**(11): 2277-2287. DOI: 10.1016/S0017-9310

[7] Springer TE, Zawodzinski TA, Gottesfeld S. Polymer electrolyte fuel

Electrochemical Society. 1991;**138**: 2334-2341. DOI: 10.1149/1.2085971

[8] Sequeira C, Santos D. Polymer Electrolytes – Fundamentals and Applications. Abington, Cambridge, MA: Woodhead Publication; 2010

[9] Tsushima S, Hirai S. In situ diagnostics for water transport in proton exchange membrane fuel cell. Progress in Energy and Combustion

cell model. Journal of the

[4] Bard AJ, Faulkner LR. Electrochemical methods. In: Fundamentals and Applications. New York: John Wiley & Sons; 2006

**144**:83-93. DOI: 10.1016/j. jpowsour.2004.12.024